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.
SYNOPSIS
Mutations in the nuclear envelope protein lamin B1 are linked to cellular aging and senescence. Here, lamin B1 ablation in adult neural stem/progenitor cells (ANSPCs) in mice is shown to disrupt neurogenesis, leading to aging‐related behavioral changes.
Lamin B1 is highly expressed in ANSPCs, but selectively declines with age.
Conditional knockout of lamin B1 in ANSPCs leads to premature differentiation and reduction of adult hippocampal neurogenesis.
Lamin B1 knockout leads to age‐related anxiety‐like behaviours in mice.
Overexpression of lamin B1 suppresses ANSPC differentiation.
Ablation of the nuclear envelope protein lamin B1 in neural stem/progenitor cells disrupts neurogenesis and causes aging‐associated behavioral changes in mice.
Comparative analyses of neuronal phenotypes in closely related species can shed light on neuronal changes occurring during evolution. The study of post-mortem brains of nonhuman primates (NHPs) has ...been limited and often does not recapitulate important species-specific developmental hallmarks. We utilize induced pluripotent stem cell (iPSC) technology to investigate the development of cortical pyramidal neurons following migration and maturation of cells grafted in the developing mouse cortex. Our results show differential migration patterns in human neural progenitor cells compared to those of chimpanzees and bonobos both in vitro and in vivo, suggesting heterochronic changes in human neurons. The strategy proposed here lays the groundwork for further comparative analyses between humans and NHPs and opens new avenues for understanding the differences in the neural underpinnings of cognition and neurological disease susceptibility between species.
Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an ...advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1β or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1β. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration.
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•Reliable method for generation of astrocytes from human iPSCs and ESCs•Generated astrocytes are functional and inflammation-responsive•Generated astrocytes share properties with primary astrocytes in vitro•This method is a valuable tool for disease modeling of neuroinflammation
In this article, Gage and colleagues developed a reliable method for generating functional astrocytes from human pluripotent stem cells. This protocol uses an intermediate glial progenitor stage and generates inflammation-responsive astrocytes, providing an important tool to model neurological diseases in-a-dish, enabling the study of neurological disorders with an inflammatory component.
Several mutations that cause Parkinson's disease (PD) have been identified over the past decade. These account for 15-25% of PD cases; the rest of the cases are considered sporadic. Currently, it is ...accepted that PD is not a single monolithic disease but rather a constellation of diseases with some common phenotypes. While rodent models exist for some of the PD-causing mutations, research on the sporadic forms of PD is lagging due to a lack of cellular models. In our study, we differentiated PD patient-derived dopaminergic (DA) neurons from the induced pluripotent stem cells (iPSCs) of several PD-causing mutations as well as from sporadic PD patients. Strikingly, we observed a common neurophysiological phenotype: neurons derived from PD patients had a severe reduction in the rate of synaptic currents compared to those derived from healthy controls. While the relationship between mutations in genes such as the SNCA and LRRK2 and a reduction in synaptic transmission has been investigated before, here we show evidence that the pathogenesis of the synapses in neurons is a general phenotype in PD. Analysis of RNA sequencing results displayed changes in gene expression in different synaptic mechanisms as well as other affected pathways such as extracellular matrix-related pathways. Some of these dysregulated pathways are common to all PD patients (monogenic or idiopathic). Our data, therefore, show changes that are central and convergent to PD and suggest a strong involvement of the tetra-partite synapse in PD pathophysiology.
We report the first isolation of progenitor cells from the hypothalamus, a derivative of the embryonic basal plate that does not exhibit neurogenesis postnatally. Neurons derived from hypothalamic ...progenitor cells were compared with those derived from progenitor cultures of hippocampus, an embryonic alar plate derivative that continues to support neurogenesis in vivo into adulthood. Aside from their different embryonic origins and their different neurogenic potential in vivo, these brain regions were chosen because they are populated with cells of three different categories: Category I cells are generated in both hippocampus and hypothalamus, Category II cells are generated in the hypothalamus but are absent from the hippocampus, and Category III is a cell type generated in the olfactory placode that migrates into the hypothalamus during development. Stem-like cells isolated from other brain regions, with the ability to generate neurons and glia, produce neurons of several phenotypes including gabaergic, dopaminergic, and cholinergic lineages. In the present study, we extended our observations into neuroendocrine phenotypes. The cultured neural precursors from 7-week-old rat hypothalamus readily generated neuropeptide-expressing neurons. Hippocampal and hypothalamic progenitor cultures converged to indistinguishable populations and produced neurons of all three categories, confirming that even short-term culture confers or selects for immature progenitors with enough plasticity to elaborate neuronal phenotypes usually inhibited in vivo by the local microenvironment. The range of phenotypes generated from neuronal precursors in vitro now includes the peptides found in the neuroendocrine system: corticotropin-releasing hormone, growth hormone-releasing hormone, gonadotropin-releasing hormone, oxytocin, somatostatin, thyrotropin-releasing hormone, and vasopressin.
The Cre/loxP system is increasingly showing promise for investigating genes involved in neural function. Here, we demonstrate that in vivo modification of genes in the mouse brain can be accomplished ...in a spatial- and temporal-specific manner by targeted delivery of an adeno-associated virus (AAV) encoding a green fluorescent protein/Cre recombinase (GFP/Cre) fusion protein. By using a reporter mouse, in which Cre recombinase activates β-galactosidase expression, we demonstrate long-term recombination of neurons in the hippocampus, striatum, and septum as early as 7 days after stereotaxic injection of virus. Recombined cells were observed for at least 6 months postinjection without evidence of cell loss or neural damage. AAV-mediated delivery of GFP/Cre provides a valuable approach to alter the mouse genome, as AAV delivers genes efficiently to neurons with low toxicity. This approach will greatly facilitate the study of genetic modifications in the mouse brain.
Currently, researchers rely on generalized methods to quantify transposable element (TE) RNA expression, such as RT-qPCR and RNA-seq, that do not distinguish between TEs expressed from their own ...promoter (bona fide) and TEs that are transcribed from a neighboring gene promoter such as within an intron or exon. This distinction is important owing to the differing functional roles of TEs depending on whether they are independently transcribed. Here we report a simple strategy to examine bona fide TE expression, termed BonaFide-TEseq. This approach can be used with any template-switch based library such as Smart-seq2 or the single-cell 5' gene expression kit from 10x, extending its utility to single-cell RNA-sequencing. This approach does not require TE-specific enrichment, enabling the simultaneous examination of TEs and protein-coding genes. We show that TEs identified through BonaFide-TEseq are expressed from their own promoter, rather than captured as internal products of genes. We reveal the utility of BonaFide-TEseq in the analysis of single-cell data and show that short-interspersed nuclear elements (SINEs) show cell type-specific expression profiles in the mouse hippocampus. We further show that, in response to a brief exposure of home-cage mice to a novel stimulus, SINEs are activated in dentate granule neurons in a time course that is similar to that of protein-coding immediate early genes. This work provides a simple alternative approach to assess bona fide TE transcription at single-cell resolution and provides a proof-of-concept using this method to identify SINE activation in a context that is relevant for normal learning and memory.
Bipolar disorder (BD) is a psychiatric condition characterized by depressive and manic episodes that affect 2% of the world population. The first-line long-term treatment for mood stabilization is ...lithium (Li). Induced pluripotent stem cell modeling of BD using hippocampal dentate gyrus-like neurons derived from Li-responsive (LR) and Li-non-responsive (NR) patients previously showed neuronal hyperexcitability. Li treatment reversed hyperexcitability only on the LR neurons. In this study we searched for specific targets of Li resistance in NR neurons and found that the activity of Wnt/β-catenin signaling pathway was severely affected, with a significant decrease in expression of LEF1. Li targets the Wnt/β-catenin signaling pathway by inhibiting GSK-3β and releasing β-catenin that forms a nuclear complex with TCF/LEF1, activating the Wnt/β-catenin transcription program. Therefore, we propose that downregulation of LEF1 may account for Li resistance in NR neurons. Our results show that valproic acid (VPA), a drug used to treat NR patients that also acts downstream of GSK-3β, upregulated LEF1 and Wnt/β-catenin gene targets, increased transcriptional activity of complex β-catenin/TCF/LEF1, and reduced excitability in NR neurons. In addition, decreasing LEF1 expression in control neurons using shLEF1 caused hyperexcitability, confirming that the impact of VPA on excitability in NR neurons was connected to changes in LEF1 and in the Wnt/β-catenin pathway. Our results suggest that LEF1 may be a useful target for the discovery of new drugs for BD treatment.
Approximately 1 in every 50 to 100 people is affected with bipolar disorder (BD), making this disease a major economic burden. The introduction of induced pluripotent stem cell methodology enabled ...better modeling of this disorder.
Having previously studied the phenotype of dentate gyrus granule neurons, we turned our attention to studying the phenotype of CA3 hippocampal pyramidal neurons of 6 patients with BD compared with 4 control individuals. We used patch clamp and quantitative polymerase chain reaction to measure electrophysiological features and RNA expression by specific channel genes.
We found that BD CA3 neurons were hyperexcitable only when they were derived from patients who responded to lithium; they featured sustained activity with large current injections and a large, fast after-hyperpolarization, similar to what we previously reported in dentate gyrus neurons. The higher amplitudes and faster kinetics of fast potassium currents correlated with this hyperexcitability. Further supporting the involvement of potassium currents, we observed an overexpression of KCNC1 and KCNC2 in hippocampal neurons derived from lithium responders. Applying specific potassium channel blockers diminished the hyperexcitability. Long-term lithium treatment decreased the hyperexcitability observed in the CA3 neurons derived from lithium responders while increasing sodium currents and reducing fast potassium currents. When differentiating this cohort into spinal motor neurons, we did not observe any changes in the excitability of BD motor neurons compared with control motor neurons.
The hyperexcitability of BD neurons is neuronal type specific with the involvement of altered potassium currents that allow for a sustained, continued firing activity.
Unique aspects of human behavior are often attributed to differences in the relative size and organization of the human brain: these structural aspects originate during early development. Recent ...studies indicate that human neurodevelopment is considerably slower than that in other nonhuman primates, a finding that is termed neoteny. One aspect of neoteny is the slow onset of action potentials. However, which molecular mechanisms play a role in this process remain unclear. To examine the evolutionary constraints on the rate of neuronal maturation, we have generated transcriptional data tracking five time points, from the neural progenitor state to 8-week-old neurons, in primates spanning the catarrhine lineage, including Macaca mulatta, Gorilla gorilla, Pan paniscus, Pan troglodytes, and Homo sapiens. Despite finding an overall similarity of many transcriptional signatures, species-specific and clade-specific distinctions were observed. Among the genes that exhibited human-specific regulation, we identified a key pioneer transcription factor, GATA3, that was uniquely upregulated in humans during the neuronal maturation process. We further examined the regulatory nature of GATA3 in human cells and observed that downregulation quickened the speed of developing spontaneous action potentials, thereby modulating the human neotenic phenotype. These results provide evidence for the divergence of gene regulation as a key molecular mechanism underlying human neoteny.
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