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Loss of Postnatal Quiescence of Neural Stem Cells Through Mtor Activation Upon Genetic Removal of Cysteine String Protein-Α

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Loss of Postnatal Quiescence of Neural Stem Cells Through Mtor Activation Upon Genetic Removal of Cysteine String Protein-Α Loss of postnatal quiescence of neural stem cells through mTOR activation upon genetic removal of cysteine string protein-α Jose L. Nieto-Gonzáleza,b,c,1,2, Leonardo Gómez-Sáncheza,b,c,1, Fabiola Mavillarda,b,c, Pedro Linares-Clementea,b,c, María C. Riveroa,b,c, Marina Valenzuela-Villatoroa,b,c, José L. Muñoz-Bravoa,b,c, Ricardo Pardala,b,c, and Rafael Fernández-Chacóna,b,c,2 aInstituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/Consejo Superior de Investigaciones Científicas/Universidad de Sevilla, 41013 Sevilla, Spain; bDepartamento de Fisiología Médica y Biofísica, Universidad de Sevilla, 41009 Sevilla, Spain; and cCentro Investigación Biomédica en Red Enfermedades Neurodegenerativas, 41013 Sevilla, Spain Edited by Thomas C. Südhof, Stanford University School of Medicine, Stanford, CA, and approved March 4, 2019 (received for review October 9, 2018) Neural stem cells continuously generate newborn neurons that protein-A (SGTA) (13). Interestingly, CSP-α is required to maintain integrate into and modify neural circuitry in the adult hippocam- the stability of the SNARE protein SNAP25 at synaptic termi- pus. The molecular mechanisms that regulate or perturb neural nals (14–16). SNAP25 cannot maintain its function without a stem cell proliferation and differentiation, however, remain poorly chaperone, which is probably due to the molecular stress induced understood. Here, we have found that mouse hippocampal radial during cycles of assembly and disassembly of the SNARE com- glia-like (RGL) neural stem cells express the synaptic cochaperone plex (15, 17). Remarkably, knockout (KO) mice lacking CSP-α cysteine string protein-α (CSP-α). Remarkably, in CSP-α knockout suffer from a devastating and early synaptic degeneration (18) mice, RGL stem cells lose quiescence postnatally and enter into a that is particularly evident in highly active neurons (19). The high-proliferation regime that increases the production of neural early lethality of CSP-α KO mice has so far impaired the per- intermediate progenitor cells, thereby exhausting the hippocam- forming of functional in vivo studies of CSP-α in adulthood. pal neural stem cell pool. In cell culture, stem cells in hippocampal Molecular mechanisms of neurodegeneration, although not yet neurospheres display alterations in proliferation for which hyper- well understood, are linked to damage of SNAP25 (20) when activation of the mechanistic target of rapamycin (mTOR) signaling CSP-α is absent. Interestingly, CSP-α is linked to human disease pathway is the primary cause of neurogenesis deregulation in the (21). The Parkinson’s disease-linked protein, α-synuclein, cooper- absence of CSP-α. In addition, RGL cells lose quiescence upon spe- ates with CSP-α to chaperone the SNARE complex (14). Moreover, cific conditional targeting of CSP-α in adult neural stem cells. Our mutations in the human gene encoding CSP-α (DNAJC5) cause findings demonstrate an unanticipated cell-autonomic and circuit- adult-onset neuronal ceroid lipofuscinosis (22). independent disruption of postnatal neurogenesis in the absence Although the expression of CSP-α in nonneural tissues was α α of CSP- and highlight a direct or indirect CSP- /mTOR signaling reported long ago (23), little attention has been paid to its interaction that may underlie molecular mechanisms of brain dys- functions beyond the synapse. Here, we have found that hippo- function and neurodegeneration. campal RGL stem cells express CSP-α, which prompted us to adult neurogenesis | DNAJC5 | adult-onset neuronal ceroid lipofuscinosis | Significance synaptic neurodegeneration | lysosome Neural stem cells generate newborn neurons in the postnatal ippocampal postnatal neurogenesis is a remarkable form of brain by a process known as neurogenesis. Cysteine string Hneural plasticity based on the integration of newborn neu- protein-α (CSP-α) maintains healthy nerve terminals and, when rons in preexisting neural circuits in the adult brain (1). Multiple mutated in humans, causes a serious disease known as neu- studies support the notion that adult neurogenesis is important ronal ceroid lipofuscinosis related to lysosomal pathologies. for learning and memory recall, particularly in the performance α – We have now found that neural stem cells without CSP- of behavioral pattern separation (2 4). Significant progress has hyperproliferate, leading to depletion of the neural stem cell been made in recent years in understanding the molecular, cel- pool in the mouse hippocampus. Biochemically, the hyper- lular, and synaptic mechanisms that regulate and perturb adult proliferation occurs through the hyperactivation of the mech- neurogenesis (5). In the dentate gyrus, radial glia-like (RGL) anistic target of rapamycin (mTOR) signaling pathway. Our stem cells constitute a relatively quiescent cell population that findings demonstrate the disruption of postnatal neurogenesis undergoes self-renewal and commits to either an astrocytic or neu- in the absence of CSP-α and unveil an intriguing signaling in- ronal cell fate (6, 7). The generation of new neurons occurs through teraction between CSP-α and mTOR that may underlie molec- sequential differentiation steps of proliferating intermediate neural ular mechanisms of brain dysfunction and neurodegeneration. progenitors that evolve into postmitotic immature neurons before finally becoming mature hippocampal granule cells (5). Critical Author contributions: J.L.N.-G., L.G.-S., F.M., P.L.-C., R.P., and R.F.-C. designed research; points of regulation of postnatal neurogenesis are the maintenance J.L.N.-G., L.G.-S., F.M., P.L.-C., M.C.R., and M.V.-V. performed research; J.L.M.-B. of quiescence (8) and neuronal survival (9). The molecular mech- contributed new reagents/analytic tools; J.L.N.-G., L.G.-S., F.M., and P.L.-C. analyzed data; anisms underlying these key phenomena, and factors that can per- and J.L.N.-G. and R.F.-C. wrote the paper. turb them, nevertheless remain poorly understood. Recent studies The authors declare no conflict of interest. have emphasized the importance of molecular chaperones to con- This article is a PNAS Direct Submission. trol factors that regulate neural stem cell quiescence (10, 11) and to Published under the PNAS license. promote the survival of newborn neurons (12). 1J.L.N.-G. and L.G.-S. contributed equally to this work. Cysteine string protein-α (CSP-α) is a molecular cochaperone 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. that belongs to the heat shock protein-40 (Hsp40) family; it This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. forms a trimeric chaperone complex with heat shock cognate-70 1073/pnas.1817183116/-/DCSupplemental. (Hsc70) and small glutamine-rich tetratricopeptide containing Published online March 29, 2019. 8000–8009 | PNAS | April 16, 2019 | vol. 116 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1817183116 Downloaded by guest on September 29, 2021 investigate the potential role of CSP-α in postnatal neurogenesis. cells, have not yet been studied. To precisely identify these cells, Interestingly, we have found striking alterations in neurogenesis we used CSP-α KO mice and control littermates expressing GFP in the dentate gyrus of CSP-α KO mice: RGL escape from the under the nestin promoter (25) (Fig. 1A). We examined the quiescent state, inducing hyperproliferation of intermediate hippocampal subgranular zone (SGZ) with antibodies against neural progenitors and exhausting the neural stem cell pool. CSP-α and found that in control mice, beyond the prominent Importantly, this deregulation of neurogenesis is caused by synaptic localization, CSP-α was present in RGL neural stem + hyperactivation of the mechanistic target of rapamycin (mTOR) cells. These cells were easily identified as GFP cells exhibiting a + pathway and can be rescued in vitro and in vivo upon pharma- characteristic glial fibrillary acidic protein-positive (GFAP ) cological blocking of the kinase activity of mTOR-complex 1 process extending perpendicularly into the molecular layer of the (mTORC1) with rapamycin. The use of conditional KO mice dentate gyrus (Fig. 1A, squares and Movie S1). As expected, we shows that the phenotype also arises upon inducing the genetic did not detect any CSP-α expression in CSP-α KO mice (SI removal of CSP-α in RGL cells in adulthood. Our study reveals a Appendix, Fig. S1). Next, we looked for potential alterations in key unforeseen implication of a molecular cochaperone whose cell division by monitoring cell cycle activity through the in- absence disrupts cell cycle progression in neural stem cells. corporation of the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) at the SGZ (Fig. 1 B–E). CSP-α KO and littermate Results control mice received a single i.p. injection of BrdU on postnatal Increased Number of Hippocampal Newborn Neurons in CSP-α KO day (P) 10 and were killed on P15 (Fig. 1C). The number of Mice. CSP-α is a synaptic vesicle protein universally expressed BrdU-labeled nuclei in the hippocampus was significantly higher in central and peripheral nerve terminals (24); however, its ex- in CSP-α KO mice (Fig. 1B) [120.9 ± 3.5 cells per section for WT pression and function in nonneuronal cells, such as neural stem (n = 3) and 146.4 ± 4.7 cells per section for CSP-α KO (n = 3); A NestinGFP DAPI/GFAP NestinGFP DAPI//CSP GFAP XZ CSP- WT CSP- WT NEUROSCIENCE DAPI/CSP XY YZ GFAP g Granule Granule g cell layer cell layer g 10 m 10 m 2m NestinGFP GFP NestinGFP DAPI/GFP XZ CSP- WT CSP- WT CSP/GFAP Fig. 1. Postnatal increase of newborn neurons at the hippocampal neurogenic niche in CSP-α KO mice. DAPI/GFP XY YZ (A, Left and Center) Maximum intensity projections CSP/GFAP of a z-stack of confocal images from nestin-GFP g transgenic CSP-α WT mouse hippocampal slices la- Granule Granule g beled with DAPI (dark blue), anti-GFP (light blue), cell layer cell layer g and anti-GFAP (green) antibodies.
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