Lifelong persistence of nuclear RNAs in the mouse brain.

Genomic DNA that resides in the nuclei of mammalian neurons can be as old as the organism itself. The life span of nuclear RNAs, which are critical for proper chromatin architecture and transcription regulation, has not been determined in adult tissues. In this work, we identified and characterized nuclear RNAs that do not turn over for at least 2 years in a subset of postnatally born cells in the mouse brain. These long-lived RNAs were stably retained in nuclei in a neural cell type-specific manner and were required for the maintenance of heterochromatin. Thus, the life span of neural cells may depend on both the molecular longevity of DNA for the storage of genetic information and also the extreme stability of RNA for the functional organization of chromatin.

Loss of individualized behavioral trajectories in adult neurogenesis-deficient cyclin D2 knockout mice.

There is still limited mechanistic insight into how the interaction of individuals with their environment results in the emergence of individuality in behavior and brain structure. Nevertheless, the idea that personal activity shapes the brain is implicit in strategies for healthy cognitive aging as well as in the idea that individuality is reflected in the brain's connectome. We have shown that even isogenic mice kept in a shared enriched environment (ENR) developed divergent and stable social and exploratory trajectories. As these trajectories-measured as roaming entropy (RE)-positively correlated with adult hippocampal neurogenesis, we hypothesized that a feedback between behavioral activity and adult hippocampal neurogenesis might be a causal factor in brain individualization. We used cyclin D2 knockout mice with constitutively extremely low levels of adult hippocampal neurogenesis and their wild-type littermates. We housed them for 3 months in a novel ENR paradigm, consisting of 70 connected cages equipped with radio frequency identification antennae for longitudinal tracking. Cognitive performance was evaluated in the Morris Water Maze task (MWM). With immunohistochemistry we confirmed that adult neurogenesis correlated with RE in both genotypes and that D2 knockout mice had the expected impaired performance in the reversal phase of the MWM. But whereas the wild-type animals developed stable exploratory trajectories with increasing variance, correlating with adult neurogenesis, this individualizing phenotype was absent in D2 knockout mice. Here the behaviors started out more random and revealed less habituation and low variance. Together, these findings suggest that adult neurogenesis contributes to experience-dependent brain individualization.

Epigenetic aging in adult neurogenesis.

Neural stem cells (NSCs) in the hippocampus generate new neurons throughout life, which functionally contribute to cognitive flexibility and mood regulation. Yet adult hippocampal neurogenesis substantially declines with age and age-related impairments in NSC activity underlie this reduction. Particularly, increased NSC quiescence and consequently reduced NSC proliferation are considered to be major drivers of the low neurogenesis levels in the aged brain. Epigenetic regulators control the gene expression programs underlying NSC quiescence, proliferation and differentiation and are hence critical to the regulation of adult neurogenesis. Epigenetic alterations have also emerged as central hallmarks of aging, and recent studies suggest the deterioration of the NSC-specific epigenetic landscape as a driver of the age-dependent decline in adult neurogenesis. In this review, we summarize the recently accumulating evidence for a role of epigenetic dysregulation in NSC aging and propose perspectives for future research directions.

The individuality paradigm: Automated longitudinal activity tracking of large cohorts of genetically identical mice in an enriched environment.

Personalized medicine intensifies interest in experimental paradigms that delineate sources of phenotypic variation. The paradigm of environmental enrichment allows for comparisons among differently housed laboratory rodents to unravel environmental effects on brain plasticity and related phenotypes. We have developed a new longitudinal variant of this paradigm, which allows to investigate the emergence of individuality, the divergence of individual behavioral trajectories under a constant genetic background and in a shared environment. We here describe this novel method, the "Individuality Paradigm," which allows to investigate mechanisms that drive individuality. Various aspects of individual activity are tracked over time to identify the contribution of the non-shared environment, that is the extent to which the experience of an environment differs between individual members of a population. We describe the design of this paradigm in detail, lay out its scientific potential beyond the published studies and discuss how it differs from other approaches to study individuality. The custom-built cage system, commercially marketed as "ColonyRack", allows mice to roam freely between 70 cages through connector tubes equipped with ring antennas that detect each animal's ID from an RFID transponder implanted in the animal's neck. The system has a total floor area of 2.74 m2 and its spatial resolution corresponds to the size of the individual cages. Spatiotemporally resolved antenna contacts yield longitudinal measures of individual behavior, including the powerful measure of roaming entropy (RE). The Individuality Paradigm provides a rodent model of the making of individuality and the impact of the 'non-shared' environment on life-course development.

Selenium mediates exercise-induced adult neurogenesis and reverses learning deficits induced by hippocampal injury and aging.

Although the neurogenesis-enhancing effects of exercise have been extensively studied, the molecular mechanisms underlying this response remain unclear. Here, we propose that this is mediated by the exercise-induced systemic release of the antioxidant selenium transport protein, selenoprotein P (SEPP1). Using knockout mouse models, we confirmed that SEPP1 and its receptor low-density lipoprotein receptor-related protein 8 (LRP8) are required for the exercise-induced increase in adult hippocampal neurogenesis. In vivo selenium infusion increased hippocampal neural precursor cell (NPC) proliferation and adult neurogenesis. Mimicking the effect of exercise through dietary selenium supplementation restored neurogenesis and reversed the cognitive decline associated with aging and hippocampal injury, suggesting potential therapeutic relevance. These results provide a molecular mechanism linking exercise-induced changes in the systemic environment to the activation of quiescent hippocampal NPCs and their subsequent recruitment into the neurogenic trajectory.

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.

Environmental enrichment preserves a young DNA methylation landscape in the aged mouse hippocampus.

The decline of brain function during aging is associated with epigenetic changes, including DNA methylation. Lifestyle interventions can improve brain function during aging, but their influence on age-related epigenetic changes is unknown. Using genome-wide DNA methylation sequencing, we here show that experiencing a stimulus-rich environment counteracts age-related DNA methylation changes in the hippocampal dentate gyrus of mice. Specifically, environmental enrichment prevented the aging-induced CpG hypomethylation at target sites of the methyl-CpG-binding protein Mecp2, which is critical to neuronal function. The genes at which environmental enrichment counteracted aging effects have described roles in neuronal plasticity, neuronal cell communication and adult hippocampal neurogenesis and are dysregulated with age-related cognitive decline in the human brain. Our results highlight the stimulating effects of environmental enrichment on hippocampal plasticity at the level of DNA methylation and give molecular insights into the specific aspects of brain aging that can be counteracted by lifestyle interventions.

Generation of mouse hippocampal neural precursor cell lines with CRISPR/Cas9-mediated gene knockouts.

Genetic manipulation of neural precursor cells is an important tool to study mechanisms underlying proliferation, fate specification, and neuron formation. The CRISPR/Cas9 system enables efficient genome editing but requires the clonal expansion of cells containing the desired mutation. Here, we describe a protocol for the effective generation of clonal mouse hippocampal neural precursor lines with CRISPR/Cas9-based gene knockouts. Edited cell lines can be used to investigate gene regulatory networks driving neuronal differentiation and for modeling of diseases that involve hippocampal neurogenesis. For complete details on the use and execution of this protocol, please refer to Pötzsch et al. (2021).

L-lactate exerts a pro-proliferative effect on adult hippocampal precursor cells in vitro.

L-lactate has energetic and signaling properties, and its availability is modulated by activity-dependent stimuli, which also regulate adult hippocampal neurogenesis. Studying the effects of L-lactate on neural precursor cells (NPCs) in vitro, we found that L-lactate is pro-proliferative and that this effect is dependent on the active lactate transport by monocarboxylate transporters. Increased proliferation was not linked to amplified mitochondrial respiration. Instead, L-lactate deviated glucose metabolism to the pentose phosphate pathway, indicated by increased glucose-6-phosphate dehydrogenase activity while glycolysis decreased. Knockout of Hcar1 revealed that the pro-proliferative effect of L-lactate was not dependent on receptor activity although phosphorylation of ERK1/2 and Akt was increased following L-lactate treatment. Together, we show that availability of L-lactate is linked to the proliferative potential of NPCs and add evidence to the hypothesis that lactate influences cellular homeostatic processes in the adult brain, specifically in the context of adult hippocampal neurogenesis.

ROS Dynamics Delineate Functional States of Hippocampal Neural Stem Cells and Link to Their Activity-Dependent Exit from Quiescence.

Cellular redox states regulate the balance between stem cell maintenance and activation. Increased levels of intracellular reactive oxygen species (ROS) are linked to proliferation and lineage specification. In contrast to this general principle, we here show that in the hippocampus of adult mice, quiescent neural precursor cells (NPCs) maintain the highest ROS levels (hiROS). Classifying NPCs on the basis of cellular ROS content identified distinct functional states. Shifts in ROS content primed cells for a subsequent state transition, with lower ROS content marking proliferative activity and differentiation. Physical activity, a physiological activator of adult hippocampal neurogenesis, recruited hiROS NPCs 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 provide a metabolic classification of NPC functional states and describe a mechanism linking the modulation of cellular ROS by behavioral cues to the activation of adult NPCs.

Early-life environmental enrichment generates persistent individualized behavior in mice.

Individuals differ in their response to environmental stimuli, but the stability of individualized behaviors and their associated changes in brain plasticity are poorly understood. We developed a novel model of enriched environment to longitudinally monitor 40 inbred mice exploring 35 connected cages over periods of 3 to 6 months. We show that behavioral individuality that emerged during the first 3 months of environmental enrichment persisted when mice were withdrawn from the enriched environment for 3 additional months. Behavioral trajectories were associated with stable interindividual differences in adult hippocampal neurogenesis and persistent epigenetic effects on neuronal plasticity genes in the hippocampus. Using genome-wide DNA methylation sequencing, we show that one-third of the DNA methylation changes were maintained after withdrawal from the enriched environment. Our results suggest that, even under conditions that control genetic background and shared environment, early-life experiences result in lasting individualized changes in behavior, brain plasticity, and epigenetics.

FASN-Dependent Lipid Metabolism Links Neurogenic Stem/Progenitor Cell Activity to Learning and Memory Deficits.

Altered neural stem/progenitor cell (NSPC) activity and neurodevelopmental defects are linked to intellectual disability. However, it remains unclear whether altered metabolism, a key regulator of NSPC activity, disrupts human neurogenesis and potentially contributes to cognitive defects. We investigated links between lipid metabolism and cognitive function in mice and human embryonic stem cells (hESCs) expressing mutant fatty acid synthase (FASN; R1819W), a metabolic regulator of rodent NSPC activity recently identified in humans with intellectual disability. Mice homozygous for the FASN R1812W variant have impaired adult hippocampal NSPC activity and cognitive defects because of lipid accumulation in NSPCs and subsequent lipogenic ER stress. Homozygous FASN R1819W hESC-derived NSPCs show reduced rates of proliferation in embryonic 2D cultures and 3D forebrain regionalized organoids, consistent with a developmental phenotype. These data from adult mouse models and in vitro models of human brain development suggest that altered lipid metabolism contributes to intellectual disability.

Selective increases in inter-individual variability in response to environmental enrichment in female mice.

One manifestation of individualization is a progressively differential response of individuals to the non-shared components of the same environment. Individualization has practical implications in the clinical setting, where subtle differences between patients are often decisive for the success of an intervention, yet there has been no suitable animal model to study its underlying biological mechanisms. Here we show that enriched environment (ENR) can serve as a model of brain individualization. We kept 40 isogenic female C57BL/6JRj mice for 3 months in ENR and compared these mice to an equally sized group of standard-housed control animals, looking at the effects on a wide range of phenotypes in terms of both means and variances. Although ENR influenced multiple parameters and restructured correlation patterns between them, it only increased differences among individuals in traits related to brain and behavior (adult hippocampal neurogenesis, motor cortex thickness, open field and object exploration), in agreement with the hypothesis of a specific activity-dependent development of brain individuality.

Transcriptome sequencing during mouse brain development identifies long non-coding RNAs functionally involved in neurogenic commitment.

Transcriptome analysis of somatic stem cells and their progeny is fundamental to identify new factors controlling proliferation versus differentiation during tissue formation. Here, we generated a combinatorial, fluorescent reporter mouse line to isolate proliferating neural stem cells, differentiating progenitors and newborn neurons that coexist as intermingled cell populations during brain development. Transcriptome sequencing revealed numerous novel long non-coding (lnc)RNAs and uncharacterized protein-coding transcripts identifying the signature of neurogenic commitment. Importantly, most lncRNAs overlapped neurogenic genes and shared with them a nearly identical expression pattern suggesting that lncRNAs control corticogenesis by tuning the expression of nearby cell fate determinants. We assessed the power of our approach by manipulating lncRNAs and protein-coding transcripts with no function in corticogenesis reported to date. This led to several evident phenotypes in neurogenic commitment and neuronal survival, indicating that our study provides a remarkably high number of uncharacterized transcripts with hitherto unsuspected roles in brain development. Finally, we focussed on one lncRNA, Miat, whose manipulation was found to trigger pleiotropic effects on brain development and aberrant splicing of Wnt7b. Hence, our study suggests that lncRNA-mediated alternative splicing of cell fate determinants controls stem-cell commitment during neurogenesis.

The domain of unknown function DUF1521 exhibits metal ion-inducible autocleavage activity - a novel example from a putative effector protein of Vibrio coralliilyticus ATCC BAA-450.

Vibrio coralliilyticus ATCC BAA-450 is a pathogen causing coral bleaching at elevated seawater temperatures. Based on the available genome sequence, the strain has a type III secretion system. Within the corresponding gene cluster, VIC_001052 is encoded, which contains a conserved domain of unknown function DUF1521. In this study, we show that the purified domain exhibits autocleavage activity in the presence of several divalent metal ions, for example, calcium and manganese but not with magnesium or zinc. Autocleavage is not affected by temperatures between 0 and 30 °C, indicating that seawater temperature is not a critical factor for this activity. The DUF1521 domain and the cleavage site are conserved in several proteins from proteobacteria, suggesting a similar cleavage activity for these proteins.

Acute inflammation initiates the regenerative response in the adult zebrafish brain.

The zebrafish regenerates its brain after injury and hence is a useful model organism to study the mechanisms enabling regenerative neurogenesis, which is poorly manifested in mammals. Yet the signaling mechanisms initiating such a regenerative response in fish are unknown. Using cerebroventricular microinjection of immunogenic particles and immunosuppression assays, we showed that inflammation is required and sufficient for enhancing the proliferation of neural progenitors and subsequent neurogenesis by activating injury-induced molecular programs that can be observed after traumatic brain injury. We also identified cysteinyl leukotriene signaling as an essential component of inflammation in the regenerative process of the adult zebrafish brain. Thus, our results demonstrate that in zebrafish, in contrast to mammals, inflammation is a positive regulator of neuronal regeneration in the central nervous system.