De novo DNA methylation controls neuronal maturation during adult hippocampal neurogenesis.

Adult neurogenesis enables the life-long addition of functional neurons to the hippocampus and is regulated by both cell-intrinsic molecular programs and behavioral activity. De novo DNA methylation is crucial for embryonic brain development, but its role during adult hippocampal neurogenesis has remained unknown. Here, we show that de novo DNA methylation is critical for maturation and functional integration of adult-born neurons in the mouse hippocampus. Bisulfite sequencing revealed that de novo DNA methyltransferases target neuronal enhancers and gene bodies during adult hippocampal neural stem cell differentiation, to establish neuronal methylomes and facilitate transcriptional up-regulation of neuronal genes. Inducible deletion of both de novo DNA methyltransferases Dnmt3a and Dnmt3b in adult neural stem cells did not affect proliferation or fate specification, but specifically impaired dendritic outgrowth and synaptogenesis of newborn neurons, thereby hampering their functional maturation. Consequently, abolishing de novo DNA methylation modulated activation patterns in the hippocampal circuitry and caused specific deficits in hippocampus-dependent learning and memory. Our results demonstrate that proper establishment of neuronal methylomes during adult neurogenesis is fundamental for hippocampal function.

Genome binding properties of Zic transcription factors underlie their changing functions during neuronal maturation.

BACKGROUND: The Zic family of transcription factors (TFs) promote both proliferation and maturation of cerebellar granule neurons (CGNs), raising the question of how a single, constitutively expressed TF family can support distinct developmental processes. Here we use an integrative experimental and bioinformatic approach to discover the regulatory relationship between Zic TF binding and changing programs of gene transcription during postnatal CGN differentiation. RESULTS: We first established a bioinformatic pipeline to integrate Zic ChIP-seq data from the developing mouse cerebellum with other genomic datasets from the same tissue. In newborn CGNs, Zic TF binding predominates at active enhancers that are co-bound by developmentally regulated TFs including Atoh1, whereas in mature CGNs, Zic TF binding consolidates toward promoters where it co-localizes with activity-regulated TFs. We then performed CUT&RUN-seq in differentiating CGNs to define both the time course of developmental shifts in Zic TF binding and their relationship to gene expression. Mapping Zic TF binding sites to genes using chromatin looping, we identified the set of Zic target genes that have altered expression in RNA-seq from Zic1 or Zic2 knockdown CGNs. CONCLUSIONS: Our data show that Zic TFs are required for both induction and repression of distinct, developmentally regulated target genes through a mechanism that is largely independent of changes in Zic TF binding. We suggest that the differential collaboration of Zic TFs with other TF families underlies the shift in their biological functions across CGN development.

Identification and characterization of functional modules reflecting transcriptome transition during human neuron maturation.

BACKGROUND: Neuron maturation is a critical process in neurogenesis, during which neurons gain their morphological, electrophysiological and molecular characteristics for their functions as the central components of the nervous system. RESULTS: To better understand the molecular changes during this process, we combined the protein-protein interaction network and public single cell RNA-seq data of mature and immature neurons to identify functional modules relevant to the neuron maturation process in humans. Among the 109 functional modules in total, 33 showed significant gene expression level changes (discriminating modules) which participate in varied functions including energy consumption, synaptic functions and housekeeping functions such as translation and splicing. Based on the identified modules, we trained a neuron maturity index (NMI) model for the quantification of maturation states of single neurons or purified bulk neurons. Applied to multiple single neuron transcriptome data sets of neuron development in humans and mice, the NMI model made estimation of neuron maturity states which were significantly correlated with the neuron maturation trajectories in both species, implying the reproducibility and conservation of the identified transcriptome transition. CONCLUSION: We identified 33 discriminating modules whose activities were significantly correlated with single neuron maturity states, which may play important roles in the neuron maturation process.

New insights into the trophic and cytoprotective effects of creatine in in vitro and in vivo models of cell maturation.

A growing body of scientific reports indicates that the role of creatine (Cr) in cellular biochemistry and physiology goes beyond its contribution to cell energy. Indeed Cr has been shown to exert multiple effects promoting a wide range of physiological responses in vitro as well as in vivo. Included in these, Cr promotes in vitro neuron and muscle cell differentiation, viability and survival under normal or adverse conditions; anabolic, protective and pro-differentiative effects have also been observed in vivo. For example Cr has been shown to accelerate in vitro differentiation of cultured C2C12 myoblasts into myotubes, where it also induces a slight but significant hypertrophic effect as compared to unsupplemented cultures; Cr also prevents the anti-differentiation effects caused by oxidative stress in the same cells. In trained adults, Cr increases the mRNA expression of relevant myogemic factors, protein synthesis, muscle strength and size, in cooperation with physical exercise. As to neurons and central nervous system, Cr favors the electrophysiological maturation of chick neuroblasts in vitro and protects them from oxidative stress-caused killing; similarly, Cr promotes the survival and differentiation of GABA-ergic neurons in fetal spinal cord cultures in vitro; in vivo, maternal Cr supplementation promotes the morpho-functional development of hippocampal neurons in rat offsprings. This article, which presents also some new experimental data, focuses on the trophic, pro-survival and pro-differentiation effects of Cr and examines the ensuing preventive and therapeutic potential in pathological muscle and brain conditions.

Adult newborn neurons are involved in learning acquisition and long-term memory formation: the distinct demands on temporal neurogenesis of different cognitive tasks.

There is evidence that adult hippocampal neurogenesis influences hippocampal function, although the role these neurons fulfill in learning and consolidation processes remains unclear. Using a novel fast X-ray ablation protocol to deplete neurogenic cells, we demonstrate that immature adult hippocampal neurons are required for hippocampal learning and long-term memory formation. Moreover, we found that long-term memory formation in the object recognition and passive avoidance tests, two paradigms that involve circuits with distinct emotional components, had different temporal demands on hippocampal neurogenesis. These results reveal new and unexpected aspects of neurogenesis in cognitive processes.

Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation.

NGN2-driven induced pluripotent stem cell (iPSC)-to-neuron conversion is a popular method for human neurological disease modeling. In this study, we present a standardized approach for generating neurons utilizing clonal, targeted-engineered iPSC lines with defined reagents. We demonstrate consistent production of excitatory neurons at scale and long-term maintenance for at least 150 days. Temporal omics, electrophysiological, and morphological profiling indicate continued maturation to postnatal-like neurons. Quantitative characterizations through transcriptomic, imaging, and functional assays reveal coordinated actions of multiple pathways that drive neuronal maturation. We also show the expression of disease-related genes in these neurons to demonstrate the relevance of our protocol for modeling neurological disorders. Finally, we demonstrate efficient generation of NGN2-integrated iPSC lines. These workflows, profiling data, and functional characterizations enable the development of reproducible human in vitro models of neurological disorders.

Neocortex neurogenesis and maturation in the African greater cane rat.

BACKGROUND: Neocortex development has been extensively studied in altricial rodents such as mouse and rat. Identification of alternative animal models along the "altricial-precocial" spectrum in order to better model and understand neocortex development is warranted. The Greater cane rat (GCR, Thyronomys swinderianus) is an indigenous precocial African rodent. Although basic aspects of brain development in the GCR have been documented, detailed information on neocortex development including the occurrence and abundance of the distinct types of neural progenitor cells (NPCs) in the GCR are lacking. METHODS: GCR embryos and fetuses were obtained from timed pregnant dams between gestation days 50-140 and their neocortex was analyzed by immunofluorescence staining using characteristic marker proteins for NPCs, neurons and glia cells. Data were compared with existing data on closely related precocial and altricial species, i.e. guinea pig and dwarf rabbit. RESULTS: The primary sequence of neuro- and gliogenesis, and neuronal maturation is preserved in the prenatal GCR neocortex. We show that the GCR exhibits a relatively long period of cortical neurogenesis of 70 days. The subventricular zone becomes the major NPC pool during mid-end stages of neurogenesis with Pax6 + NPCs constituting the major basal progenitor subtype in the GCR neocortex. Whereas dendrite formation in the GCR cortical plate appears to initiate immediately after the onset of neurogenesis, major aspects of axon formation and maturation, and astrogenesis do not begin until mid-neurogenesis. Similar to the guinea pig, the GCR neocortex exhibits a high maturation status, containing neurons with well-developed dendrites and myelinated axons and astrocytes at birth, thus providing further evidence for the notion that a great proportion of neocortex growth and maturation in precocial mammals occurs before birth. CONCLUSIONS: Together, this work has deepened our understanding of neocortex development of the GCR, of the timing and the cellular differences that regulate brain growth and development within the altricial-precocial spectrum and its suitability as a research model for neurodevelopmental studies. The timelines of brain development provided by this study may serve as empirical reference data and foundation in future studies in order to model and better understand neurodevelopment and associated alterations.

Developmental Differences in Neocortex Neurogenesis and Maturation Between the Altricial Dwarf Rabbit and Precocial Guinea Pig.

Mammals are born on a precocial-altricial continuum. Altricial species produce helpless neonates with closed distant organs incapable of locomotion, whereas precocial species give birth to well-developed young that possess sophisticated sensory and locomotor capabilities. Previous studies suggest that distinct patterns of cortex development differ between precocial and altricial species. This study compares patterns of neocortex neurogenesis and maturation in the precocial guinea pig and altricial dwarf rabbit, both belonging to the taxon of Glires. We show that the principal order of neurodevelopmental events is preserved in the neocortex of both species. Moreover, we show that neurogenesis starts at a later postconceptional day and takes longer in absolute gestational days in the precocial than the altricial neocortex. Intriguingly, our data indicate that the dwarf rabbit neocortex contains a higher abundance of highly proliferative basal progenitors than the guinea pig, which might underlie its higher encephalization quotient, demonstrating that the amount of neuron production is determined by complex regulation of multiple factors. Furthermore, we show that the guinea pig neocortex exhibits a higher maturation status at birth, thus providing evidence for the notions that precocial species might have acquired the morphological machinery required to attain their high functional state at birth and that brain expansion in the precocial newborn is mainly due to prenatally initiating processes of gliogenesis and neuron differentiation instead of increased neurogenesis. Together, this study reveals important insights into the timing and cellular differences that regulate mammalian brain growth and maturation and provides a better understanding of the evolution of mammalian altriciality and presociality.

Reprogramming an energetic AKT-PAK5 axis boosts axon energy supply and facilitates neuron survival and regeneration after injury and ischemia.

Mitochondria supply adenosine triphosphate (ATP) essential for neuronal survival and regeneration. Brain injury and ischemia trigger acute mitochondrial damage and a local energy crisis, leading to degeneration. Boosting local ATP supply in injured axons is thus critical to meet increased energy demand during nerve repair and regeneration in adult brains, where mitochondria remain largely stationary. Here, we elucidate an intrinsic energetic repair signaling axis that boosts axonal energy supply by reprogramming mitochondrial trafficking and anchoring in response to acute injury-ischemic stress in mature neurons and adult brains. P21-activated kinase 5 (PAK5) is a brain mitochondrial kinase with declined expression in mature neurons. PAK5 synthesis and signaling is spatiotemporally activated within axons in response to ischemic stress and axonal injury. PAK5 signaling remobilizes and replaces damaged mitochondria via the phosphorylation switch that turns off the axonal mitochondrial anchor syntaphilin. Injury-ischemic insults trigger AKT growth signaling that activates PAK5 and boosts local energy supply, thus protecting axon survival and facilitating regeneration in in vitro and in vivo models. Our study reveals an axonal mitochondrial signaling axis that responds to injury and ischemia by remobilizing damaged mitochondria for replacement, thereby maintaining local energy supply to support central nervous system (CNS) survival and regeneration.

Fully Characterized Mature Human iPS- and NMP-Derived Motor Neurons Thrive Without Neuroprotection in the Spinal Contusion Cavity.

Neural cell interventions in spinal cord injury (SCI) have focused predominantly on transplanted multipotent neural stem/progenitor cells (NSPCs) for animal research and clinical use due to limited information on survival of spinal neurons. However, transplanted NSPC fate is unpredictable and largely governed by injury-derived matrix and cytokine factors that are often gliogenic and inflammatory. Here, using a rat cervical hemicontusion model, we evaluate the survival and integration of hiPSC-derived spinal motor neurons (SMNs) and oligodendrocyte progenitor cells (OPCs). SMNs and OPCs were differentiated in vitro through a neuromesodermal progenitor stage to mimic the natural origin of the spinal cord. We demonstrate robust survival and engraftment without additional injury site modifiers or neuroprotective biomaterials. Ex vivo differentiated neurons achieve cervical spinal cord matched transcriptomic and proteomic profiles, meeting functional electrophysiology parameters prior to transplantation. These data establish an approach for ex vivo developmentally accurate neuronal fate specification and subsequent transplantation for a more streamlined and predictable outcome in neural cell-based therapies of SCI.

Mitochondrial dysfunction increases fatty acid ß-oxidation and translates into impaired neuroblast maturation.

The metabolic transition from anaerobic glycolysis and fatty acid ß-oxidation to glycolysis coupled to oxidative phosphorylation is a key process for the transition of quiescent neural stem cells to proliferative neural progenitor cells. However, a full characterization of the metabolic shift and the involvement of mitochondria during the last step of neurogenesis, from neuroblasts to neuron maturation, is still elusive. Here, we describe a model of neuroblasts, Neuro2a cells, with impaired differentiation capacity due to mitochondrial dysfunction. Using a detailed biochemical characterization consisting of steady-state metabolomics and metabolic flux analysis, we find increased fatty acid ß-oxidation as a peculiar feature of neuroblasts with altered mitochondria. The consequent metabolic switch favors neuroblast proliferation at the expense of neuron maturation.

High content imaging quantification of multiple in vitro human neurogenesis events after neurotoxin exposure.

BACKGROUND: Our objective was to test neural active compounds in a human developmental neurotoxicity (DNT) model that represents neural tube stages of vulnerability. Previously we showed that 14 days in vitro (DIV 14) was sufficient to generate cryopreserved neuronal cells for post thaw neurite recovery assays. However, short exposure and assessment may not detect toxicants that affect an early neurogenesis continuum, from a mitotic human neural progenitor (hNP) cell population through the course of neurite outgrowth in differentiating neurons. Therefore, we continuously exposed differentiating hNP cells from DIV 0 through DIV 14 to known toxicants and endocrine active compounds in order to assess at DIV 14 effects of these compounds in a human DNT maturation model for neurogenesis. METHODS: The Human DNT continuum (DIV 0 to DIV 14) was determined using immunocytochemistry for SOX1+ (proliferating hNP) and HuC/D+ (post mitotic neurons). The cumulative effects of five compounds was observed on neurite outgrowth in (ßIII-tubulin+) and (HuC/D+) cells using high content imaging. All data were analyzed using a one-way ANOVA with a significance threshold of p < 0.05. RESULTS: During maturation in vitro, the neural cultures transitioned from uniform hNP cells (DIV 0) to predominantly mature post mitotic neuronal neurons (HuC/D+, 65%; DIV14) but also maintained a smaller population of hNP cells (SOX1+). Using this DNT maturation model system, Bis-1, testosterone, and ß-estradiol inhibited neuronal maturation at micromolar levels but were unaffected by acetaminophen. ß-estradiol also disrupted neurite extension at 10 µM. Treating cells in this window with Bisphenol A (BPA) significantly inhibited neurite outgrowth and branching in these continuum cultures but only at the highest concentrations tested (10 µM). CONCLUSIONS: Cumulative effects of neurotoxicant exposure during a maturation continuum altered human neurogenesis at lower exposure levels than observed in acute exposure of static cryopreserved neurite recovery neurons cultures. Unlike prior acute studies, ß-estradiol was highly toxic when present throughout the continuum and cytotoxicity was manifested starting early in the continuum via a non-estrogen receptor α (ER α) mechanism. Therefore, the effect of neural developmental neurotoxins can and should be determined during the dynamic process of human neural maturation.

Neural stem cells isolated from amyloid precursor protein-mutated mice for drug discovery.

AIM: To develop an in vitro model based on neural stem cells derived from transgenic animals, to be used in the study of pathological mechanisms of Alzheimer's disease and for testing new molecules. METHODS: Neural stem cells (NSCs) were isolated from the subventricular zone of Wild type (Wt) and Tg2576 mice. Primary and secondary neurosphere generation was studied, analysing population doubling and the cell yield per animal. Secondary neurospheres were dissociated and plated on MCM Gel Cultrex 2D and after 6 d in vitro (DIVs) in mitogen withdrawal conditions, spontaneous differentiation was studied using specific neural markers (MAP2 and TuJ-1 for neurons, GFAP for astroglial cells and CNPase for oligodendrocytes). Gene expression pathways were analysed in secondary neurospheres, using the QIAGEN PCR array for neurogenesis, comparing the Tg2576 derived cell expression with the Wt cells. Proteins encoded by the altered genes were clustered using STRING web software. RESULTS: As revealed by 6E10 positive staining, all Tg2576 derived cells retain the expression of the human transgenic Amyloid Precursor Protein. Tg2576 derived primary neurospheres show a decrease in population doubling. Morphological analysis of differentiated NSCs reveals a decrease in MAP2- and an increase in GFAP-positive cells in Tg2576 derived cells. Analysing the branching of TuJ-1 positive cells, a clear decrease in neurite number and length is observed in Tg2576 cells. The gene expression neurogenesis pathway revealed 11 altered genes in Tg2576 NSCs compared to Wt. CONCLUSION: Tg2576 NSCs represent an appropriate AD in vitro model resembling some cellular alterations observed in vivo, both as stem and differentiated cells.