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.

Long interspersed nuclear elements safeguard neural progenitors from precocious differentiation.

Long interspersed nuclear element-1 (L1 or LINE-1) is a highly abundant mobile genetic element in both humans and mice, comprising almost 20% of each genome. L1s are silenced by several mechanisms, as their uncontrolled expression has the potential to induce genomic instability. However, L1s are paradoxically expressed at high levels in differentiating neural progenitor cells. Using in vitro and in vivo techniques to modulate L1 expression, we report that L1s play a critical role in both human and mouse brain development by regulating the rate of neural differentiation in a reverse-transcription-independent manner.

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.

A Nuclear Belt Fastens on Neural Cell Fate.

Successful embryonic and adult neurogenesis require proliferating neural stem and progenitor cells that are intrinsically and extrinsically guided into a neuronal fate. In turn, migration of new-born neurons underlies the complex cytoarchitecture of the brain. Proliferation and migration are therefore essential for brain development, homeostasis and function in adulthood. Among several tightly regulated processes involved in brain formation and function, recent evidence points to the nuclear envelope (NE) and NE-associated components as critical new contributors. Classically, the NE was thought to merely represent a barrier mediating selective exchange between the cytoplasm and nucleoplasm. However, research over the past two decades has highlighted more sophisticated and diverse roles for NE components in progenitor fate choice and migration of their progeny by tuning gene expression via interactions with chromatin, transcription factors and epigenetic factors. Defects in NE components lead to neurodevelopmental impairments, whereas age-related changes in NE components are proposed to influence neurodegenerative diseases. Thus, understanding the roles of NE components in brain development, maintenance and aging is likely to reveal new pathophysiological mechanisms for intervention. Here, we review recent findings for the previously underrepresented contribution of the NE in neuronal commitment and migration, and envision future avenues for investigation.

Improved Method for Efficient Generation of Functional Neurons from Murine Neural Progenitor Cells.

Neuronal culture was used to investigate neuronal function in physiological and pathological conditions. Despite its inevitability, primary neuronal culture remained a gold standard method that requires laborious preparation, intensive training, and animal resources. To circumvent the shortfalls of primary neuronal preparations and efficiently give rise to functional neurons, we combine a neural stem cell culture method with a direct cell type-conversion approach. The lucidity of this method enables the efficient preparation of functional neurons from mouse neural progenitor cells on demand. We demonstrate that induced neurons (NPC-iNs) by this method make synaptic connections, elicit neuronal activity-dependent cellular responses, and develop functional neuronal networks. This method will provide a concise platform for functional neuronal assessments. This indeed offers a perspective for using these characterized neuronal networks for investigating plasticity mechanisms, drug screening assays, and probing the molecular and biophysical basis of neurodevelopmental and neurodegenerative diseases.

Lamin B1 decline underlies age-related loss of adult hippocampal neurogenesis.

Neurogenesis in the adult hippocampus declines with age, a process that has been implicated in cognitive and emotional impairments. However, the mechanisms underlying this decline have remained elusive. Here, we show that the age-dependent downregulation of lamin B1, one of the nuclear lamins in adult neural stem/progenitor cells (ANSPCs), underlies age-related alterations in adult hippocampal neurogenesis. Our results indicate that higher levels of lamin B1 in ANSPCs safeguard against premature differentiation and regulate the maintenance of ANSPCs. However, the level of lamin B1 in ANSPCs declines during aging. Precocious loss of lamin B1 in ANSPCs transiently promotes neurogenesis but eventually depletes it. Furthermore, the reduction of lamin B1 in ANSPCs recapitulates age-related anxiety-like behavior in mice. Our results indicate that the decline in lamin B1 underlies stem cell aging and impacts the homeostasis of adult neurogenesis and mood regulation.

When function follows form: Nuclear compartment structure and the epigenetic landscape of the aging neuron.

The human brain is affected by cellular aging. Neurons are primarily generated during embryogenesis and early life with a limited capacity for renewal and replacement, making them some of the oldest cells in the human body. Our present understanding of neurodegenerative diseases points towards advanced neuronal age as a prerequisite for the development of these disorders. While significant progress has been made in understanding the relationship between aging and neurological disease, it will be essential to delve further into the molecular mechanisms of neuronal aging in order to develop therapeutic interventions targeting age-related brain dysfunction. In this mini review, we highlight recent findings on the relationship between the aging of nuclear structures and changes in the epigenetic landscape during neuronal aging and disease.

The role of adult hippocampal neurogenesis in brain health and disease.

Adult neurogenesis in the dentate gyrus of the hippocampus is highly regulated by a number of environmental and cell-intrinsic factors to adapt to environmental changes. Accumulating evidence suggests that adult-born neurons may play distinct physiological roles in hippocampus-dependent functions, such as memory encoding and mood regulation. In addition, several brain diseases, such as neurological diseases and mood disorders, have deleterious effects on adult hippocampal neurogenesis, and some symptoms of those diseases can be partially explained by the dysregulation of adult hippocampal neurogenesis. Here we review a possible link between the physiological functions of adult-born neurons and their roles in pathological conditions.

Adult Hippocampal Neurogenesis: A Coming-of-Age Story.

What has become standard textbook knowledge over the last decade was a hotly debated matter a decade earlier: the proposition that new neurons are generated in the adult mammalian CNS. The early discovery by Altman and colleagues in the 1960s was vulnerable to criticism due to the lack of technical strategies for unequivocal demonstration, quantification, and physiological analysis of newly generated neurons in adult brain tissue. After several technological advancements had been made in the field, we published a paper in 1996 describing the generation of new neurons in the adult rat brain and the decline of hippocampal neurogenesis during aging. The paper coincided with the publication of several other studies that together established neurogenesis as a cellular mechanism in the adult mammalian brain. In this Progressions article, which is by no means a comprehensive review, we recount our personal view of the initial setting that led to our study and we discuss some of its implications and developments that followed. We also address questions that remain regarding the regulation and function of neurogenesis in the adult mammalian brain, in particular the existence of neurogenesis in the adult human brain.

Review: adult neurogenesis contributes to hippocampal plasticity.

Adult hippocampal neurogenesis is the process by which new functional neurons are added to the adult dentate gyrus of the hippocampus. Animal studies have shown that the degree of adult hippocampal neurogenesis is regulated by local environmental cues as well as neural network activities. Furthermore, accumulating evidence has suggested that adult hippocampal neurogenesis plays prominent roles in hippocampus-dependent brain functions. In this review, we summarize the mechanisms underlying the regulation of adult hippocampal neurogenesis at various developmental stages and propose how adult-born neurons contribute to structural and functional hippocampal plasticity.

Nup153 Interacts with Sox2 to Enable Bimodal Gene Regulation and Maintenance of Neural Progenitor Cells.

Neural progenitor cells (NeuPCs) possess a unique nuclear architecture that changes during differentiation. Nucleoporins are linked with cell-type-specific gene regulation, coupling physical changes in nuclear structure to transcriptional output; but, whether and how they coordinate with key fate-determining transcription factors is unclear. Here we show that the nucleoporin Nup153 interacts with Sox2 in adult NeuPCs, where it is indispensable for their maintenance and controls neuronal differentiation. Genome-wide analyses show that Nup153 and Sox2 bind and co-regulate hundreds of genes. Binding of Nup153 to gene promoters or transcriptional end sites correlates with increased or decreased gene expression, respectively, and inhibiting Nup153 expression alters open chromatin configurations at its target genes, disrupts genomic localization of Sox2, and promotes differentiation in vitro and a gliogenic fate switch in vivo. Together, these findings reveal that nuclear structural proteins may exert bimodal transcriptional effects to control cell fate.

An essential role of SVZ progenitors in cortical folding in gyrencephalic mammals.

Because folding of the cerebral cortex in the mammalian brain is believed to be crucial for higher brain functions, the mechanisms underlying its formation during development and evolution are of great interest. Although it has been proposed that increased neural progenitors in the subventricular zone (SVZ) are responsible for making cortical folds, their roles in cortical folding are still largely unclear, mainly because genetic methods for gyrencephalic mammals had been poorly available. Here, by taking an advantage of our newly developed in utero electroporation technique for the gyrencephalic brain of ferrets, we investigated the role of SVZ progenitors in cortical folding. We found regional differences in the abundance of SVZ progenitors in the developing ferret brain even before cortical folds began to be formed. When Tbr2 transcription factor was inhibited, intermediate progenitor cells were markedly reduced in the ferret cerebral cortex. Interestingly, outer radial glial cells were also reduced by inhibiting Tbr2. We uncovered that reduced numbers of SVZ progenitors resulted in impaired cortical folding. When Tbr2 was inhibited, upper cortical layers were preferentially reduced in gyri compared to those in sulci. Our findings indicate the biological importance of SVZ progenitors in cortical folding in the gyrencephalic brain.

Functional Implications of miR-19 in the Migration of Newborn Neurons in the Adult Brain.

Altered microRNA profiles have been implicated in human brain disorders. However, the functional contribution of individual microRNAs to neuronal development and function is largely unknown. Here, we report biological functions for miR-19 in adult neurogenesis. We determined that miR-19 is enriched in neural progenitor cells (NPCs) and downregulated during neuronal development in the adult hippocampus. By manipulating miR-19 in NPCs for gain- and loss-of-function studies, we discovered that miR-19 regulates cell migration by directly targeting Rapgef2. Concordantly, dysregulation of miR-19 in NPCs alters the positioning of newborn neurons in the adult brain. Furthermore, we found abnormal expression of miR-19 in human NPCs generated from schizophrenic patient-derived induced pluripotent stem cells (iPSCs) that have been described as displaying aberrant migration. Our study demonstrates the significance of posttranscriptional gene regulation by miR-19 in preventing the irregular migration of adult-born neurons that may contribute to the etiology of schizophrenia.

Sox11 Balances Dendritic Morphogenesis with Neuronal Migration in the Developing Cerebral Cortex.

UNLABELLED: The coordinated mechanisms balancing promotion and suppression of dendritic morphogenesis are crucial for the development of the cerebral cortex. Although previous studies have revealed important transcription factors that promote dendritic morphogenesis during development, those that suppress dendritic morphogenesis are still largely unknown. Here we found that the expression levels of the transcription factor Sox11 decreased dramatically during dendritic morphogenesis. Our loss- and gain-of-function studies using postnatal electroporation and in utero electroporation indicate that Sox11 is necessary and sufficient for inhibiting dendritic morphogenesis of excitatory neurons in the mouse cerebral cortex during development. Interestingly, we found that precocious suppression of Sox11 expression caused precocious branching of neurites and a neuronal migration defect. We also found that the end of radial migration induced the reduction of Sox11 expression. These findings indicate that suppression of dendritic morphogenesis by Sox11 during radial migration is crucial for the formation of the cerebral cortex. SIGNIFICANCE STATEMENT: Because dendritic morphology has profound impacts on neuronal information processing, the mechanisms underlying dendritic morphogenesis during development are of great interest. Our loss- and gain-of-function studies indicate that Sox11 is necessary and sufficient for inhibiting dendritic morphogenesis of excitatory neurons in the mouse cerebral cortex during development. Interestingly, we found that precocious suppression of Sox11 expression caused a neuronal migration defect. These findings indicate that suppression of dendritic morphogenesis by Sox11 during radial migration is crucial for the formation of the cerebral cortex.

Pathophysiological analyses of cortical malformation using gyrencephalic mammals.

One of the most prominent features of the cerebral cortex of higher mammals is the presence of gyri. Because malformations of the cortical gyri are associated with severe disability in brain function, the mechanisms underlying malformations of the cortical gyri have been of great interest. Combining gyrencephalic carnivore ferrets and genetic manipulations using in utero electroporation, here we successfully recapitulated the cortical phenotypes of thanatophoric dysplasia (TD) by expressing fibroblast growth factor 8 in the ferret cerebral cortex. Strikingly, in contrast to TD mice, our TD ferret model showed not only megalencephaly but also polymicrogyria. We further uncovered that outer radial glial cells (oRGs) and intermediate progenitor cells (IPs) were markedly increased. Because it has been proposed that increased oRGs and/or IPs resulted in the appearance of cortical gyri during evolution, it seemed possible that increased oRGs and IPs underlie the pathogenesis of polymicrogyria. Our findings should help shed light on the molecular mechanisms underlying the formation and malformation of cortical gyri in higher mammals.

Directly Reprogrammed Human Neurons Retain Aging-Associated Transcriptomic Signatures and Reveal Age-Related Nucleocytoplasmic Defects.

Aging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging.

The development of suckling behavior of neonatal mice is regulated by birth.

BACKGROUND: Although the function of the sensory system rapidly develops soon after birth in newborn pups, little is known about the mechanisms triggering this functional development of the sensory system. RESULTS: Here we show that the birth of pups plays an active role in the functional development of the sensory system. We first optimized the experimental procedure for suckling behavior using neonatal mouse pups. Using this procedure, we found that preterm birth selectively accelerated the development of suckling behavior in neonatal pups, but not that of motor performance, suggesting that the birth of pups regulates the functional development of the sensory system soon after birth. CONCLUSIONS: Taken together with our recent findings that birth itself regulates the initiation of sensory map formation in the somatosensory and visual systems, these results support the idea that the birth of pups actively regulates the anatomical and functional development of the sensory system.

Birth regulates the initiation of sensory map formation through serotonin signaling.

Although the mechanisms underlying the spatial pattern formation of sensory maps have been extensively investigated, those triggering sensory map formation during development are largely unknown. Here we show that the birth of pups instructively and selectively regulates the initiation of barrel formation in the somatosensory cortex by reducing serotonin concentration. We found that preterm birth accelerated barrel formation, whereas it did not affect either barreloid formation or barrel structural plasticity. We also found that serotonin was selectively reduced soon after birth and that the reduction of serotonin was triggered by birth. The reduction of serotonin was necessary and sufficient for the effect of birth on barrel formation. Interestingly, the regulatory mechanisms described here were also found to regulate eye-specific segregation in the visual system, suggesting that they are utilized in various brain regions. Our results shed light on roles of birth and serotonin in sensory map formation.

In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation.

Brain structures such as the outer subventricular zone (OSVZ) and the inner fiber layer (IFL) in the developing cerebral cortex are especially prominent in higher mammals. However, the molecular mechanisms underlying the formation of the OSVZ are still largely unknown, mainly because genetic manipulations that can be applied to the OSVZ in higher mammals had been poorly available. Here we developed and validated a rapid and efficient genetic manipulation technique for germinal zones including the OSVZ using in utero electroporation in developing gyrencephalic carnivore ferrets. We also determined the optimal conditions for using in utero electroporation to express transgenes in germinal zones. Using our electroporation procedure, the morphology of GFP-positive cells in the OSVZ was clearly visible even without immunostaining, and multiple genes were efficiently co-expressed in the same cells. Furthermore, we uncovered that fibers, which seemed to correspond to those in the IFL of monkeys, also existed in ferrets, and were derived from newly generated cortical neurons. Our technique promises to be a powerful tool for investigating the fundamental mechanisms underlying the formation and abnormalities of the cerebral cortex in higher mammals.

Whisker-related axonal patterns and plasticity of layer 2/3 neurons in the mouse barrel cortex.

Elucidating neuronal circuits and their plasticity in the cerebral cortex is one of the important questions in neuroscience research. Here we report novel axonal trajectories and their plasticity in the mouse somatosensory barrel cortex. We selectively visualized layer 2/3 neurons using in utero electroporation and examined the axonal trajectories of layer 2/3 neurons. We found that the axons of layer 2/3 neurons preferentially run in the septal regions of layer 4 and named this axonal pattern "barrel nets." The intensity of green fluorescent protein in the septal regions was markedly higher compared with that in barrel hollows. Focal in utero electroporation revealed that the axons in barrel nets were indeed derived from layer 2/3 neurons in the barrel cortex. During development, barrel nets became visible at postnatal day 10, which was well after the initial appearance of barrels. When whisker follicles were cauterized within 3 d after birth, the whisker-related pattern of barrel nets was altered, suggesting that cauterization of whisker follicles results in developmental plasticity of barrel nets. Our results uncover the novel axonal trajectories of layer 2/3 neurons with whisker-related patterns and their developmental plasticity in the mouse somatosensory cortex. Barrel nets should be useful for investigating the pattern formation and axonal reorganization of intracortical neuronal circuits.