Differential vulnerability of adult neurogenic niches to dosage of the neurodevelopmental-disorder linked gene Foxg1.

The transcription factor FOXG1 serves pleiotropic functions in brain development ranging from the regulation of precursor proliferation to the control of cortical circuit formation. Loss-of-function mutations and duplications of FOXG1 are associated with neurodevelopmental disorders in humans illustrating the importance of FOXG1 dosage for brain development. Aberrant FOXG1 dosage has been found to disrupt the balanced activity of glutamatergic and GABAergic neurons, but the underlying mechanisms are not fully understood. We report that FOXG1 is expressed in the main adult neurogenic niches in mice, i.e. the hippocampal dentate gyrus and the subependymal zone/olfactory bulb system, where neurogenesis of glutamatergic and GABAergic neurons persists into adulthood. These niches displayed differential vulnerability to increased FOXG1 dosage: high FOXG1 levels severely compromised survival and glutamatergic dentate granule neuron fate acquisition in the hippocampal neurogenic niche, but left neurogenesis of GABAergic neurons in the subependymal zone/olfactory bulb system unaffected. Comparative transcriptomic analyses revealed a significantly higher expression of the apoptosis-linked nuclear receptor Nr4a1 in FOXG1-overexpressing hippocampal neural precursors. Strikingly, pharmacological interference with NR4A1 function rescued FOXG1-dependent death of hippocampal progenitors. Our results reveal differential vulnerability of neuronal subtypes to increased FOXG1 dosage and suggest that activity of a FOXG1/NR4A1 axis contributes to such subtype-specific response.

Transcription factor Tcf4 is the preferred heterodimerization partner for Olig2 in oligodendrocytes and required for differentiation.

Development of oligodendrocytes and myelin formation in the vertebrate central nervous system is under control of several basic helix-loop-helix transcription factors such as Olig2, Ascl1, Hes5 and the Id proteins. The class I basic helix-loop-helix proteins Tcf3, Tcf4 and Tcf12 represent potential heterodimerization partners and functional modulators for all, but have not been investigated in oligodendrocytes so far. Using mouse mutants, organotypic slice and primary cell cultures we here show that Tcf4 is required in a cell-autonomous manner for proper terminal differentiation and myelination in vivo and ex vivo. Partial compensation is provided by the paralogous Tcf3, but not Tcf12. On the mechanistic level Tcf4 was identified as the preferred heterodimerization partner of the central regulator of oligodendrocyte development Olig2. Both genetic studies in the mouse as well as functional studies on enhancer regions of myelin genes confirmed the relevance of this physical interaction for oligodendrocyte differentiation. Considering that alterations in TCF4 are associated with syndromic and non-syndromic forms of intellectual disability, schizophrenia and autism in humans, our findings point to the possibility of an oligodendroglial contribution to these disorders.

Sox11 is an Activity-Regulated Gene with Dentate-Gyrus-Specific Expression Upon General Neural Activation.

Neuronal activity initiates transcriptional programs that shape long-term changes in plasticity. Although neuron subtypes differ in their plasticity response, most activity-dependent transcription factors (TFs) are broadly expressed across neuron subtypes and brain regions. Thus, how region- and neuronal subtype-specific plasticity are established on the transcriptional level remains poorly understood. We report that in young adult (i.e., 6-8 weeks old) mice, the developmental TF SOX11 is induced in neurons within 6 h either by electroconvulsive stimulation or by exploration of a novel environment. Strikingly, SOX11 induction was restricted to the dentate gyrus (DG) of the hippocampus. In the novel environment paradigm, SOX11 was observed in a subset of c-FOS expressing neurons (ca. 15%); whereas around 75% of SOX11+ DG granule neurons were c-FOS+, indicating that SOX11 was induced in an activity-dependent fashion in a subset of neurons. Environmental enrichment or virus-mediated overexpression of SOX11 enhanced the excitability of DG granule cells and downregulated the expression of different potassium channel subunits, whereas conditional Sox11/4 knock-out mice presented the opposite phenotype. We propose that Sox11 is regulated in an activity-dependent fashion, which is specific to the DG, and speculate that activity-dependent Sox11 expression may participate in the modulation of DG neuron plasticity.

Enriched environment ameliorates adult hippocampal neurogenesis deficits in Tcf4 haploinsufficient mice.

BACKGROUND: Transcription factor 4 (TCF4) has been linked to human neurodevelopmental disorders such as intellectual disability, Pitt-Hopkins Syndrome (PTHS), autism, and schizophrenia. Recent work demonstrated that TCF4 participates in the control of a wide range of neurodevelopmental processes in mammalian nervous system development including neural precursor proliferation, timing of differentiation, migration, dendritogenesis and synapse formation. TCF4 is highly expressed in the adult hippocampal dentate gyrus - one of the few brain regions where neural stem / progenitor cells generate new functional neurons throughout life. RESULTS: We here investigated whether TCF4 haploinsufficiency, which in humans causes non-syndromic forms of intellectual disability and PTHS, affects adult hippocampal neurogenesis, a process that is essential for hippocampal plasticity in rodents and potentially in humans. Young adult Tcf4 heterozygote knockout mice showed a major reduction in the level of adult hippocampal neurogenesis, which was at least in part caused by lower stem/progenitor cell numbers and impaired maturation and survival of adult-generated neurons. Interestingly, housing in an enriched environment was sufficient to enhance maturation and survival of new neurons and to substantially augment neurogenesis levels in Tcf4 heterozygote knockout mice. CONCLUSION: The present findings indicate that haploinsufficiency for the intellectual disability- and PTHS-linked transcription factor TCF4 not only affects embryonic neurodevelopment but impedes neurogenesis in the hippocampus of adult mice. These findings suggest that TCF4 haploinsufficiency may have a negative impact on hippocampal function throughout adulthood by impeding hippocampal neurogenesis.

The transcription factor prospero homeobox protein 1 is a direct target of SoxC proteins during developmental vertebrate neurogenesis.

The high-mobility-group domain containing SoxC transcription factors Sox4 and Sox11 are expressed and required in the vertebrate central nervous system in neuronal precursors and neuroblasts. To identify genes that are widely regulated by SoxC proteins during vertebrate neurogenesis we generated expression profiles from developing mouse brain and chicken neural tube with reduced SoxC expression and found the transcription factor prospero homeobox protein 1 (Prox1) strongly down-regulated under both conditions. This led us to hypothesize that Prox1 expression depends on SoxC proteins in the developing central nervous system of mouse and chicken. By combining luciferase reporter assays and over-expression in the chicken neural tube with in vivo and in vitro binding studies, we identify the Prox1 gene promoter and two upstream enhancers at -44 kb and -40 kb relative to the transcription start as regulatory regions that are bound and activated by SoxC proteins. This argues that Prox1 is a direct target gene of SoxC proteins during neurogenesis. Electroporations in the chicken neural tube furthermore show that Prox1 activates a subset of SoxC target genes, whereas it has no effects on others. We propose that the transcriptional control of Prox1 by SoxC proteins may ensure coupling of two types of transcription factors that are both required during early neurogenesis, but have at least in part distinct functions. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

SoxC transcription factors: multifunctional regulators of neurodevelopment.

During development, generation of neurons is coordinated by the sequential activation of gene expression programs by stage- and subtype-specific transcription factor networks. The SoxC group transcription factors, Sox4 and Sox11, have recently emerged as critical components of this network. Initially identified as survival and differentiation factors for neural precursors, SoxC factors have now been linked to a broader array of developmental processes including neuronal subtype specification, migration, dendritogenesis and establishment of neuronal projections, and are now being employed in experimental strategies for neuronal replacement and axonal regeneration in the diseased central nervous system. This review summarizes the current knowledge regarding SoxC factor function in CNS development and disease and their promise for regeneration.

Mitochondria modify exercise-induced development of stem cell-derived neurons in the adult brain.

Neural stem cells in the adult mammalian hippocampus continuously generate new functional neurons, which modify the hippocampal network and significantly contribute to cognitive processes and mood regulation. Here, we show that the development of new neurons from stem cells in adult mice is paralleled by extensive changes to mitochondrial mass, distribution, and shape. Moreover, exercise-a strong modifier of adult hippocampal neurogenesis-accelerates neuronal maturation and induces a profound increase in mitochondrial content and the presence of mitochondria in dendritic segments. Genetic inhibition of the activity of the mitochondrial fission factor dynamin-related protein 1 (Drp1) inhibits neurogenesis under basal and exercise conditions. Conversely, enhanced Drp1 activity furthers exercise-induced acceleration of neuronal maturation. Collectively, these results indicate that adult hippocampal neurogenesis requires adaptation of the mitochondrial compartment and suggest that mitochondria are targets for enhancing neurogenesis-dependent hippocampal plasticity.

RNA interference machinery-mediated gene regulation in mouse adult neural stem cells.

BACKGROUND: Neurogenesis in the brain of adult mammals occurs throughout life in two locations: the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus in the hippocampus. RNA interference mechanisms have emerged as critical regulators of neuronal differentiation. However, to date, little is known about its function in adult neurogenesis. RESULTS: Here we show that the RNA interference machinery regulates Doublecortin levels and is associated with chromatin in differentiating adult neural progenitors. Deletion of Dicer causes abnormal higher levels of Doublecortin. The microRNA pathway plays an important role in Doublecortin regulation. In particular miRNA-128 overexpression can reduce Doublecortin levels in differentiating adult neural progenitors. CONCLUSIONS: We conclude that the RNA interference components play an important role, even through chromatin association, in regulating neuron-specific gene expression programs.

Prospero-related homeobox 1 gene (Prox1) is regulated by canonical Wnt signaling and has a stage-specific role in adult hippocampal neurogenesis.

Neural stem cells (NSCs) generate new granule cells throughout life in the mammalian hippocampus. Canonical Wnt signaling regulates the differentiation of NSCs towards the neuronal lineage. Here we identified the prospero-related homeodomain transcription factor Prox1 as a target of ß-catenin-TCF/LEF signaling in vitro and in vivo. Prox1 overexpression enhanced neuronal differentiation whereas shRNA-mediated knockdown of Prox1 impaired the generation of neurons in vitro and within the hippocampal niche. In contrast, Prox1 was not required for survival of adult-generated granule cells after they had matured, suggesting a role for Prox1 in initial granule cell differentiation but not in the maintenance of mature granule cells. The data presented here characterize a molecular pathway from Wnt signaling to a transcriptional target leading to granule cell differentiation within the adult brain and identify a stage-specific function for Prox1 in the process of adult neurogenesis.

SoxC transcription factors are required for neuronal differentiation in adult hippocampal neurogenesis.

Neural stem cells (NSCs) generate new hippocampal dentate granule neurons throughout adulthood. The genetic programs controlling neuronal differentiation of adult NSCs are only poorly understood. Here we show that, in the adult mouse hippocampus, expression of the SoxC transcription factors Sox4 and Sox11 is initiated around the time of neuronal commitment of adult NSCs and is maintained in immature neurons. Overexpression of Sox4 and Sox11 strongly promotes in vitro neurogenesis from adult NSCs, whereas ablation of Sox4/Sox11 prevents in vitro and in vivo neurogenesis from adult NSCs. Moreover, we demonstrate that SoxC transcription factors target the promoters of genes that are induced on neuronal differentiation of adult NSCs. Finally, we show that reprogramming of astroglia into neurons is dependent on the presence of SoxC factors. These data identify SoxC proteins as essential contributors to the genetic network controlling neuronal differentiation in adult neurogenesis and neuronal reprogramming of somatic cells.

Crybb2 coding for ßB2-crystallin affects sensorimotor gating and hippocampal function.

ßB2-crystallin (gene symbol: Crybb2/CRYBB2) was first described as a structural protein of the ocular lens. This gene, however, is also expressed in several regions of the mammalian brain, although its function in this organ remains entirely unknown. To unravel some aspects of its function in the brain, we combined behavioral, neuroanatomical, and physiological analyses in a novel Crybb2 mouse mutant, O377. Behavioral tests with male O377 mutants revealed altered sensorimotor gating, suggesting modified neuronal functions. Since these mouse mutants also displayed reduced hippocampal size, we concentrated further investigations on the hippocampus. Free intracellular Ca(2+) levels were increased and apoptosis was enhanced in the hippocampus of O377 mutants. Moreover, the expression of the gene encoding calpain 3 (gene symbol Capn3) was elevated and the expression of genes coding for the NMDA receptor subunits was downregulated. Additionally, the number of parvalbumin-positive interneurons was decreased in the hippocampus but not in the cortex of the mutants. High-speed voltage-sensitive dye imaging demonstrated an increased translation of input-to-output neuronal activity in the dentate gyrus of this Crybb2 mutant. These results point to an important function of ßB2-crystallin in the hippocampal network. They indicate pleiotropic effects of mutations in the Crybb2 gene, which previously had been considered to be specific to the ocular lens. Moreover, our results are the first to demonstrate that ßB2-crystallin has a role in hippocampal function and behavioral phenotypes. This model can now be further explored by future experiments.

CRISPR/Cas9-mediated generation of hESC lines with homozygote and heterozygote p.R331W mutation in CTBP1 to model HADDTS syndrome.

C-terminal Binding Protein 1 (CTBP1) is a ubiquitously expressed transcriptional co-repressor and membrane trafficking regulator. A recurrent de novo c.991C>T mutation in CTBP1 leads to expression of p.R331W CTBP1 and causes hypotonia, ataxia, developmental delay, and tooth enamel defects syndrome (HADDTS), a rare early onset neurodevelopmental disorder. We generated hESCs lines with heterozygote and homozygote c.991C>T in CTBP1 using CRISPR/Cas9 genome editing and validated them for genetic integrity, off-target mutations, and pluripotency. They will be useful for investigation of HADDTS pathophysiology and for screening for potential therapeutics.

Voluntary wheel running in mice increases the rate of neurogenesis without affecting anxiety-related behaviour in single tests.

BACKGROUND: The role played by adult neurogenesis in anxiety is not clear. A recent study revealed a surprising positive correlation between increased anxiety and elevated neurogenesis following chronic voluntary wheel running and multiple behavioural testing in mice, suggesting that adult hippocampal neurogenesis is involved in the genesis of anxiety. To exclude the possible confounding effect of multiple testing that may have occurred in the aforementioned study, we assessed (1) the effects of mouse voluntary wheel running (14 vs. 28 days) on anxiety in just one behavioural test; the open field, and (2), using different markers, proliferation, differentiation, survival and maturation of newly born neurons in the dentate gyrus immediately afterwards. Effects of wheel running on anxiety-related behaviour were confirmed in a separate batch of animals tested in another test of anxiety, the light/dark box test. RESULTS: Running altered measures of locomotion and exploration, but not anxiety-related behaviour in either test. 14 days running significantly increased proliferation, and differentiation and survival were increased after both running durations. 28 day running mice also exhibited an increased rate of maturation. Furthermore, there was a significant positive correlation between the amount of proliferation, but not maturation, and anxiety measures in the open field of the 28 day running mice. CONCLUSIONS: Overall, this evidence suggests that without repeated testing, newly born mature neurons may not be involved in the genesis of anxiety per se.

CREB signalling regulates early survival, neuronal gene expression and morphological development in adult subventricular zone neurogenesis.

Neural stem cells in the subventricular zone (SVZ) of the lateral ventricles give rise to new interneurons of the olfactory bulb (OB) throughout life. SVZ/OB neurogenesis is influenced by olfactory network activity, which modulates the survival of new neurons during their integration into the OB network. Previous work suggested that such activity-dependent survival is regulated via the CREB signalling pathway. Curiously, CREB signalling is already active during the early developmental stages of adult SVZ/OB neurogenesis. To investigate the role of cell autonomous CREB signalling during early stages of adult SVZ/OB neurogenesis, we ablated CREB-pathway activity in the SVZ/OB neurogenic lineage using a retroviral strategy. Surprisingly, loss of CREB signalling resulted in increased cell death and loss of expression of the neurogenic transcription factor Pax 6, and of a subset of neuronal proteins in migrating neurons of the RMS. Moreover, post-migratory neurons in the OB displayed impaired dendritic development. These results demonstrate an essential role for CREB signalling in maturation of newborn neurons in the OB and uncover a novel role for CREB signalling in the survival and maintenance of neuronal gene expression during the early stages of SVZ/OB neurogenesis.

RBPJkappa-dependent signaling is essential for long-term maintenance of neural stem cells in the adult hippocampus.

The generation of new neurons from neural stem cells in the adult hippocampal dentate gyrus contributes to learning and mood regulation. To sustain hippocampal neurogenesis throughout life, maintenance of the neural stem cell pool has to be tightly controlled. We found that the Notch/RBPJκ-signaling pathway is highly active in neural stem cells of the adult mouse hippocampus. Conditional inactivation of RBPJκ in neural stem cells in vivo resulted in increased neuronal differentiation of neural stem cells in the adult hippocampus at an early time point and depletion of the Sox2-positive neural stem cell pool and suppression of hippocampal neurogenesis at a later time point. Moreover, RBPJκ-deficient neural stem cells displayed impaired self-renewal in vitro and loss of expression of the transcription factor Sox2. Interestingly, we found that Notch signaling increases Sox2 promoter activity and Sox2 expression in adult neural stem cells. In addition, activated Notch and RBPJκ were highly enriched on the Sox2 promoter in adult hippocampal neural stem cells, thus identifying Sox2 as a direct target of Notch/RBPJκ signaling. Finally, we found that overexpression of Sox2 can rescue the self-renewal defect in RBPJκ-deficient neural stem cells. These results identify RBPJκ-dependent pathways as essential regulators of adult neural stem cell maintenance and suggest that the actions of RBPJκ are, at least in part, mediated by control of Sox2 expression.

CREB in adult neurogenesis--master and partner in the development of adult-born neurons?

The generation of new neurons in the adult brain is modulated by complex stimuli and a broad range of extrinsic signals. It remains a mystery how stem cells and their progeny integrate this wealth of regulatory input to generate a precise number of neurons that matches the physiological needs of the olfactory and hippocampal network. cAMP response element binding protein (CREB)-dependent signalling is controlling essential developmental steps in adult neurogenesis, i.e. survival, maturation and integration of new neurons. Here, we summarize the current knowledge on the function of CREB in adult neurogenesis and discuss the potential of CREB to integrate complex stimuli and to translate these into precise developmental processes in adult neurogenesis. The complex modulation of CREB-signalling may allow the adult neurogenic system to respond to stimuli in a fine-tuned rather than in an on-off fashion.

Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus.

Newborn granule cells become functionally integrated into the synaptic circuitry of the adult dentate gyrus after a morphological and electrophysiological maturation process. The molecular mechanisms by which immature neurons and the neurites extending from them find their appropriate position and target area remain largely unknown. Here we show that single-cell-specific knockdown of cyclin-dependent kinase 5 (cdk5) activity in newborn cells using a retrovirus-based strategy leads to aberrant growth of dendritic processes, which is associated with an altered migration pattern of newborn cells. Even though spine formation and maturation are reduced in cdk5-deficient cells, aberrant dendrites form ectopic synapses onto hilar neurons. These observations identify cdk5 to be critically involved in the maturation and dendrite extension of newborn neurons in the course of adult neurogenesis. The data presented here also suggest a mechanistic dissociation between accurate dendritic targeting and subsequent synapse formation.

A Case-Control Study Evaluating the Impact of Dedicated Palliative Care Training on Critical Care Interventions at the end of Life.

Many patients admitted to intensive care units (ICUs) are at high risk of dying. We hypothesize that focused training sessions for ICU providers by palliative care (PC) certified experts will decrease aggressive medical interventions at the end of life. We designed and implemented a 6-session PC training program in communication skills and goals of care (GOC) meetings for ICU teams, including house staff, critical care fellows, and attendings. We then reviewed charts of ICU patients treated before and after the intervention. Forty-nine of 177 (28%) and 63 of 173 (38%) patients were identified to be at high risk of death in the pre- and postintervention periods, respectively, and were included based on the study criteria. Inpatient mortality (45% vs 33%; P = .24) and need for mechanical ventilation (59% vs 44%, P = .13) were slightly higher in the preintervention population, but the difference was not statistically significant. The proportion of patients in whom the decision not to initiate renal replacement therapy was made because of poor prognosis was significantly higher in the postintervention population (14% vs 67%, P = .05). There was a nonstatistically significant trend toward earlier GOC discussions (median time from ICU admission to GOC 4 vs 3 days) and fewer critical care interventions such as tracheostomies (17% vs 4%, P = .19). Our study demonstrates that directed PC training of ICU teams has a potential to reduce end of life critical care interventions in patients with a poor prognosis.

GABA-cAMP response element-binding protein signaling regulates maturation and survival of newly generated neurons in the adult hippocampus.

Survival and integration of new neurons in the hippocampal circuit are rate-limiting steps in adult hippocampal neurogenesis. Neuronal network activity is a major regulator of these processes, yet little is known about the respective downstream signaling pathways. Here, we investigate the role of cAMP response element-binding protein (CREB) signaling in adult hippocampal neurogenesis. CREB is activated in new granule neurons during a distinct developmental period. Loss of CREB function in a cell-autonomous manner impairs dendritic development, decreases the expression of the neurogenic transcription factor NeuroD and of the neuronal microtubule-associated protein, doublecortin (DCX), and compromises the survival of newborn neurons. In addition, GABA-mediated excitation regulates CREB activation at early developmental stages. Importantly, developmental defects after loss of GABA-mediated excitation can be compensated by enhanced CREB signaling. These results indicate that CREB signaling is a central pathway in adult hippocampal neurogenesis, regulating the development and survival of new hippocampal neurons downstream of GABA-mediated excitation.

Epigenetic regulation of neurogenesis in the adult hippocampus.

The dentate gyrus of the hippocampus is an exception to a 'neurogenesis-unfriendly' environment of the adult brain. New functional neurons generated in this region contribute to learning and mood regulation, and thus represent a unique form of neural plasticity. The rate of hippocampal neurogenesis significantly changes on physiological or pathological influences, such as physical activity, environmental enrichment, stress, and aging. We suggest that epigenetic mechanisms could be sensors of environmental changes and fine modulators of adult hippocampal neurogenesis. Here, we examine the role of DNA methylation and methylation of core histones mediated by the Polycomb and Trithorax complexes in the regulation of adult neurogenesis. Given the recent surprising discovery of dynamic and reversible DNA methylation in the hippocampus, we speculate regarding its regulation and its role in adult neurogenesis.