Posters 2019

Poster presentation will take place during coffee breaks.
See in the attached file (pdf file below) the presenting day for each poster.
Presenting day 1: Tuesday 11th June 2019
Presenting day 2 : Wednesday 12th June 2019
Presenting day 3: Thursday 13th June 2019
Posters in red have been selected for short talks.
Poster size: A0 (Max width 1 meter, Max height 2 meters; No landscape format please!)

Presenting Day Poster Number First Name Last Name Affiliation Title Authors Abstract (250 words max) Email address
1 1 Rita Soares Instituto de Medicina Molecular | João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa and Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Lisboa. Characterizing the role of mitochondrial dynamics in adult neural stem cells differentiation Rita Soares, Ana M. Sebastião, Sara Xapelli, Vanessa A. Morais Neural stem/progenitor cells (NSPCs) are found in discrete regions of the mammalian brain. During adulthood, NSCs can be a source of new neurons in neurological disorders, such as Parkinson’s disease and Alzheimer’s disease, and oligodendrocytes in demyelinating disorders such as Multiple Sclerosis. However, these newborn cells are not sufficient to overcome the neurological deficits involved by neuronal loss. Therefore, the identification of novel mechanisms responsible for modulating NSC fate represents a key issue for future brain repair strategies. Several studies suggest that mitochondria have an important role in regulating NSC differentiation and lineage determination. However, the molecular mechanisms involved in this regulation remain unknown. Hence, our work aims to dissect how mitochondria biogenesis, morphology and bioenergetics can modulate the NSC differentiation into neuronal or oligodendroglial lineages. For this, NSPCs were obtained by isolating subventricular zone (SVZ) and dentate gyrus (DG) cells from P1-3 mouse models. The isolated cells, grown in neurospheres, were plated under specific differentiation conditions giving raise to neurons, astrocytes and oligodendrocytes. Different parameters namely seeding density, culture conditions and number of passages were determined. Moreover, the multipotency of SVZ/DG-derived NSPCs, obtained from different passages, was also accessed. At present, our data reveals that expression of proteins involved in mitochondrial biogenesis and fusion/fission tend to change during NSPC differentiation, while mitochondrial network appears to become more fragmented with NSPCs differentiation. These results will pave the road towards novel findings concerning the role of mitochondrial dynamics in NSC fate.
1 2 Annina Denoth-Lippuner Brain Research Institute, University of Zurich, Zurich, Switzerland The iCOUNT: a novel tool to study the division history of stem cells Denoth-Lippuner A., Liang T., Jaeger B. N., Jessberger S. Cell proliferation is required for organ development, tissue homeostasis and repair. To allow for visualization and counting of previous cell divisions on a single cell level, we developed the iCOUNT: a system that is based on the recombination-induced tag exchange (RITE) of a stable and cell cycle-dependent protein. Using CRISPR/Cas9 we endogenously tagged different proteins with the iCount cassette encoding an inducible color switch. Thereby, the cells change their color in a cell cycle-dependent manner, allowing for live analysis of individual cell division history.

Testing the iCOUNT system in mouse and human embryonic and neural stem cells revealed a robust color exchange enabling precise counting for several cell divisions. Therefore, we generated a CRISPR knock-in mouse expressing the iCOUNT. Inducing Cre led to a change in color in all tissues analyzed. In the embryonic brain a gradual change was observed corresponding to the localization of the cells. Therefore, the iCOUNT provides a novel technique to analyze the division history of stem cells in vivo.

Using the iCount, we sorted neural stem cells based on their division history and used single nuclei RNA sequencing to identify transcriptional changes that occur with progressive cell divisions. Furthermore, we will use the iCOUNT mouse to study other stem cell compartments.

Taken together, the iCOUNT-system is a unique genetic tool that will not only allow to count divisions of somatic stem cells but also to identify cellular changes occurring with cell divisions and to compare those changes among different types of stem cells.
1 3 Aixa V. Morales Instituto Cajal (CSIC) SOXD TRANSCRIPTION FACTORS PROMOTE ACTIVATION OF QUIESCENT NEURAL STEM CELLS IN THE ADULT HIPPOCAMPUS Lingling Li1, Sara Muñoz1, Cristina Medina1, Carmen Córdoba1,María Ciorraga1, Elena Calleja1, Silvia Nicolis4, Véronique Lefebvre3, Helena Mira2 and Aixa V. Morales1.

1Instituto Cajal, (C.S.I.C), Madrid, Spain.
2Instituto de Biomedicina de Valencia, (C.S.I.C), Valencia, Spain.
3Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA.
4University of Milano-Bicocca, Milano, Italy.

Adult neurogenesis, a process of generating functionally integrated neurons throughout life, constitutes an important strategy to generate plasticity in the mature central nervous system. In the adult subgranular zone (SGZ) of the hippocampus, neural stem cells (NSCs) generate new granule cells via a well characterized cell lineage that includes a succession of intermediate progenitor cells (IPCs). The majority of NSCs in the SGZ niche are in a reversible state of quiescence, a situation that protects the cells from DNA damage and the population from depletion. However, little is known, of how the transition from quiescence to an active mitotic state is regulated.
Genes of the Sox family of transcription factors are essential during neurogenesis. In the developing spinal cord, Sox5 controls cell cycle exit of neural progenitors and dorsal interneurons specification, counteracting the Wnt signalling pathway. More recently, we have characterized that both SoxD genes (Sox5 and Sox6) are expressed in the majority of NSCs and in IPCs in the SGZ of adult mouse hippocampus and that Sox5 levels are increased in the activated proliferating NSCs.
Using inducible conditional mutant mice to specifically delete Sox5 and Sox6 expression in the adult neurogenic niches, we have determined that both factors are required for radial glial-like NSCs proliferation/activation and for the generation of new neurons in the adult SGZ. Moreover, we have determined that proneural gene Ascl1 (esential for NSCs activation) is severly downregulated in NSCs of the SGZ in Sox5 or Sox6 adult mutant hippocampus and that could be directly controlled by SoxD transciption factors. In summary, SoxD genes are required in NSCs for the transition from the quiescence to the activated mitotic state, an step essential to promote neurogenesis in a tightly regulated manner throughout adulthood.
1 4 Lachlan Harris The Francis Crick Institute Progressive changes to hippocampal neural stem cell quiescence ensure lifelong neurogenesis Lachlan Harris*, Marco Ortiz*, Francois Guillemot
*equal contributions

In most mammals, neural stem cells (NSCs) persist in the hippocampus where they continue to generate neurons (neurogenesis). The general pattern of hippocampal neurogenesis is that it occurs at high levels in young animals, rapidly declines and then is maintained at low levels throughout life.  In mice, the rapid decline in neurogenesis in juveniles, is due to the self-consuming divisions of NSCs. However, the mechanisms that underpin the relative maintenance of NSCs and neurogenesis in older animals are unknown. Here, we demonstrate that with age, NSCs acquire the capacity to return to quiescence – instead of differentiating. And secondly, NSCs that have never activated, progress into a deeper state of quiescence. Both of these processes are controlled by a common mechanism: the relative abundance of the transcription factor Ascl1, which in turn, is controlled by sonic hedgehog signalling and the activity of the ubiquitin ligase Huwe1. In the absence of these cellular and molecular changes, the NSC pool exhausts and neurogenesis ends prematurely. Together, these findings provide insights into how the regulation of NSC quiescence ensures lifelong neurogenesis.
1 5 Dorothea Schulte Institute of Neurology, University Hospital Frankfurt, Goethe University Posttranslational control of a neuronal fate determinant in the SVZ adult neurogenic niche  Jasmine Kolb, Tanja Müller, Ann-Christin Hau, Marie Anders-Maurer, Christian Behrends and Dorothea Schulte Adult neurogenesis is regulated by stem cell niche-derived extrinsic factors and cell-intrinsic regulators, yet the mechanisms by which niche signals impinge on the activity of intrinsic neurogenic transcription factors remain poorly defined. The atypical TALE-homeodomain transcription factor MEIS2 is an essential regulator of adult subventricular zone (SVZ) neurogenesis that enables chromatin decompaction and effective transcription of neuron-specific genes by facilitating the assembly of a PBX1-PAX6-PARP1/ARTD1 containing complex at promoter-proximal regions of these genes. Here we show that MEIS2 itself is under dual posttranslational regulation in the SVZ / olfactory bulb neurogenic system. Firstly, MEIS2 protein is cytoplasmic in undifferentiated neural cells but accumulates in the cell nucleus in response to down-regulation of EGFR signaling. Nuclear accumulation is mediated by methylation of MEIS2 on a conserved arginine and involves direct competition between the nuclear export receptor CRM1 and the MEIS dimerization partner PBX1 for association with MEIS2. Secondly, cytoplasmic MEIS2 is fragmented in a process that is sensitive to phosphorylation at several positions within the MEIS2 polypeptide and blocked by heterodimer formation with PBX1. We further link these processes to the formation of glioma, highly aggressive glial tumors that are thought to originate from the SVZ stem cell niche. Collectively, our results establish MEIS2 as a hub that integrates extrinsic signals and translates them into a neurogenic transcriptional program in the SVZ and suggest that MEIS2 deactivation may indirectly favor gliomagenesis.
1 6 Nathalie Coré CNRS UMR7288 Control of neural stem cell specification in the postnatal forebrain by antagonist function of Vax1 and Pax6  Nathalie Coré, Andrea Erni, Christophe Beclin, Eva Hennen and Harold Cremer  Neural stem cells (NSCs) in the postnatal mouse ventricular/subventricular zone (V/SVZ), that generate different types of interneurons for the olfactory bulb, are highly heterogeneous. Depending on their location along the ventro-dorsal axis of the lateral ventricles, they preferentially give rise to distinct neuronal subclasses with defined positions, connectivity or neurotransmitter phenotypes. Understanding the molecular mechanisms that determine the generation of the distinct neuron populations at the NSC level will be essential for directing the differentiation of NSCs into defined neuronal cell populations in therapeutic contexts.
We found that the homeodomain transcription factor (TF) Vax1 is expressed in the lateral V-SVZ stem cell compartment in a ventro-dorsal gradient, suggesting a role in stem cell patterning and determination of neuronal phenotype. We used postnatal in vivo electroporation to miss-express Vax1 in the dorsal NSC aspects of the V-SVZ. Overexpressing Vax1 in dorsal or dorso-lateral NSCs led to a decrease in the dopaminergic interneuron population in the olfactory bulb. This phenotype is accompanied by a reduction of the pro-dopaminergic TF Pax6 in the stem cell compartment, suggesting that dopaminergic fate repression by Vax1 occurs through repression of Pax6. Moreover, conditional inactivation of Vax1 in NSCs along the lateral SVZ led to a reduced number of Calbindin+ neurons, showing that Vax1 is necessary for the production of this sub-set of OB neurons.
This shows that antagonistic interactions between Vax1 and Pax6 control neuronal phenotype along the dorso-ventral axis of the forebrain stem cell compartment. If these interactions are direct or implicate intermediate regulators like, for example, miR-7a is currently under investigation.
1 7 Luca Bonfanti University of Turin Greater occurrence of cortical layer II “immature” neurons in mammals with expanded neocortex Chiara La Rosa, Francesca Cavallo, Alessandra Pecora, Ugo Ala, Juan Nacher, Bruno Cozzi, Chet Sherwood, Irmgard Amrein, Luca Bonfanti Doublecortin-positive (DCX+), PSA-NCAM+ cortical neurons were discovered in the rodent piriform cortex. They are generated pre-natally, then continue to express markers of immaturity (non-newly generated immature neurons, nng-INs), thus potentially representing a reservoir of young neurons in the adult brain (Gomez-Climent et al, 2008; Piumatti et al, 2018). In some mammals, they extend into neocortex; the hypothesis has been advanced that nng-INs might be more important in large-brained mammals characterized by decline of active adult neurogenesis (Palazzo et al., 2018; Parolisi et al. 2018).
We collected brains from 12 mammals (4 entire hemispheres in 10 animal species, at different ages; total: 84 brains) belonging to 8 orders and endowed with different neuroanatomy, lifespan, ecological niche. Occurrence of nng-INs in paleo- and neo-cortex, their morphology (type 1, small-bipolar; type 2 cells, large-ramified), and amount (linear density: cells/mm of cortical layer II perimeter) were evaluated at 4 representative brain levels, previously established to be comparable. Cell proliferation (Ki-67 antigen, PCNA, BrdU), and immaturity/maturity markers (DCX, PSA-NCAM, NeuN) were also employed. All non-rodents species hosted nng-INs in the neocortex, with remarkable heterogeneity as to their relative amount: cortical layer II linear densities increase in neocortex more than in paleocortex in mammals with a large, gyrencephalic brain (almost an order of magnitude from mice to chimpanzee). By contrast, morphology, phenotype, non-proliferative and immaturity features were rather constant. We show that nng-INs are a well preserved feature in mammals, with far higher importance in the expanded cortex of large-brained species.
1 8 Farah Alammari Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Building, S Parks Road, Oxford OX1 3PT, UK The role of the transcription co-regulator KAP1 and the lncRNA Paupar in postnatal/adult subventricular zone development Farah Alammari, Francis Szele The function of many transcriptional regulators and of long non-coding RNAs in neural development and in the SVZ are poorly understood. KAP1 is an important transcriptional and epigenetic regulator that mediates silencing and heterochromatin formation. Here, we studied the function of KAP1 on postnatal and adult SVZ neurogenesis. We showed that knockdown of KAP1 in vivo disrupted normal SVZ neurogenesis and reduced the number of newborn neurons in the olfactory bulb.  We also showed that KAP1 directly binds to Paupar, a vertebrate conserved lncRNA and forms a trimeric complex with Pax6. Similar to Kap1, we showed that Paupar is necessary for OB neurogenesis as Paupar knockdown decreased the number of newborn OB neurons. In both loss-of-function studies newborn OB granule neurons appeared to have delayed morphological differentiation. Neither Kap1 or Paupar loss affected cell death. We have however generated data suggesting Paupar and Kap1 may regulate stem cell maintenance and self-renewal in vivo. Finally, we found that KAP1 and Paupar co-regulated genes important in self-renewal and differentiation. Together, these results provide understanding about the role of the transcription coregulator KAP1 and the lncRNA Paupar in SVZ neurogenesis.
1 9 Isabelle Blomfield The Francis Crick Institute Id4 eliminates the pro-activation factor Ascl1 to maintain quiescence of adult hippocampal stem cells Isabelle Blomfield, Brenda Rocamonde, Maria del Mar Masdeu, Eskeatnaf Mulugeta, Stefania Vaga, Debbie L. C. van den Berg, Emmanuelle Huillard, François Guillemot, Noelia Urbán Quiescence is essential for the long-term maintenance of adult stem cells and tissue homeostasis. However, how stem cells maintain quiescence is still poorly understood. Here we show that neural stem cells in the dentate gyrus of the adult hippocampus actively transcribe the pro-activation factor Ascl1 regardless of their activated or quiescent states. We found that the Inhibitor of DNA binding protein Id4 eliminates Ascl1 protein in neural stem cell cultures. Id4 sequesters Ascl1 heterodimerisation partner E47, promoting Ascl1 protein degradation and neural stem cell quiescence. Accordingly, elimination of Id4 from stem cells in the adult hippocampus results in abnormal accumulation of Ascl1 protein and premature stem cell activation. Our results highlight the importance of non-transcriptional mechanisms for the maintenance of neural stem cell quiescence and reveal a crucial role for Id4 as a quiescence-inducing factor, in sharp contrast with its role of promoting the proliferation of embryonic neural progenitors.
1 10 Sebastian  Arredondo Institute of Biomedical Biosciences, Universdad Andrés Bello Wnt5a promotes adult hippocampal neurogenesis by non-canonical Wnt signaling Sebastian B. Arredondo, Fernanda G. Guerrero, Andrea Herrera-Soto, Joaquin Jensen-Flores, Daniel B. Bustamante, Alejandro Oñate-Ponce, Pablo Henny, Manuel Varas-Godoy, Nibaldo C. Inestrosa and Lorena Varela-Nallar Adult hippocampal neurogenesis is regulated by the Wnt signaling pathway, but little is known about the endogenous Wnt ligands involved. The binding of a Wnt ligand to its receptor and co-receptor can trigger the canonical Wnt/β-catenin signaling pathway or the non-canonical Wnt/PCP or Wnt/Ca+2 signaling cascades. Here, we investigated the role of Wnt5a on adult hippocampal neurogenesis. This ligand is present in the adult hippocampus and is required to maintain dendritic architecture of hippocampal neurons. We determined that Wnt5a knockdown in the adult mouse dentate gyrus by lentivirus-mediated shRNA reduced neurogenesis and impaired dendritic development of adult-born neurons. In addition, in cultured adult neural progenitor cells (aNPCs) isolated from the hippocampus of adult mice, Wnt5a knockdown reduced neuronal differentiation and morphological development of aNPCs-derived neurons, while treatment with recombinant Wnt5a promoted neuronal differentiation and induced morphological development of newborn neurons. Interestingly, astrocytic differentiation was not affected by expression of shWnt5a or by treatment with recombinant Wnt5a, suggesting that Wnt5a does not affect fate commitment. By using specific inhibitors, we determined that Wnt5a signals through CaMKII to induce neurogenesis, and promotes dendritic development of newborn neurons through activating Wnt/JNK and Wnt/CamKII signaling. Our results indicate that Wnt5a ligand is a niche factor in the adult dentate gyrus that promotes neuronal differentiation and development through activation of non-canonical Wnt signaling pathways.

Supported by: Fondecyt 1190461 and Núcleo UNAB DI-4-17/N (LVN); Conicyt 21151115 (SBA).
1 11 Diogo Lourenço (1) Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal; 
(2) Instituto de Medicina Molecular | João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal;
Cannabidivarin and Capsaicin promote neuronal differentiation of neural stem cells Diogo M. Lourenço (1,2), R. Soares (1,2), Rui S. Rodrigues (1,2), A. M. Sebastião (1,2), S. Xapelli (1,2) Adult neural stem/progenitor cells (NSPC) with multipotent and self-renewing properties can be mostly found in two neurogenic niches, the Subventricular Zone (SVZ) and the Dentate Gyrus (DG) of the hippocampus. Cannabinoids have been shown to play pivotal roles in different neurogenic stages, namely in differentiation and maturation of NSPC. In this work we aimed at unravelling the role of cannabidivarin (CBDV), a non-psychoactive phytocannabinoid, homolog of cannabidiol, with high affinity for the vanilloid receptor 1 (TRPV1), on SVZ postnatal neurogenesis.
SVZ neurospheres were prepared from C57BL/6J (WT) mice pups (P1-3) and were incubated for 2 days with CBDV and Capsaicin (TRPV1 agonist). Three groups were tested: 1) control (no drugs); 2) CBDV (100nM; 300nM; 1µM); 3) Capsaicin (3µM; 10µM; 30µM). Immunocytochemistry against mature neurons (NeuN) and oligodendrocyte progenitor cells (NG2) was used to evaluate the effect of CBDV and Capsaicin on neuronal differentiation.
Here we show that SVZ neurospheres treated for 2 days in vitro (n=4) with 1µM of CBDV or 30µM Capsaicin show a significant increase in the number of NeuN-positive cells (p<0.05). No differences were found regarding the number of NG2-positive cells at any concentration when compared with the control condition. These results show that CBDV and Capsaicin promote neuronal differentiation at 2 days in vitro in SVZ cultures.
This work will allow determining whether CBDV or Capsaicin, most probably trough the activation of TRPV1, can modulate postnatal neurogenesis or oligodendrogenesis and will be important for future brain repair strategies.
1 12 Alexandra Pötzsch DZNE L-lactate acts as a pro-proliferative cue for adult hippocampal precursor cells in vitro Alexandra Pötzsch, Odette Leiter, Sara Zocher, Stefanie Bernas, Gerd Kempermann
We add new evidence to the hypothesis that in the adult brain, the metabolite L-lactate can influence cellular homeostasis by proposing such a role in the context of adult hippocampal neurogenesis. Neural precursor cells (NPCs) in the adult hippocampus can adapt their behavior upon changes in their microenvironment, for instance the availability of metabolites. L-lactate has both energetic and signaling properties and shows locally fluctuating concentrations governed by diverse stimuli (i.e. exercise, neuronal excitation), which also regulate adult neurogenesis. Studying how L-lactate affects NPCs in vitro, we demonstrated that L-lactate had a pro-proliferative effect on NPCs that depended on both active transport via monocarboxylate transporters (MCTs) and the activation of lactate receptor HCAR1. The increased proliferation was not linked to amplified mitochondrial respiration. Instead, L-lactate deviated glucose metabolism in that glycolysis was decreased and G6PD activity increased indicating augmented pentose phosphate pathway action. Furthermore, Erk1/2 phosphorylation and HCAR1 dependent PI3K/Akt signaling were induced by L-lactate. Our results show that L-lactate availability is linked to the proliferative potential of NPCs and demonstrate that a broader understanding of metabolite signaling properties is crucial to comprehend the link between metabolism, signaling, cellular behavior and plasticity in the adult hippocampus.
1 13 Gontzal García del Caño Universidad del País Vasco / Euskal Herriko Unibertsitatea Silencing of Phospholipase C-β1 expression impairs AraC-induced neuronal differentiation of NT2 cells Isasti A, González-Burguera I, Borrega L, Saumell M, Barrondo S, Sallés J, García del Caño G, López de Jesús M Phospholipase C-β1 (PLCβ1) is the predominant PLCβ1 in human cerebral cortex. The time course of PLCβ1 expression in the human cortex during pre- and postnatal development (1) and the fact that knocking out Plcb1 gene in mice disrupts cortical development and alters neurogenesis and migration of new born granule neurons of the adult hippocampus (2,3) indicates a critical role for PLCβ1 in neuronal development and differentiation. Using the NT2 cell line as a well characterized model of neuronal differentiation (4), we have recently shown that acquisition of neuronal fate and upregulation of the neuron-specific markers NF200 and β-III-tubulin after exposure to cytosine-β-D-arabinofuranoside (AraC) is preceded by a drastic increase of PLCβ1 protein expression levels (5), suggesting that neuronal differentiation of NT2 progenitors requires PLCβ1 upregulation. To test this hypothesis, we have investigated the effects of PLCβ1 silencing on the expression of neuronal markers during AraC-induced neuronal differentiation. Our results show that knocking down of PLCB1 gene, using either shRNAs or siRNAs, impairs AraC-induced cell morphological differentiation and upregulation of NF200 and β-III-tubulin proteins, which demonstrates that PLCβ1 is necessary for AraC-induced neuronal differentiation of NT2 cells.
1. López De Jesús et al. (2006). Neurochem Int, 49:72-79.
2. Spires et al. (2005). Cerebral Cortex, 15:385.393.
3. Manning et al (2012). Hippocampus, 22:309-319.
4. Gonzalez-Burguera et al. (2015). Stem Cell Res, 16:541-551.
5. Gonzalez-Burguera et al. (2011). SENC congress, 28-30 september 2011.
1 14 Gregor-Alexander Pilz University of Zurich Diversity of neural stem cells in the adult hippocampus Gregor-Alexander Pilz1, Sara Bottes1, Baptiste N. Jaeger1, David J. Jörg2, John D. Cole1, Merit Kruse1, Fritjof Helmchen3, Benjamin D. Simons2, Sebastian Jessberger1#

1Laboratory of Neural Plasticity, Faculties of Medicine and Science, Brain Research Institute, University of Zurich, 8057 Zurich, Switzerland. 3Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge CB3 0HE, UK; Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK; Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 1QR, UK. 3Laboratory of Neural Circuit Dynamics, Faculties of Medicine and Science, Brain Research Institute, University of Zurich, 8057 Zurich, Switzerland. 
Neural stem cells (NSCs) generate new granule neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. To address this stem cell property, we here used chronic in vivo 2-photon microscopy and followed single NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI Family Zinc Finger 1 (Gli1) or Achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny for up to 3 months, we identify a population of Gli1-targeted NSCs showing long-term self-renewal. In contrast, Ascl1-targeted NSCs undergo limited cell divisions before they become exhausted, as previously described by our lab. Using protein expression profiling (4i iterative stainings) and single cell RNA sequencing (sc-RNAseq) we show that Gli1- and Ascl1-targeted NSCs and their progeny have highly similar transcriptional profiles, supporting the presence of heterogeneous NSC populations with distinct behavioral properties. Using longitudinal observations of single NSCs and their progeny we here identify functional diversity of NSCs and provide the cellular framework how NSCs generate new neurons throughout life in the adult hippocampus.
1 15 Filippo Michelon 1 Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano (Turin), Italy; Department of Life Sciences and Systems Biology (DBIOS), University of Turin, Turin, Italy

2 Université de Lille, Inserm, CHU Lille, UMR-S 1172, laboratoire du développement et plasticité du cerveau neuroendocrine, centre de recherche Jean-Pierre Aubert, Lille, France

3 Université Côte d’Azur (UCA), CNRS, Inserm, iBV, Nice, France

COUP-TFI/Nr2f1 overexpression in the GLAST-lineage perturbs the migration and morphology of a subpopulation of hippocampal granule neurons Michelon F 1,2*, Bonzano S 1*, Crisci I 1, Ternier G 2, Giacobini P 2, Studer M 3, De Marchis S 1 *equal contribution In the mammalian brain, newborn granule neurons develop throughout life from multipotent adult neural stem cells (NSCs) located in the subgranular zone of the dentate gyrus. Intrinsic/extrinsic factors and external cues control their multistep developmental process, eventually leading to their functional integration in hippocampal circuits. Among the intrinsic factors regulating this process, we recently demonstrated that the transcriptional regulator COUP-TFI (also known as Nr2f1) acts as a central player in controlling adult hippocampal NSC fate choice. Here we used a tamoxifen-inducible mouse line to study the phenotype of newborn neurons following conditional COUP-TFI overexpression in the GLAST-lineage. Our data suggest that COUP-TFI regulates migration and dendritic development of newborn neurons within the adult mouse dentate gyrus. Indeed, COUP-TFI overexpression results in the mislocalization of a subpopulation of newborn Prox1+ granule neurons that migrate to ectopic sub-regions (i.e. the outer granule cell layer and the hilus). Moreover, morphological analysis showed that misplaced cells bear an altered dendritic architecture compared to “normotopic” granule cells. Overall, these findings indicate a new role of COUP-TFI in adult neurogenesis by controlling the proper positioning and morphological maturation of newborn granule neurons. Future studies will be aimed to dissect the molecular mechanisms through which COUP-TFI exerts its role and to investigate the possible involvement of COUP-TFI in neurological disorders (e.g. epilepsy) characterized by ectopic granule cell migration and alterations in granule neuron morphological features.
1 16 Sophie Austin The Francis Crick Institute Dose Dependent Effects of Canonical Wnt Signalling in Quiescent Adult Hippocampal Stem Cells Sophie Austin, Noelia Urbán, Isabelle Blomfield, François Guillemot  Adult hippocampal stem cells (AHSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood/memory. AHSCs are largely quiescent and stimulated to activate and produce neurons by surrounding signals. Although canonical Wnt signalling modulates adult neurogenesis it is unclear if/how it directly affects AHSCs. Our aim is to characterise the role of canonical Wnt signalling in active and quiescent AHSCs.

Using BATGAL Wnt reporter mice we find that both active and quiescent AHSCs respond heterogeneously to Wnt signalling in vivo. Similarly, using an in vitro model of active and quiescent AHSCs, we find that AHSCs in both states respond heterogeneously to Wnt. This suggests that AHSCs’ response to Wnt signaling is independent of their activation state both in vivo and in vitro. In vitro we find that intact canonical Wnt signaling is not essential for quiescent AHSC activation or for the maintenance of their quiescent state. Strong Wnt activation initiates neuronal differentiation in both active and quiescent culture conditions. Milder Wnt activation also promotes differentiation of active AHSCs but, interestingly stimulates proliferation in quiescent AHSCs. Thus, lower levels of Wnt signalling can promote quiescence exit. Overall this indicates that levels of canonical Wnt signalling are important in determining proliferation vs neuronal differentiation responses in quiescent AHSCs. We are now investigating the molecular mechanisms driving these differential effects in response to different canonical Wnt signalling levels. We are also currently modulating Wnt in AHSCs in vivo to determine how active and quiescent AHSCs are affected by different Wnt levels.
1 17 Miguel V. Guerra 1Instituto de Ciencias Biomedicas, Facultad de Medicina y Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago, Chile. 2Centro de Envejecimiento y Regeneración (CARE-UC), Facultad de Ciencias Biológicas, P. Universidad Católica de Chile Epigenetic changes during adult hippocampal neurogenesis Miguel V. Guerra 1, Brigitte van Zundert 1,2* and Lorena Varela-Nallar 1*  In the dentate gyrus of the adult hippocampus, the generation of new neurons from neural stem cells (NSCs) involves proliferation, differentiation and maturation of newborn cells. These stages are controlled by transcriptional and epigenetic mechanisms. Regarding the latter, little is known about the role of histone post-translational modifications (HPTMs) during hippocampal neurogenesis. HPTMs can either promote or inhibit transcription depending on the modification, and the histone residue being modified. Here we studied HPTMs during the stages of neurogenesis in the dentate gyrus of 2 month-old mice. A strong increase in epigenetic repressive marks was observed in progenitor cells and proliferating neuroblasts compared to quiescent NSCs and mature neurons. No changes were observed in histone activation marks studied. In addition, HPTMs were evaluated in proliferating and differentiated cultured adult neural progenitor cells (aNPCs), isolated from adult mouse hippocampus. A decrease in repressive marks was observed during neuronal differentiation by immunostaining and immunoblot. Interestingly, no changes in repressive histone marks were observed in aNPC-derived astrocytes. Finally, we determined a significant decrease in neuronal differentiation of aNPCs in the presence of a small molecule that inhibits histone methyltransferases. Our results suggest that epigenetic repression through HPTMs regulates early stages of adult hippocampal neurogenesis.
Supported by: Fondecyt 1190461 (LVN), Fondecyt 1181645 and CARE-UC AFB 170005 (BvZ), Nucleo UNAB DI-4-17/N, Conicyt 21181497 (MGV).
1 18 Sofia Madsen Department of Physiology, University of Lausanne Generating an endogenous reporter mouse to study lipid droplets in neural stem cells Sofia Madsen, Mergim Ramosaj, Denise Tavel and Marlen Knobloch Lipid metabolism plays an important role in the regulation of neural stem cells (NSCs): Build-up of lipids through de novo lipogenesis seems to be crucial for NSC proliferation (Knobloch et al. 2013), whereas the break-down of lipids via fatty acid beta-oxidation has recently been shown to regulate NSC quiescence (Knobloch et al 2017). Lipid droplets (LDs) are the intracellular organelles that store lipids, mainly triacylglycerols and sterol esters. LDs have classically been seen as inert storage organelles, however studies over the recent years have revealed them as highly dynamic organelles that fulfil crucial metabolic functions. How LDs are involved in the regulation of adult NSCs is currently poorly understood.
We are investigating the role of LDs in NSCs during development and in adulthood, both in vitro and in vivo. As LDs are very sensitive to the common staining methods used for visualization, we are using CRISPR/Cas9 to create an endogenous LD reporter mouse. This mouse will allow us to visualize LDs in a staining-free manner and will also enable us to follow LD dynamics using live imaging. Here we present the target selection, validation and tagging approach for this novel endogenous LD reporter mouse.
1 19 Sara Bonzano NICO - University of Turin The transcriptional regulator COUP-TFI/Nr2f1 exerts an anti-astrogliogenic function on adult mouse hippocampal NSCs/progenitors enabling adult neurogenesis Bonzano Sara 1,4, Crisci Isabella 1, Stajano Daniele 1, Podlesny-Drabiniok Anna 2, Rolando Chiara 3, Krezel Wojciech 2, Studer Michèle 4, De Marchis Silvia 1

1 Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano (Turin), Italy; Department of Life Sciences and Systems Biology (DBIOS), University of Turin, Turin, Italy; 
2 Université de Strasbourg, CNRS, Inserm, IGBMC, Illkirch, France;
3 Department of Biomedicine, University of Basel, Basel, Switzerland;
4 Université Côte d’Azur (UCA), CNRS, Inserm, iBV, Nice, France.

In the adult hippocampal dentate gyrus (DG), radial glial-like neural stem cells (NSCs) are multipotent (generating both neurons and astrocytes) while progenitors are fate-restricted to the neuronal lineage. Despite the importance of a tight control of neurogenic versus astrogliogenic potential, the underlying transcriptional program is still largely unknown.
In this study, we found that a large subset of NSCs/progenitors co-expressed the transcription factor COUP-TFI (also known as Nr2f1) in the healthy DG, whereas neuroinflammation led to its downregulation. By combining inducible knockouts to fate mapping approaches we showed that COUP-TFI deletion from adult DG NSCs reduced neurogenesis and increased astrocyte production likely by inducing the pro-astrogliogenic factor NFIA. Remarkably, this shift also occurred upon COUP-TFI loss by retroviral targeting of mitotic progenitors, indicating that these cells might still be bipotent and need COUP-TFI to limit their potential to the neuronal fate. Moreover, complementary experiments clearly demonstrated that COUP-TFI overexpression abolished the production of new astrocytes under physiological conditions and was sufficient to abate the inflammation-induced gain in astrogliogenesis and to restore proper neurogenesis levels, thus revealing a crucial function for COUP-TFI in protecting the DG niche from inflammatory insults. Finally, downregulation of COUP-TFI takes place within the mouse hippocampal niche early during physiological aging concomitantly with the drop in neurogenesis, further supporting COUP-TFI as a central regulator of the adult DG neurogenic niche.
1 20 Ariane Sharif UMRS1172 JPArc Development and Plasticity of the Neuroendocrine Brain Lab, 1 place de Verdun 59045 Lille France Characterization of the neural stem cell niche in the adult human hypothalamus Giuliana Pellegrino, Claire Trubert, Jérémy Terrien, Fabien Pifferi, Danièle Leroy, Anne Loyens, Martine Migaud, Marc Baroncini, Claude-Alain Maurage, Christian Fontaine, Vincent Prévot, Ariane Sharif The adult brain contains niches of neural stem cells that continuously add new neurons to selected circuits throughout life. Besides the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus, which have been extensively studied in various mammalian species including humans, a third neurogenic niche has recently been identified in the adult hypothalamus of several animal models, mostly rodents. To evaluate whether a neural stem cell niche also exists in the adult hypothalamus in humans, we performed multiple immunofluorescent stainings to assess the expression of a panel of neural stem/progenitor cell (NPC) markers (Sox2, nestin, vimentin, GLAST, GFAP) in the human hypothalamus in comparison with the mouse, rat and a non-human primate species, the grey mouse lemur (Microcebus murinus). Our results show that the adult human hypothalamus contains four populations of cells co-expressing the five NPC markers: i) a ribbon of small stellate cells that lines the third ventricular wall behind a hypocellular gap and is similar to that found along the lateral ventricles, ii) ependymal cells, iii) tanycytes, which line the floor of the third ventricle in the tuberal region, and iv) a population of small stellate cells in the suprachiasmatic nucleus. In the mouse, rat and mouse lemur hypothalamus, co-expression of NPC markers is essentially restricted to tanycytes and these species lack a ventricular ribbon. Altogether, we identify in the adult human hypothalamus four distinctive cell populations harbouring an antigenic profile of neural stem cells, three of which appear specific to humans.
1 21 Anja Urbach Jena University Hospital, Dept. for Neurology Differential effects of cyclin D2 ablation on the self-renewal capacity of postnatal and adult hippocampal stem cells and dynamic expression of D-cyclins during precursor differentiation in vitro Syeda A. Zahra, Otto W. Witte, Anja Urbach The neurogenic potential of the adult dentate gyrus (DG) relies on a pool of quiescent neural stem cells (NSCs) that become activated to generate new granule cells on demand. Our studies on cyclin D2 knockout (D2KO) mice suggest that cyclin D2 is required for the establishment of the adult NSC pool during the second to third postnatal week, and that cyclin D2 becomes increasingly important for hippocampal neurogenesis during this period. We showed that cyclin D2 is the only D-type cyclin of adult NSCs, suggesting an important role for their proliferation and self-renewal. On the contrary, developmental NSCs in the postnatal hilar germinative matrix as well as in the emerging subgranular zone also express cyclin D1, which might compensate for the loss of cyclin D2 during postnatal neurogenesis.  To further examine these findings, we isolated neural precursors from postnatal day 7 (P7) and adult mouse dentate gyri and cultured them as neurospheres. We found that neurospheres could be established from both, P7 and adult D2KO precursors. However, precursors from D2KO mice formed less and in adults also smaller spheres during primary culture as compared to wildtype mice. The spheres of P7 D2KO mice were able to expand exponentially over at least ten passages, demonstrating that their DG contains bona fide self-renewing NSCs that do not require D2 for proliferation. On the contrary, the precursor population from adult D2KO mice displayed no long-term neurosphere-forming capacity and stopped growing after the third passage, suggesting that it is composed of only progenitor cells. Proliferation of D2KO precursors is likely accomplished by cyclin D1 which is expressed in spheres of both, P7 and adult mice (qRT-PCR and Western blot). To further evaluate the dynamics of D-cyclin expression during adult neurogenesis, we cultured DG precursors from wildtype mice as monoadherent cultures (with FGF2 and EGF) and differentiated them in mitogen-free media for 96 hrs. We observed similar quantities of cyclin D1 and D2 transcripts and proteins which were high in proliferating cultures and sharply decreased during the first 24 hrs of differentiation, simultaneously with a drop of the proliferation marker Ki67 and increasing levels of differentiation markers DCX, Fox3 and GFAP. Together, these findings confirm our in vivo data suggesting that cyclin D2 is dispensable for proliferation of developmental NSCs but required for the formation and maintenance of the adult NSC population.
1 22 Iris Schäffner Universität Erlangen FoxO-dependent control of lysosome function in adult neurogenesis Iris Schäffner, Georgia Minakaki, Ursula Schlötzer-Schrehardt, Jihye Paik, Wolfgang Wurst, Jochen Klucken, and D. Chichung Lie The Autophagy-Lysosome Pathway (ALP) is a highly conserved catabolic pathway with emerging functions in adult neurogenesis. The mechanisms controlling ALP activity in adult neural stem cells and subsequent generated neurons are largely unknown. We recently identified transcription factors of the FoxO family as central regulators of ALP activity in adult hippocampal neurogenesis. Conditional deletion of FoxO transcription factors led to a progressing loss of adult neural stem cells and strongly impaired autophago-lysosomal flux in developing neurons of the adult hippocampus. Furthermore, FoxO-deficiency altered dendritic morphology, elevated spine density and led to aberrant spine positioning in adult-generated hippocampal neurons. How FoxOs impact on autophago-lysosomal flux remains to be determined. We now observed insufficient acidification of lysosomes in FoxO-deficient cells, which in turn lead to the accumulation of fused autophagolysosomes and the block of the following degradation process. Additional data point to a decreased expression of Tfeb, a master regulator of lysosomal biogenesis, and of specific subunits of the vacuolar-ATPase (v-ATPase) – a multiple-subunit composed proton pump residing in the lysosomal membrane, which is responsible for the acidification of lysosomes. Collectively, our new findings suggest a new link between FoxO transcription factors and v-ATPase-dependent acidification of lysosomes in adult neurogenesis.
1 23 Carmen Castro University of Cadiz NOVEL PROTEIN KINASE C ACTIVATION PROMOTES MIGRATION OF NEUROBLASTS TOWARD BRAIN INJURIES: A ROLE IN NEUREGULIN RELEASE Ricardo Gómez-Oliva1,6, Noelia Geribaldi-Doldán 1,6, Samuel Domínguez-García1,6, Felix A. Ruiz2,6, Pedro A. Nunez-Abades3,6, Livia Carrascal3,6, Jorge Bolívar-Pérez 4,6, Antonio J. Macías-Sánchez5,6, Rosario Hernández-Galán5,6 and Carmen Castro1,6

1 Área de Fisiología, Facultad de Medicina, Universidad Cádiz, Spain;
2 Área de Nutrición, Facultad de Medicina, Universidad Cádiz, Spain;
3 Departamento Fisiología, Facultad de Farmacia, Universidad Sevilla, Spain;
4 Área de Bioquímica y Biología Molecular. Facultad de Ciencias, Universidad de Cádiz, Spain;
5 Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, Spain;
6 Instituto de Investigación Biomédica de Cádiz (INIBICA), Cádiz, Spain.

Neural stem cells (NSC) are activated within neurogenic niches in response to brain injuries. These response results in the production of neuroblasts, which attempt to migrate towards the injured area, yet this neuroblast do not contribute to the generation of new neurons within the damaged tissue. Injuries constitute a gliogenic/non-neurogenic niche in which neuronal production is impaired. Signaling molecules within the injury released in response to inflammation prevent neuroblast migration, differentiation and/or survival. The introduction of neurogenic signaling molecules in injuries may facilitate neuronal enrichment. Kinases of the protein kinase C (PKC) family mediate the release of growth factors that participate in different steps of the neurogenic process. Activation of novel PKC isozymes facilitates release of neurogenic growth factors such as neuregulin, however, specific novel PKC activators have not been described so far. We have isolated a plant derived diterpene, 7,8,12-tri-O-acetyl-3-O-(4-methoxyphenyl) acetylingol (EOF2; CAS number 944799-48-8), with the capacity to specifically activate novel PKC. We have found in here that in mice with controlled mechanical injuries, intranasal administration of EOF2 facilitates migration of neuroblasts from neurogenic regions towards the perilesional area. We also demonstrated, using fusion proteins in which membrane-anchored growth factors neuregulin 1 and transforming growth factor a (TGFα) were fused to a mCherry and GFP in both N-terminal and C-terminal ends, that EOF2 selectively mediated the release of neuregulin 1 without affecting the release of TGFα. Our findings highlight the role of novel PKC in brain injury regeneration. Funded by Spanish Ministerio de Ciencia, Innovación y Universidades (BFU2015-68652-R).
1 24 David Morizet Institut Pasteur Unveiling the heterogeneity of vertebrate adult Neural Stem Cells David Morizet, Alessandro Alunni, Laure Bally-Cuif The vertebrate brain harbors neural stem cells (NSCs) that persist into adulthood to ensure the life-long production of functional neurons. These NSCs are mainly quiescent, a feature important for their maintenance into old age. NSCs however do not constitute a homogeneous population and different subtypes can contribute differently to maintaining long-term neurogenic ability.
The adult zebrafish pallium hosts NSCs with similar characteristics to their mammalian counterparts but present in large number, making it a powerful model to unravel NSC properties. Using this model, our lab recently demonstrated the existence of subpopulations of NSCs that differ in their quiescence depths and/or are hierarchically organized along a cascade leading to activation and neuronal production. However the molecular signature and functionally relevant markers for these subpopulations remain unknown.
In order to get a better description of the heterogeneity of quiescent NSCs we used single-cell RNA-sequencing with the 10x Genomics platform.  We enriched for NSCs via flow-assisted cell-sorting with a transgenic line and obtained over 17k cells, with over 3k of them being quiescent NSCs. Our analysis confirms the existence of different clusters of quiescent NSCs and highlights markers that can be used to study these cells in situ. These results also allow us to order the cells along a differentiation trajectory using pseudotime and obtain insights about the physiological functions of these clusters. We are in the process of validating in situ the expression of diagnostic markers prior to initiating functional assays. My most recent results on these fronts will be presented.
1 25 Fabienne Grolig University Erlangen FoxO transcription factors and mitophagy in adult neural stem cells Fabienne Grolig, Ursula Schlötzer-Schrehardt, D.Chichung Lie, Iris Schäffner Maintenance of the adult neural stem cell pool is crucial to sustain neurogenesis-dependent plasticity throughout life.
The FoxO transcription factors serve as regulators of adult neural stem cell maintenance at least in part through the control of autophago-lysosomal activity in adult neural stem/progenitor cells (NSPC). In general, degradation of aged or damaged mitochondria via the autophago-lysosomal pathway - a process termed mitophagy - is critical for mitochondrial homeostasis. Interestingly, it was recently found that mitochondrial function is vital to maintain NSPC proliferation and neurogenesis.
We hypothesize that mitochondria homeostasis in NSPCs is dependent on FoxO-mediated mitophagy. Preliminary results revealed differences in number, size and morphology of mitochondria between wildtype and FoxO cKO NSPCs. Additionally, mitochondrial membrane potential was reduced in FoxO cKO cells, indicating an accumulation of a fraction of aged or damaged mitochondria. Furthermore, markers involved in the induction of mitophagy, as e.g. PINK1, were down-regulated in FoxO cKO NSPCs.
These preliminary data support the hypothesis, that the FoxO transcription factors might be involved in the regulation of mitophagy in adult NSPCs.
1 26 Aneta Grymanowska Mossakowski Medical Research Centre, PAS, 02-106 Warsaw, 5 Pawińskiego Str., Poland Distinct nuclear architecture of neural stem cells among other neural cell types in adult hippocampal neurogenesis Aneta W. Grymanowska, Ana Martin, Krzysztof H. Olszyński, Krzysztof Nowiński, Bartosz Borucki, Piotr Majka, Sylwia Bednarek, Grzegorz M. Wilczyński, Adriana Magalska, Robert K. Filipkowski Three-dimensional reconstruction of the nuclear shape of every cell type in adult hippocampal neurogenesis was performed along with semi-automated shape analysis that quantitatively measures volume, height, surface area and additional metrics of nuclear morphology such as the density of nuclear DNA. Also, we are introducing a novel, high throughput nuclear shape analysis with automatic nuclei segmentation and cellular type classification, with the application of U-Net based Deep Convolutional Neural Network (DCNN), modified to accommodate 3D multichannel images.
Four channels correspond to four types of staining used: GFP-Nestin, NeuN, DAPI and DCX. Cells are assigned probabilities of belonging to each of five possible types: type 1 (GFP-Nestin+/neural process), type 2a (GFP-Nestin+), type 2b (GFP-Nestin+/DCX+), type 3 (DCX+), type 4 (NeuN). We are showing that neural stem cells in the dentate gyrus of adult murine hippocampus exhibit a distinct nuclear architecture. Obtained results also show that the chromatin in the nucleus of cell type 1 and type 2 is more condensed compared to other neural cell types in the dentate gyrus. This work was supported by National Science Centre, Poland, grant no. 2014/14/M/NZ4/00561 (for RKF)
1 27 Nesrin Sharif Institute of Physiological Chemistry, University Medical Center, Mainz Neurogenin2-induced neurons under the scope: from activity-dependent gene regulation to synaptic plasticity Nesrin Sharif, Filippo Calzolari, Nicolas Marichal, Benedikt Berninger Recovery of brain function following damage depends on the successful functional integration of newly generated neurons and their ability to plastically contribute to pre-existing neuronal network activity. Direct astroglia-to-neuron conversion by ectopic expression of the neuronal fate determining transcription factor neurogenin-2 has emerged as a powerful tool to generate induced glutamatergic cortical neurons (iNs) in vitro as well as in vivo. However, the extent to which iNs may display long-term synaptic plasticity as well as contribute to homeostatic synaptic scaling in comparison to endogenous neurons has not yet been investigated. This study aims to investigate the extent to which iNs undergo transcriptional modulation in an activity-dependent manner, in comparison to primary cortical neurons in vitro. My current results demonstrate that functionally active synaptic connections can be established among iNs. iNs can be stimulated by several stimulation paradigms, resulting in upregulation of multiple immediate-early genes in vitro. Motivated by these findings, I am modulating activity (e.g. long-term exposure to or complete blockage of activity) in cultures of iNs with cortical neurons in order to investigate activity-dependent transcriptomic changes by performing RNA-sequencing on FACS-sorted iN nuclei. These data will provide a clearer understanding of the authenticity of iNs, potentially paving the way for further improvements of the astroglia-to-neuron conversion process.
1 28 Jorge Valero Achucarro Basque Center for Neuroscience | Ikerbasque Basque Foundation for Science Microglia actively remodels adult hippocampal neurogenesis through the phagocytosis secretome  J. Valero(1,2,8), I. Diaz-Aparicio(1,2), I. Paris(1,2), V. Sierra-Torre(1,2), A. Plaza-Zabala(1), N. Rodríguez-Iglesias(1,2), M. Márquez-Ropero(1,2), S. Beccari(1,2), O. Abiega(1,2), E. Alberdi(1,2), C. Matute(1,2), I. Bernales(2), A. Schulz(3), L. Otrokocsi(4), B. Sperlagh(4), K. E. Happonen(5), G. Lemke(5), M. Maletic-Savatic(6,7), A. Sierra(1,2,8)

1 Achucarro Basque Center for Neuroscience, Leioa, Bizkaia, Spain.
2 University of the Basque Country UPV/EHU, Leioa, Bizkaia, Spain.
3 University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
4 Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.
5 Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
6 Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, TX, USA.
7 Baylor College of Medicine, Houston, TX, USA.
8 Ikerbasque Foundation, Bilbao, Bizkaia, Spain.

During adult hippocampal neurogenesis, the majority of newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia in order to avoid disturbing the surrounding neurons. Here, we propose that phagocytosis is not merely a passive process of corpse removal but has an active role in maintaining adult hippocampal neurogenesis. First, we found that neurogenesis was disrupted in mice chronically deficient for two microglial phagocytosis pathways (P2Y12, MerTK/Axl), but was transiently increased in mice in which MerTK expression was conditionally downregulated. We then followed an in vitro approach to perform a transcriptomic analysis of microglial phagocytosis and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we determined that the phagocytic microglia secretome limits the production of new neurons both in vivo and in vitro. Our data suggest that reprogrammed phagocytic microglia acts as a sensor of local cell death, modulating the balance between cell proliferation and cell survival in the neurogenic niche, supporting the long-term maintenance of adult hippocampal neurogenesis.

This work was supported by grants from the Spanish Ministry of Economy and Competitiveness with FEDER funds to AS (BFU2012-32089 and RYC-2013-12817), to AS and JV (BFU2015-66689); a Leonardo Award from the BBVA Foundation to AS; a Basque Government project (PI_2016_1_0011); a Hungarian Research and Development Fund Grant (K116654) to B.S; a Hungarian Brain Research Program grant (2017-1.2.1-NKP-2017-00002) to B.S. and Ikerbasque start-up funds to JV.
1 29 Luke Foulser Wellcome Sanger Institute  Dechiphering Developmental Disorders Sebastian Gerety, Andy Day, Adam Hunter, Luke Foulser, Neophytos Kouphou, Alex Neaverson and Malin Andersson   The deciphering developmental disorders project aims to advance clinical genetic practice for children with developmental disorders by the systematic application of the latest microarray and sequencing methods. This has led to the identification of a number of implicated genes which have since sought to be explored.  CRISPR/Cas9 KO iPSCs are used to produce homozygous and heterozygous mutations of 100 genes of known importance in biological duplicate. Here, we attempt to understand the biological function of these mutations potentially leading to these disorders. The KO iPSCs will be used to model brain development by differentiating to a neural stem cell population in vitro. These NSCs will then be characterised by immunocytochemistry for NSC markers to ensure a successful differentiation and homogenous population. NSCs with mutations in these developmental genes will then be used to analyse the genome-wide expression profile to identify disrupted signalling pathways underlying the disorders observed in children. Moreover, future differentiation into cortical neurons may prove key, due to high prevalence of cognitive disorders within the cohort.  In turn, this may lead to future target pathways that could be treated to obviate the disorder’s phenotype.
1 30 Giuseppe Lupo Sapienza University of Rome Epigenomic profiling of aged mouse neural stem/progenitor cells identifies Dbx2 as a candidate regulator of age-associated neurogenic decline Giuseppe Lupo, Paola S. Nisi, Mario Fiore, Pilar Esteve, Yu-Lee Paul, Clara Lopes Novo, Ben Sidders, Muhammad A. Khan, Stefano Biagioni, Hai-Kun Liu, Paola Bovolenta, Emanuele Cacci, Peter J. Rugg-Gunn Rodent adult neurogenesis declines with ageing, but the underlying molecular mechanisms are poorly understood. To investigate the intrinsic molecular changes that occur upon neural stem/progenitor cell (NSPC) ageing, we compared the transcriptional, histone methylation and DNA methylation signatures of NSPCs derived from the subventricular zone (SVZ) of young adult (3 months old) and aged (18 months old) mice. Surprisingly, the genome-wide transcriptional and epigenetic profiles of SVZ-derived NSPCs were largely unchanged in aged cells. Despite the global similarities, we detected robust age-dependent changes at several genes and their regulatory elements, thereby identifying putative regulators of neurogenic decline. Among them, the homeobox gene Dbx2 was upregulated in vitro and in vivo, and its promoter region had altered histone and DNA methylation levels, in aged NSPCs. Using functional in vitro assays, we found that elevated Dbx2 expression in adult NSPCs promotes age-related phenotypes. In particular, Dbx2 overexpression reduced NSPC proliferation by impairing the G1/S and G2/M cell cycle transitions, and caused the dysregulation of age-associated modulators of NSPC self-renewal and differentiation. Depleting Dbx2 in aged NSPCs led to the reverse gene expression changes. Taken together, these results provide new insights into the molecular programmes that are affected during NSPC ageing, and uncover a new functional role for Dbx2 in promoting age-related neurogenic decline.
1 31 Hyunah Lee Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology, and Neuroscience, King's College London, LONDON, SE5 9RX, UK ¹ | Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, USA ² The effects of apolipoprotein E (APOE) polymorphism on human hippocampal neurogenesis Hyunah Lee ¹, Graham Cocks ¹, Lucia Dutan Polit ¹, Maria Carolina Marchetto ², Fred H. Gage ², Jack Price ¹, Deepak Srivastava ¹, Sandrine Thuret ¹ Apolipoprotein E4 (APOE4) allele is the most common genetic risk factor for Alzheimer's disease, which is known to both increase the overall onset of the disease and decrease the age of onset. Growing body of evidence suggests that APOE4 may negatively affect adult hippocampal neurogenesis in the dentate gyrus, which has been implicated in AD progression. However, the impact of APOE isoforms on hippocampal neurogenesis at a cellular/molecular level is not fully understood. In this study, time-course characterization of neurogenic properties was performed on human isogenic APOE induced pluripotent stem cells (iPSCs). Using an in vitro model of hippocampal neurogenesis that was previously optimized in the lab, isogenic APOE iPSCs were differentiated to hippocampal neural progenitors and dentate gyrus granule cell-like neurons. Compared to neutral APOE3 homozygous cells, APOE4 homozygous cells showed significantly different expression patterns for markers of hippocampal neural progenitors, and PROX1, the marker for mature dentate gyrus granule cells. The gene expression pattern of APOE and protein expression patterns of MAP2 and DCX were comparable between APOE3 and APOE4 cells. The isogenic lines also showed differential phenotypes for the expression of Ki-67, a marker for cell proliferation, during neuronal differentiation. Taken together, our findings suggest that APOE genotype can impact the course of hippocampal neurogenesis, and that sustaining normal hippocampal neurogenesis can be a potential target for early intervention against AD. Whether APOE genotype interacts with environmental factors to either exacerbate or ameliorate the phenotypes we have characterized thus far is currently under investigation.  
2 1 Curie Kim King's College London Investigating the impact of intermittent energy restriction and mastication on adult hippocampal neurogenesis-associated cognition in an ageing population  Curie Kim, Sophie Miquel, Wendy Hall and Sandrine Thuret Adult hippocampal neurogenesis (AHN) is needed for pattern separation, the ability to differentially encode small changes derived from similar inputs, and recognition memory, the ability to recognise previously encountered stimuli. Intermittent energy restriction (IER) may increase neurogenesis as a “cellular relic” of feeding patterns during evolution in response to alternating famine and abundant food. Human trials have found significant improvements in verbal recognition memory after 30% calorie reduction. IER has been associated with significant increases in human brain activation volume in areas involved in brain function control and plasticity. Food texture and mastication have also been implicated. Decreased mastication in humans and animals have a negative impact on AHN and associated cognition. There is a close association between masticatory function, cognitive status and age-related neurodegeneration in human elderly. Previous findings show that a 4-week IER intervention can improve pattern separation performance in 43 individuals aged 35-75 years. It also resulted in increased levels of serum α-klotho, an anti-ageing protein. A mastication intervention carried out in 54 individuals aged 45-70 years old required participants to consume chewing gum for 10 minutes, 3 times a day over 3 months. There was a significantly bigger improvement in recognition memory compared to a non-chewing control group. Following on from these findings, a 3-month intermittent ER and mastication intervention trial is being carried out on healthy human participants aged 60 years and older. The impact of IER and mastication, independently and synergistically, on AHN-associated factors is measured using the mnemonic similarity task and serum α-klotho.
2 2 Shane Marie Ohline Department of Psychology, University of Otago, Dunedin, New Zealand Adult-born dentate granule cell excitability depends on the interaction of neuron age, ontogenetic age and experience S.M. Ohline1,3, K.L. Wake1,3, M.-V. Hawkridge4, M.F. Dinnunhan1,3, R.U. Hegemann1,3, A. Wilson1,3, L. Schoderboeck1,2,3, B.J. Logan1,3, T. Jungenitz4, S.W. Schwarzacher4,  S.M. Hughes2,3 and W.C. Abraham1,3

1Department of Psychology, and 2Department of Biochemistry, 3Brain Health Research Centre and Brain Research New Zealand, University of Otago, Dunedin, New Zealand

4Institute of Clinical Neuroanatomy, Goethe-University of Frankfurt, Frankfurt am Main, Germany

Early during their maturation, adult-born dentate granule cells (aDGCs) are particularly excitable, but eventually develop the electrophysiologically quiet properties of mature cells. However, the stability versus plasticity of this quiet state across time and experience remains unresolved. By birthdating two populations of aDGCs across different animal ages, we found for 10-month-old rats the expected reduction in excitability across cells aged 4-12 weeks, as determined by Egr1 immunoreactivity. Unexpectedly, cells 35 weeks old (after genesis at an animal age of 2 months) were as excitable as 4-week-old cells, in the dorsal hippocampus. This high level of excitability at maturity was specific for cells born in animals 2 months of age, as cells born later in life did not show this effect. Importantly, excitability states were not fixed once maturity was gained, but were enhanced by enriched environment exposure or LTP induction, indicating that any maturational decrease in excitability can be compensated by experience. These data reveal the importance of the animal’s age for aDGC excitability, and emphasize their prolonged capability for plasticity during adulthood.
This work was made possible by a grant from the Marsden Fund administered by the Royal Society of NZ.
2 3 Rupert Overall DZNE (German Center for Neurodegenerative Diseases) The Adult Neurogenesis Map Rupert W Overall The hippocampus is a key brain structure for learning and memory. It not only processes input from the environment, but also fundamentally influences behaviour. This means that the neural network in the hippocampus is a core part of an information loop connecting environmental stimulus and response. It is particularly intriguing that this special brain region is also home to a population of neural stem cells which allow the environmentally-regulated creation of new neurons, throughout the life of the organism, that add an extra level of flexibility to hippocampal performance. We have previously shown that the regulation of the stem cell pool and the generation of new neurons are under complex genetic control. We also maintain a structured database of all genes reported to affect adult hippocampal neurogenesis in some way. We are now extending this effort to encompass behavioural phenotypes and environmental stimuli. The resulting information is being organised into a structured SBML map to enable interactive browsing and complex searching of the knowledgebase, as well as to provide a platform for predictive modelling. We present here an outline and working draft of the Adult Neurogenesis Map and look forward to community feedback as the project expands.
2 4 Julia Schneider Institute of Biochemistry, Friedrich-Alexander-University Erlangen-Nürnberg Astrogenesis and dynamic of astrocytes in the mouse hippocampus Julia Schneider, Julian Karpf, Ruth Beckervordersandforth The brain comprises two broad classes of cells: neurons and glia cells. Astrocytes are ranked to the glial family and fulfil a variety of functions. In the dentate gyrus (DG) of the hippocampus, specialized astrocytes, known as radial glia-like cells, function as neural stem cells and generate new neurons and astrocytes. Non-radial astrocytes, which are major components of the DG, contribute to the generation and survival of newborn neurons. Astrocytes were believed to be homogenous within one region, but recent events suggest that astrocytes are molecularly and functionally different. Using a genetic labelling strategy, we found that the adult hippocampal dentate gyrus is populated by morphologically distinct astrocytes that are localized to specific compartments and associated to diverse processes. Surprisingly, experiments revealed the proliferation of non-radial astrocytes, which is in contrast to the prevailing assumption that astrocytes are postmitotic in the non-injured brain. Based on these findings we aim to describe hippocampal astrogenesis not only in the adult but also during embryonic and fetal states. We attempt to answer the following questions: What is the origin of embryonic and adult-born astrocytes? When does astrogenesis start during embryogenesis? How do hippocampal astrocytes develop and become morphologically and functionally different? In addition to the proliferation potential of non-radial astrocytes, we could observe that the proliferation could be influenced by the DG is composed of morphologically and functionally distinct astrocytes, whose dynamics are critical modulators for hippocampal adaption to changing conditions. Further analysis revealed that adult astrogenesis and the survival of adult-born astrocytes are increased under exercising conditions. As adult neurogenesis is important for brain plasticity, a contributing role of adult astrogenesis has to be investigated with an in vivo knock out model of dividing astrocytes. Collectively, our study revealed structural heterogeneity and subtype-specific dynamics of hippocampal astrocytes in response to physiological stimuli. It will also enlighten the role of adult astrogenesis on hippocampal plasticity. Furthermore, we aim to be the first who will investigate adult and embryonic astrogenesis in the hippocampal neurogenic niche.
2 5 Jariya Welbat Department of Anatomy,  Faculty of Medicine, Khon Kaen University Effects of Hesperidin on oxidative stress and hippocampal neurogenesis treatment in adult rats treated with methotrexate Jariya Umka Welbat, Salinee Naewla, Kornrawee Suwannakot, Anusara Aranarochana, Apiwat Sirichoat, Wanassanan Pannangrong Methotrexate (MTX) is a folic acid antagonist widely used a chemotherapeutic agent. MTX induces formation of reactive oxygen species (ROS) and leads to neurotoxicity, which is associated with a reduction neurogenesis. Hesperidin (Hsd) is a flavanone glycoside found in citrus fruits. Hsd has antioxidant activities and consequently prevent neuronal cell death. This study was designed to evaluate the neuroprotective effect of Hsd against MTX-induced oxidative stress and reductions of neurogenesis in adult rats. Sprague Dawley rats were randomly divided into four groups. The vehicle group received saline and propylene glycol. The Hsd group received Hsd (100 mg/kg) by oral gavage for 21 days. The MTX group received MTX (75 mg/kg intravenous) by intravenous injection on day 8 and 15 of the study and the MTX+Hsd group received both MTX and Hsd. After treatment, oxidative stress [malondialdehyde (MDA)] and antioxidant activities [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)] were determined. Cell proliferation, survival and immature neurons in the hippocampal dentate gyrus were quantified using Ki67 and bromodeoxyuridine (BrdU) and doublecortin (DCX), respectively. Increases in MDA and decreases in the antioxidant activities in the hippocampus and prefrontal cortex were significantly observed in the MTX-treated rats. In addition, MTX significantly reduced neurogenesis, including cell proliferation, survival and immature neurons. However, co-treatment with Hsd ameliorated the negative effects of MTX on oxidative stress, antioxidant enzymes and neurogenesis. These findings suggest that Hsd could prevent neurotoxicity effect of MTX by inhibiting oxidative stress and increasing neuroprotective properties.
2 6 Apiwat Sirichoat Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand Neuroprotective effect of melatonin on impairments of spatial working memory and neurogenesis caused by 5-fluorouracil in adult rats. Apiwat Sirichoat, Kornrawee Suwannakot, Pornthip Chaisawang, Wanassanun Pannangrong, Peter Wigmore, Jariya Umka Welbat Melatonin is a hormone originally produced by the pineal gland. Melatonin can modulate neurogenesis both in vitro and in vivo and protect deterioration of neurons in the hippocampus. The objective of the present study was to investigate the neuroprotective effects of melatonin on a reduction of spatial memory and hippocampal neurogenesis caused by 5-fluorouracil chemotherapy in male Spraque-Dawley rats. Animals were separated into 6 groups, including vehicle, melatonin, 5-FU and co-administration with melatonin and 5-FU (preventive, recovery and throughout) groups. 5-FU (25 mg/kg/day) was administered by i.v. injection 5 times every 3 days, whereas melatonin (8 mg/kg/day) was given 1 time/day by intraperitoneal (i.p.) injection for 21 days and 42 days. Spatial memory test was determined using the novel object location (NOL) test. Ki-67, bromodeoxyuridine (BrdU) and doublecortin (DCX) were used to quantify the numbers of cell proliferation, cell survival and immature neurons, respectively. The results demonstrated that 5-FU-treated animals did not prefer either objects in familiar or novel locations. Moreover, 5-FU significantly reduced numbers of Ki-67, BrdU and DCX positive cells. These results indicate that 5-FU treatment caused impairments of spatial working memory, cell proliferation, survival and immature neurons in the SGZ of the hippocampal DG. However, these aberrations were restored to control levels by co-treatment with melatonin in all time periods. These data suggest that melatonin could prevent and restore the spatial working memory and neurogenesis impairments caused by 5-FU.
2 7 Odette Leiter The University of Queensland; Technische Universität Dresden Exercise-induced activated platelets increase adult hippocampal neurogenesis Odette Leiter, Suse Seidemann, Rupert W. Overall, Beáta Ramasz, Nicole Rund, Sonja Schallenberg, Tatyana Grinenko, Ben Wielockx, Gerd Kempermann and Tara L. Walker Systemic factors contribute to the regulation of adult hippocampal neural precursor cells. Physical exercise is associated with systemic changes and represents a strong positive physiological stimulus of neural precursor cell proliferation. However, the mechanisms underlying these effects are largely unknown. We show that dentate gyrus primary cultures form significantly more neurospheres when treated with serum from running mice. Using proteomic screening of mouse plasma following exercise, we identified platelets and their released factors as potential mediators of this effect. We found that platelets are activated after short periods of exercise and that activated platelets promote neurogenesis. Similarly, treatment with platelet factor 4, a protein released after platelet activation and elevated in the plasma of running mice, increased neurogenesis. Ex vivo, the neurogenesis-promoting effects of activated platelets and platelet factor 4 were exclusively observed in dentate gyrus primary cultures but not in cultures derived from the subventricular zone. Moreover, in mice depleted of circulating platelets, the dentate gyrus-specific running-induced increase in precursor cell proliferation was absent. Our data demonstrate that platelets and their released factors can modulate adult neural precursor cells under physiological conditions and provide an intriguing link between running-induced platelet activation and the regulation of neural precursor cells following exercise.
2 8 Victor Luna Columbia University/RFMH Adult-born Neurons Monosynaptically Inhibit and Excite Mature Granule Cells in the Dentate Gyrus Victor M. Luna, Christoph Anacker, Nesha S. Burghardt, Hameda Khandaker, Valentine Andreu, Amira Millette, Paige Leary, Rebecca Ravenelle, Jessica C. Jimenez, Alessia Mastrodonato, Christine A. Denny, Andre A. Fenton, Helen E. Scharfman, Rene Hen Young adult-born granule cells (abGCs) in the dentate gyrus (DG) of the hippocampus have a profound impact on cognition and mood. However, it remains unclear how abGCs uniquely contribute to local DG information processing. Using a combination of electrophysiology, optogenetics, pharmacology, and behavior, we show that the actions of abGCs in the DG depend on the origin of incoming afferents. In response to lateral entorhinal cortex (LEC) inputs, abGCs surprisingly exert monosynaptic inhibition of developmentally-born mature granule cells (mGCs) via group II metabotropic glutamate receptors. In contrast, in response to medial entorhinal cortex (MEC) inputs, abGCs directly excite mGCs via glutamatergic N-methyl-D-aspartate receptors likely containing the atypical NR3 subunit. These modulatory mechanisms are utilized exclusively by abGCs and not mGCs. Moreover, we find that neurogenesis-dependent inhibition and excitation appear to contribute to the differences in sub-regional activity levels within the DG during an active place avoidance task and a novel object recognition task. Our results suggest that a critical function of abGCs may be to regulate the relative synaptic strengths of LEC-driven contextual information versus MEC-driven spatial information to shape distinct neural representations in the DG. We therefore propose that abGC-driven inhibition and excitation depends on the demands of the environment, which can be reflected in the activity of cortical as well as sub-cortical regions sending inputs to the DG.
2 9 Jadna Bogado Lopes German Center for Neurodegenerative Diseases (DZNE) Dresden, Center for Regenerative Therapies TU Dresden (CRTD), Technische Universität Dresden, Dresden, Germany.  What is the role of new-neurons in the development of individual behaviour? J Bogado-Lopes, F Ehret, AN Grzyb, AE Rünker, G Kempermann The interaction of individuals with their environment leads to the development of distinct behavioural patterns. Previously, we showed that isogenic mice kept in an enriched environment (ENR) established divergent and stable social and exploratory trajectories. Remarkably, the amount of exploratory activity, measured as roaming entropy (RE), correlated positively with adult hippocampal neurogenesis (AHN), a cellular plasticity mechanism in the hippocampus. We hypothesised that the feedback between activity (and hence experience of the environment) and brain plasticity, including AHN, is a core mechanism underlying brain individualization in ENR. To investigate whether disruption of AHN would compromise the exploratory activity and individualisation processes, we here used cyclin D2-ko mice with constitutively suppressed AHN (but normal hippocampal development), and their wild-type littermates (n = 40). Animals were housed together for 3-months in a novel large ENR enclosure consisting of 70 connected cages equipped with radio antennae for longitudinal tracking. Their cognitive performance was evaluated in the Morris Water Maze task (MWM) and AHN levels were assessed using BrdU labeling. We confirmed that the number of BrdU positive cells correlated with RE in the wild-type animals, and cyclinD2-ko mice had impaired performance in the reversal phase of MWM. Whereas wild-type animals developed stable exploratory trajectories, the behaviour of cyclinD2-ko mice remained more random. Furthermore, different patterns of correlations between exploratory behaviour, cognitive performance and AHN were observed in wild-type and knockout mice. Together, these results suggest that adult neurogenesis may have a crucial role at the individualisation of brain-related phenotypes.
2 10 Tara Walker The University of Queensland Selenium increases hippocampal neurogenesis and rescues the age-related cognitive decline Tara L Walker, Ruslan Rust, Lisa Grönnert, Joanna M Wasielewska, Susann Ruhwald, Rupert W Overall, Vijay S Adusumilli, Daniel G Blackmore, Nicole Rund, Alexander M Sykes, Annette E Rünker, Odette Leiter, Perry F Bartlett and Gerd Kempermann Adult hippocampal neurogenesis, the lifelong generation of new neurons decreases with age, and results in a concomitant decline in particular forms of learning and memory. This process can be positively regulated by a wide range of external stimuli, one of which is diet. We show that the trace element selenium increased the number of proliferating neural precursor cells and newly born neurons in the hippocampus of young adult and aged mice. Importantly, dietary selenium supplementation also rescued the age-related decline in hippocampus-associated learning and memory. Selenium is the core regulator of the ferroptotic cell death pathway. We found that selenium treatment reduced levels of intracellular reactive oxygen species and blocked lipid peroxidation, hallmarks of ferroptotic cell death, specifically in nestin+ precursor cells. In addition, in vivo infusion of the ferroptosis inhibitor liproxstatin-1 significantly increased the number of proliferating hippocampal precursor cells.  Conversely, infusion of the ferroptosis inducer RSL-3 reduced precursor proliferation, and imaging by electron microscopy revealed alterations in mitochondrial morphology that are characteristic of cells undergoing ferroptotic cell death. Together this data suggests that selenium acts to increase hippocampal neurogenesis via a reduction in ferroptotic cell death at the precursor cell stage.
2 11 Thomas Kerloch Neurocentre Magendie INSERM U1215 Rnd2: a new player in the regulation of adult hippocampal neurogenesis Thomas Kerloch, Fanny Farrugia, Marlène Maître, Muriel Koehl, Mylène Blanchard, Hélène Doat, Thierry Leste-Lasserre, Julian Ik-Tsen Heng, Adeline Goron, Delphine Gonzales, François Guillemot, Djoher Nora Abrous, Emilie Pacary  In most areas of the brain, neurons are born during embryogenesis. In contrast, the majority of granule neurons in the dentate gyrus (DG) of the hippocampus are born postnatally and their generation continues throughout adulthood. This finding that new neurons are generated in the adult mammalian brain has opened novel avenues for brain repair and has initiated, in the last 20 years, tremendous efforts to characterize how new neurons differentiate and integrate into adult neural circuitries. However, further studies are needed to better understand the mechanisms and signaling cascades involved in this process.
Although neurogenesis is a highly choreographed process that requires an extensive and dynamic remodeling of the cytoskeleton, very little is known about the role of cytoskeletal regulators during adult neurogenesis. In particular, few studies have addressed the roles played by RhoGTPases, the master regulators of cytoskeleton rearrangements. In this context, we focused on Rnd2, a Rho GTPase particularly enriched in the temporal part of the adult mouse DG and expressed in endosomes. We found, using retrovirus in vivo, that the deletion of Rnd2 specifically in adult-born hippocampal neurons decreases the survival of these cells, and in the surviving ones, leads to soma hypertrophy, increases dendritic arborization and induces mispositioning. Moreover, we show that Rnd2 is critical for adult hippocampal newborn neuron survival, only during a defined period of their development at the immature stage when they integrate the hippocampal circuitry. Importantly, Rnd2 deletion in adult-born hippocampal neurons also increases anxiety-like behavior in mice, thus identifying Rnd2 as a potential novel target for anxiety disorders. In addition, our data show that Rnd2 does not play the same functions in granule neurons born at P0, highlighting a differential regulation of developmental and adult neurogenesis in the DG and demonstrating that Rnd2 has specific functions in adult-born neurons.
Altogether our data demonstrate that Rnd2 is a critical regulator of adult newborn neuron development and function in the hippocampus and highlight new important functions for this RhoGTPase in vivo.
2 12 Elena P. Moreno Jiménez Centro de Biología Molecular Severo Ochoa (CBMSO) ADULT HIPPOCAMPAL NEUROGENESIS IS A ROBUST PHENOMENON DURING PHYSIOLOGICAL AGING IN HUMANS E. P. Moreno-Jiménez, M. Flor-García, J. Terreros-Roncal and M. Llorens-Martín The hippocampus hosts one of the most unique phenomena of the adult mammalian brain, namely, the addition of new neurons throughout life. This process, named adult hippocampal neurogenesis (AHN), confers an unparalleled degree of plasticity to the entire hippocampal circuitry. Nonetheless, direct evidence of AHN in humans has remained elusive. Thus, determining whether new neurons are continuously incorporated into the human dentate gyrus (DG) during physiological aging is a crucial question with outstanding therapeutic potential. By combining brain samples from neurologically healthy subjects (between 42 and 87 years of age) obtained under tightly controlled conditions, and state-of-the-art tissue processing methods, we identified thousands of immature neurons in the DG of neurologically healthy human subjects up to the ninth decade of life. Our results strongly support the persistence of AHN during physiological aging in humans. Therefore, we addressed whether cell subpopulations at distinct stages of maturity could also be distinguished among DCX+ cells in the human DG. To this end, we first analyzed the expression of cell markers characteristic of different maturation stages of AHN in human DCX+ cells. Moreover, we analyzed morphometric features of double-labeled cells. Based on both the percentages of double-labeled cells and their morphological features, we have outlined the first proposed model of the differentiation stages of AHN in humans. Our data bring to light the existence of a dynamic population of immature neurons in the human DG throughout physiological aging until the tenth decade of life. These findings point to unexplored mechanisms of circuit plasticity in the aging human hippocampus.
2 13 Vanessa Charrier Neurocentre Magendie INSERM U1215 Influence of spatial learning on the connectome of adult-born neurons in rats Charrier V, Masachs N, Farrugia F, Blin N, Proton V, van Praag H, Gage FH, Callaway ME, Abrous DN. Experience such as learning enhances adult hippocampal neurogenesis in the dentate gyrus, a brain region essential for learning and memory, by promoting newborn neurons integration into the established network and by increasing the complexification of their dendritic arborisation. In return, adult-born neurons play an important role in the encoding of new memories, especially during spatial learning. Yet, it is still unknown how the connectome of adult-born neurons is influenced by spatial learning, though these connections are the source of their activation and role in memory. Here, using an approach allowing the visualization of the glutamatergic post-synaptic densities and a retroviral retrograde monosynaptic tracing method, we characterized the connectome of adult-born neurons following spatial learning at different time points of their maturation. First, we showed that the total glutamatergic post-synaptic density of immature adult-born neurons as well as the one of each sub-part of the molecular layer increased following spatial learning, suggesting an enhanced excitatory innervation. We demonstrated that this enhancement first corresponds to a transient increase in the innervation from other mature granule cells during the first days of life of newborn neurons and would be needed for a successful integration into the network. This transient increase is followed later in the maturation process by an enhanced innervation from the lateral entorhinal cortex and then from the CA3 region. Once adult-born neurons reach full maturity, we showed that spatial learning similarly increased their glutamatergic innervation. Our results suggest a modulation by spatial learning of the adult-born neurons connectome during their maturation.
2 14 Miguel de la Flor García Centro de Biología Molecular Severo Ochoa (CBMSO) UNMASKING NEWBORN DENTATE GRANULE CELLS IN ADULT HUMANS  M. Flor-García, J. Terreros-Roncal, E. P. Moreno-Jiménez and M. Llorens-Martín Hippocampal dentate gyrus (DG) hosts one of the most unique phenomena of the adult mammalian brain, namely adult hippocampal neurogenesis (AHN). AHN results in the generation of dentate granule cells (DGCs). However, direct evidence of human AHN remains elusive. Thus, we sought to determine the existence of AHN in human samples. To this end, we tested different combinations of NaBH4, glycine and heat-mediated citrate buffer antigen retrieval (HC-AR) using brain samples obtained from neurologically healthy subjects until 87 years of age. We chose 0.5% NaBH4 plus a HC-AR as the best sample treatment to unmask newborn DGCs in immunofluorescence assays. Then, we performed stereological counts of DGCs positive for validated doublecortin (DCX) and polysialylated-neural cell adhesion molecule (PSA-NCAM) markers in 4 non-unmasked control samples fixed during 1, 2, 4, 6, 12, 24 and 48 hours in 4% PFA. We observed that the density of DGCs for each marker decreased as fixation times increased, though, a recovery in the densities for both markers was reached by pre-unmasking samples. Also, we studied the influence of other fixative solutions using samples fixed in 3.7% formalin, but DGCs densities for any marker could be recovered by this novel method. Finally, we analysed pre-unmasked samples from 13 neurologically healthy subjects correlating both markers densities with post-mortem delay (PMD). Nevertheless, PMD showed not to affect DCX+ or PSA-NCAM+ DGCs densities. Taken together, these observations demonstrate the great importance of fixative solutions and unmasking procedures used instead of PMD for the newborn DGCs analysis in human samples.
2 15 Mariela Trinchero Leloir Institute A critical period for experience-dependent plasticity in neurons born in the aged hippocampus Mariela F. Trinchero, Magalí  Herrero, M. Cristina Monzón-Salinas, and Alejandro F. Schinder
The aging brain displays a generalized decline in cognitive capacity and circuit plasticity, including a decline in the production of adult-born hippocampal neurons. Morphological development of new dentate granule cells (GCs) is also affected by age. However, their functional properties and integration to the circuit along maturation remains unclear. We performed whole-cell recordings in 8-month old (8M) Ascl1(CreERT2);CAG(floxStopTom) mice to assess intrinsic properties, firing behavior and afferent excitatory connectivity in adult-born GCs labeled with tdTomato. We found that functional properties and connectivity of these neurons develop very slowly. Despite the delayed maturation, new GCs in aging mice display a remarkable potential for structural plasticity. Retrovirally labeled 3-week-old GCs in middle-aged mice are small, underdeveloped and disconnected. Notably, we found that a 7-day exposure to an enriched environment (EE) induced substantial dendritic growth, spine formation and increase in the number of filopodia of mossy fiber boutons in CA3, indicating that experience boosts both input and output connectivity.  EE was particularly effective in promoting integration during the second week of GCs development, but not earlier or later. Moreover, EE accelerated maturation of intrinsic membrane properties and functional integration of new GCs. This short period of increased responsiveness to hippocampal activity induced by experience unveils high levels of neuronal plasticity in the aging brain.
2 16 Frédéric Cassé Center for psychiatric neurosciences, Lausanne University Hospital, Switzerland An astrocyte-secreted peptide that increase adult hippocampal neurogenesis Frédéric Cassé, Charline Carron, Kevin Richetin, Thomas Larrieu and Nicolas Toni Adult hippocampal neurogenesis is regulated by astrocytes, which provides a structural and molecular support for adult neuronal stem cell (aNSCs) proliferation and differentiation and for the functional integration of new neurons.
 In this study, we investigated the effect of astrocytic-released molecules on mouse hippocampal adult neurogenesis. By using the doxycyclin-dependent inducible expression of a dominant-negative form of synaptobrevin-2 (dnSNARE) in astrocytes we blocked astrocytic vesicular release. Interestingly, we found a decrease of aNSCs proliferation in the dentage gyrus (DG) of the dnSNARE mice suggesting that astrocytic molecules released by SNARE dependent process control adult neurogenesis.
Using a combination of in vitro approaches, biochemistry and mass spectrometry, we identified a peptide released by astrocytes that regulate adult neurogenesis. The secreted peptide, that we named peptide P9, is derived from the phospoprotein enriched in astrocyte protein (PEA15) which is a cytoplasmic protein involved in intracellular signaling and can regulates diverse cellular processes, such as proliferation and apoptosis. Interestingly, P9 increased aNSCs proliferation both in vitro and in vivo and increased the number of newborn hippocampal neuron. However, despite its effect on adult hippocampal neurogenesis, P9 neither enhanced memory performance nor affected basal anxiety-like behavior.
These results indicate that astrocytes release peptides that regulate hippocampal neurogenesis in the adult brain. Understanding the mechanism of action of the astrocyte-secreted peptide P9 on adult neurogenesis in the hippocampus could be an interesting therapeutic strategy in order to control neurogenesis in pathological conditions.
2 17 Vijay S  Adusumilli German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany Redox potential defines functional states of adult hippocampal stem cells
Vijay S Adusumilli*, Tara L Walker*, Rupert W Overall*, Gesa M Klatt , Salma A Zeidan, Tim J Fischer, Sara Zocher, Alex M Sykes, Susanne Reinhardt, Andreas Dahl, Dilyana G Kirova, Jörg Mansfeld, Annette E Rünker, and Gerd Kempermann  Intracellular redox states regulate the balance between stem cell maintenance and activation. Increased levels of reactive oxygen species (ROS) are linked to proliferation and lineage specification. In contrast to this general principle, we show that in the hippocampus of adult mice it is the quiescent neural stem cells (NSCs) that maintain the highest ROS levels (hiROS). Classifying NSCs based on intracellular ROS content identified subpopulations with distinct molecular profiles, corresponding to functional states. Shifts in ROS content primed cells for a subsequent transition of cellular state, with lower cellular ROS content marking activity and differentiation. Physical activity, a known physiological activator of adult hippocampal neurogenesis, recruited the quiescent hiROS NSCs into proliferation via a transient Nox2-dependent ROS surge. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity-induced increase in proliferation disappeared. These results describe a novel mechanism linking the modulation of cellular ROS by behavioral cues to the maintenance and activation of adult NSCs.
2 18 Meredith Lodge Department of Biomedicine, University of Basel, Switzerland Differential Synaptic Activation of α5-containing GABAA Receptors in Adult-born Young and Mature Hippocampal Granule Cells Lodge, M.E., Schulz, J. M., Bischofberger, J. γ-Aminobutyric acid (GABA), the main inhibitory transmitter in the adult brain, acts as a trophic factor during the development of adult-born granule cells in the dentate gyrus. GABAergic synaptic currents have been observed in young cells from as early as 4 days post-mitosis, however the molecular composition of GABAA receptors in somatic and dendritic synapses are largely unknown . Here we investigated soma-targeting inhibition using either electrical stimulation of the granule cell layer, or optogenetic stimulation of parvalbumin-positive GABAergic interneurons. Dendritic inhibition was examined using either electrical stimulation of the molecular layer, or optogenetic stimulation of somatostatin-positive interneurons. We found that both forms of inhibition were present from as early as 9 days post-mitosis. Furthermore, we investigated the voltage dependence of synaptic GABAA receptors in young granule cells and found that the inputs are highly non-linear, with strong outward rectification. While both dendritic and somatic inputs onto young cells showed rectifying properties,  mature granules cells only showed rectification in dendritic inputs, suggesting that GABAA receptors present in peri-somatic synapses in young granule cells are different from those in mature. Due to the slow kinetic properties of IPSCs seen in young cells, we tested for the presence of α5-containing GABAA receptors. We found that young granule cells have α5-GABARs present in both dendritic and peri-somatic synapses, while in mature neurons these are  specifically targeted into dendritic synapses.
Supported by Swiss National Science Foundation (SNSF 31003A-176321)
2 19 Samuel Domínguez García Universidad de Cádiz NEUROGENESIS, LEARNING AND MEMORY FACILITATED BY CHRONICAL ADMINISTRATION OF A PROTEIN KINASE C ACTIVATING COMPOUND Samuel Dominguez-García, Carmen Hierro-Bujalance, Noelia Geribaldi-Doldán, A.J. Macías-Sánchez , Rosario Hernández-Galán, Mónica García-Alloza and Carmen Castro  A therapeutic option to achieve neuronal renewal in damaged adult brains, is promoting endogenous neurogenesis. We have previously seen that 12-deoxyphorbols activate PKC promoting the proliferation of adult neural progenitor cell (NPC) in vitro and in vivo, with the advantage that they lack tumorogenic activity. Intracerebroventricular administration of 12-deoxyphorbols such as ER272, induces the proliferation of NPC within the subventricular zone and the dentate gyrus of the hippocampus facilitating the generation of new neurons. In order to find a non-invasive method to deliver this compound in the adult brain overcoming the blood brain barrier, we have tested whether intranasal administration of ER272 exerts a similar effect on proliferation. We have analyzed in here the effect of intranasal administration during 3, 7 and 28 days. After treatment, an increase in the proliferation of NPCs was observed in both SVZ and DG compared with the control. Chronical intranasal administration of ER272 for 28 days increased the number of neuroblasts and neurons within the DG of the hippocampus. In addition, intranasal administration of ER272 for 28 days improved performance of mice in the Morris Water Maze and in new object discrimination tasks indicating it improved memory and learning tasks. These results suggest that intranasal administration is a good non-invasive method of administration of 12-desoxyphorbols, which can be use to promote neuronal replacement for the treatment of neurological disorder. 
Funded by the Spanish Consejería de Innovación, Ciencia y Empleo, Junta de Andalucía (P10CTS6639), and by Ministerio de Economía y Competitividad (BFU2015-68652-R, and BFU2016-75038-R MINECO/FEDER)
2 20 Ricardo Gomez Oliva Universidad de Cadiz CLASSICAL PKC ACTIVATION FACILITATES TGF-alpha RELEASE PROMOTING UNDIFFERENTIATED PROGENITOR CELL PROLIFERATION IN THE SUBVENTRICULAR ZONE Ricardo Gómez-Oliva, Pedro A. Nunez-Abades, Noelia Geribaldi-Doldán, Samuel Domínguez-García, Livia Carrascal, Félix A. Ruiz and Carmen Castro Neurogenic niches such as the subventricular zone (SVZ) harbor neural stem cells (NSC), which generate new neurons and glial cells throughout a lifetime. Upon activation NSC give rise to transit amplifying undifferentiated progenitors, which generate neuroblasts that migrate to the olfactory bulb. Activated NSC and undifferentiated progenitors are colony forming cells when isolated and cultured in vitro. Both types of cells express the epidermal growth factor receptor (EGFR) and proliferate in response to the epidermal growth factor (EGF). Activation of protein kinase C (PKC) by non-tumorigenic diterpenes such as 12-deoxyphorbols promotes proliferation of SVZ-isolated cells in vitro in a PKC-dependent manner and in vivo when infused into the SVZ. Here, we have elucidated the cellular and molecular mechanisms underlying the proliferative effect of 12-deoxyphorbols. We show herein that SVZ isolated cells cultured as neurospheres in the presence of 12-deoxyphorbols maintain a proliferation rate similar to cultures treated with the epidermal growth factor and this effect is dependent on PKC-alpha activation. Moreover, 12-deoxyphorbols specifically promote proliferation of EGFR+ undifferentiated progenitors when infused in the SVZ. Using cells transfected with a construct expressing TGF-alpha fused to Cherry and green fluorescent protein in opposite ends, we show that 12-deoxyphorbols facilitate the release of the EGFR ligand transforming growth factor alpha (TGF-alpha) upon activation of a PKC of the classical subfamily. These results highlight the role of 12-deoxyphorbols as potential drugs to induce neurogenesis for the treatment of neurological disorders.
Funded by Spanish Ministerio de Ciencia, Innovación y Universidades (BFU2015-68652-R).
2 21 Chiara La Rosa Neuroscience Institute Cavalieri Ottolenghi  Cell proliferation and amount of doublecortin-positive neurons in the dentate gyrus of different mammalian species Chiara La Rosa, Chiara Olmeo, Marco Ghibaudi, Irmgard Amrein, Luca Bonfanti Adult neurogenesis extension, rate, time course and functions can be quite heterogeneous among mammals. No systematic, fully comparable analyses are available on a wide range of animal species. Knowledge is restricted to the “extremes”: high rate of neurogenesis in small-brained rodents versus reduced/vestigial in large-brained humans and dolphins. A debate is ongoing due to reports with different conclusions about the rate and temporal extension of adult human hippocampal neurogenesis (e.g., Sorrells et al. 2018; Moreno-Jiménez et al. 2019); in all cases, doublecortin-positive (DCX+) “immature” neurons were observed during adulthood, while no substantial cell proliferation was detectable.
Here, the optical Fractionator with StereoInvestigator software was used to estimate the number of granule cells, DCX+ and Ki-67+ cells in three adjacent sections (of corresponding levels) of the dentate gyrus (DG) of young-adult rabbits, cats, and sheep. Preliminary data indicate that in brain sheep, the largest/most gyrencephalic considered, the number of DCX+ cells is 80-fold higher with respect to proliferating cells, while in rabbit, the smallest/most lissencephalic brain, the ratio is about 9.
These data suggest that in gyrencephalic brains hippocampal immature neurons might persist for a long time in a state of protracted maturation. Previous studies carried out in the hippocampus of sheep and monkeys showed that newly born neurons mature far slower than in rodents (3 to 6 months: Brus et al. 2013). This trend is reminiscent of what has been described in adult humans, and might be shared by newly generated and non-newly generated immature neurons in large-brained mammals.
2 22 Joana Mateus 1-Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal; 2-Instituto de Medicina Molecular João Lobo Antunes (iMM), Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal. Adenosine A2AR as modulators of adult oligodendrogenesis: a potential target for MS therapy Joana Moura Mateus1,2, Marta Alonso Gomes1,2, Rita Soares1,2, Sara L Paulo1,2, Ângelo F Chora2, Ana M Sebastião1,2, Sara Xapelli1,2 Under Multiple Sclerosis (MS) pathological conditions, there is a depletion of oligodendrocytes (OLs), but oligodendrocyte precursor cells (OPCs) present in the brain parenchyma or derived from subventricular zone (SVZ) neural stem cells (NSCs) can differentiate, migrate and partially remyelinate the lesioned areas. Previous data from our group demonstrated that activation of adenosine A2A receptors (A2AR) promoted SVZ-NSCs oligodendroglial differentiation, both in vitro and in vivo. Hence, we aimed at understanding the role of A2AR in adult oligodendrogenesis derived from SVZ-NSCs in an in vivo mouse model of MS. For this, the EAE mouse model was developed, and behavioural tests (open field, rotarod, pole test) were performed to evaluate motor function. Cellular differentiation was assessed by immunohistochemistry assays for bromodeoxyuridine (BrdU) colocalization with oligodendrocytic markers in brain regions of interest. Western blot and ELISA assays were used for myelin protein levels and inflammatory cytokine quantification. Our results for EAE model characterization suggested that motor impairment was proportional to the score of the disease and cellular and molecular data showed an increase in the levels of the pro-inflammatory cytokine TNFα (n=5, p<0.01). A significant increase in NG2+BrdU+ cells in the corpus callosum (CC) of EAE mice was observed (n=3, p<0.05), hinting at the migration of precursor cells from the SVZ to the CC.  Ongoing studies encompass the administration of A2AR agonist CGS21680 in the lateral ventricle for 28 days and assessing its effect on EAE phenotype and adult oligodendrogenesis, ultimately unveiling the modulation of adult oligodendrogenesis as a putative therapy for MS.
2 23 Muriel D. Mardones 1Institute of Biomedical Sciences, Faculty of Medicine and Faculty of Life Sciences, Universidad Andres Bello; 2McGovern Institute for Brain Research, MIT, Cambridge, MA, United States; 3Instituto de Ciencias Biomédicas, Facultad de Ciencias de la Salud, Universidad Autónoma de Chile; 4Centro de Envejecimiento y Regeneración (CARE), P. Universidad Católica de Chile.  PSD95 regulates maturation of adult-born neurons in the mouse hippocampus Muriel D. Mardones1, Patricia V. Jorquera1, Andrea Herrera-Soto1, Fernando J. Bustos1,2, Estibaliz Ampuero3, Brigitte van Zundert1,4 and Lorena Varela-Nallar1. The generation of new neurons in the dentate gyrus of the adult mouse hippocampus involves proliferation of neural stem cells, differentiation, neuronal maturation and integration of new neurons into the hippocampal network. The scaffold postsynaptic density protein 95 (PSD95) regulates dendritogenesis, synaptic formation, and spine morphogenesis of hippocampal neurons during embryonic development. Here we evaluated the role of PSD95 in adult hippocampal neurogenesis. Retroviruses expressing a control shRNA or an shRNA targeting PSD95 (shPSD95), and the ZsGreen reporter protein were stereotaxically injected into the dentate gyrus of 2-month-old mice. Animals were sacrificed 2 or 4 weeks post injection (wpi). PSD95 knockdown did not affect differentiation of newborn cells into neurons as assessed 2 wpi by double immunostaining for ZsGreen and the immature neuronal marker DCX. At 4 wpi, PSD95-deficient neurons expressing the mature neuronal marker NeuN showed an increase in total dendritic length, and in the number of high-order dendrites. Moreover, dendrites from shPSD95-expressing newborn granule neurons showed a reduction in the density of dendritic spines. Our results indicate that PSD95 is required for the final stages of morphological maturation of adult-born granule neurons.

Supported by: FONDECYT N°1190461 (LVN); FONDECYT Nº1181645 and CARE-UC AFB 170005 (BvZ); Nucleo UNAB DI-4-17/N (LVN, BvZ); FONDECYT Nº 11180540 (FJB). FONDECYT N°11180777 (EA)
2 24 Carmen Vivar Laboratory of Neurogenesis and Neuroplasticity. Department of Physiology, Biophysics, and Neuroscience, Center for Research and Advanced Studies of the National Polytechnic Institute, Mexico City Long-term exercise modifies the neuronal network of adult-born granule cells  Carmen Vivar The adult mammalian brain continuously generates new neurons in the hippocampus. The integration of newborn neurons into the existing hippocampal neuronal network is physiologically important for learning and memory. Adult neurogenesis is upregulated by exercise and this increment correlates with improved synaptic plasticity and memory functions. In addition, exercise evokes robust effects on neural plasticity with a rapid onset that evolves over time. We previously described that adult-born hippocampal neurons are sequentially innervated by structures important for memory function forming a unique neuronal circuit and that this circuit is remodeled by one month of physical activity. To understand how exercise enhances learning and memory, we evaluated the long-term modifications to the circuitry of adult-born neurons. Using a combination of retrovirus to label dividing cells and rabies virus as a retrograde tracer we identified the monosynaptic inputs to the adult-born hippocampal neurons in young male C57Bl/6 mice (5-6 weeks old) in sedentary control or exercise conditions. Stereotaxic surgeries were performed to deliver the retrovirus into the right dentate gyrus (DG) of the hippocampus, and 3 or 5 months thereafter rabies virus was injected into the same DG location. Preliminary histological analysis shows transsynaptic labeling of both subcortical and cortical afferents to the DG. These projections were modified by voluntary wheel running in a time-dependent manner suggesting that exercise induces long-lasting modifications to the adult-born hippocampal circuit.
2 25 Hiyaa Ghosh National Center for Biological Science, Tata Institute of Fundamental Research, Bangalore, India A novel regulator of adult neurogenesis and fate-decision in the dentate gyrus     hiyaa@ncbs
2 26 Jana Heppt University Erlangen-Nürnberg, Institute of Biochemistry The role of canonical Wnt-signaling in maturation of adult hippocampal neurons Jana Heppt1, Marie-Theres Wittmann2, Benjamin Häberle1, Nilima Prakash3, D. Chichung Lie1
1Institute of Biochemistry, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany
2Institute of Human Genetics, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany
3Institute of Developmental Genetics, Helmholtz Zentrum München, Germany
It has been established that precise regulation of maturation and functional integration of new-born neurons is essential for adult hippocampal neurogenesis-dependent memory formation, however molecular regulation of this key developmental step remains largely unknown.
Using reporter mouse lines for canonical Wnt-signaling activity, we could show a drop of canonical Wnt-activity after fate specification and re-activation at the timepoint of functional integration, which points towards the importance of canonical Wnt-signaling for maturation.
To investigate the function of canonical Wnt-signaling in maturation we generated a transgenic mouse line that allowed activation of canonical Wnt-signaling via expression of stabilized β-catenin. While continuous Wnt-signaling activity throughout development led to neurons with aberrant morphology, timed activation of canonical Wnt-signaling in immature neurons resulted in downregulation of DCX, enhanced expression of mature neuronal markers, enhanced dendritic refinement and increase in spine density. This indicates that premature canonical Wnt-signaling activity accelerated maturation, however a transient downregulation is necessary for neuronal patterning.
With increasing age mice exhibit impaired maturation of adult-born hippocampal neurons. Intriguingly, adult-born neurons in the aging hippocampus also display a decrease in canonical Wnt-signaling activity, suggesting that impaired Wnt-signaling may contribute to the age-associated maturation deficit. Indeed, expression of stabilized β-catenin in immature neurons of aged mice promoted maturation of adult-born neurons.
In summary, the data emphasize that tight regulation of canonical Wnt-activity plays a crucial role in maturation of adult-born neurons and also suggest that the age-related deceleration of neurogenesis is partly mediated by a decrease in Wnt-signaling activity.
2 27 Agathe Athena Grandcolas Institute of Molecular Biotechnology (IMBA) Potential role of the ECM as a regulator of adult neurogenesis in the hippocampal neurogenic niche  Agathe Grandcolas, Tatjana Kepcija, Rut Gabarró-Solanas, Debora de Azevedo Pires, Iván Crespo and Noelia Urbán Adult neural stem cells (aNSCs) give rise to new neurons and astrocytes in the dentate gyrus (DG) of the hippocampus. The majority remain in a quiescent state, not actively proliferating. Quiescent aNSCs are tightly regulated by niche components like extracellular matrix (ECM) proteins. The regulation of the transition between quiescence and proliferation is essential to balance generation of new neurons with long-term preservation of the aNSC pool. We have analysed RNAseq and Mass Spectrometry data from quiescent versus proliferative aNSCs in vivo and in vitro and selected ECM components differentially expressed between these conditions. One of such candidates is the apolipoprotein Clusterin, strongly expressed in quiescence, that could play a role in maintaining this state through regulation of proliferation and apoptosis pathways. Another candidate expressed in quiescence is Tmem47, a membrane protein of unknown function in the brain. To confirm expression differences, we use quantitative PCR and compare proliferative versus quiescent aNSCs using an in vitro system that can mimic these states in aNSCs. We have already started to characterise several candidates using immunostaining and WB for protein profiling, first in vitro then in vivo. In the future, we will assess the functional role of these candidates in controlling quiescence/activation transitions with loss and gain of function experiments in vitro. Ultimately, we aim to unravel how ECM proteins modulate the effects of systemic metabolism on aNSC quiescence and adult neurogenesis.
2 28 Marion Martin 1 Jean‐Pierre Aubert Research Centre, UMR‐S 1172, Développement et plasticité du cerveau neuroendocrine, Lille Cedex, France;
2 University of Lille, School of Medicine, Place de Verdun, 59045 Lille Cedex, France
Cell neogenesis in the rat maternal brain Marion Martin1,2, Giuliana Pellegrino1,2, Clémentine Maurice1, Vincent Prevot1,2, Ariane Sharif1,2 The success of pregnancy involves a high degree of brain plasticity, including increased neurogenesis in the subventricular zone/olfactory bulb system that promotes maternal behaviour. The hypothalamus has recently emerged as another germinal niche producing new neurons and glial cells in the postnatal brain. Given the central role of the hypothalamus in the control of reproduction and physiological adaptations to pregnancy, we explored whether cell neogenesis occurs in the rat maternal hypothalamus.
Analysis of cell proliferation using the thymidine analogue BrdU showed that hypothalamic proliferation varies across the estrous cycle, with a peak in diestrus 2, which precedes the pre-ovulatory surge leading to ovulation. Cells born in diestrus 2 preferentially survived if females became pregnant. This differential survival was selectively seen in the medial preoptic area (mPOA), a hypothalamic region acting as a hub in the onset of maternal behaviour. Co-immunofluorescent labelings showed that most BrdU+ cells in the mPOA expressed the oligodendroglial marker Olig2 but were negative for the astrocyte markers GFAP and S100b, and for the neuronal marker HuC/D. The fraction of BrdU+ cells co-expressing Olig2 was higher in pregnant versus non-pregnant rats. Moreover, BrdU+ newborn cells were frequently found morphologically associated with neuron cell bodies, some of which expressed parvalbumin and/or ERa.
 Altogether, our data show that pregnancy is associated with the production of oligodendroglial lineage cells in the mPOA that associate with neurons, and raise the possibility that gliogenesis may contribute to modulating the activity of neuronal networks involved in the onset of maternal behaviour.
2 29 Davide De Pietri Tonelli Fondazione Istituto Italiano di Tecnologia (IIT) miR-135a-5p is critical for exercise-induced adult neurogenesis Meritxell Pons-Espinal; Caterina Gasperini; Matteo J Marzi; Clarissa Braccia; Andrea Armirotti; Alexandra Pötzsch; Tara L Walker; Klaus Fabel; Francesco Nicassio; Gerd Kempermann
 and Davide De Pietri Tonelli
Physical exercise stimulates adult hippocampal neurogenesis and is considered a relevant strategy for preventing age-related cognitive decline in humans. The underlying mechanisms remains controversial. Here, we show that exercise increases proliferation of neural precursor cells (NPCs) of the mouse dentate gyrus (DG) via downregulation of microRNA 135a-5p (miR-135a). MiR-135a inhibition stimulates NPC proliferation leading to increased neurogenesis, but not astrogliogenesis, in DG of resting mice and intriguingly it re-activates NPC proliferation in aged mice. We identify 17 proteins (11 putative targets) modulated by miR-135 in NPCs. Of note, inositol 1,4,5-trisphosphate (IP3) receptor 1(ITPR1) and inositol polyphosphate-4-phosphatase type I (INPP4A) are among the modulated proteins, suggesting that IP3-signaling may act downstream miR-135.
miR-135 is the first noncoding RNA essential modulator of the brain’s response to physical exercise. Prospectively, the miR-135-IP3-axis might represent a novel target of therapeutic intervention to prevent pathological brain ageing.
2 30 Marie Lods 1 INSERM U1215, Neurocentre Magendie, “Neurogenesis and Pathophysiology” Group, 146 rue Leo Saignat 33077 Bordeaux-CEDEX, France
2 INSERM U1215, Neurocentre Magendie, “Endocannabinoïds and neuroadaptation” Group, 146 rue Leo Saignat 33077 Bordeaux-CEDEX, France
3 INSERM U1215, Neurocentre Magendie, “Energy balance and obesity” Group, 146 rue Leo Saignat 33077 Bordeaux-CEDEX, France

Pharmacogenetic stimulation of hippocampal adult-born neurons increases remote spatial memory accuracy Marie Lods1 | Emilie Pacary1 | Geoffrey Terral2 | Wilfried Maziers3 | Fanny Farrugia1 | Vanessa Charrier1 | Federico Massa2 | Daniela Cota3 | Djoher Nora Abrous1 | Sophie Tronel1 Adult neurogenesis refers to the creation of new neurons in the dentate gyrus of the adult hippocampus. A decade of research has established the role of adult neurogenesis in memory formation, in particular in spatial learning. However, the role of newborn neurons in long-term processes remains unclear.

Here we investigate the role of neurons that were either immature (1 week old) or more functionally mature (6 weeks old) at the time of learning during long-term memory retrieval, in rats.
Using immediate early gene expression analysis, we first show that 6 weeks-old neurons are activated by learning, which is not the case of immature neurons. In order to stimulate adult-born neurons during remote memory retrieval, we used a retrovirus coupled with a DREADD Gs protein. When injected into the dentate gyrus, this retrovirus allows the specific and reversible activation of new neurons a long time after memory acquisition. Our data show that activating adult-born neurons during retrieval improves remote memory retention. This improvement is observed when both populations, immature and mature, are stimulated. This effect is specific of adult-born neurons because stimulating neurons born during the development had no effect on remote memory retention. These results suggest that new neurons are involved in spatial remote memory during retrieval. Furthermore, these results underline the role of a population of new neurons that is not activated by learning but becomes nevertheless involved in the long term fate of this memory.
2 31 Rocio Foltran Inst. de Biología Celular y Neurociencias (IBCN) CONICET-UBA. Buenos Aires, Argentina Exploring the role of serotonin depletion in the BDNF pathway and the ability for pattern separation Rocío B Foltran, Karen Stefani, Silvina L Diaz Modulation of serotonergic neurotransmission has revealed as an exciting tool to study the process of neurogenesis in the adult hippocampus (HC). Chronic inhibition of tryptophan hydroxylase by para-chlorophenylalanine (PCPA), induces a decrease of serotonin (5-HT) levels and promotes survival of newborn neurons in the HC. We analyzed the molecular pathway of the Brain derived neurotrophin factor (BDNF) in mice treated with PCPA. C57BL/6J male 6-week old mice were treated during 8 weeks by giving aprox PCPA 250mg/kg/day inside yeast and jelly cubes, an oral administration we set up. Control group received cubes without PCPA. After treatments, proteins were extracted from hippocampi and levels of BDNF, TrkB, p75, proBDNF, BCL2 were quantified by Western Blot. P75 was found to be enhanced after PCPA treatment, and a tendency for a decrease was found for TrkB and proBDNF. When animals were administered a 5-HT1A receptor agonist for 1 week after 4 weeks of PCPA, a protocol that reestablishes the neurogenical phenotype, an increase was observed for proBDNF and p75. As we intend to study the role of the new neurons in the HC, we also set up the Object Pattern Separation (OPS), a test recently developed that allows finding subtle differences compared to classical tests. Although no significance was achieved, hyposerotonergic mice showed a tendency to a minor discrimination index. Our results show a clear participation of the BDNF pathway in the regulation of neurogenesis induced by PCPA, but the lack of improved performance in the OPS casts doubts about the functional role of these newborn neurons.
2 32 Edina Silajdžić 1Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, UK.
2Department of Public Health Sciences, King’s College London, London, UK.
3Department of Psychiatry, University of Oxford, UK.
4Department of Neurology, Akershus University Hospital, Lørenskog, Norway.
5Institute of Clinical Medicine, University of Oslo, Campus Ahus, Oslo, Norway.
6Center for Age‐Related Diseases, Stavanger University Hospital, Stavanger, Norway.
7Department of Old Age Psychiatry, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, London, UK.

Validation study of a prognostic biomarker for Alzheimer's disease Edina Silajdžić1a, Aleksandra Maruszak1, Tytus Murphy1a, Benjamine Liu3, Chiara de Lucia1, Dr Abdel Douiri2, Alejo J Nevado3, Jack Price1, Simon Lovestone3, Tormod Fladby4,5, Dag Aarsland,4, 6,7, Sandrine Thuret1a Efficacy trials for Alzheimer’s Disease (AD) will be more successful if conducted in prodromal or preclinical stages necessitating the use of biomarkers that reflect and detect the early disease process. Hippocampal neurogenesis, which is important for learning and memory, is altered early in AD.  We have previously developed a prognostic biomarker for AD that predicts conversion from Mild Cognitive Impairment (MCI) to AD by using an in vitro assay capable of measuring the neurogenic process.  In this assay, HPC0A07/03C human hippocampal progenitor cells are treated with 1 % serum from a prospective cohort including: cognitively healthy individuals, and individuals that were diagnosed with mild cognitive impairment (MCI), some of whom will not develop any disease (MCI-stable) and some will subsequently convert to AD (MCI-converters).  Serum of individuals with MCI differentially alters human hippocampal progenitor cell fate to predict conversion to AD.  The in vitro neurogenesis assay predicted the conversion of MCI patients to AD up to 3.5 years before the AD clinical diagnosis with 92 % sensitivity, 94 % specificity, 97 % positive predictive value, and 84 % negative predictive value. We are currently validating the initial predictive power of the neurogenesis assay, in a blinded fashion using a larger, independent cohort of baseline samples from elderly cognitively healthy, MCI-stable and MCI-progressive individuals.  Once validated in a larger independent cohort, this assay has the potential to be further automated using cell-culture robots to qualify as an affordable and minimally invasive biomarker test to identify individuals at an earlier stage of AD for trial enrichment for disease-modifying therapy interventions.
3 1 Kunimasa Ohta Kumamoto University DISRUPTION OF TSUKUSHI FUNCTION LEADS TO HYDROCEPHALUS BY ABERRANT NEUROGENESIS IN THE BRAIN K Ohta, SAI Ahmad, MB Anam, and N Ito We have been studying the molecular function of Tsukushi (TSK), which is a soluble molecule belonging to the Small Leucine-Rich Proteoglycan family (Ahmad et al., 2018). TSK is expressed in pericytes on the blood vessels and ependymal cells in the subventricular zone (SVZ) of the lateral ventricle (LV) of the mouse brain. We analyzed brain morphology and expression of marker genes in TSK knockout mice (TSK-/-) brain. TSK-/- mice developed hydrocephalus after birth and this phenotype became even more severe at later stages. We found aberrant cell proliferation and cell death at SVZ in TSK-/-. Both overexpression of TSK protein using transgenic mice in TSK-/- background and direct injection of TSK protein into the LV of TSK-/- brain rescued the LV expansion in TSK-/-. We performed sequencing of peripheral blood DNA from 13 hydrocephalus patients with unknown reason and found 3 heterozygous nucleotides changes within TSK coding region, which introduce missense mutations that are predicted to cause amino acid changes in TSK protein sequence.
Our results suggest that TSK is involved in the pathogenesis of hydrocephalus in human patients. We would like to discuss the developmental mechanism of hydrocephalus in the absence of TSK, therapeutic potential of TSK to rescue hydrocephalus and determine the percentage of patients carrying mutated TSK gene to establish TSK as a marker for hydrocephalus diagnosis.
3 2 Rui Rodrigues Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Cannabinoids and Adenosine A2A receptors: impact on postnatal neurogenesis R.S. Rodrigues 1,2, A. Armada-Moreira 1,2, F.F Ribeiro 1,2, A.M Sebastião 1,2, S. Xapelli 1,2

1 - Instituto de Farmacologia e Neurociências, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal;
2 - Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
Postnatal neurogenesis operates in specialized niches of the mammalian brain in a process modulated by cannabinoid type 1 and 2 receptors (CB1Rs and CB2Rs). Recent evidence sheds light on the interaction of adenosine A2A receptors (A2ARs) with cannabinoid receptors. Herein, we aimed at understanding the putative role of A2ARs on cannabinoid-mediated cell fate, cell proliferation and neuronal differentiation of rat subventricular zone (SVZ) and dentate gyrus (DG) neural stem/progenitor cell cultures. CB2R or A2AR activation was found to promote self-renewing divisions of DG cells. Importantly, A2AR antagonist blocked the effect mediated by CB2R activation, while CB1R or CB2R antagonists blocked A2AR-mediated effect. SVZ cell proliferation was only affected by CB1R activation, an effect blocked in the presence of an A2AR antagonist. Although CB1R, CB2R or A2AR activation alone did not alter DG cell proliferation, CB1R or CB2R co-activation with A2ARs promoted a significant increase in DG cell proliferation. Lastly, CB1R and/or CB2R activation promoted SVZ and DG neuronal differentiation, while A2AR activation only promoted DG neuronal differentiation. In both cases, the proneurogenic effect mediated by CB1R or CB2R agonists was blocked by an A2AR antagonist, while in DG the A2AR-mediated actions on neuronal differentiation were blocked by CB1R or CB2R antagonists. Preliminary in vivo evidence suggests that blockage of A2ARs may be implicated in cannabinoid-mediated regulation of hippocampal neurogenesis, affecting cognitive performance.
Taken together, our findings suggest an interaction between the adenosinergic and cannabinergic systems, cross-antagonism being evident, responsible for controlling early stages of postnatal neurogenesis.
3 3 Julia Terreros Roncal Centro de Biología Molecular Severo Ochoa (CBMSO) ADULT HIPPOCAMPAL NEUROGENESIS IS DRAMATICALLY IMPAIRED IN ALZHEIMER´S DISEASE PATIENTS J. Terreros-Roncal, E. P. Moreno-Jiménez, M. Flor-García, and M. Llorens-Martín  Alzheimer´s disease (AD) is a devastating disease which is considered to be the most common type of dementia in industrialized countries. One of the brain areas primarily affected by disease progression, namely the hippocampus, hosts one of the most unique phenomena that occurs in the adult mammalian brain. This phenomenon, named adult hippocampal neurogenesis (AHN) encompasses the addition of new neurons throughout life. This process is crucial for hippocampal-dependent learning and confers an unparalleled degree of plasticity to the entire hippocampal circuitry.
By using a collection of hippocampal samples obtained from 58 control subjects and AD patients distributed among the 6 neuropathological Braak and Braak stages of the disease, we have studied the survival and maturation of immature neurons in the dentate gyrus of these individuals. Strikingly, our data reveal a progressive reduction in the number immature neurons as the disease progresses. Furthermore, the maturation of these cells is blocked at multiple levels. Importantly, these alterations are observed at early stages of the disease, although a progressive worsening is observed as the disease advances.
Therefore, the putative detection of AHN impairments by non-invasive techniques might turn this phenomenon into a relevant biomarker for AD progression. Moreover, given that AHN can be regulated extrinsically (at least in rodents), increasing AHN in humans emerges as a potential therapeutic target for AD or other neurodegenerative diseases.
3 4 Soraya Martín Suárez Achucarro Basque Center for Neuroscience.
University of the Basque Country (UPV/EHU). 
ATP links the Induction of Reactive Neural Stem Cells by Neuronal Hyperexcitation Soraya Martín-Suárez; Oier Pastor-Alonso José R Pineda; Juan M Encinas. Besides generating neurons, the NSCs dwelling in the adult hippocampus generate astrocytes, more copies of themselves and even olygodendrocytes after genetic manipulation. The balance between excitatory and inhibitory input regulates the behavior and fate of hippocampal NSCs. Thus, in normal conditions NSCs generate mostly neurons and astrocytes and symmetric self-renewing division cannot compensate for the natural decline of the population. As we demonstrated using a model of temporal lobe epilepsy, when neuronal hyperexcitation is high enough to trigger seizures NSCs become reactive (React-NSCs): they lose their broccoli-like apical arborization, become multibranched and hypertrophic, also entering massively into mitosis and overexpressing proinflammatory cytokines. React-NSCs divide symmetrically generating more copies of themselves that will ultimately differentiate into reactive astrocytes. Therefore NSCs are able to contribute to the neuroinflammatory response at the expense of abandoning their neurogenic potential. We show here how the induction of React-NSCs requires neuronal hyperexcitation and is cannot be only a consequence of inflammation. Thus our hypothesis is that NSCs could become React-NSCs in parallel to the astroglial and microglial reactive response, but is independent of it. Also our work suggests that ATP is a direct link between hyperactive/excitotoxic neurons and NSCs and mediates the induction of React-NSCs.  Purinergic P2X receptors, specific for ATP, are present in NSCs in vivo and in vitro and in both cases administrating ATP triggers the conversion of NSCs into React-NSCs by increasing the intracelullar concentration of calcium. This mechanism for NSC regulation in pathological conditions could represent a potential therapeutic target to preserve NSCs and adult neurogenesis in epilepsy.
3 5 Barbara Planchez Inserm U1253 iBrain, Tours, France.
Neurogenesis and resilience to Post-Traumatic Stress Disorder

Barbara Planchez, Mohamed-Lyes Kaci, Bruno Brizard, Anne-Marie Le Guisquet, René Hen, Catherine Belzung Post-Traumatic Stress Disorder (PTSD) is a neuropsychiatric illness which can occur after being exposed to a traumatic event, inducing typical symptom clusters in patients such as intrusive revival memories, constant avoiding of stimuli associated with the trauma and an overdrive of the autonomous nervous system. This traumatic event also triggers morphological and functional alterations in brain regions mediating emotions and stress-related situations. Thus, the pathophysiology of PTSD is characterized by decreased volume and activity in the medial prefrontal cortex and the hippocampus, a hyper-activity of the amygdala and an alteration of the neuroendocrinian system (Hypothalamus Pituitary Adrenal axis overdrive). Studies showed that the alteration of the hippocampus, and more precisely the dentate gyrus, where adult neurogenesis stem from, could produce abnormalities in stress regulation, fear response extinction and contextual memorization. This knowledge made ground for an hypothesis, that is, integrity of new neurons could be an important factor for developing a PTSD. To answer this very question, we worked on mice having an increase in adult-generated neurogenesis (Ibax mice) and confronted, at different time-points after induction of neurogenesis, to acute traumatic stress (an electric footshock). We show a decrease in anxiety-related behaviors after an increase of newborn neurons, suggesting that it is sufficient to reverse the trauma-related phenotype. Moreover, neurobiological modifications in brain regions implicated in fear response along with these behavioural changes were found. Thus, adult hippocampal neurogenesis could impact the activity of extra-hippocampal brain areas.
3 6 Hoda Eliwa PhD student Estimation of Adult Hippocampal Neurogenesis (AHN) sufficiency for anti-depressive like effects Hoda Eliwa, Anne-Marie Le Guisquet, René Hen, Catherine Belzung, Alexandre Surget Major depressive disorders (MDDs) are the most up-growing diseases among millions of people globally. However, the treatment options and approaches required for drug development are still restricted. Deteriorated adult hippocampal neurogenesis (AHN) has been identified as a putative contributor to the evolution of MDDs. In addition, it has been revealed that AHN is crucial for several behavioral effects of antidepressants (ADs).  In our study, we investigate whether increasing AHN during 8 weeks of unpredictable chronic mild stress (UCMS), a mouse model of depression, could be sufficient to produce antidepressant-like effects in a transgenic mouse line (iBax mice). In this mouse model, the pro-apoptotic gene Bax can be inducibly ablated in the neural stem cells by tamoxifen (Tam) intraperitoneal injection, in turn, this is assumed to ameliorate the survival of adult born neurons whose 60% cells undergo apoptosis naturally. Depressive-like behaviors (changes in coat state, despair, anhedonia, and anxiety) have been evaluated within this study in addition to neuro-histological evaluations including BrdU immunohistochemical staining within the hippocampal dentate gyrus. UCMS induced several behavioral effects that are related to depression-like state. These effects were partly reversed by tamoxifen treatment. In addition, AHN alterations may be related to some of these behavioral changes. Finally, our results are discussed in relation to the adult neurogenesis hypothesis of depression and antidepressant effects.
3 7 Ruth Beckervordersandforth 1 Institute of Biochemistry, Friedrich-Alexander-University of Erlangen-Nuremberg, Germany
2 Department of Physiology, University of Lausanne, Switzerland
Adult astrogenesis and functional astrocyte heterogeneity in the adult mouse hippocampus Julia Schneider1, Julian Karpf1, Marlen Knobloch2, and Ruth Beckervordersandforth1 The brain works as a functional co-operation unit between neurons and glial cells. This unit differs in its physiological properties in distinct brain regions and developmental stages. Neuronal diversity has been extensively investigated in the last decades. Recent works now suggests that also astrocytes are molecularly and functionally distinct, yet still little is known about astrocyte heterogeneity. We found that the adult hippocampal dentate gyrus is populated by morphologically distinct astrocytes that are localized to specific compartments. In order to assess structural astrocyte heterogeneity we carried out a detailed morphological analysis of distinct astrocyte subtypes and assessed their “connectome”, i.e. which other niche cells and structures are in direct contact to the astrocytes. As structural heterogeneity of astrocytes may be a reflection of their functional properties, we performed single-cell sequencing of astrocyte subtypes to analyze distinct molecular properties of astrocytes and to identify new markers for targeting astrocyte subtypes.
In contrast to the prevailing assumption that astrocytes are postmitotic in the non-injured adult brain, our work revealed proliferation of non-radial astrocytes in the adult dentate gyrus. Even more surprising was the finding that morphologically distinct astrocytes show a differential proliferation response in the context of specific stimuli (voluntary exercise and ageing). These observations led to the hypothesis that the dentate gyrus is composed of molecularly and functionally distinct astrocytes whose dynamics are critical modulators for hippocampal adaption to changing conditions. Collectively, our study provides the first description of adult astrogenesis, structural heterogeneity and subtype-specific dynamics of astrocytes in the hippocampus.
3 8 CHIH-WEI WU INSTITUTE FOR TRANSLATIONAL RESEARCH IN BIOMEDICINE Maternal high fructose intake suppressed cell proliferation and neuronal differentiation in dentate
gyrus of female offspring via histone deacetylase 4-mediated neuroinflammation
Chih-Wei Wu§, Wen-Chung Liu§, Pi-Lien Hung, You-Lin Tain Lee-Wei Chen, Mu-Hui Fu, Chih-Kuang Liang, Chun-Ying Hung
, Chou-Hwei Lee, and Kay L.H. Wu
Histone deacetylase 4 (HDAC4) mediates maternal high fructose diet (HFD)-impaired learning and memory capacity of adult female offspring. However, the underlying mechanism is largely unknown. Hippocampal adult neurogenesis is important for supporting the cognition. Peroxisome proliferatoractivated receptors γ (PPAR γ) enhances cell proliferation and differentiation while nuclear factor-κB (NF-κB)-mediated neuroinflammation possess opposite effects. In this study, we investigated the role of HDAC4 in PPAR γ, NF-κB and the hippocampal neural stem cells (NSCs) proliferation and neuronal differentiation of adult female offspring of maternal HFD during gestation and lactation. The protein expressions and cell count of Ki67 and doublecortin (DCX) signals indicated that maternal HFD suppressed hippocampal NSCs proliferation and neuronal differentiation. Hippocampal ionized calcium binding adaptor molecule 1 (Iba-1) and IL-1β were increased. Moreover, the nuclear expression of PPAR γ was downregulated concurrent with increased HDAC4 and NF-κB in the HFD group. Intracerebroventricular infusion with HDAC4 inhibitor (Mc1568, 4 weeks) upregulated the PPARγ, Ki67 and DCX alongside with the downregulation of NF-κB and neuroinflammation. Further, PPARγ activator (pioglitazone, 4 weeks) effectively decreased neuroinflammation and reversed NSCs proliferation and neuronal differentiation. Together, these results demonstrated that HDAC4-suppressed PPARγ to initiate neuroinflammation resulting in decreased hippocampal NSCs proliferation and neuronal differentiation of HFD female offspring.
3 9 Romain Troubat Inserm U1253 « Imaging and Brain » (iBrain)

Team Neuro-functional Psychiatry
“Impact of P2X7 receptor Knock out mice on Neuroinflammation and Neurogenesis in animal model of depression: research for new potential therapeutic targets.” Troubat Romain, Brizard Bruno, Pinchaud Katleen, Roger Sébastien, Leman Samuel, Camus Vincent Major depressive disorder (MDD) is one of the most complex and frequent neuropsychiatric condition with 300 million affected patients worldwide. Despite its obvious importance to global disease burden, the last decades have seen a persistent lack of progress in therapeutic research relating to MDD. Actual standard treatments use antidepressants (ADs), efficient by targeting monoamines neurotransmission and exhibit strong link with hippocampal neurogenesis. Even if ADs display a good efficacy, it appears that 40% of MDD’s patient develop a resistance to this treatment. Comprehensive investigation of inflammatory pathways in depressed people have characterized neuroinflammation (NI) as a potential pathophysiological mechanism of MDD. Furthermore, converging data have already associated the purinergic receptor P2X7 (P2X7R) with mood disorder due to its important involvements in maturation and release of interleukin-1 β, a main actor in NI. In our study, we investigate the P2X7 receptor gene deletion in mice model of MDD to improve our understanding of NI’s mechanism in the depressive pathophysiology and its potential interactions with hippocampal neurogenesis. Unpredictable chronic mild stress (UCMS) protocol has been used as animal model for depression follow by behavioral tests to evaluate depressive like behavior (anxiety, anhedonia, resignation). In addition, we performed immunostaining to study NI processes and neurogenesis on mice brain. As expected P2X7R deletion elicit antidepressant effect in behavioral component potentially explain by reduced NI and elevated neurogenesis observed by immunohistochemical study. Our findings suggest that P2X7R can be used as putative therapeutic strategies for MDD.
3 10 Anthony Carrard Center for Psychiatric Neurosciences Role of adult hippocampal neurogenesis in the antidepressant effects of Lactate Anthony Carrard, Frédéric Cassé, Sophie Burlet-Godino, Nicolas Toni, Pierre J. Magistretti and Jean-Luc Martin Growing evidence indicates that astrocytes are involved in the pathophysiology and treatment of depression. For instance, SSRIs stimulate lactate release from cortical astrocytes. Recently, we showed that acute lactate administration increased lactate concentration in the hippocampus and reduced immobility in the forced swim test (Carrard et al, Mol Psychiatry 2018). We further investigated the antidepressant effects of lactate in the corticosterone model of depression and the open-space forced swim model of depression, two animal models that respond to chronic antidepressant treatment. Chronic administration of lactate improved depressive-like behavior. In particular, chronic lactate injection reversed the corticosterone-induced anhedonia and partially restored mobility in the open-space forced swim model of depression. The antidepressant effects of lactate are associated with changes in the expression of Hes5, a transcription factor involved in adult hippocampal neurogenesis. These findings led us to investigate the role of adult hippocampal neurogenesis in the antidepressant effects of lactate.
The involvement of hippocampal neurogenesis in the antidepressant effects of lactate was assessed in the corticosterone model of depression. Chronic peripheral injections of lactate counteracted the decreased neural progenitor proliferation and survival induced by corticosterone treatment. In contrast, chronic administration of pyruvate did not produce antidepressant effects and did not prevent the inhibition of neural progenitor proliferation and survival induced by corticosterone. Importantly, depletion of hippocampal neurogenesis by the antimitotic drug temozolomide suppressed the antidepressant effects of lactate in the chronic corticosterone paradigm. Collectively, these data emphasize the importance of adult hippocampal neurogenesis in the antidepressant effects of lactate.
3 11 Pascal Bielefeld University of Amsterdam Single cell RNA sequencing of hippocampal neural stem and progenitor cell populations after Cortical Traumatic Brain Injury  P. Bielefeld1, A. Martirosyan2, E. Toledo1, J.M. Encinas2, M.G. Holt3, Fitzsimons C.P1 Traumatic brain injury (TBI) is associated with long-term cognitive deficits that are often dependent on hippocampal functioning. Even when not directly mechanically affected by the trauma, the hippocampus undergoes atrophy and synaptic alterations after TBI. Furthermore, neural stem/progenitor cells (NSPCs) in the hippocampus, which are crucial for adult hippocampal neurogenesis, are also affected.
Here we aim to identify the effect of cortical TBI on different subpopulations of NSPCs. To this aim we make use of the controlled cortical impact (CCI) model of TBI, in combination with the Nestin-GFP NSPC reporter mouse strain. We use single cell RNA sequencing to further identify alterations within hippocampal NSPC populations isolated from the dentate gyrus using FACS.
Initial immunohistochemistry data indicates that cortical TBI quickly affects the NSPCs. Severe astrogliosis can be seen in the hippocampus 3 days post TBI, as well as an increase in two astrogenic NSC populations, the Type B NSCs and the reactive NSCs.
Ongoing single cell RNA sequencing analysis will help describe changes in specific NSPC populations after TBI, and possibly identify potential genetic mechanisms underlying changes in NSC fate.

This work is financed by the Horizon 2020 cofund initiative ERA-NET NEURON, Hersenstichting Nederland and The Netherlands Organization for Scientific Research (NWO) grants to C.P.F.
3 12 Valentine Golzné CHUV/ Center of psychiatric Neurosciences Bugs and the brain: Is adult neurogenesis sensitive to changes in gut microbiota ?  Valentine Golzné1, Frédéric Cassé1, , Manuel Oliveira2, Amy Pooler2, Chloé Picq3, Sebastien Sultan2, Gabriela Bergonzelli3, Pascal Steiner2 and Nicolas Toni1 The gut microbiota is involved in general health and an increasing number of studies suggest that its impairment may participate to the development of psychiatric diseases such as depression. Modulating the gut microbiota with probiotics may therefore offer a novel therapeutic target for mood and anxiety disorders.

In the present study, we tested the possibility that adult neurogenesis may be sensitive to changes in the gut microbiota.

To this aim, cell proliferation using BrdU administration after Bifidobacterium feeding was assess firstly. Secondly, survival, differentiation and differentiation of newborn hippocampal neurons using confocal microscopy were examined. Different administration protocols and durations were tested to assess effect duration and stability.
3 13 Vanessa Oliveira Moreira Collège de France OTX2 signals from the choroid plexus to regulate adult neurogenesis Anabelle Planques*, Vanessa Oliveira Moreira*, Chantal Dubreuil, Alain Prochiantz and Ariel A. Di Nardo Proliferation and migration during adult neurogenesis are regulated by a microenvironment of signaling molecules originating from local vasculature, from cerebrospinal fluid produced by the choroid plexus, and from local supporting cells including astrocytes. Here, we focus on the function of OTX2 homeoprotein transcription factor in the mouse adult ventricular-subventricular zone (V-SVZ) which generates olfactory bulb neurons. We find that OTX2 secreted by choroid plexus is transferred to supporting cells of the V-SVZ and rostral migratory stream. Deletion of Otx2in choroid plexus affects neuroblast migration and reduces the number of olfactory bulb newborn neurons. Adult neurogenesis was also decreased by expressing secreted single-chain antibodies to sequester OTX2 in the cerebrospinal fluid, demonstrating the importance of non-cell autonomous OTX2. We show that OTX2 activity modifies extracellular matrix components and signaling molecules produced by supporting astrocytes. Thus, we reveal a multi-level and non-cell autonomous role of a homeoprotein and reinforce the choroid plexus and astrocytes as key niche compartments affecting adult neurogenesis.
3 14 Farah Chamaa American University of Beirut Modulation of Hippocampal Neurogenesis in Awake Rats by Electrical and Chemical Thalamic Stimulation Farah Chamaa, Batoul Darwish, Elie Al-Chaer, Ziad Nahas, Nayef Saade, and Wassim Abou-Kheir  Deep brain stimulation (DBS) provides clinical benefit for a variety of neurological disorders, but the underlying mechanism of how it alters neural activity remains ill-understood. Our group showed that DBS to the anteromedial thalamic nucleus (AMN) in awake rats modulates adult hippocampal neurogenesis. As DBS might induce off-targets effects, we sought to specifically stimulate the cell bodies of AMN by chemical stimulation using low doses of Kainic acid (KA). (KA). This study included two groups of adult male sprague-dawley rats: Group 1 received 6-sessions of unilateral DBS in the right AMN and group 2 received implants of mini-osmotic pumps in same region releasing KA (500pM) at a rate of 1ml/injection/hr/7days. Sham animals were included for both groups. All rats received BrdU injections during stimulation and were followed for 4 weeks. Novel arm exploration was examined using the Y-maze, and co-labeling of BrdU/NeuN cells was counted in the dentate gyrus. Four weeks after DBS, BrdU+/NeuN+ mature neurons were 3-folds higher than sham. Continuous micro-perfusion of KA increased the number of mature neurons to 4-folds higher than vehicle. The Y-maze test showed that both electrical and chemical stimulation to the AMN enhanced novel arm exploration at 4-weeks after stimulation. The current study presents hippocampal neurogenic responses to electrical and chemical stimulation and reveals a translational behavioral enhancement of hippocampal-related skills following stimulation. It highlights the importance of glutamic kainate receptor activation in the AMN nucleus in modulating hippocampal neurogenesis.
3 15 Nuria Masachs Neurocentre Magendie INSERM U1215 Influence of ontogenetic age of dentate granule neurons in spatial memory Masachs N, Charrier V, Farrugia F, Mazier W, Blin N, Tronel S, Montaron MF, Cota D, Deroche-Gamonet V, Herry C, Abrous DN The mammalian dentate gyrus presents the peculiarity to generate granule neurons throughout the life. Although the contribution of neurons born during adulthood to spatial navigation has been demonstrated, the implication of developmentally born ones’ is unknown. Here, we analyzed the implication of embryonic and, neonatal, early postnatal and juvenile neurogenesis in spatial leaning. We have shown that meanwhile embryonic and postnatal do not play a role in spatial navigation, granule cells born during adolescence play a transient role in spatial navigation.
3 16 Louis Foucault Univ Lyon, Université Claude Bernard Lyon 1, Inserm, Stem Cell and Brain Research Institute U1208, 69500 Bron, France. Pharmacological  Wnt/β-catenin activation promotes cellular regeneration within the neonatal cortex while preserving stem cell maintenance Louis Foucault, Diane Angonin, Vanessa Donega, Guillaume Marcy, Olivier Raineteau
Germinal activity persists throughout life within the subventricular zone (SVZ) of the postnatal forebrain, due to the presence of quiescent neural stem cells (NSCs) that gradually reactivate throughout life. Accumulating evidences point at a role for these cells during tissue repair following premature brain injuries (Fagel et al., 2006; Salmaso et al., 2014), and suggest their amenability to pharmacological manipulations (Azim et al., 2017 Scafidi et al., 2014). The extent of this repair and its long-term consequences on forebrain germinal activity however remain to be explored.
We used chronic neonatal hypoxia as a rodent model of premature brain injury, to investigate the contribution of SVZ NSCs to cellular regeneration within the cortex. Our results reveal an increased proliferation and production of TBR2+ & OLIG2+ progenitors within the dorsal SVZ, which was paralleled by an activation of the Wnt canonical pathway. Fate mapping of SVZ NSCs demonstrates their contribution to de novo oligodendrogenesis and cortical neurogenesis following hypoxia, while confirming a delay of oligodendrocytes maturation. Remarkably, a pharmacological activation of the Wnt/β-catenin pathway by intranasal administration of a Gsk3β inhibitor following hypoxia, promotes neurogenesis and oligodendrogenesis as well as their specification and maturation. Importantly, labeling of NSCs in different states of activation, demonstrates that neither hypoxia nor pharmacological NSCs activation have adverse effects on the reservoir of SVZ NSCs and on their long-term germinal activity.
Altogether, our work highlights the potential of pharmacological approaches to promote cellular regeneration within the neonatal forebrain, while demonstrating no detrimental long-term effect on forebrain germinal activity.

1 Achucarro Basque Center for Neuroscience, Leioa, Bizkaia,Spain.
2 University of the Basque Country (UPV/EHU), Leioa, Bizkaia, Spain.
3 Ikerbasque, The Basque Science Foundation, Bilbao, Bizkaia, Spain.
Functional plasticity of synaptic networks in the hippocampus has been implicated in the development of posttraumatic epilepsy after traumatic brain injury (TBI). Within the hippocampus Dentate Gyrus (DG) acts as “gatekeeper” and “filter” of aberrant or excessive input information. DG function is directly determined by a delicate balance between neuronal excitation and inhibition and TBI can cause changes of this state of equilibrium. Moreover TBI can affect adult hippocampal neurogenesis (AHN) and induce long-term changes in both neural stem cells (NSCs) and newborn neurons and those alterations can contribute to hippocampal dysfunction.
We aim to understand what particular changes TBI induces at the cellular, molecular and electrophysiological level in existing granule cells (GCs), NSCs and newborn neurons by using a model of controlled cortical impact. 
We have observed an increase in neurogenesis up to two months after the injury. These newborn neurons however present altered morphology and migration. In addition, we have found that NSCs get activated in higher numbers and acquire a reactive-like phenotype that is most likely caused by hiperexcitation. Observed changes in spontaneous excitatory currents (sEPSCs) frequency indicate remodeling of excitatory input likely expressed as an increase in the number of excitatory synapses. Those changes are accompanied by a decrease in spontaneous inhibitory currents (sIPSCs) frequency indicating a loss of GABAergic neurons.
This project has received founding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska – Curie grant agreement No. 799384.
3 18 Rut Gabarró Solanas Institute of Molecular Biotechnology (IMBA) Diet as a regulator of adult neurogenesis Rut Gabarró Solanas, Tatjana Kepcija, Agathe Grandcolas, Débora Pires, Rebecca McDonald, Iván Crespo, Noelia Urbán Adult neural stem cells (aNSCs) generate new neurons throughout life that regulate memory and emotions in the hippocampus. This process is subjected to strict intrinsic regulation and responds to extrinsic signals such as diet. For instance, high-fat diet (HFD) decreases neurogenesis, while intermittent fasting (IF) has the opposite effect. The lifelong potential to produce newly-born neurons is determined by the long-term maintenance of aNSCs, which switch between a quiescent or proliferating state to preserve the pool. This transition is, in fact, the most important regulatory step for aNSCs, however, very little is known about its systemic regulation. Combining lineage tracing and DNA label retention experiments, I showed that one month of IF does not affect neuronal production. On the other hand, I have observed changes in behaviour of NSCs upon IF that I am currently characterizing. My next step will be to look at the long-term consequences IF has on the aNSC pool and neurogenesis. I have also investigated the effects of HFD and have not found differences in neurogenesis with its control. However, both the HFD and the control diet reduced neurogenesis compared to a regular chow diet, highlighting the importance of using nutrient matched control diets. Moreover, I want to identify the circulating factors that bridge diet and adult neurogenesis and dissect the molecular mechanism by which they modulate aNSCs. Lessons learned from aNSCs could be applied to other adult stem cell systems and provide new therapeutic approaches for regenerative medicine.
3 19 Marco Fogli 1 Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Italy
2 Department of Life Sciences and System Biology, University of Turin, Italy
3 Mathematical Sciences, University of Southampton, United Kingdom
4 Department of Neurosciences “Rita Levi Montalcini”, University of Turin, Italy
* These authors contributed equally to the study

Transient neurogenic niches are generated by the sparse and asynchronous activation of striatal astrocytes after excitotoxic lesion Marco Fogli 1,2, Nato Giulia 1,2, Philip Greulich 3, Paolo Peretto 1,2, Annalisa Buffo 1,4,* & Federico Luzzati 1,2,* In the adult brain, subsets of astrocytes act as neural stem cells in two anatomically defined neurogenic niches: the sub-ventricular zone and hippocampal dentate gyrus. Surprisingly, after excitotoxic lesion striatal astrocytes acquire stem cell properties and generate a large amount of neuroblasts for at least six months. Yet the presence and organization of striatal neurogenic niches and the spatio-temporal dynamics of striatal astrocytes activation and lineage progression remain by large unclear.
Here, through genetic lineage-tracing experiments and 3D reconstructions coupled with mathematical modelling and computer simulations we dissected the transition of striatal astrocytes toward neurogenesis. In the striatum, neurogenic astrocytes are scattered throughout the parenchyma and expand locally, generating clusters of clonally related cells, that we define as striatal niches. These structures are initially composed only of activated astrocytes and transient amplifying progenitors. These latter cells subsequently expand and generate proliferating neuroblasts following a stochastic mode of division and differentiation. Post-mitotic neuroblasts accumulate in the cluster before dispersing as individual cells. Interestingly, striatal astrocytes become activated at a constant rate, resulting in the continuous addition of new striatal niches with time. Nevertheless, the total number of niches does not increase with time indicating that these structures have a transient existence. Thus, continuous striatal neurogenesis occurs through the asynchronous transition of scattered neurogenic astrocytes from quiescence to an active state.
Overall, these data suggest that the neurogenic potential is widespread among striatal astrocytes, and that the striatal parenchyma is largely permissive for de-novo establishment of neurogenic niches.
3 20 Giulia Nato 1 Neuroscience Institute Cavalieri Ottolenghi, University of Turin, Orbassano, Italy
2 Department of Life Sciences and Systems Biology, University of Turin, Italy.
3 Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany; and Focus Program Translational Neuroscience, Johannes Gutenberg University Mainz, Germany.
4 Centre for Developmental Neurobiology, King's College London, United Kingdom.
5 Department of Neuroscience Rita Levi-Montalcini, University of Turin, Italy.
‡These authors contributed equally to this work
Astrocyte-generated neurons functionally integrate into the lesioned striatum Giulia Nato 1,2, Marco Fogli 1,2, Nicolás Marichal 3,4, Ilaria Ghia 1, Paolo Peretto 1,2, Benedikt Berninger 3,4, Annalisa Buffo 1,5,‡ and Federico Luzzati1,2,‡ After excitotoxic lesion, subsets of striatal astrocytes undergo a spontaneous neurogenic activation leading to the local generation of a large amount of neuroblasts for at least six months post-lesion. Yet, the identity of the lesion induced neurons and their functional integration remain unclear. Fate mapping and 3D reconstruction analyses show that striatal neuroblasts undergo a maturation process in which initially they organize in clusters, subsequently disperse as individual cells, and gradually attain complex morphologies often showing dendritic spines. These neurons fail to express typical markers of striatal neurons and live transiently, similar to other models of physiological and pathological striatal neurogenesis. Surprisingly, rabies virus-based monosynaptic tracing indicated that despite their transient life, striatal neuroblasts receive local inputs from striatal projection neurons and interneurons as well as long-range connections from different cortical and thalamic areas. Electrophysiological recordings in acute brain slices showed that many of these local-generated cells acquired membrane properties similar to immature neurons, displaying transient inward currents and generating single action potentials in response to depolarizing current steps. Further, some individual neuroblasts received spontaneous excitatory synaptic inputs while others, likely consistent with a more mature status, generated action potentials repetitively and exhibited inhibitory postsynaptic currents. These results indicate that striatal neuroblasts functionally interact with pre-existing circuits, thus potentially taking part in post-lesion network plasticity supporting functional recovery after damage.
3 21 Roberta Gioia Department of Biology and Biotechnology “Charles Darwin”, Sapienza University, Rome, Italy. Neural stem cell properties and adult hippocampal neurogenesis in a knock-in mice model, expressing an autism-associated mutation.
Roberta Gioia, Raimondo Sollazzo, Marina Vitale, Laura Trobiani, Miriam Di Mattia, Stefano Biagioni, Antonella De Jaco and Emanuele Cacci. The alteration of adult neurogenesis has been associated with neuropsychiatric disorders, including autism spectrum disorders (ASDs). Particularly, in the hippocampus of few ASD mice models, the properties of adult neural stem/progenitor cells (aNSPC) pool and the formation of new neurons have been found altered, suggesting a link between deregulated neurogenesis and some of the behavioural deficits found in these mice.
In order to investigate neurogenesis in association to ASDs, we have been using the R451C Neuroligin3 (NLG3) knock-in mice, a model of a monogenic form of ASDs carrying the R451C substitution found in autistic patients. NLG3 is a postsynaptic protein involved in maturation, specification and plasticity of neural networks and the R451C knock-in mice display excitatory/inhibitory balance alterations in different brain regions, behavioural deficits, and structural brain abnormalities.
We focused our study on the subgranular zone of the hippocampal dentate gyrus (DG), a neurogenic niche of the adult brain. Specifically, we compared proliferation and differentiation of new neurons between knock-in and wild-type mice, both in vivo and in vitro.
In vitro data demonstrate that NSPC cultures derived from the DG of two-month-old knock-in mice contained a higher number of cells compared to the wild-type. However, BrdU cell number in the DGs was unchanged between KI and WT mice. In vivo data also show a decrease in the number of newly formed differentiated mature neurons (BrdU+/NeuN+ cells) in the hippocampus of the knock-in compared to wild-type mice.
The mechanisms underlying the neurogenesis reduction in the knock-in mice are currently under investigation.
3 22 Julia Leschik University Medical Center Mainz, Institute of Physiological Chemistry BDNF overexpression in serotonergic neurons increases adult stem cell proliferation and confers protection against chronic stress by acting in an antidepressive-like manner Julia Leschik, Antonietta Gentile, Cigdem Cicek, Beat Lutz The neurotrophin brain-derived neurotrophic factor (BDNF) influences structural plasticity and function of serotonergic neurons. Both, the serotonergic system and BDNF/TrkB signaling modulate behavioral responses to stress and can lead to pathological states when dysregulated. Both system have been shown to mediate the therapeutic efficacy of antidepressant drugs. Herein, one mechanism could be the upregulation of adult neurogenesis in the hippocampus, which is suggested to play a role in stress resilience. Furthermore, BDNF and serotonin individually upregulate neurogenesis in the subgranular zone (SGZ) of the dentate gyrus. The details on how both systems work together and influence each other on the cellular and behavioral level still remain elusive. Therefore, we have generated a transgenic mouse line which overexpresses BDNF in serotonergic neurons in an inducible manner. In these mice, we observe increased neural stem/progenitor cell proliferation in the SGZ and an enhanced hippocampus-dependent context fear learning, whereas cued fear learning is unchanged. Furthermore, transgenic mice are less affected by chronic social defeat stress (CSD) compared to wild-type animals, likely through upregulated neurogenesis specifically in the ventral part of the hippocampus. In the forced swim test (FST), BDNF-overexpressing mice behave similarly as wild-type mice treated with the antidepressant fluoxetine. Our data indicate an antidepressant role for serotonergic BDNF by enhancing adult neurogenesis.
3 23 Monika Małż German Center for Neurodegenerative Diseases (DZNE)  Effect of ALS/FTD-related mutation in FUS protein on the adult hippocampal neurogenesis Monika Małż, Vijay Adusumilli, Annette Rünker, Zeina Nicola,
Gerd Kempermann

In the adult brain new neurons are continuously generated from neural stem cells (NSCs) in the dentate gyrus of the hippocampus, providing structural plasticity and playing role in learning and memory. This process of adult neurogenesis is altered in the neurodegenerative diseases but still, there is a lack of knowledge pertaining to adult neurogenesis in Amyotrophic Lateral Sclerosis (ALS)/ Frontotemporal Dementia (FTD) spectrum. Therefore, we aim to characterize how the ALS/FTD-related mutation in the Fus gene affects adult hippocampal neurogenesis during aging and how that may contribute to brain function.
We use a mouse model of ALS/FTD in which one copy of FUS harbors complete nuclear localization signal deletion and their wild type littermates as a control. In vivo results indicate that the baseline adult neurogenesis stays on a similar level in mice of both genotypes. However, in vitro proliferation level of cells containing mutated FUS is significantly higher than control’s cells during early passages and this difference declines later on. Moreover, our results indicate that the mutation of FUS protein alters the ability of NSCs to respond to external stimuli such as hypoxia or physical activity, which are known as strong, positive regulators of neurogenesis. Now, our research focuses on investigation what is the underlying mechanism of change in activation ability of NSCs and to test multiple phenotypic aspects, including differentiation potential and response to stress conditions.
Understanding the pathomechanisms of ALS/FTD-FUS in relation to adult hippocampal neurogenesis may provide fundamental bases to consider new therapy based on NSCs.
3 24 Mahmoud Dahab Hans-Berger-Department of Neurology, Jena University Hospital, Jena, Germany  Deletion of the chloride transporter NKCC1 increases radial glia-like stem cell pool in the hippocampus and impairs learning and memory performance in adult mice M. Dahab, E. Göller, K. Reiche, M. Günther, L. Wohlsperger, I. Schaeffner, CW. Schmeer, CA. Hübner, OW. Witte, K. Holthoff, DC. Lie, S. Keiner Aging is the most important contributing factor for decreasing number of radial glia-like stem cells (RGLs) and impaired neurogenesis in the healthy brain. The underlying determinants of this age-dependent decline are not fully understood but may comprise an increase in RGL quiescence or a reduction of RGL self-renewal. One important intrinsic regulator of adult neurogenesis is the inhibitory neurotransmitter GABA. The mode of GABA action depends on intracellular chloride levels, which are determined the differential expression of chloride transporters NKCC1 and KCC2. NKCC1 is predominantly expressed in neural precursor cells and drives cellular Cl- influx. The role of these transporters in RGLs activity in the dentate gyrus remains unknown. In our study we used a transgenic mouse model (NestinCreERT2/NKCC1fl/fl/tdtomato mice) to specifically delete the NKCC1 transporter in nestin+ RGLs. Our data show that NKCC1 knockout strongly promotes the expansion of the RGL neural stem cell population in the hippocampal dentate gyrus during aging. Detailed morphometric analyses of branched and unbranched RGLs indicated an increased cellular complexity in adult and aged mice. Learning and memory performance were assessed with a modified version of the Morris water maze including the re-learning paradigm, as well as hippocampus-dependent and -independent search strategies. KO mice showed higher latency to find the platform. This impairment in flexible learning was also reflected by reduced use of hippocampus-dependent strategies. Our study shows for the first time that the self-renewal capacity of RGLs in the adult and aged hippocampal dentate gyrus is strongly dependent on the chloride importer NKCC1. 
3 25 Jeff Davies Swansea University Medical School Circulating unacylated-ghrelin impairs hippocampal neurogenesis and memory in mice and is altered in human Parkinson's disease dementia Amanda K. E. Hornsby1, Luke Buntwal1, Vanessa V. Santos2, Fionnuala Johnston3, Luke D. Roberts1, Romana Stark2, Alex Reichenbach2, Mario Siervo3, Timothy Wells4, Zane B. Andrews2, David J. Burn3, Jeffrey S. Davies1*. New neurones are formed from neural stem/progenitor cells (NSPCs) in the adult dentate gyrus (DG) throughout life and contribute to spatial pattern separation memory. Factors that promote neurogenesis may attenuate age-related cognitive decline.
Calorie restriction (CR) has been shown to modulate the DG and improve cognitive function, albeit via unknown mechanisms. Previously, we showed that the stomach hormone, acyl-ghrelin (AG), which is elevated during CR, increases neurogenesis in the DG and enhances pattern-separation memory (Kent et al.2015). The ghrelin-receptor (GHSR) is expressed in mature DG neurones in close proximity to DG NSPCs of adult mice, suggesting a non-cell autonomous mechanism of action. We also show that CR enhances neurogenesis in WT but not in GHSR-null mice, demonstrating that CR induces AHN in a GHSR-dependent manner (Hornsby et al.2016).
Here, to determine whether unacylated-ghrelin (UAG), the so-called inactive form of ghrelin, regulates neurogenesis, WT and ghrelin-O-acyl transferase null mice (GOAT-ko) - that lack circulating acyl-ghrelin - were treated with vehicle or UAG for 7-days. Surprisingly, UAG-treated WT mice had reduced proliferating (Ki67+) cells, (DCX+) neurones and newborn (BrdU+/DCX+) neurones. GOAT-ko mice had similar reductions in neurogenic markers and impairments in hippocampal-dependent memory that were restored by acyl-ghrelin treatment.
Finally, we show that circulating AG:UAG in Parkinson's disease dementia was significantly reduced compared to both age-matched healthy controls and a cognitively normal PD group.
These data identify a novel role for UAG in regulating hippocampal plasticity and memory, and suggest that AG:UAG may be a biomarker of dementia in humans.
3 26 Friederike Klempin Max Delbrueck Center Berlin Novel genetic tool to manipulate brain serotonin levels: a new window on the neurobiology of affective disorders  Maria Sidorova, Susann Matthes, Natalia Alenina, Golo Kronenberg, and Friederike Klempin Serotonin is a crucial signal in the neurogenic niche microenvironment, and involved in antidepressant action. Studies of modified animal models constitutively depleted of brain serotonin support the importance of the neurotransmitter in adult neurogenesis. However, one cannot discriminate between phenotypes induced by serotonin per se and compensatory processes provoked by its life-long depletion. Here, we employ a new transgenic rat model (TetO-shTph2), where brain serotonin levels can be acutely depleted, or repeatedly stimulated, which recapitulate the human condition more adequately. Based on doxycycline-inducible shRNA-expression, we measure serotonin levels, and its metabolite, in various brain areas, and specifically examine precursor cell proliferation and survival in the dentate gyrus. Surprisingly, we found that decreased serotonin levels in the raphe and prefrontal cortex of TetO-shTph2 rats at 14 days (Tph2 knockdown) are associated with increased numbers of BrdU-labeled cells in the hippocampus. A result that remains, for the moment, counterintuitive, considering that the mechanism of antidepressants targeting the serotonin system is believed to be increased neurogenesis. Furthermore, reduced brain serotonin in TetO-shTph2 rats leads to a more anxious phenotype in the Open field test. At 5 weeks after serotonin replenishment, the anxious phenotype remains; yet, no differences on cell proliferation and survival were observed compared with control groups using the CldU/IdU treatment paradigm. This study will yield insights into the role of serotonin in the etiology and pathogenesis of affective syndromes. We speculate that decreased serotonin concentrations might represent a compensatory mechanism. Further research will be required to address the new questions raised by these findings.
3 27 Magdalena Zaniewska 1Department of Drug Addiction Pharmacology, Maj Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland
2Laboratory of Pharmacology and Brain Biostructure, Department of Pharmacology, Maj Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland

Effects of the long-term physical activity on depression-like symptoms, drug-seeking behavior and changes in hippocampal neurogenesis during nicotine cessation Magdalena Zaniewska1,2,*, Sabina Brygider1,2, Dawid Gawliński1, Głowacka Urszula2, Glińska Sława3, Balcerzak Łucja3, Mateusz Wątroba1, Krzysztof Wędzony2, Marzena Maćkowiak2

1Department of Drug Addiction Pharmacology, Maj Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland
2Laboratory of Pharmacology and Brain Biostructure, Department of Pharmacology, Maj Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland
3Laboratory of Microscopic Imaging and Specialized Biological Techniques, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland.
Literature data indicate that chronic exposure to nicotine impairs the adult hippocampal neurogenesis in rats, while the long-term exercise may enhance this process. Since the abnormal hippocampal neurogenesis seems to contribute to depression, it can therefore be assumed that disturbances in this process observed in animals self-administering nicotine may be associated with mood disorders occurring during nicotine withdrawal, and that their modification could prevent relapses.
The present study aimed at investigating the effect of running on nicotine withdrawal symptoms and hippocampal neurogenesis. Rats were subjected to nicotine (0.03 mg/kg/inf) self-administration under an increasing schedule of reinforcement. After 21 self-administration sessions, animals were subjected to a withdrawal phase during which they were kept in cages equipped with running wheels (for 14 days).
We reported that exposure to the running wheels during nicotine withdrawal attenuated depression (day 14), but was devoid of the effect on nicotine craving (day 15). We found that on day 14 of nicotine withdrawal there was a significant attenuation of neuronal maturation and decrease in the number of DCX-positive cells, but no change in the number of BrdU- or Ki-67-positive cells. The long-term physical activity during nicotine withdrawal ameliorated the attenuated number of DCX-positive cells.
In summary, the long-term exposure to the running wheels reduced depression-like behavior in nicotine-weaned rats. One possible mechanism underlying the positive effect of running on the affective state during nicotine withdrawal may be the attenuation of deficits in DCX-positive cells in the hippocampus. Research supported by grant HARMONIA 2014/14/M/N24/00561.
3 28 Filippo Calzolari Institute of Physiological Chemistry, University Medical Center, Mainz Increased quiescence and NSC division asymmetry during ageing, in the adult subependymal zone Filippo Calzolari* , Lisa Bast*, Michael Strasser, Jan Hasenauer, Fabian Theis, Jovica Ninkovic and Carsten Marr Neural stem cells in the adult murine brain have only a limited capacity to self-renew, and the number of neurons they generate drastically declines with age. How cellular dynamics sustain neurogenesis and how alterations with age may result in this decline, are both unresolved issues. Therefore, we clonally traced neural stem cell lineages using confetti reporters in young and middle-aged adult mice. To understand the underlying mechanisms, we derived mathematical population models of adult
neurogenesis that explain observed clonal cell type abundances. Models fitting the data best, consistently show self renewal of transit amplifying progenitors and rapid neuroblast cell cycle exit. Most importantly, we identified an increased probability of asymmetric stem cell divisions at the expense of symmetric differentiation, accompanied by an extended persistence into quiescence between activation phases,
with age. Our model explains existing longitudinal population data and identifies particular cellular strategies underlying adult neural stem cell homeostasis and the aging of this stem cell compartment. Moreover, we provide a quantitative framework to facilitate the interpretation of reported and novel neurogenesis-related phenotypes.
3 29 Evgenia Salta VIB-KULEUVEN MicroRNA-132 restores adult hippocampal neurogenesis and memory deficits in Alzheimer's disease Evgenia Salta, Hannah Walgrave, Katleen Craessaerts, Nicky Thrupp, Sriram Balusu, Edina Silajdzic, Henrik Zetterberg, Sandrine Thuret, Mark Fiers, Bart De Strooper Adult hippocampal neurogenesis (AHN) is functionally linked to mnemonic and cognitive plasticity in humans and rodents. In Alzheimer’s disease (AD), the process of generating new neurons at the hippocampal neurogenic niche is altered, but the mechanisms involved are unknown. Here we identify miR-132, one of the most consistently downregulated microRNAs in AD, as a potent regulator of AHN, exerting complex proneurogenic effects in several resident niche cell populations in vivo. Using distinct AD mouse models, cultured human primary and established neural stem cells, and human patient material, we demonstrate that AHN is directly impacted by AD pathology. MiR-132 replacement in adult mouse AD hippocampus restores AHN and relevant memory deficits. Our findings corroborate the significance of AHN in AD and reveal the possible therapeutic significance of targeting miR-132 in neurodegeneration.
3 30 Angelica Zepeda Universidad Nacional Autónoma de México Neurogenesis as a correlate of functional reorganization in the damaged dentate gyrus Andrea Aguilar-Arredondo; Laura Ramos Languren; Martha Escobar; Clorinda Arias and Angélica Zepeda The dentate gyrus (DG) displays enhanced neurogenesis along functional recovery after damage. It is unknown whether new cells become functional with time and if its activation correlates with recovery. To evaluate this, we induced a focal lesion in the DG of young adult rats and analyzed long-term potentiation in the mossy fiber pathway as well as memory function and activation of new neurons in response either to contextual fear memory (CFM) or to a control spatial exploratory task. We analyzed the number of BrdU+ cells that co-localized with doublecortin (DCX) or with NeuN within the damaged DG and evaluated the number of cells in each population that co-labeled with the activity marker c-fos after either task.
At 10 days post-lesion (dpl), a region of the granular cell layer was devoid of cells, evidencing the damaged area, whereas at 30 dpl this region was significantly smaller. At 10 dpl, LTP could not be elicited and CFM was impaired while the number of BrdU+/DCX+/c-fos+ cells had increased compared to the sham-lesion group. At 30 dpl, a significantly greater number of BrdU+/NeuN+/cfos+ cells was observed compared to 10 dpl and LTP could be induced. Activation of BrdU+/NeuN+ cells correlated with CFM recovery, while performance in the spatial exploratory task induced marginal c-fos immunoreactivity in this cell population. Our results show that neurons born after DG damage, survive and become activated in a time- and task-dependent manner, while activation of new neurons occurs along functional recovery.
Funding: CONACyT 282470, PAPIIT IN208518
3 31 Thomas  Berger 1Institute of Psychiatry, Psychology and Neuroscience, King’s College London, The Maurice Wohl Clinical Neuroscience Institute,125 Coldharbour Lane, SE5 9NU London, United Kingdom 2Social, Genetic & Developmental Psychiatry Centre, King’s College London, 16 De Crespigny Park, SE5 9NU London, United Kingdom  Adult hippocampal neurogenesis – A potential converging mechanism for Major Depressive Disorder & Alzheimer’s Disease  Thomas Berger1, Hyunah Lee1, Marc-David Ruepp1, Timothy R. Powell2, Sandrine Thuret1  Mental health disorders are widespread throughout society. Two of the most common neurological or psychiatric disorders are dementia and depression. About 50 million people suffer from dementia (70 % Alzheimer’s Disease) worldwide. Clinical studies report correlations between Alzheimer’s Disease (AD) and Major Depressive Disorder (MDD). AD patients are significantly more likely to develop depressive symptoms and impaired cognition is a common symptom of both disorders. One brain region where the production of new neurons continues throughout life and which plays an important role in memory and cognitive function is the hippocampus. The generation of those new neurons and their integration is crucial for the manifestation of novel memories and cognitive performance and the regulation of mood. Previous studies have already suggested that hippocampal neurogenesis is altered in MDD and AD. However, no cellular mechanism has been proposed yet to connect these two disorders. Therefore, this project aims to investigate the role of hippocampal neurogenesis in MDD and AD as a shared mechanism and as a potential therapeutic target. Therefore, using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) we will generate isogenic induced pluripotent stem cell (iPSC) lines with mutations that are most likely to have an impact on neurogenesis based on significance levels in genome-wide association studies (GWAS) and bioinformatic predictions.

eurogenesis Bordeaux,
Jun 6, 2019, 2:37 AM