Utilizing AAV-mediated LEAPER 2.0 for programmable RNA editing in non-human primates and nonsense mutation correction in humanized Hurler syndrome mice.

BACKGROUND: The endogenous adenosine deaminases acting on RNA (ADAR) have been harnessed to facilitate precise adenosine-to-inosine editing on RNAs. However, the practicability of this approach for therapeutic purposes is still ambiguous due to the variable expression of intrinsic ADAR across various tissues and species, as well as the absence of all-encompassing confirmation for delivery methods. RESULTS: In this study, we demonstrate that AAV-mediated delivery of circular ADAR-recruiting RNAs (arRNAs) achieves effective RNA editing in non-human primates at dosages suitable for therapy. Within a time frame of 4 to 13 weeks following infection, the editing efficiency in AAV-infected cells can reach approximately 80%, with no discernible toxicity, even at elevated dosages. In addition, when AAV-delivered circular arRNAs are systematically administered to a humanized mouse model of Hurler syndrome, it rectifies the premature stop codon precisely and restores the functionality of IDUA enzyme encoded by the Hurler causative gene in multiple organs. CONCLUSIONS: These discoveries considerably bolster the prospects of employing AAV-borne circular arRNAs for therapeutic applications and exploratory translational research.

Abnormal chromatin remodeling caused by ARID1A deletion leads to malformation of the dentate gyrus.

ARID1A, an SWI/SNF chromatin-remodeling gene, is commonly mutated in cancer and hypothesized to be a tumor suppressor. Recently, loss-of-function of ARID1A gene has been shown to cause intellectual disability. Here we generate Arid1a conditional knockout mice and investigate Arid1a function in the hippocampus. Disruption of Arid1a in mouse forebrain significantly decreases neural stem/progenitor cells (NSPCs) proliferation and differentiation to neurons within the dentate gyrus (DG), increasing perinatal and postnatal apoptosis, leading to reduced hippocampus size. Moreover, we perform single-cell RNA sequencing (scRNA-seq) to investigate cellular heterogeneity and reveal that Arid1a is necessary for the maintenance of the DG progenitor pool and survival of post-mitotic neurons. Transcriptome and ChIP-seq analysis data demonstrate that ARID1A specifically regulates Prox1 by altering the levels of histone modifications. Overexpression of downstream target Prox1 can rescue proliferation and differentiation defects of NSPCs caused by Arid1a deletion. Overall, our results demonstrate a critical role for Arid1a in the development of the hippocampus and may also provide insight into the genetic basis of intellectual disabilities such as Coffin-Siris syndrome, which is caused by germ-line mutations or microduplication of Arid1a.

Acetate supplementation restores cognitive deficits caused by ARID1A haploinsufficiency in excitatory neurons.

Mutations in AT-rich interactive domain-containing protein 1A (ARID1A) cause Coffin-Siris syndrome (CSS), a rare genetic disorder that results in mild to severe intellectual disabilities. However, the biological role of ARID1A in the brain remains unclear. In this study, we report that the haploinsufficiency of ARID1A in excitatory neurons causes cognitive impairment and defects in hippocampal synaptic transmission and dendritic morphology in mice. Similarly, human embryonic stem cell-derived excitatory neurons with deleted ARID1A exhibit fewer dendritic branches and spines, and abnormal electrophysiological activity. Importantly, supplementation of acetate, an epigenetic metabolite, can ameliorate the morphological and electrophysiological deficits observed in mice with Arid1a haploinsufficiency, as well as in ARID1A-null human excitatory neurons. Mechanistically, transcriptomic and ChIP-seq analyses demonstrate that acetate supplementation can increase the levels of H3K27 acetylation at the promoters of key regulatory genes associated with neural development and synaptic transmission. Collectively, these findings support the essential roles of ARID1A in the excitatory neurons and cognition and suggest that acetate supplementation could be a potential therapeutic intervention for CSS.

Surviving winter on the Qinghai-Tibetan Plateau: Pikas suppress energy demands and exploit yak feces to survive winter.

The Qinghai-Tibetan Plateau, with low precipitation, low oxygen partial pressure, and temperatures routinely dropping below -30 °C in winter, presents several physiological challenges to its fauna. Yet it is home to many endemic mammalian species, including the plateau pika (Ochotona curzoniae). How these small animals that are incapable of hibernation survive the winter is an enigma. Measurements of daily energy expenditure (DEE) using the doubly labeled water method show that pikas suppress their DEE during winter. At the same body weight, pikas in winter expend 29.7% less than in summer, despite ambient temperatures being approximately 25 °C lower. Combined with resting metabolic rates (RMRs), this gives them an exceptionally low metabolic scope in winter (DEE/RMRt = 1.60 ± 0.30; RMRt is resting metabolic rate at thermoneutrality). Using implanted body temperature loggers and filming in the wild, we show that this is achieved by reducing body temperature and physical activity. Thyroid hormone (T3 and T4) measurements indicate this metabolic suppression is probably mediated via the thyroid axis. Winter activity was lower at sites where domestic yak (Bos grunniens) densities were higher. Pikas supplement their food intake at these sites by eating yak feces, demonstrated by direct observation, identification of yak DNA in pika stomach contents, and greater convergence in the yak/pika microbiotas in winter. This interspecific coprophagy allows pikas to thrive where yak are abundant and partially explains why pika densities are higher where domestic yak, their supposed direct competitors for food, are more abundant.

Overexpression of serotonin receptor 5b expression rescues neuronal and behavioral deficits in a mouse model of Kabuki syndrome.

5-hydroxytryptamine receptor 5B (5-HT5B) is a gene coding for a G protein-coupled receptor (GPCR) that plays key roles in several neurodevelopmental disorders. Our previous study showed that disruption of 5-HT5B induced by lysine (K)-specific demethylase 6A (Kdm6a, also known as Utx) conditional knockout (cKO) in mouse hippocampus was associated with cognition deficits underlying intellectual disability in Kabuki syndrome (KS), a rare disease associated with multiple congenital and developmental abnormalities, especially neurobehavioral features. Here we show that Utx knockout (KO) in cultured hippocampal neurons leads to impaired neuronal excitability and calcium homeostasis. In addition, we show that 5-HT5B overexpression reverses dysregulation of neuronal excitability, intracellular calcium homeostasis, and long-term potentiation (LTP) in cultured Utx KO hippocampal neurons and hippocampal slices. More importantly, overexpression of 5-HT5B in Utx cKO mice results in reversal of abnormal anxiety-like behaviors and impaired spatial memory ability. Our findings therefore indicate that 5-HT5B, as a downstream target of Utx, functions to modulate electrophysiological outcomes, thereby affecting behavioral activities in KS mouse models.

Partial loss of psychiatric risk gene Mir137 in mice causes repetitive behavior and impairs sociability and learning via increased Pde10a.

Genetic analyses have linked microRNA-137 (MIR137) to neuropsychiatric disorders, including schizophrenia and autism spectrum disorder. miR-137 plays important roles in neurogenesis and neuronal maturation, but the impact of miR-137 loss-of-function in vivo remains unclear. Here we show the complete loss of miR-137 in the mouse germline knockout or nervous system knockout (cKO) leads to postnatal lethality, while heterozygous germline knockout and cKO mice remain viable. Partial loss of miR-137 in heterozygous cKO mice results in dysregulated synaptic plasticity, repetitive behavior, and impaired learning and social behavior. Transcriptomic and proteomic analyses revealed that the miR-137 mRNA target, phosphodiesterase 10a (Pde10a), is elevated in heterozygous knockout mice. Treatment with the Pde10a inhibitor papaverine or knockdown of Pde10a ameliorates the deficits observed in the heterozygous cKO mice. Collectively, our results suggest that MIR137 plays essential roles in postnatal neurodevelopment and that dysregulation of miR-137 potentially contributes to neuropsychiatric disorders in humans.

Wnt3 and Gata4 regulate axon regeneration in adult mouse DRG neurons.

Neurons in the adult central nervous system (CNS) have a poor intrinsic axon growth potential after injury, but the underlying mechanisms are largely unknown. Wingless-related mouse mammary tumor virus integration site (WNT) family members regulate neural stem cell proliferation, axon tract and forebrain development in the nervous system. Here we report that Wnt3 is an important modulator of axon regeneration. Downregulation or overexpression of Wnt3 in adult dorsal root ganglion (DRG) neurons enhances or inhibits their axon regeneration ability respectively in vitro and in vivo. Especially, we show that Wnt3 modulates axon regeneration by repressing mRNA translation of the important transcription factor Gata4 via binding to the three prime untranslated region (3'UTR). Downregulation of Gata4 could restore the phenotype exhibited by Wnt3 downregulation in DRG neurons. Taken together, these data indicate that Wnt3 is a key intrinsic regulator of axon growth ability of the nervous system.

Polycomb protein family member CBX7 regulates intrinsic axon growth and regeneration.

Neurons in the central nervous system (CNS) lose their intrinsic ability and fail to regenerate, but the underlying mechanisms are largely unknown. Polycomb group (PcG) proteins, which include PRC1 and PRC2 complexes function as gene repressors and are involved in many biological processes. Here we report that PRC1 components (polycomb chromobox (CBX) 2, 7, and 8) are novel regulators of axon growth and regeneration. Especially, knockdown of CBX7 in either embryonic cortical neurons or adult dorsal root ganglion (DRG) neurons enhances their axon growth ability. Two important transcription factors GATA4 and SOX11 are functional downstream targets of CBX7 in controlling axon regeneration. Moreover, knockdown of GATA4 or SOX11 in cultured DRG neurons inhibits axon regeneration response from CBX7 downregulation in DRG neurons. These findings suggest that targeting CBX signaling pathway may be a novel approach for promoting the intrinsic regenerative capacity of damaged CNS neurons.

The Histone H3K27 Demethylase UTX Regulates Synaptic Plasticity and Cognitive Behaviors in Mice.

Histone demethylase UTX mediates removal of repressive trimethylation of histone H3 lysine 27 (H3K27me3) to establish a mechanistic switch to activate large sets of genes. Mutation of Utx has recently been shown to be associated with Kabuki syndrome, a rare congenital anomaly syndrome with dementia. However, its biological function in the brain is largely unknown. Here, we observe that deletion of Utx results in increased anxiety-like behaviors and impaired spatial learning and memory in mice. Loss of Utx in the hippocampus leads to reduced long-term potentiation and amplitude of miniature excitatory postsynaptic current, aberrant dendrite development and defective synapse formation. Transcriptional profiling reveals that Utx regulates a subset of genes that are involved in the regulation of dendritic morphology, synaptic transmission, and cognition. Specifically, Utx deletion disrupts expression of neurotransmitter 5-hydroxytryptamine receptor 5B (Htr5b). Restoration of Htr5b expression in newborn hippocampal neurons rescues the defects of neuronal morphology by Utx ablation. Therefore, we provide evidence that Utx plays a critical role in modulating synaptic transmission and cognitive behaviors. Utx cKO mouse models like ours provide a valuable means to study the underlying mechanisms of the etiology of Kabuki syndrome.

MiR-203 Interplays with Polycomb Repressive Complexes to Regulate the Proliferation of Neural Stem/Progenitor Cells.

The polycomb repressive complexes 1 (PRC1) and 2 (PRC2) are two distinct polycomb group (PcG) proteins that maintain the stable silencing of specific sets of genes through chromatin modifications. Although the PRC2 component EZH2 has been known as an epigenetic regulator in promoting the proliferation of neural stem/progenitor cells (NSPCs), the regulatory network that controls this process remains largely unknown. Here we show that miR-203 is repressed by EZH2 in both embryonic and adult NSPCs. MiR-203 negatively regulates the proliferation of NSPCs. One of PRC1 components, Bmi1, is a downstream target of miR-203 in NSPCs. Conditional knockout of Ezh2 results in decreased proliferation ability of both embryonic and adult NSPCs. Meanwhile, ectopic overexpression of BMI1 rescues the proliferation defects exhibited by miR-203 overexpression or EZH2 deficiency in NSPCs. Therefore, this study provides evidence for coordinated function of the EZH2-miR-203-BMI1 regulatory axis that regulates the proliferation of NSPCs.

Leptin regulates energy intake but fails to facilitate hibernation in fattening Daurian ground squirrels (Spermophilus dauricus).

Body fat storage before hibernation affects the timing of immergence in Daurian ground squirrels (Spermophilus dauricus). Leptin is an adipose signal and plays vital role in energy homeostasis mainly by action in brain. To test the hypothesis that leptin plays a role in facilitating the process of hibernation, squirrels were administrated with recombinant murine leptin (1µg/day) through intracerebroventricular (ICV) injection for 12 days during fattening. From day 7 to 12, animals were moved into a cold room (5±1°C) with constant darkness which functioned as hibernaculum. Energy intake, body mass and core body temperature (Tb) were continuously monitored throughout the course of experiment. Resting metabolic rate (RMR) was measured under both warm and cold conditions. At the end of leptin administration, we measured the serum concentration of hormones related to energy regulation, mRNA expression of hypothalamic neuropeptides and uncoupling protein 1 (UCP1) levels in brown adipose tissue (BAT). Our results showed that during leptin administration, the cumulative food intake and increase of body mass were suppressed while Tb and RMR were unaltered. The proportion of torpid squirrels was not different between two groups. At the end of leptin administration, the expressions of hypothalamic neuropeptide Y and agouti gene-related protein were suppressed. There were no differences in UCP1 mRNA expression or protein content in BAT between groups. Our data suggest that leptin can affect energy intake via hypothalamic neuropeptides, but is not involved in the initiation of hibernation in fattening Daurian ground squirrels.

Intracerebroventricular administration of leptin increase physical activity but has no effect on thermogenesis in cold-acclimated rats.

Most small homotherms display low leptin level in response to chronic cold exposure. Cold-induced hypoleptinemia was proved to induce hyperphagia. However, it is still not clear whether hypoleptinemia regulates energy expenditure in cold condition. We try to answer this question in chronic cold-acclimated rats. Results showed that 5-day intracerebroventricular(ICV) infusion of leptin (5 µg/day) had no effects on basal and adaptive thermogenesis and uncoupling protein 1 expression. Physical activity was increased by leptin treatment. We further determined whether ghrelin could reverse the increasing effect of leptin on physical activity. Coadministration of ghrelin (1.2 µg/day) completely reversed the effect of leptin on physical activity. Collectively, this study indicated the regulation of leptin on energy expenditure during cold acclimation may be mainly mediated by physical activity but not by thermogenesis. Our study outlined behavioral role of leptin during the adaptation to cold, which adds some new knowledge to promote our understanding of cold-induced metabolic adaptation.

Effects of leptin infusion during peak lactation on food intake, body composition, litter growth, and maternal neuroendocrine status in female Brandt's voles (Lasiopodomys brandtii).

During lactation, female small mammals frequently reduce their fat reserves to very low levels. The function of this reduction is unclear, as calculations suggest that the contribution of the withdrawn energy from fat to the total energy balance of lactation is trivial. An alternative hypothesis is that reducing fat leads to a reduction in circulating adipokines, such as leptin, that play a role in stimulating the hyperphagia of lactation. We investigated the role of circulating leptin in lactation by repleting leptin levels using miniosmotic pumps during the last 7 days of lactation in Brandt's voles (Lasiopodomys brandtii), a model small wild mammal we have extensively studied in the context of lactation energy demands. Repletion of leptin resulted in a dose-dependent reduction of body mass and food intake in lactating voles. Comparisons to nonreproducing individuals suggests that the reduced leptin in lactation, due to reduced fat stores, may account for ∼16% of the lactational hyperphagia. Reduced leptin in lactation may, in part, cause lactational hyperphagia via stimulatory effects on hypothalamic orexigenic neuropeptides (neuropeptide Y and agouti-related peptide) and inhibition of the anorexigenic neuropeptide (proopiomelanocortin). These effects were reversed by the experimental repletion of leptin. There was no significant effect of leptin treatment on daily energy expenditure, milk production or pup growth, but leptin repletion did result in a reversal of the suppression of uncoupling protein-1 levels in brown adipose tissue, indicating an additional role for reducing body fat and leptin during peak lacation.

Role of hypoleptinemia during cold adaptation in Brandt's voles (Lasiopodomys brandtii).

Brandt's voles Lasiopodomys brandtii exhibit large increases in nonshivering thermogenesis to cope with chronic cold exposure, resulting in compensatory hyperphagia and fat mobilization. These physiological events are accompanied by a remarkable reduction in serum leptin levels. However, the role of hypoleptinemia in cold adaptation in this species is still unknown. In the present study, we tested the hypothesis that hypoleptinemia contributes to increases in food intake and brown adipose tissue (BAT) thermogenesis by modifying hypothalamic neuropeptides in cold-exposed Brandt's voles. Adult male voles were transferred to 5 degrees C for 28 days. Accompanied by a decrease in serum leptin levels, hypothalamic agouti-related protein (AgRP) mRNA levels were significantly increased, but there were no changes in the long form of leptin receptor (Ob-Rb), suppressor of cytokine signaling 3 (SOCS3), neuropeptide Y (NPY) mRNA, proopiomelanocortin (POMC), and cocaine- and amphetamine-regulated peptide (CART) mRNA levels in the hypothalamus. When cold-exposed voles were returned to warm (23 degrees C) for 28 days, body mass, food intake, serum leptin, and AgRP mRNA were restored to control levels. Leptin administration in cold-exposed voles decreased food intake as well as hypothalamic AgRP mRNA levels. There were no significant effects of leptin administration on hypothalamic Ob-Rb, SOCS3, NPY, POMC, CART mRNA, and uncoupling protein 1 levels under cold conditions. These results suggest that hypoleptinemia partially contributes to cold-induced hyperphagia, which might involve the elevation of hypothalamic AgRP gene expression.