Transcriptome analysis identifies an ASD-Like phenotype in oligodendrocytes and microglia from C58/J amygdala that is dependent on sex and sociability.

BACKGROUND: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. METHODS: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. RESULTS: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using Bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. LIMITATIONS: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its' potential as an ASD therapeutic. CONCLUSIONS: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

Evaluation of neurological behavior alterations and metabolic changes in mice under chronic glyphosate exposure.

Glyphosate is a widely used active ingredient in agricultural herbicides, inhibiting the biosynthesis of aromatic amino acids in plants by targeting their shikimate pathway. Our gut microbiota also facilitates the shikimate pathway, making it a vulnerable target when encountering glyphosate. Dysbiosis in the gut microbiota may impair the gut-brain axis, bringing neurological outcomes. To evaluate the neurotoxicity and biochemical changes attributed to glyphosate, we exposed mice with the reference dose (RfD) set by the U.S. EPA (1.75 mg/Kg-BW/day) and its hundred-time-equivalence (175 mg/Kg-BW/day) chronically via drinking water, then compared a series of neurobehaviors and their fecal/serum metabolomic profile against the non-exposed vehicles (n = 10/dosing group). There was little alteration in the neurobehavior, including motor activities, social approach, and conditioned fear, under glyphosate exposure. Metabolomic differences attributed to glyphosate were observed in the feces, corresponding to 68 and 29 identified metabolites with dysregulation in the higher and lower dose groups, respectively, compared to the vehicle-control. There were less alterations observed in the serum metabolome. Under 175 mg/Kg-BW/day of glyphosate exposure, the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were reduced in the feces but not in the serum of mice. We further focused on how tryptophan metabolism was dysregulated based on the pathway analysis, and identified the indole-derivatives were more altered compared to the serotonin and kynurenine derivatives. Together, we obtained a three-dimensional data set that records neurobehavioral, fecal metabolic, and serum biomolecular dynamics caused by glyphosate exposure at two different doses. Our data showed that even under the high dose of glyphosate irrelevant to human exposure, there were little evidence that supported the impairment of the gut-brain axis.

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

Developmental Regulation of Basket Interneuron Synapses and Behavior through NCAM in Mouse Prefrontal Cortex.

Parvalbumin (PV)-expressing basket interneurons in the prefrontal cortex (PFC) regulate pyramidal cell firing, synchrony, and network oscillations. Yet, it is unclear how their perisomatic inputs to pyramidal neurons are integrated into neural circuitry and adjusted postnatally. Neural cell adhesion molecule NCAM is expressed in a variety of cells in the PFC and cooperates with EphrinA/EphAs to regulate inhibitory synapse density. Here, analysis of a novel parvalbumin (PV)-Cre: NCAM F/F mouse mutant revealed that NCAM functions presynaptically in PV+ basket interneurons to regulate postnatal elimination of perisomatic synapses. Mutant mice exhibited an increased density of PV+ perisomatic puncta in PFC layer 2/3, while live imaging in mutant brain slices revealed fewer puncta that were dynamically eliminated. Furthermore, EphrinA5-induced growth cone collapse in PV+ interneurons in culture depended on NCAM expression. Electrophysiological recording from layer 2/3 pyramidal cells in mutant PFC slices showed a slower rise time of inhibitory synaptic currents. PV-Cre: NCAM F/F mice exhibited impairments in working memory and social behavior that may be impacted by altered PFC circuitry. These findings suggest that the density of perisomatic synapses of PV+ basket interneurons is regulated postnatally by NCAM, likely through EphrinA-dependent elimination, which is important for appropriate PFC network function and behavior.

Common Pathophysiology in Multiple Mouse Models of Pitt-Hopkins Syndrome.

Mutations or deletions of the transcription factor TCF4 are linked to Pitt-Hopkins syndrome (PTHS) and schizophrenia, suggesting that the precise pathogenic mutations dictate cellular, synaptic, and behavioral consequences. Here, we generated two novel mouse models of PTHS, one that mimics the most common pathogenic TCF4 point mutation (human R580W, mouse R579W) and one that deletes three pathogenic arginines, and explored phenotypes of these lines alongside models of pan-cellular or CNS-specific heterozygous Tcf4 disruption. We used mice of both sexes to show that impaired Tcf4 function results in consistent microcephaly, hyperactivity, reduced anxiety, and deficient spatial learning. All four PTHS mouse models demonstrated exaggerated hippocampal long-term potentiation (LTP), consistent with deficits in hippocampus-mediated behaviors. We further examined R579W mutant mice and mice with pan-cellular Tcf4 heterozygosity and found that they exhibited hippocampal NMDA receptor hyperfunction, which likely drives the enhanced LTP. Together, our data pinpoint convergent neurobiological features in PTHS mouse models and provide a foundation for preclinical studies and a rationale for testing whether NMDAR antagonists might be used to treat PTHS.SIGNIFICANCE STATEMENT Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder associated with TCF4 mutations/deletions. Despite this genetic insight, there is a need to identify the function of TCF4 in the brain. Toward this goal, we developed two mouse lines, including one harboring the most prevalent pathogenic point mutation, and compared them with two existing models that conditionally delete Tcf4 Our data identify a set of overlapping phenotypes that may serve as outcome measures for preclinical studies of PTHS treatments. We also discovered penetrant enhanced synaptic plasticity across mouse models that may be linked to increased NMDA receptor function. These data reveal convergent neurobiological characteristics of PTHS mouse models and support the further investigation of NMDA receptor antagonists as a possible PTHS treatment.

Enhanced Operant Extinction and Prefrontal Excitability in a Mouse Model of Angelman Syndrome.

Angelman syndrome (AS), a neurodevelopmental disorder associated with intellectual disability, is caused by loss of maternal allele expression of UBE3A in neurons. Mouse models of AS faithfully recapitulate disease phenotypes across multiple domains, including behavior. Yet in AS, there has been only limited study of behaviors encoded by the prefrontal cortex, a region broadly involved in executive function and cognition. Because cognitive impairment is a core feature of AS, it is critical to develop behavioral readouts of prefrontal circuit function in AS mouse models. One such readout is behavioral extinction, which has been well described mechanistically and relies upon prefrontal circuits in rodents. Here we report exaggerated operant extinction in male AS model mice, concomitant with enhanced excitability in medial prefrontal neurons from male and female AS model mice. Abnormal behavior was specific to operant extinction, as two other prefrontally dependent tasks (cued fear extinction and visuospatial discrimination) were largely normal in AS model mice. Inducible deletion of Ube3a during adulthood was not sufficient to drive abnormal extinction, supporting the hypothesis that there is an early critical period for development of cognitive phenotypes in AS. This work represents the first formal experimental analysis of prefrontal circuit function in AS, and identifies operant extinction as a useful experimental paradigm for modeling cognitive aspects of AS in mice.SIGNIFICANCE STATEMENT Prefrontal cortex encodes "high-level" cognitive processes. Thus, understanding prefrontal function is critical in neurodevelopmental disorders where cognitive impairment is highly penetrant. Angelman syndrome is a neurodevelopmental disorder associated with speech and motor impairments, an outwardly happy demeanor, and intellectual disability. We describe a behavioral phenotype in a mouse model of Angelman syndrome and related abnormalities in prefrontal cortex function. We hypothesize that robust and reliable prefrontally encoded behavior may be used to model cognitive impairments in Angelman syndrome.

Modulation of CA2 neuronal activity increases behavioral responses to fear conditioning in female mice.

Activity of hippocampal pyramidal cells is critical for certain forms of learning and memory, and work from our lab and others has shown that CA2 neuronal activity is required for social cognition and behavior. Silencing of CA2 neurons in mice impairs social memory, and mice lacking Regulator of G-Protein Signaling 14 (RGS14), a protein that is highly enriched in CA2 neurons, learn faster than wild types in the Morris water maze spatial memory test. Although the enhanced spatial learning abilities of the RGS14 KO mice suggest a role for CA2 neurons in at least one hippocampus-dependent behavior, the role of CA2 neurons in fear conditioning, which requires activity of hippocampus, amygdala, and possibly prefrontal cortex is unknown. In this study, we expressed excitatory or inhibitory DREADDs in CA2 neurons and administered CNO before the shock-tone-context pairing. On subsequent days, we measured freezing behavior in the same context but without the tone (contextual fear) or in a new context but in the presence of the tone (cued fear). We found that increasing CA2 neuronal activity with excitatory DREADDs during training resulted in increased freezing during the cued fear tests in males and females. Surprisingly, we found that only females showed increased freezing during the contextual fear memory tests. Using inhibitory DREADDs, we found that inhibiting CA2 neuronal activity during the training phase also resulted in increased freezing in females during the subsequent contextual fear memory test. Finally, we tested fear conditioning in RGS14 KO mice and found that female KO mice had increased freezing on the cued fear memory test. These three separate lines of evidence suggest that CA2 neurons are actively involved in both intra- and extra-hippocampal brain processes and function to influence fear memory. Finally, the intriguing and consistent findings of enhanced fear conditioning only among females is strongly suggestive of a sexual dimorphism in CA2-linked circuits.

Noradrenergic dysfunction accelerates LPS-elicited inflammation-related ascending sequential neurodegeneration and deficits in non-motor/motor functions.

The loss of central norepinephrine (NE) released by neurons of the locus coeruleus (LC) occurs with aging, and is thought to be an important factor in producing the many of the nonmotor symptoms and exacerbating the degenerative process in animal models of Parkinson's disease (PD). We hypothesize that selectively depleting noradrenergic LC neurons prior to the induction of chronic neuroinflammation may not only accelerate the rate of progressive neurodegeneration throughout the brain, but may exacerbate nonmotor and motor behavioral phenotypes that recapitulate symptoms of PD. For this reason, we used a "two-hit" mouse model whereby brain NE were initially depleted by DSP-4 one week prior to exposing mice to LPS. We found that pretreatment with DSP-4 potentiated LPS-induced sequential neurodegeneration in SNpc, hippocampus, and motor cortex, but not in VTA and caudate/putamen. Mechanistic study revealed that DSP-4 enhanced LPS-induced microglial activation and subsequently elevated neuronal oxidative stress in affected brain regions in a time-dependent pattern. To further characterize the effects of DSP-4 on non-motor and motor symptoms in the LPS model, physiological and behavioral tests were performed at different time points following injection. Consistent with the enhanced neurodegeneration, DSP-4 accelerated the progressive deficits of non-motor symptoms including hyposmia, constipation, anxiety, sociability, exaggerated startle response and impaired learning. Furthermore, notable decreases of motor functions, including decreased rotarod activity, grip strength, and gait disturbance, were observed in treated mice. In summary, our studies provided not only an accelerated "two-hit" PD model that recapitulates the features of sequential neuron loss and the progression of motor/non-motor symptoms of PD, but also revealed the critical role of early LC noradrenergic neuron damage in the pathogenesis of PD-like symptoms.

Trim9 Deletion Alters the Morphogenesis of Developing and Adult-Born Hippocampal Neurons and Impairs Spatial Learning and Memory.

UNLABELLED: During hippocampal development, newly born neurons migrate to appropriate destinations, extend axons, and ramify dendritic arbors to establish functional circuitry. These developmental stages are recapitulated in the dentate gyrus of the adult hippocampus, where neurons are continuously generated and subsequently incorporate into existing, local circuitry. Here we demonstrate that the E3 ubiquitin ligase TRIM9 regulates these developmental stages in embryonic and adult-born mouse hippocampal neurons in vitro and in vivo Embryonic hippocampal and adult-born dentate granule neurons lacking Trim9 exhibit several morphological defects, including excessive dendritic arborization. Although gross anatomy of the hippocampus was not detectably altered by Trim9 deletion, a significant number of Trim9(-/-) adult-born dentate neurons localized inappropriately. These morphological and localization defects of hippocampal neurons in Trim9(-/-) mice were associated with extreme deficits in spatial learning and memory, suggesting that TRIM9-directed neuronal morphogenesis may be involved in hippocampal-dependent behaviors. SIGNIFICANCE STATEMENT: Appropriate generation and incorporation of adult-born neurons in the dentate gyrus are critical for spatial learning and memory and other hippocampal functions. Here we identify the brain-enriched E3 ubiquitin ligase TRIM9 as a novel regulator of embryonic and adult hippocampal neuron shape acquisition and hippocampal-dependent behaviors. Genetic deletion of Trim9 elevated dendritic arborization of hippocampal neurons in vitro and in vivo Adult-born dentate granule cells lacking Trim9 similarly exhibited excessive dendritic arborization and mislocalization of cell bodies in vivo These cellular defects were associated with severe deficits in spatial learning and memory.

Bcl-xL Is Essential for the Survival and Function of Differentiated Neurons in the Cortex That Control Complex Behaviors.

UNLABELLED: Apoptosis plays an essential role during brain development, yet the precise mechanism by which this pathway is regulated in the brain remains unknown. In particular, mammalian cells are known to express multiple anti-apoptotic Bcl-2 family proteins. However, the cells of the developing brain could also exist in a primed state in which the loss of a single anti-apoptotic Bcl-2 family protein is sufficient to trigger apoptosis. Here, we examined the critical role of Bcl-xL, an anti-apoptotic protein, during brain development. Using conditional knock-out mice in which Bcl-xL is deleted in neural progenitor cells (Bcl-xL(Emx1-Cre)), we show that the loss of Bcl-xL is not sufficient to trigger apoptosis in these proliferating progenitors. In contrast, specific populations of postmitotic neurons derived from these progenitors, including upper layer cortical neurons and the CA1-CA3 regions of the hippocampus, were acutely dependent on Bcl-xL. Consistent with this finding, deletion of Bcl-xL selectively in the postmitotic neurons in the brain (Bcl-xL(Nex-Cre)) also resulted in similar patterns of apoptosis. This Bcl-xL deficiency-induced neuronal death was a consequence of activation of the apoptotic pathway, because the cell death was rescued with codeletion of the proapoptotic proteins Bax and Bak. Importantly, the loss of these Bcl-xL-dependent neurons led to severe neurobehavioral abnormalities, including deficits in motor learning, hyperactivity, and increased risk-taking and self-injurious behaviors. Together, our results identify a population of neurons in the developing brain that are acutely dependent on Bcl-xL during the peak period of synaptic connectivity that are important for the establishment of higher-order complex behaviors. SIGNIFICANCE STATEMENT: Although Bcl-xL is known to inhibit apoptosis, exactly which cells in the brain are dependent on Bcl-xL has remained unclear because of the embryonic lethality of mice globally deleted for Bcl-xL. Here, we conditionally deleted Bcl-xL in the brain and found that this did not result in widespread apoptosis in the proliferating progenitors. Instead, Bcl-xL deficiency induced apoptosis in a select population of differentiated neurons predominantly in the early postnatal stages. Importantly, these Bcl-xL-dependent neurons are not essential for survival of the organism but instead regulate complex behaviors. Our results show that the selective loss of these Bcl-xL-dependent neurons results in mice exhibiting severe neurobehavioral abnormalities, including self-injurious and risk-taking behaviors, hyperactivity, and learning and memory defects.

Imbalanced glucocorticoid and mineralocorticoid stress hormone receptor function has sex-dependent and independent regulatory effects in the mouse hippocampus.

Many stress-related neuropsychiatric disorders display pronounced sex differences in their frequency and clinical symptoms. Glucocorticoids are primary stress hormones that have been implicated in the development of these disorders but whether they contribute to the observed sex bias is poorly understood. Glucocorticoids signal through two closely related nuclear receptors, the glucocorticoid (GR) and mineralocorticoid receptor (MR). To elucidate the sex-specific and independent actions of glucocorticoids in the hippocampus, we developed knockout mice lacking hippocampal GR, MR, or both GR and MR. Mice deficient in hippocampal MR or both GR and MR showed an altered molecular phenotype of CA2 neurons and reduced anxiety-like behavior in both sexes, but altered stress adaptation behavior only in females and enhanced fear-motivated cue learning only in males. All three knockout mouse models displayed reduced sociability but only in male mice. Male and female mice deficient in both hippocampal GR and MR exhibited extensive neurodegeneration in the dentate gyrus. Global transcriptomic analysis revealed a marked expansion in the number of dysregulated genes in the hippocampus of female knockout mice compared to their male counterparts; however, the overall patterns of gene dysregulation were remarkably similar in both sexes. Within and across sex comparisons identified key GR and MR target genes and associated signaling pathways underlying the knockout phenotypes. These findings define major sex-dependent and independent effects of GR/MR imbalances on gene expression and functional profiles in the hippocampus and inform new strategies for treating men and women with stress-related neuropsychiatric disorders.

Sleep Disruption Precedes Forebrain Synaptic Tau Burden and Contributes to Cognitive Decline in a Sex-Dependent Manner in the P301S Tau Transgenic Mouse Model.

Sleep disruption and impaired synaptic processes are common features in neurodegenerative diseases, including Alzheimer's disease (AD). Hyperphosphorylated Tau is known to accumulate at neuronal synapses in AD, contributing to synapse dysfunction. However, it remains unclear how sleep disruption and synapse pathology interact to contribute to cognitive decline. Here, we examined sex-specific onset and consequences of sleep loss in AD/tauopathy model PS19 mice. Using a piezoelectric home-cage monitoring system, we showed PS19 mice exhibited early-onset and progressive hyperarousal, a selective dark-phase sleep disruption, apparent at 3 months in females and 6 months in males. Using the Morris water maze test, we report that chronic sleep disruption (CSD) accelerated the onset of decline of hippocampal spatial memory in PS19 males only. Hyperarousal occurs well in advance of robust forebrain synaptic Tau burden that becomes apparent at 6-9 months. To determine whether a causal link exists between sleep disruption and synaptic Tau hyperphosphorylation, we examined the correlation between sleep behavior and synaptic Tau, or exposed mice to acute or chronic sleep disruption at 6 months. While we confirm that sleep disruption is a driver of Tau hyperphosphorylation in neurons of the locus ceruleus, we were unable to show any causal link between sleep loss and Tau burden in forebrain synapses. Despite the finding that hyperarousal appears earlier in females, female cognition was resilient to the effects of sleep disruption. We conclude sleep disruption interacts with the synaptic Tau burden to accelerate the onset of cognitive decline with greater vulnerability in males.

Evaluating Fatty Acid Amide Hydrolase as a Suitable Target for Sleep Promotion in a Transgenic TauP301S Mouse Model of Neurodegeneration.

Sleep disruption is an expected component of aging and neurodegenerative conditions, including Alzheimer's disease (AD). Sleep disruption has been demonstrated as a driver of AD pathology and cognitive decline. Therefore, treatments designed to maintain sleep may be effective in slowing or halting AD progression. However, commonly used sleep aid medications are associated with an increased risk of AD, highlighting the need for sleep aids with novel mechanisms of action. The endocannabinoid system holds promise as a potentially effective and novel sleep-enhancing target. By using pharmacology and genetic knockout strategies, we evaluated fatty acid amide hydrolase (FAAH) as a therapeutic target to improve sleep and halt disease progression in a transgenic Tau P301S (PS19) model of Tauopathy and AD. We have recently shown that PS19 mice exhibit sleep disruption in the form of dark phase hyperarousal as an early symptom that precedes robust Tau pathology and cognitive decline. Acute FAAH inhibition with PF3845 resulted in immediate improvements in sleep behaviors in male and female PS19 mice, supporting FAAH as a potentially suitable sleep-promoting target. Moreover, sustained drug dosing for 5-10 days resulted in maintained improvements in sleep. To evaluate the effect of chronic FAAH inhibition as a possible therapeutic strategy, we generated FAAH-/- PS19 mice models. Counter to our expectations, FAAH knockout did not protect PS19 mice from progressive sleep loss, neuroinflammation, or cognitive decline. Our results provide support for FAAH as a novel target for sleep-promoting therapies but further indicate that the complete loss of FAAH activity may be detrimental.

Transcriptome Analysis Identifies An ASD-Like Phenotype In Oligodendrocytes And Microglia From C58/J Amygdala That Is Dependent On Sex and Sociability.

Background: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. Methods: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. Results: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. Limitations: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its potential as an ASD therapeutic. Conclusions: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

Loss of neuronal lysosomal acid lipase drives amyloid pathology in Alzheimer's disease.

Underlying drivers of late-onset Alzheimer's disease (LOAD) pathology remain unknown. However, multiple biologically diverse risk factors share a common pathological progression. To identify convergent molecular abnormalities that drive LOAD pathogenesis we compared two common midlife risk factors for LOAD, heavy alcohol use and obesity. This revealed that disrupted lipophagy is an underlying cause of LOAD pathogenesis. Both exposures reduced lysosomal flux, with a loss of neuronal lysosomal acid lipase (LAL). This resulted in neuronal lysosomal lipid (NLL) accumulation, which opposed Aß localization to lysosomes. Neuronal LAL loss both preceded (with aging) and promoted (targeted knockdown) Aß pathology and cognitive deficits in AD mice. The addition of recombinant LAL ex vivo and neuronal LAL overexpression in vivo prevented amyloid increases and improved cognition. In WT mice, neuronal LAL declined with aging and correlated negatively with entorhinal Aß. In healthy human brain, LAL also declined with age, suggesting this contributes to the age-related vulnerability for AD. In human LOAD LAL was further reduced, correlated negatively with Aß 1-42 , and occurred with polymerase pausing at the LAL gene. Together, this finds that the loss of neuronal LAL promotes NLL accumulation to impede degradation of Aß in neuronal lysosomes to drive AD amyloid pathology. Summary: Cellular and molecular drivers of late-onset Alzheimer's disease (LOAD) are unknown, though several risk factors account for the majority of disease incidence 1-5 . Though diverse in their biological natures, each of these risk exposures converge on a shared pathological progression with the accumulation of amyloid early in the disease. Human genetic and transcriptomic studies suggest a role for altered lipid metabolism 6-9 , though the mechanism has been unknown. Here, using two common midlife risk exposures for LOAD, we found that dysfunctional lipophagy caused by the loss of lysosomal acid lipase (LAL) promotes early LOAD pathogenesis. Both midlife obesity and heavy alcohol reduced neuronal LAL, causing an increase in neuronal lysosomal lipid, and a subsequent accumulation of Aß in the extra-lysosomal cytosol. This loss of LAL preceded and promoted Aß pathology and cognitive deficits in AD mice. The addition of recombinant LAL ex vivo and neuronal LAL overexpression in vivo prevented increases in amyloid and improved cognition. In human brain, LAL declined with age in healthy subjects, similar to rodents, showing robust losses in LOAD subjects with polymerase pausing. Together, this implicates neuronal LAL loss in LOAD pathogenesis and presents LAL as a promising diagnostic, preventative, and/or therapeutic target for AD.

Interleukin-6 (IL-6) receptor/IL-6 fusion protein (Hyper IL-6) effects on the neonatal mouse brain: possible role for IL-6 trans-signaling in brain development and functional neurobehavioral outcomes.

Adverse neurodevelopmental outcomes are linked to perinatal production of inflammatory mediators, including interleukin 6 (IL-6). While a pivotal role for maternal elevation in IL-6 has been established in determining neurobehavioral outcomes in the offspring and considered the primary target mediating the fetal inflammatory response, questions remain as to the specific actions of IL-6 on the developing brain. CD-1 male mice received a subdural injection of the bioactive fusion protein, hyper IL-6 (HIL-6) on postnatal-day (PND)4 and assessed from preweaning until adulthood. Immunohistochemical evaluation of astrocytes and microglia and mRNA levels for pro-inflammatory cytokines and host response genes indicated no evidence of an acute neuroinflammatory injury response. HIL-6 accelerated motor development and increased reactivity to stimulation and number of entries in a light/dark chamber, decreased ability to learn to withhold a response in passive avoidance, and effected deficits in social novelty behavior. No changes were observed in motor activity, pre-pulse startle inhibition, or learning and memory in the Morris water maze or radial arm maze, as have been reported for models of more severe developmental neuroinflammation. In young animals, mRNA levels for MBP and PLP/DM20 decreased and less complexity of MBP processes in the cortex was evident by immunohistochemistry. The non-hydroxy cerebroside fraction of cerebral lipids was increased. These results provide evidence for selective effects of IL-6 signaling, particularly trans-signaling, in the developing brain in the absence of a general neuroinflammatory response. These data contribute to our further understanding of the multiple aspects of IL-6 signaling in the developing brain.

Prenatal stress unmasks behavioral phenotypes in genetic mouse models of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) are complex conditions characterized by heterogeneous clinical profiles and symptoms that arise in infancy and childhood. NDDs are often attributed to a complicated interaction between genetic risk and environmental factors, suggesting a need for preclinical models reflecting the combined impact of heritable susceptibility and environmental effects. A notable advantage of "two-hit" models is the power to reveal underlying vulnerability that may not be detected in studies employing only genetic or environmental alterations. In this review, we summarize existing literature that investigates detrimental interactions between prenatal stress (PNS) and genes associated with NDDs, with a focus on behavioral phenotyping approaches in mouse models. A challenge in determining the overall role of PNS exposure in genetic models is the diversity of approaches for inducing stress, variability in developmental timepoints for exposure, and differences in phenotyping regimens across laboratories. Identification of optimal stress protocols and critical windows for developmental effects would greatly improve the use of PNS in gene × environment mouse models of NDDs.

Neonatal Behavioral Screen for Mouse Models of Neurodevelopmental Disorders.

Behavioral phenotyping approaches for neonatal mice are important for investigating early alterations in brain development and function, relevant to neurodevelopmental disorders in humans. This chapter describes a behavioral screen that can provide an overall profile of function across the neonatal and preweaning period while also minimizing pup stress and disturbance of the maternal environment. Testing begins when mice are between 6 and 8 days in age, with additional evaluations at discrete time points until postnatal day (PD) 20-21, using tests for negative geotaxis, surface righting reflex, activity in an open field, acoustic startle responses and sensorimotor gating, and limb clasp.

Sleep disruption precedes forebrain synaptic Tau burden and contributes to cognitive decline in a sex-dependent manner in the P301S Tau transgenic mouse model.

Background: Sleep is an essential process that supports brain health and cognitive function in part through the modification of neuronal synapses. Sleep disruption, and impaired synaptic processes, are common features in neurodegenerative diseases, including Alzheimer's disease (AD). However, the casual role of sleep disruption in disease progression is not clear. Neurofibrillary tangles, made from hyperphosphorylated and aggregated Tau protein, form one of the major hallmark pathologies seen in AD and contribute to cognitive decline, synapse loss and neuronal death.Tau has been shown to aggregate in synapses which may impair restorative synapse processes occurring during sleep. However, it remains unclear how sleep disruption and synaptic Tau pathology interact to drive cognitive decline. It is also unclear whether the sexes show differential vulnerability to the effects of sleep loss in the context of neurodegeneration. Methods: We used a piezoelectric home-cage monitoring system to measure sleep behavior in 3-11month-old transgenic hTau P301S Tauopathy model mice (PS19) and littermate controls of both sexes. Subcellular fractionation and Western blot was used to examine Tau pathology in mouse forebrain synapse fractions. To examine the role of sleep disruption in disease progression, mice were exposed to acute or chronic sleep disruption. The Morris water maze test was used to measure spatial learning and memory performance. Results: PS19 mice exhibited a selective loss of sleep during the dark phase, referred to as hyperarousal, as an early symptom with an onset of 3months in females and 6months in males. At 6months, forebrain synaptic Tau burden did not correlate with sleep measures and was not affected by acute or chronic sleep disruption. Chronic sleep disruption accelerated the onset of decline of hippocampal spatial memory in PS19 males, but not females. Conclusions: Dark phase hyperarousal is an early symptom in PS19 mice that precedes robust Tau aggregation. We find no evidence that sleep disruption is a direct driver of Tau pathology in the forebrain synapse. However, sleep disruption synergized with Tau pathology to accelerate the onset of cognitive decline in males. Despite the finding that hyperarousal appears earlier in females, female cognition was resilient to the effects of sleep disruption.

RNA-binding deficient TDP-43 drives cognitive decline in a mouse model of TDP-43 proteinopathy.

TDP-43 proteinopathies including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders characterized by aggregation and mislocalization of the nucleic acid-binding protein TDP-43 and subsequent neuronal dysfunction. Here, we developed endogenous models of sporadic TDP-43 proteinopathy based on the principle that disease-associated TDP-43 acetylation at lysine 145 (K145) alters TDP-43 conformation, impairs RNA-binding capacity, and induces downstream mis-regulation of target genes. Expression of acetylation-mimic TDP-43K145Q resulted in stress-induced nuclear TDP-43 foci and loss of TDP-43 function in primary mouse and human-induced pluripotent stem cell (hiPSC)-derived cortical neurons. Mice harboring the TDP-43K145Q mutation recapitulated key hallmarks of FTLD, including progressive TDP-43 phosphorylation and insolubility, TDP-43 mis-localization, transcriptomic and splicing alterations, and cognitive dysfunction. Our study supports a model in which TDP-43 acetylation drives neuronal dysfunction and cognitive decline through aberrant splicing and transcription of critical genes that regulate synaptic plasticity and stress response signaling. The neurodegenerative cascade initiated by TDP-43 acetylation recapitulates many aspects of human FTLD and provides a new paradigm to further interrogate TDP-43 proteinopathies.