restrepressor element 1 ( re1 ) silencing transcription factornpcneural progenitor cellhekhuman embryonic kidneyctdsp1c - terminal domain small phosphatase 1egfepidermal growth factorh - rasharvey rat sarcoma viral oncogene homologerkextracellular signal - regulated kinaseck1casein kinase 1sserineeglutamatepin1peptidylprolyl cis / trans isomerasetrcp-transducin repeat containing e3 ubiquitin protein ligasefgffibroblast growth factorvegfvascular endothelial growth factorbmpbone morphogenetic proteinpax6paired box repressor element 1 ( re1 ) silencing transcription factor neural progenitor cell human embryonic kidney c - terminal domain small phosphatase 1 epidermal growth factor harvey rat sarcoma viral oncogene homolog extracellular signal - regulated kinase peptidylprolyl cis / trans isomerase -transducin repeat containing e3 ubiquitin protein ligase fibroblast growth factor vascular endothelial growth factor bone morphogenetic protein deoxyribonucleic acid guanosine triphosphate central nervous system the food and drug administration . new neurons are generated from neural progenitor cells ( npcs ) via a process called neurogenesis , which in vertebrates occurs in restricted brain regions . neurogenesis is associated with biological functions such as learning , memory , and other cognitive functions . inhibition of neurogenesis via antimitotic agents , radiation , or genetic manipulations has been demonstrated to impair hippocampus - dependent forms of memory in rodents . defects in neurogenesis have been linked with many disease states , with cognitive etiologies including developmental disorders ( e.g. , microcephaly , megalencephaly , and autism ) as well as neurodegenerative diseases ( e.g. , dementia and alzheimer disease ) . ultimately , resolving the signaling mechanisms that regulate neurogenesis is key to advancing our understanding of these biological processes . neurogenesis is orchestrated by several signaling pathways originating at the plasma membrane , including wnt , egf , fgf , vgef , and bmp , and terminating in the cell nucleus . these signaling cascades initiate the progressive expression of many transcription factors , including pax6 , ngn2 , tbr2 , neurod , and tbr1 . despite this list of implicated proteins , many gaps remain in our knowledge regarding the signaling mechanisms in the nucleus that leads to transcriptional changes that occur during neuronal differentiation . these nuclear signaling components are attractive targets for treating neurological disorders because they directly regulate cellular differentiation . our recent report provides new insight into the signaling mechanisms regulating the repressor element 1 ( re1 ) silencing transcription factor ( rest ) , a master regulator of neuronal differentiation . rest acts by binding to the dna chromatin at the re1 sites near the regulatory regions of neuronal genes to repress their expression . consistent with its function , rest is present in most non - neuronal tissues including stem cells . many target genes of rest repression have been identified , including those required for the terminally differentiated neuronal phenotype such as receptors , ion channels , growth factors , and axonal - guidance proteins . consistent with this role , rest is aggressively degraded in neural stem / progenitor cells , and the clearance of rest from the chromatin allows for the expression of neuronal genes , enabling terminal differentiation . prior studies of rest degradation identified a mechanism that involves the phosphorylation of 2 c - terminus sites of rest ( e1009/s1013 and s1024a / s1027a / s1030a ) by casein kinase 1 , ck1 . phosphorylation of these 2 sites facilitates binding of a chaperone protein , -transducin repeat containing e3 ubiquitin protein ligase ( trcp ) , which then shuttles rest to the proteasome for degradation . however , both ck1 and trcp are present in stem cells where rest is abundant , and phosphorylation of residues s1013/1024/1030 does not explain the observed temporal regulation of rest degradation . our objective was to elucidate the cell signals that are consistent with the timing of rest degradation . using mass - spectrometry cycloheximide degradation assays in hek cells expressing rest truncations and point mutations revealed that a proline - directed phosphorylation motif at serines 861 and 864 regulates rest stability / activity . coincidentally , these amino acids were found to be the most frequently phosphorylated . in light of the previously identified rest degradation sites accordingly , we mutated serines 861 and 864 and assessed trcp binding at the previously identified down stream rest degradation sites . we found mutating serines 861 and 864 indirectly regulated binding of trcp to the downstream rest degradation sites . we hypothesized that the mechanism governing trcp binding was through either a change in protein - protein interactions or conformation . consistent with this hypothesis , we identified serines 861 and 864 as a pin1 binding motif . pin1 is a well - established modulator of protein - protein interactions ( including trcp ) , and phosphorylation dependent conformational changes . we found pin1 recognized phosphorylated serines 861 and 864 , and that inhibiting pin1 activity inhibited the binding of trcp . our data show that serines 861 and 864 shared the same mechanism for regulating rest stability as the previously identified downstream sites . given that pin1 binding requires a phosphorylated serine or threonine adjacent to a proline , we sought to identify the kinase that phosphorylates these residues . both serine 861 and 864 are highly predicted for recognition by the extracellular signal - regulated kinases , erk , 1 and 2 . we hypothesized that erk and its canonical upstream activators , epidermal growth factor ( egf ) and the small gtpase harvey rat sarcoma viral oncogene homolog ( h - ras ) would promote phosphorylation at these serines in rest . we demonstrated that serine 861 and 864 are terminal targets of egf - ras - erk signaling and that erk2 can directly phosphorylate rest . the egf - ras - erk pathway is implicated in both embryonic and adult neurogenesis . in embryonic culture models of neuronal differentiation , therefore , our findings implicate phosphorylated serines 861 and 864 as an early biomarker for neurogenesis . in differentiating neural progenitor cells , targeting rest for degradation leads to the removal of rest from the chromatin , allowing for the expression of neuronal genes . in non - neuronal cell types , rest protein is more stable - likely a critical factor contributing to its retention time on the chromatin . we were interested in identifying how rest stability because we found that kinase activity at serines 861 and 864 to mediate rest degradation , we hypothesized that phosphatase activity may protect rest from degradation . consistent with this hypothesis , rest has been detected colocalized on neuronal gene chromatin with the protein phosphatase ctdsp1 . additionally , it is known that the expression of ctdsp1 decreases dramatically as neural progenitor cells differentiate into mature neurons , while knockdown of ctdsp1 in a neural progenitor cell line accelerates neuronal differentiation . we found that ctdsp1 dephosphorylates seines 861 and 864 , resulting in stabilization of rest . one of the unresolved questions in mammalian neurogenesis is how neural progenitors switch from a proliferation to differentiation state . relevant to this question is the observation that wnt/-catenin signaling has different effects on neural progenitor cells depending on when it is expressed during development . in the expansion phase of early neural progenitors , , rest is protected from degradation by ctdsp1 , and remains bound to the chromatin to repress the expression of neuronal genes that would have otherwise been induced by wnt/-catenin signaling . in subsequent neurogenic phases , this results in the removal of rest from the chromatin and the derepression of neuronal genes induced by late wnt/-catenin signaling . future investigation of this model will provide additional resolution of the early and late stage mechanics of coordinated neurogenesis . neural stem cells in the adult brain have the ability to generate and integrate new neurons . exercise and mental simulation are correlated with increased neurogenesis ; while advanced age , stress , and diseases associated with cognitive impairment are correlated with an arrest or decrease in neurogenesis . given the flexibility in the rate at which stem cells can generate new neurons , researchers have attempted to harness this potential to repair brain damage . current therapeutic approaches have identified strategies to generate new neurons in uninjured brains , including infusing or transplanting exogenous stem cells , coaxing endogenous stem cells to become neurons by using blood transfusion , and supplementing growth / neurotrophic factor . unfortunately , none of these approaches have been successful in producing new integrated neurons after traumatic brain injury in humans . brain injury induces npcs to generate astrocytes preferentially over neurons , and these astrocytes migrate to the site of injury forming an astrocytic scar . the development of strategies directing npc fate is an appealing approach to enhance neuronal regeneration after brain injury . this suggests that brain injury induces signaling cascades that promote the repression of neuronal genes . consistent with this hypothesis , studies have found that conditions that damage the brain such as a stroke or seizure can result in derepression of rest . in fact , brain injury induces npcs to generate astrocytes instead of neurons . an attractive and yet untested approach to promoting neuronal regeneration in brain injury is to remove this neuronal gene repression ( e.g. inhibit rest ) . there are many ways that inhibiting rest activity would improve neuronal regeneration therapy after cns injury . first , it would promote the differentiation of neural progenitor cells toward neurons instead of astrocytes . second , inhibiting rest would likely reduce the risk of brain cancer associated with transplanted stem cells , according to a leading hypothesis that cancer arises from such neural stem cells . in fact , insuring against tumorogenesis by transplanted cells has been a major hindrance for the adoption of brain injury - targeted stem cell therapies ( fda , http://www.fda.gov/newsevents/publichealthfocus/ucm286218.htm ) . relevant to this discussion of rest regulation is the fact that many brain cancers , including glioblastoma , meduloblastoma , and neuroblastoma have overexpressed rest which is implicated in oncogenic transformation . thus , inhibiting rest activity or expression would promote the differentiation of stem cells into neurons , reducing the risk of them transforming into tumorigenic cells . this observation indicates that inhibiting / degrading rest could protect neurons from death , improving cns recovery after injury . serines 861 and 864 determine whether rest is targeted for degradation , a critical step in neurogenesis . in the phosphorylated state serines 861 and 864 are a predicted biomarker for neurogenesis , and may help to determine the potential of neural stem cells to differentiate into neurons after brain injury . in situations where neurogenesis is repressed , it could then be reversed . in our model , we identify 2 opposing signals that regulate rest activity / stability . first , ctdsp1 protects rest from degradation by dephosphorylating serines 861 and 864 ( fig . second , egf - ras - erk signaling phosphorylates rest at serines 861 and 864 ( fig . figure 1.a model for reciprocal regulation of rest through post - translational modifications on serines 861 and 864 . in stem cells , rest sits on the chromatin and represses neuronal gene expression ( left panel ) . here , rest is protected from degradation , because ctdsp1 dephosphorylates serines 861 and 864 on rest . during neuronal differentiation , growth factor signaling ( e.g. , egf ) increases activation of h - ras and erk , resulting in phosphorylation of serines 861 and 864 . trcp binding leads to rest degradation , allowing for expression of neuronal genes ( right panel ) . 
  a model for reciprocal regulation of rest through post - translational modifications on serines 861 and 864 . in stem cells , rest sits on the chromatin and represses neuronal gene expression ( left panel ) . here , rest is protected from degradation , because ctdsp1 dephosphorylates serines 861 and 864 on rest . during neuronal differentiation , growth factor signaling ( e.g. , egf ) increases activation of h - ras and erk , resulting in phosphorylation of serines 861 and 864 . trcp binding leads to rest degradation , allowing for expression of neuronal genes ( right panel ) . in our study we , demonstrate the feasibility of using a peptidomimetic ( decoy ) containing the erk and pin1 sites to stabilizes rest and inhibit neurogenesis . it then follows that a phosphomimetic ( decoy ) version of this peptide could be used to block ctdsp1 activity on rest to promote its degradation resulting in neurogenesis . as proof - of - concept alternatively , small molecule inhibitors of ctdsp1 activity on rest could be developed . in sum , the mechanisms and reagents we have discovered for rest regulation reveal new elements regulating cell differentiation and provide us with new tools to influence the process of neurogenesis . using this exciting new strategy , it may be possible to enhance neuronal regeneration after injury and interrupt the events leading to oncogenic transformation , - 2 processes that are in dire need of novel therapeutic strategies . i thank gail mandel , anthony p. barnes , mary y. heng , and mark a. verdecia for reading the manuscript and providing helpful comments ; and wendy w. wu for illustrations .
neurogenesis begins in embryonic development and continues at a reduced rate into adulthood in vertebrate species , yet the signaling cascades regulating this process remain poorly understood . 
 plasma membrane - initiated signaling cascades regulate neurogenesis via downstream pathways including components of the transcriptional machinery . a nuclear factor that temporally regulates neurogenesis by repressing neuronal differentiation is the repressor element 1 ( re1 ) silencing transcription ( rest ) factor . 
 we have recently discovered a regulatory site on rest that serves as a molecular switch for neuronal differentiation . 
 specifically , c - terminal domain small phosphatase 1 , ctdsp1 , present in non - neuronal cells , maintains rest activity by dephosphorylating this site . 
 reciprocally , extracellular signal - regulated kinase , erk , activated by growth factor signaling in neural progenitors , and peptidylprolyl cis / trans isomerase pin1 , decrease rest activity through phosphorylation - dependent degradation . 
 our findings further resolve the mechanism for temporal regulation of rest and terminal neuronal differentiation . 
 they also provide new potential therapeutic targets to enhance neuronal regeneration after injury .