To summarize recent advances in our conceptual understanding of the epigenetic mechanisms of DNA repair contributing to autism spectrum disorder (ASD). Large-scale genetic studies indicate that genes involved in DNA repair contribute to some cases of ASD, and smaller scale studies have reported increased DNA damage in peripheral tissues of both individuals with ASD and their parents. ASD-associated chromatin remodelers, thought to contribute to ASD by regulating gene expression, also facilitate DNA repair. Recent evidence has provided some mechanistic insight into the function of ASD-associated genes in the epigenetic regulation of DNA repair. Importantly, the disruption of DNA repair is a leading candidate to explain the emerging connection between ASD and neurodegenerative disease and may also provide insight into sex differences in ASD. Here, we highlight recent evidence that dysfunction of the DNA repair machinery is an overlooked mechanism underlying ASD, one which requires further systematic study for the benefit of individuals with ASD and their support systems.

Sleep is essential for maintaining cognitive, emotional, metabolic, and immune functions. Although research on sleep homeostasis is traditionally neuron-centric, increasing evidence indicates non-neuronal cells also play critical roles. In this study, we performed transcriptomic analyses of non-neuronal nuclei from the hippocampus and prefrontal cortex of mice subjected to acute sleep deprivation (SD). We found that acute SD induces robust, cell-type- and region-specific transcriptional reprogramming in astrocytes and oligodendrocytes. The most pronounced changes occurred in astrocytes, including downregulation of cholesterol biosynthesis genes in both brain regions, accompanied by region-specific and opposing regulation of genes involved in mitochondrial function and neurodegeneration-related pathways. Notably, genes associated with primary cilia were selectively induced in cortical astrocytes. In oligodendrocytes, acute SD led to downregulation of genes encoding cell-adhesion molecules. Together, these findings provide molecular evidence that non-neuronal cells actively contribute to sleep regulation and suggest novel potential mechanisms that broaden our understanding of sleep homeostasis.

The complement system is a major component of the innate immune system and plays an important role in immune surveillance. Recent research has demonstrated that the complement system also plays pivotal roles in brain development, and dysregulation of complement is involved in neurodegenerative and neuropsychiatric disorders. However, the mechanisms by which the complement system contributes to neurodevelopmental disorders (NDDs) remain poorly understood. In this study, we find that the expression of complement components, including complement C3a receptor (C3aR), is upregulated in the striatum of mice modeling the 16p11.2 hemideletion (16p11.2 del). 16p11.2 del is among the most common copy number variations associated with NDDs including attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and intellectual disability (ID). Pharmacological inhibition of C3aR reduces hyperactivity in 16p11.2 del mice, suggesting that potentiated complement signaling contributes to NDD-relevant behavioral changes. Several cytokines are also upregulated in 16p11.2 del mice striatum, a key neural substrate for locomotor behavior, where we also observe morphological and transcriptional indications of reactive microglia. C3aR inhibition suppresses the elevated expression of interferon-stimulated genes in the 16p11.2 del striatum suggesting that crosstalk between complement and interferon signaling may influence microglial state and hyperactivity in 16p11.2 del mice. Collectively, these data indicate that increased expression of the complement system exacerbates hyperactive behavior and is associated with microglial responses in the striatum of 16p11.2 del mice. Our results suggest that inhibition of the complement system may be an effective strategy to ameliorate NDD symptoms resulting from 16p11.2 hemideletion including those associated with ADHD.

Biological sex shapes the manifestation and progression of neurodevelopmental disorders (NDDs), however, the underlying mechanisms remains unclear. Hemideletion of the 16p11.2 region (16p11.2 del/+) is associated with NDDs, and 16p11.2 del/+ mice exhibit sex-specific, striatum-related phenotypes relevant to NDDs. In this study, using snRNA-seq, we identify cell type- and sex-specific transcriptomic changes in D1- and D2-spiny projection neurons (SPNs), with greater impact in males. Fiber photometry recordings reveal reduced neuronal activity in the dorsal striatum of 16p11.2 del/+ males, but not females, with D2-SPNs identified as the primary contributors to this reduction. Behaviorally, we utilize conditional genetic approaches and find that selective hemideletion in D2-SPNs, but not D1-SPNs, induces male-specific hyperactivity, whereas cortical hemideletion increases hyperactivity in both sexes. Thus, a locus linked to NDDs acts in distinct striatal circuits, selectively impacting behavior in a sex- and cell type-specific manner. This study demonstrates that 16p11.2 hemideletion, a genetic deletion linked to neurodevelopmental disorders, affects male and female mice differently, altering specific striatal neuronal circuits and driving sex-specific behavioral changes.

Neurons utilize RNA interference in the reversible translational repression of synaptically localized mRNAs, enabling rapid translation in response to synaptic activity. Two evolutionarily conserved proteins, Translin and Trax, form an RNase complex that processes miRNAs, tRNAs, and siRNAs. To determine the specific role of the RNase activity of this complex in brain function, we employed a mouse line harboring a point mutation in Trax (E126A) that renders the Translin/Trax RNase inactive. At the molecular level, we found alterations in the levels of multiple small RNAs, including miRNAs, tsRNAs, and substantial downregulation of gene expression at the mRNA level in the hippocampus of TraxE126A mice. At the synaptic level, TraxE126A mice exhibit deficits in specific forms of long-term hippocampal synaptic plasticity. At the behavioral level, TraxE126A mice display impaired long-term spatial memory and altered open-field behavior. These studies reveal the functional role of Translin/Trax RNase in the mammalian brain.

Alzheimer's disease-related dementia (ADRD) is a progressive neurodegenerative condition characterised by cognitive decline and overlapping pathological features. Despite advances in diagnostic tools and therapies, challenges persist due to limited efficacy, high costs, and the complexity of ADRD pathophysiology. Evidence suggests that ADRD arises from a lifelong interplay of genetic, lifestyle, and environmental factors, with recent findings indicating potential neurodevelopmental origins. Proteins implicated in neurodegeneration, such as amyloid and tau, may play critical roles in early nervous system development, whereas disruptions during critical periods, such as adolescence, may increase later-life susceptibility to ADRD. The global prevalence of ADRD is projected to reach 153 million by 2050, emphasising the urgent need for prevention strategies in addition to therapeutic interventions. Reframing ADRD as a neurodevelopmental condition with a delayed onset may provide alternative insights into its aetiology, paving the way for alternative innovative therapeutic and preventive approaches.

Sleep supports a variety of physiological processes, ranging from metabolic to immune system homeostasis, and plays a critical role in cognition and memory. A brief period of sleep loss impairs memory, particularly hippocampus-dependent memories, alters molecular signaling, and synaptic plasticity in the hippocampus. Studies have shown that sleep deprivation (SD) alters neuronal activation as indicated by broad changes in gene expression signatures and by the increased expression of c-Fos, an immediate early gene that functions as a molecular marker of neuronal activity. In the present study, we examined hippocampal subregion-specific c-Fos induction patterns via immunohistochemical staining. We found that CA1 pyramidal neurons exhibit the most robust c-Fos induction after SD. Using an activity-driven ribosomal tagging system (c-Fos-RiboTag) and a repeated SD model, we labeled sleep deprivation activated CA1 neurons and observed a population of excitatory neurons in area CA1 that are reactivated by repeated SD. Using the c-Fos-RiboTag system that enables the isolation of ribosomes with attached mRNA from labeled neurons, we performed fosTRAP-seq and identified activity-dependent gene expression changes in c-Fos+ CA1 neurons. Our results revealed that synapse organization, protein dephosphorylation, cellular response to endogenous stimulus (such as insulin) are upregulated, whereas mRNA processing and splicing are downregulated. In summary, our study provides a detailed characterization of hippocampal neuronal activation following SD and identifies a subset of CA1 pyramidal neurons that are selectively reactivated by repeated SD. This SD-sensitive neuronal population enables investigation of molecular changes in neurons specifically impacted by sleep loss and suggests a potential connection between acute and chronic sleep loss at the cellular and molecular levels.

The cerebellum has recently been recognized for its role in non-motor functions, including classical fear conditioning. However, the molecular mechanisms underlying non-motor learning and memory remain largely unknown. Here, we investigate the transcriptional changes in the cerebellum associated with auditory fear conditioning. Spatial transcriptomic analysis revealed elevated expression of immediate early genes in the deep cerebellar nuclei (DCN), an output region of the cerebellum, following fear conditioning (CD) and tone retrieval (TN), suggesting that the DCN may contribute to fear memory processing. To further elucidate transcriptional changes in specific DCN cell types involved in fear processing, we performed single-nucleus RNA sequencing and identified prominent transcriptional changes in Kit+ inhibitory neurons. Collectively, our findings highlight region- and cell-type-specific molecular adaptations in the cerebellum, providing insights into its contribution to non-motor learning.

Hippocampal theta oscillations coordinate computations underlying learning and memory. The frequency of theta varies with factors such as locomotion and anxiety, but the effect of an animal's sleep history on theta frequency remains unknown. Using long-duration CA1 recordings in rats, we found that awake theta frequency progressively decreases following sleep but remains elevated during sleep deprivation. These changes were not accounted for by movement but were predicted by the proportion of sleep in the preceding 30 min. In contrast, theta frequency remained relatively stable during rapid eye movement sleep. In aged rats, sleep-dependent frequency changes were less pronounced, likely due to sleep fragmentation. Systemic administration of the phosphodiesterase inhibitor rolipram during sleep deprivation reduced theta frequency, mimicking natural sleep. Collectively, these findings reveal a robust influence of sleep history on hippocampal theta frequency, with potential implications for understanding memory function, age-related sleep disruptions, and the potential for pharmacological interventions.

Histone post-translational modifications (PTMs), particularly lysine acetylation (Kac), are critical epigenetic regulators of gene transcription underlying long-term memory consolidation. Beyond Kac, several other non-acetyl acylations have been identified, but their role in memory consolidation remains unknown. Here, we demonstrate histone lysine crotonylation (Kcr) as a key molecular switch of hippocampal memory storage. Spatial memory training induces distinct spatiotemporal patterns of Kcr induction in the dorsal hippocampus of mice. Through genetic and pharmacological manipulations, we show that reducing hippocampal Kcr levels impairs long-term memory, while increasing Kcr enhances memory. Utilizing single-nuclei multiomics, we delineate that Kcr enhancement during memory consolidation activates transcription of genes involved in neurotransmission and synaptic function within hippocampal excitatory neurons. Cell-cell communication analysis further inferred that Kcr enhancement strengthens glutamatergic signaling within principal hippocampal neurons. Our findings establish Kcr as a novel epigenetic mechanism governing memory consolidation and provide a foundation for therapeutic strategies targeting memory-related disorders.

The dysregulation of sleep-wake patterns that occurs during aging is well documented and coincides with changes in intracellular signaling pathways that regulate sleep, such as the calcium/calmodulin-dependent protein kinase (CaMKII)/cyclic-AMP response element-binding protein (CREB) pathway. However, much less is known about the relationship between other CREB-activating members of the CaMK family, such as calcium/calmodulin-dependent protein kinase IV (CaMKIV), and the regulation of sleep. Using 2- to 4-month-old (young adult) and 22- to 24-month-old (aged) male and female CaMKIV-overexpressing (CaMKIV-OE) mice, we observed that overexpression of CaMKIV in the forebrain decreased wakefulness and increased the amount of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep in aged male mice, but not young adult male mice, in comparison to age- and sex-matched controls. Conversely, female mice overexpressing CaMKIV displayed no significant differences in the percentage of time spent in each vigilance state compared to their wild-type counterparts, regardless of age. While CaMKIV overexpression also led to more sleep-wake fragmentation in young adult and aged male mice, aged female mice displayed more consolidated NREM sleep. Overall, our results suggest that CaMKIV overexpression enhances sleep in aged male mice, and differentially affects sleep-wake architecture based on sex and age, providing insights into the potential mechanism by which CaMKIV overexpression enhances memory.

Excessive glutamate receptor activation during brain pathologies causes varicose dendritic swelling, also known as “dendritic beading”, yet its impact on developing brain circuits is poorly understood. Using field electrophysiology and two-photon imaging in awake, behaving mice and acute brain slices (P11–19), we found that severe and recurrent seizure-like activity (induced by NMDA and 4-aminopyridine) resulted in widespread, long-lasting dendritic beading and spine loss in cortical and hippocampal neurons, with localization patterns distinct from those described in adults. Beads showed persistently high calcium levels and stopped the spread of dendritic calcium signals. Dendritic beads suppressed hippocampal evoked field potentials, followed by only partial recovery, and reduced hippocampal long-term potentiation. Clinically used hyperosmotic treatments (mannitol or hypertonic saline) reduced seizure-induced beading and restored dendritic signal propagation. These findings suggest that seizure-induced dendritic beading disrupts circuit function and synaptic plasticity and may contribute to cognitive deficits after early-life seizures.

Alcohol use disorder (AUD) is characterized by pathological motivation to consume alcohol and cognitive inflexibility, leading to excessive alcohol seeking and use. In this study, we investigated the molecular correlates of impaired extinction of alcohol seeking during forced abstinence using a mouse model of AUD in the automated IntelliCage social system. This model distinguished AUD-prone and AUD-resistant animals based on the presence of ≥2 or <2 criteria of AUD, respectively. We used RNA sequencing to identify genes differentially expressed in the hippocampus, a brain region implicated in alcohol motivation, seeking during abstinence, and cognitive inflexibility. Our findings revealed differences in the hippocampal genes linked to the actin cytoskeleton and synaptic function, including cofilin (Cfl), and impaired synaptic transmission in the molecular layer of the hippocampal dentate gyrus (ML-DG) in ≥2 criteria mice as compared to <2 crit animals. To complement this data, we conducted local genetic manipulations in DG. Overexpression of Cfl in the polymorphic layer of the hippocampal dentate gyrus (PoDG) inhibited ML-DG synapses, increased motivation to seek alcohol and sucrose rewards, impaired extinction of seeking, and decreased reward consumption during relapse. Reducing Cfl levels had opposite effects. We also identified three SNPs in the human CFL1 gene (rs369270402, rs2376005, rs36124259) associated with increased AUD risk and found CFL1 mRNA blood levels correlated with alcohol-related hospital admissions. Overall, our study uncovers a novel mechanism linking hippocampal Cfl expression with AUD phenotypes and identifies CFL1 polymorphisms as AUD risk factor in humans.

Sleep deprivation (SD) impairs long-term memory, but the molecular mechanisms underlying the impact of sleep loss on memory are poorly understood. Molecular changes driven by SD have thus far focused on transcription and translation. Long non-coding RNAs (lncRNAs), a class of regulatory RNAs, have recently been recognized as an important player in memory research. However, it remains unclear how sleep deprivation modulates the expression of lncRNAs or their targets to lead to memory impairment. In this study, we explored the role of lncRNAs in the disruption of spatial memory caused by SD. We examined a set of synapse-associated lncRNAs that were identified through a transcriptome analysis after SD. Among them, we discovered that the lncRNA Cerox1 is downregulated in dorsal hippocampus following SD, and its levels recover after 2.5 hours of rebound sleep. Sleep is critical for the regulation of metabolism and sleep loss impairs mitochondrial function. Both sleep deprivation and Cerox1 knockdown were found to reduce complex I activity of the mitochondrial electron transport chain. This reduction of complex I activity is linked to the decrease in expression of a subset of complex I subunits including Ndufs1, Ndufs3, Ndufa3 and Ndufs6. Overexpression of Cerox1 has the opposite effect, leading to increased complex I activity. Sleep deprivation reduced ATP levels in the dorsal hippocampus, while Cerox1 overexpression restored it. SD disrupted memory consolidation, and this impairment was rescued when Cerox1 was overexpressed. Cerox1 transcript contains multiple miRNA binding sites that regulate the activity of the lncRNA. Notably, overexpression of Cerox1 transcript lacking miRNA binding sites did not rescue the memory deficit caused by SD. Our findings demonstrated that the impairment of memory consolidation after SD is linked to lncRNA-mediated control of mitochondrial electron transport chain activity essential for sustaining energy requirements.

Neurons utilize RNA interference in the reversible translational repression of synaptically localized mRNAs, enabling rapid translation in response to synaptic activity. Two evolutionarily conserved proteins, Translin and Trax, form an RNase complex which processes miRNAs, tRNAs and siRNAs. To determine the specific role of the RNase activity of this complex in brain function, we employed a mouse line harboring a point mutation in Trax (E126A) that renders the Translin/Trax RNase inactive. At the molecular level, we found alterations in the levels of multiple small RNAs including miRNAs, tsRNAs and substantial downregulation of gene expression at the mRNA level in the hippocampus of TraxE126A mice. At the synaptic level, TraxE126A mice exhibit deficits in specific forms of long-term hippocampal synaptic plasticity. At the behavioral level, TraxE126A mice display impaired long-term spatial memory and altered openfield and acoustic-startle behavior. These studies reveal the functional role of Translin/Trax RNase in the mammalian brain.

Biological sex shapes the manifestation and progression of neurodevelopmental disorders (NDDs). These disorders often demonstrate male-specific vulnerabilities; however, the identification of underlying mechanisms remains a significant challenge in the field. Hemideletion of the 16p11.2 region (16p11.2 del/+) is associated with NDDs, and mice modeling 16p11.2 del/+ exhibit sex-specific striatum-related phenotypes relevant to NDDs. Striatal circuits, crucial for locomotor control, consist of two distinct pathways: the direct and indirect pathways originating from D1 dopamine receptor (D1R) and D2 dopamine receptor (D2R) expressing spiny projection neurons (SPNs), respectively. In this study, we define the impact of 16p11.2 del/+ on striatal circuits in male and female mice. Using snRNA-seq, we identify sex- and cell type-specific transcriptomic changes in the D1- and D2-SPNs of 16p11.2 del/+ mice, indicating distinct transcriptomic signatures in D1-SPNs and D2-SPNs in males and females, with a ∼5-fold greater impact in males. Further pathway analysis reveals differential gene expression changes in 16p11.2 del/+ male mice linked to synaptic plasticity in D1- and D2-SPNs and GABA signaling pathway changes in D1-SPNs. Consistent with our snRNA-seq study revealing changes in GABA signaling pathways, we observe distinct changes in miniature inhibitory postsynaptic currents (mIPSCs) in D1- and D2-SPNs from 16p11.2 del/+ male mice. Behaviorally, we utilize conditional genetic approaches to introduce the hemideletion selectively in either D1- or D2-SPNs and find that conditional hemideletion of genes in the 16p11.2 region in D2-SPNs causes hyperactivity in male mice, but hemideletion in D1-SPNs does not. Within the striatum, hemideletion of genes in D2-SPNs in the dorsal lateral striatum leads to hyperactivity in males, demonstrating the importance of this striatal region. Interestingly, conditional 16p11.2 del/+ within the cortex drives hyperactivity in both sexes. Our work reveals that a locus linked to NDDs acts in different striatal circuits, selectively impacting behavior in a sex- and cell type-specific manner, providing new insight into male vulnerability for NDDs. Highlights:- 16p11.2 hemideletion (16p11.2 del/+) induces sex- and cell type-specific transcriptomic signatures in spiny projection neurons (SPNs). - Transcriptomic changes in GABA signaling in D1-SPNs align with changes in inhibitory synapse function. - 16p11.2 del/+ in D2-SPNs causes hyperactivity in males but not females. - 16p11.2 del/+ in D2-SPNs in the dorsal lateral striatum drives hyperactivity in males. - 16p11.2 del/+ in cortex drives hyperactivity in both sexes. Graphic abstract:

Direct electrical stimulation has been used for decades as a gold standard clinical tool to map cognitive function in neurosurgery patients1-8. However, the molecular impact of electrical stimulation in the human brain is unknown. Here, using state-of-the-art transcriptomic and epigenomic sequencing techniques, we define the molecular changes in bulk tissue and at the single-cell level in the human cerebral cortex following direct electrical stimulation of the anterior temporal lobe in patients undergoing neurosurgery. Direct electrical stimulation surprisingly had a robust and consistent impact on the expression of genes related to microglia-specific cytokine activity, an effect that was replicated in mice. Using a newly developed deep learning computational tool, we further demonstrate cell type-specific molecular activation, which underscores the effects of electrical stimulation on gene expression in microglia. Taken together, this work challenges the notion that the immediate impact of electrical stimulation commonly used in the clinic has a primary effect on neuronal gene expression and reveals that microglia robustly respond to electrical stimulation, thus enabling these non-neuronal cells to sculpt and shape the activity of neuronal circuits in the human brain.

Protein kinase A (PKA) neuronal function is controlled by the interaction of a regulatory (R) subunit dimer with two catalytic subunits. Recently, the L50R variant in the gene encoding the RI beta subunit was identified in individuals with a novel neurodegenerative disease. However, the mechanisms driving the disease phenotype remained unknown.In this study, we generated a mouse model carrying the RI beta-L50R mutation to replicate the human disease phenotype and study its progression with age. We examined post-mortem brains of affected individuals as well as live cell cultures. Employing biochemical assays, immunohistochemistry and behavioural assessments, we investigated the impact of the mutation on PKA complex assembly, protein aggregation and neuronal degeneration.We reveal that RI beta is an aggregation-prone protein that progressively accumulates in wildtype and Alzheimer's mouse models with age, while aggregation is accelerated in the RI beta-L50R mouse model. We define RI beta-L50R as a causal mutation driving an age-dependent behavioural and disease phenotype in human and mouse models. Mechanistically, this mutation disrupts RI beta dimerization, leading to aggregation of its monomers. Intriguingly, interaction with the catalytic subunit protects the RI beta-L50R from self-aggregating, in a dose-dependent manner. Furthermore, cAMP signaling induces RI beta-L50R aggregation.The pathophysiological mechanism elucidated here for a newly recognized neurodegenerative disease, in which protein aggregation is the result of disrupted homodimerization, sheds light on a remarkably under-appreciated but potentially common mechanism across several neurodegenerative diseases. The PRKAR1B gene encodes a regulatory subunit of protein kinase A. Benjamin-Zukerman et al. generate a mouse model harbouring a variant in PRKAR1B previously detected in patients with a rare neurodegenerative disease, and identify the molecular mechanisms driving the disease phenotype.

Autism spectrum disorder (ASD) is an increasingly prevalent neurodevelopmental condition characterized by social and communication deficits as well as patterns of restricted, repetitive behavior. Abnormal brain development has long been postulated to underlie ASD, but longitudinal studies aimed at understanding the developmental course of the disorder have been limited. More recently, abnormal development of the striatum in ASD has become an area of interest in research, partially due to overlap of striatal functions and deficit areas in ASD, as well as the critical role of the striatum in early development, when ASD is first detected. Focusing on the dorsal striatum and the associated symptom domain of restricted, repetitive behavior, we review the current literature on dorsal striatal abnormalities in ASD, including studies on functional connectivity, morphometry, and cellular and molecular substrates. We highlight that observed striatal abnormalities in ASD are often dynamic across development, displaying disrupted developmental trajectories. Important findings include an abnormal trajectory of increasing corticostriatal functional connectivity with age and increased striatal growth during childhood in ASD. We end by discussing striatal findings from animal models of ASD. In sum, the studies reviewed here demonstrate a key role for developmental disruptions of the dorsal striatum in the pathogenesis of ASD. Directing attention toward these findings will improve our understanding of ASD and of how associated deficits may be better addressed.