Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q.

Although genome wide association studies (GWAS) have identified loci for sleep-related traits, they do not directly uncover the underlying causal variants and corresponding effector genes. The majority of such variants reside in non-coding regions and are therefore presumed to impact cis-regulatory elements. Our previously reported 'variant-to-gene mapping' effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia 'WDR90' GWAS locus. However, importantly that effort did not characterize the corresponding underlying causal variant. Specifically, our previous 3D genomic datasets nominated a shortlist of three neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium within an intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. We sought to investigate the influence of these SNPs collectively and then individually on PIG-Q modulation to pinpoint the causal "regulatory" variant. Starting with gross level perturbation, deletion of the entire region in NPCs via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from individual luciferase reporter assays for each SNP in iPSCs revealed that the region with the rs3752495 risk allele induced a ~2.5-fold increase in luciferase expression. Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by ~2-fold versus the non-risk allele. In conclusion, our variant-to-function approach and in vitro validation implicates rs3752495 as a causal insomnia variant embedded within WDR90 while modulating the expression of the distally located PIG-Q.

Screening effects of HCN channel blockers on sleep/wake behavior in zebrafish.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels generate electrical rhythmicity in various tissues although primarily heart, retina and brain. The HCN channel blocker compound, Ivabradine (Corlanor), is approved by the US Food and Drug Administration (FDA) as a medication to lower heart rate by blocking hyperpolarization activated inward current in the sinoatrial node. In addition, a growing body of evidence suggests a role for HCN channels in regulation of sleep/wake behavior. Zebrafish larvae are ideal model organisms for high throughput drug screening, drug repurposing and behavioral phenotyping studies. We leveraged this model system to investigate effects of three HCN channel blockers (Ivabradine, Zatebradine Hydrochloride and ZD7288) at multiple doses on sleep/wake behavior in wild type zebrafish. Results of interest included shorter latency to daytime sleep at 0.1 µM dose of Ivabradine (ANOVA, p: 0.02), moderate reduction in average activity at 30 µM dose of Zatebradine Hydrochloride (ANOVA, p: 0.024) in daytime, and increased nighttime sleep at 4.5 µM dose of ZD7288 (ANOVA, p: 0.036). Taken together, shorter latency to daytime sleep, decrease in daytime activity and increased nighttime sleep indicate that different HCN channel antagonists affected different parameters of sleep and activity.

Perturbation of the insomnia WDR90 GWAS locus pinpoints rs3752495 as a causal variant influencing distal expression of neighboring gene, PIG-Q.

Although genome wide association studies (GWAS) have been crucial for the identification of loci associated with sleep traits and disorders, the method itself does not directly uncover the underlying causal variants and corresponding effector genes. The overwhelming majority of such variants reside in non-coding regions and are therefore presumed to impact the activity of cis-regulatory elements, such as enhancers. Our previously reported 'variant-to-gene mapping' effort in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs), combined with validation in both Drosophila and zebrafish, implicated PIG-Q as a functionally relevant gene at the insomnia 'WDR90' locus. However, importantly that effort did not characterize the corresponding underlying causal variant at this GWAS signal. Specifically, our genome-wide ATAC-seq and high-resolution promoter-focused Capture C datasets generated in this cell setting brought our attention to a shortlist of three tightly neighboring single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium in a candidate intronic enhancer region of WDR90 that contacted the open PIG-Q promoter. The objective of this study was to investigate the influence of the proxy SNPs collectively and then individually on PIG-Q modulation and to pinpoint the causal "regulatory" variant among the three SNPs. Starting at a gross level perturbation, deletion of the entire region harboring all three SNPs in human iPSC-derived neural progenitor cells via CRISPR-Cas9 editing and subsequent RNA sequencing revealed expression changes in specific PIG-Q transcripts. Results from more refined individual luciferase reporter assays for each of the three SNPs in iPSCs revealed that the intronic region with the rs3752495 risk allele induced a ~2.5-fold increase in luciferase expression (n=10). Importantly, rs3752495 also exhibited an allele specific effect, with the risk allele increasing the luciferase expression by ~2-fold compared to the non-risk allele. In conclusion, our variant-to-function approach and subsequent in vitro validation implicates rs3752495 as a causal insomnia risk variant embedded at the WDR90-PIG-Q locus.

Pleiotropic effects of a high confidence Autism Spectrum Disorder gene, arid1b, on zebrafish sleep.

Sleep fulfills critical functions in neurodevelopment, such as promoting synaptic plasticity, neuronal wiring, and brain connectivity which are critical phenomena in Autism Spectrum Disorder (ASD) pathophysiology. Sleep disturbance, specifically insomnia, accompanies ASD and is associated with more severe core symptoms (e.g., social impairment). It is possible that focusing on identifying effective ways to treat sleep problems can help alleviate other ASD-related symptoms. A body of evidence indicates shared mechanisms and neurobiological substrates between sleep and ASD and investigation of these may inform therapeutic effects of improving sleep at both behavioral and molecular levels. In this study, we tested if sleep and social behavior were different in a zebrafish model with the arid1b gene mutated compared to controls. This gene was selected for study as expert curations conducted for the Simons Foundation for Autism Research Institute (SFARI) Gene database define it is as a 'high confidence' ASD gene (i.e., clearly implicated) encoding a chromatin remodeling protein. Homozygous arid1b mutants displayed increased arousability and light sleep compared to their heterozygous and wild type counterparts, based on testing a mechano-acoustic stimulus presenting different vibration frequencies of increasing intensity to detect sleep depth. In addition, decreased social preference was observed in arid1b heterozygous and homozygous mutant zebrafish. The behavioral phenotypes reported in our study are in line with findings from mouse models and human studies and demonstrate the utility of zebrafish as a vertebrate model system with high throughput phenotyping in the investigation of changes in sleep in models relevant to ASD. Furthermore, we demonstrate the importance of including assessments of arousal threshold when studying sleep using in vivo models.

Distribution and inflammatory cell response to intracranial delivery of radioluminescent Y2(SiO4)O:Ce particles.

Due to increasing advances in their manufacture and functionalization, nanoparticle-based systems have become a popular tool for in vivo drug delivery and biodetection. Recently, scintillating nanoparticles such as yttrium orthosilicate doped with cerium (Y2(SiO4)O:Ce) have come under study for their potential utility in optogenetic applications, as they emit photons upon low levels of stimulation from remote x-ray sources. The utility of such nanoparticles in vivo is hampered by rapid clearance from circulation by the mononuclear phagocytic system, which heavily restricts nanoparticle accumulation at target tissues. Local transcranial injection of nanoparticles may deliver scintillating nanoparticles to highly specific brain regions by circumventing the blood-brain barrier and avoiding phagocytic clearance. Few studies to date have examined the distribution and response to nanoparticles following localized delivery to cerebral cortex, a crucial step in understanding the therapeutic potential of nanoparticle-based biodetection in the brain. Following the synthesis and surface modification of these nanoparticles, two doses (1 and 3 mg/ml) were introduced into mouse secondary motor cortex (M2). This region was chosen as the site for RLP delivery, as it represents a common target for optogenetic manipulations of mouse behavior, and RLPs could eventually serve as an injectable x-ray inducible light delivery system. The spread of particles through the target tissue was assessed 24 hours, 72 hours, and 9 days post-injection. Y2(SiO4)O:Ce nanoparticles were found to be detectable in the brain for up to 9 days, initially diffusing through the tissue until 72 hours before achieving partial clearance by the final endpoint. Small transient increases in the presence of IBA-1+ microglia and GFAP+ astrocytic cell populations were detected near nanoparticle injection sites of both doses tested 24 hours after surgery. Taken together, these data provide evidence that Y2(SiO4)O:Ce nanoparticles coated with BSA can be injected directly into mouse cortex in vivo, where they persist for days and are broadly tolerated, such that they may be potentially utilized for remote x-ray activated stimulation and photon emission for optogenetic experiments in the near future.

Variant-to-gene mapping followed by cross-species genetic screening identifies GPI-anchor biosynthesis as a regulator of sleep.

Genome-wide association studies (GWAS) in humans have identified loci robustly associated with several heritable diseases or traits, yet little is known about the functional roles of the underlying causal variants in regulating sleep duration or quality. We applied an ATAC-seq/promoter focused Capture C strategy in human iPSC-derived neural progenitors to carry out a "variant-to-gene" mapping campaign that identified 88 candidate sleep effector genes connected to relevant GWAS signals. To functionally validate the role of the implicated effector genes in sleep regulation, we performed a neuron-specific RNA interference screen in the fruit fly, Drosophila melanogaster, followed by validation in zebrafish. This approach identified a number of genes that regulate sleep including a critical role for glycosylphosphatidylinositol (GPI)-anchor biosynthesis. These results provide the first physical variant-to-gene mapping of human sleep genes followed by a model organism-based prioritization, revealing a conserved role for GPI-anchor biosynthesis in sleep regulation.

Global loss of Neuron-specific gene 1 causes alterations in motor coordination, increased anxiety, and diurnal hyperactivity in male mice.

The Neuron-specific gene family (NSG1-3) consists of small endolysosomal proteins that are critical for trafficking multiple receptors and signaling molecules in neurons. NSG1 has been shown to play a critical role in AMPAR recycling from endosomes to plasma membrane during synaptic plasticity. However, to date nothing is known about whether NSG1 is required for normal behavior at an organismal level. Here we performed a battery of behavioral tests to determine whether loss of NSG1 would affect motor, cognitive, and/or affective behaviors, as well as circadian-related activity. Consistent with unique cerebellar expression of NSG1 among family members, we found that NSG1 was obligatory for motor coordination but not for gross motor function or learning. NSG1 knockout (KO) also altered performance across other behavioral modalities including anxiety-related and diurnal activity paradigms. Surprisingly, NSG1 KO did not cause significant impairments across all tasks within a given modality, but had specific effects within each modality. For instance, we found increases in anxiety-related behaviors in tasks with multiple stressors (e.g., elevation and exposure), but not those with a single main stressor (e.g., exposure). Interestingly, NSG1 KO animals displayed a significant increase in locomotor activity during subjective daytime, suggesting a possible impact on diurnal activity rhythms or vigilance. Surprisingly, loss of NSG1 had no effect on hippocampal-dependent learning despite previous studies showing deficits in CA1 long-term potentiation. Together, these findings do not support a role of NSG1 in hippocampal-dependent learning, but support a role in mediating proper neuronal function across amygdalar and cerebellar circuits.

A bidirectional competitive interaction between circHomer1 and Homer1b within the orbitofrontal cortex regulates reversal learning.

Although circular RNAs (circRNAs) are enriched in the brain, their relevance for brain function and psychiatric disorders is poorly understood. Here, we show that circHomer1 is inversely associated with relative HOMER1B mRNA isoform levels in both the orbitofrontal cortex (OFC) and stem-cell-derived neuronal cultures of subjects with psychiatric disorders. We further demonstrate that in vivo circHomer1 knockdown (KD) within the OFC can inhibit the synaptic expression of Homer1b mRNA. Furthermore, we show that circHomer1 directly binds to Homer1b mRNA and that Homer1b-specific KD increases synaptic circHomer1 levels and improves OFC-mediated behavioral flexibility. Importantly, double circHomer1 and Homer1b in vivo co-KD results in a complete rescue in circHomer1-associated alterations in both chance reversal learning and synaptic gene expression. Lastly, we uncover an RNA-binding protein that can directly bind to circHomer1 and promote its biogenesis. Taken together, our data provide mechanistic insights into the importance of circRNAs in brain function and disease.

Proteopathic tau primes and activates interleukin-1ß via myeloid-cell-specific MyD88- and NLRP3-ASC-inflammasome pathway.

Pathological hyperphosphorylation and aggregation of tau (pTau) and neuroinflammation, driven by interleukin-1ß (IL-1ß), are the major hallmarks of tauopathies. Here, we show that pTau primes and activates IL-1ß. First, RNA-sequence analysis suggests paired-helical filaments (PHFs) from human tauopathy brain primes nuclear factor κB (NF-κB), chemokine, and IL-1ß signaling clusters in human primary microglia. Treating microglia with pTau-containing neuronal media, exosomes, or PHFs causes IL-1ß activation, which is NLRP3, ASC, and caspase-1 dependent. Suppression of pTau or ASC reduces tau pathology and inflammasome activation in rTg4510 and hTau mice, respectively. Although the deletion of MyD88 prevents both IL-1ß expression and activation in the hTau mouse model of tauopathy, ASC deficiency in myeloid cells reduces pTau-induced IL-1ß activation and improves cognitive function in hTau mice. Finally, pTau burden co-exists with elevated IL-1ß and ASC in autopsy brains of human tauopathies. Together, our results suggest pTau activates IL-1ß via MyD88- and NLRP3-ASC-dependent pathways in myeloid cells, including microglia.

Diastereoselective Synthesis of P-Stereogenic Secondary Phosphine Oxides (SPOs) Bearing a Chiral Substituent by Ring Opening of (+)-Limonene Oxide with Primary Phosphido Nucleophiles.

Kinetic separation of the commercially available cis/trans-(+)-limonene oxide mixture by ring opening with primary phosphido nucleophiles LiPHR (R = ferrocenyl, Ph, Cy, t-Bu, Mes* (Mes* = 2,4,6-(t-Bu)3C6H2)), followed by treatment with aqueous NH4Cl and H2O2, gave unreacted cis-(+)-limonene oxide and diastereoenriched mixtures of the secondary phosphine oxides (SPOs) PHR(trans-(+)-Lim-OH)(O), which could be separated by chromatography and/or recrystallization. This one-pot synthesis uses a cheap chiral material and commercially available primary phosphines to control the configuration of the new P-stereogenic SPOs, which are potentially useful as ligands for metal complexes in asymmetric catalysis.

Neonatal Hypoxic-Ischemic Encephalopathy Yields Permanent Deficits in Learning Acquisition: A Preclinical Touchscreen Assessment.

Neonatal hypoxic-ischemic encephalopathy (HIE) remains a common problem world-wide for infants born at term. The impact of HIE on long-term outcomes, especially into adulthood, is not well-described. To facilitate identification of biobehavioral biomarkers utilizing a translational platform, we sought to investigate the impact of HIE on executive function and cognitive outcomes into adulthood utilizing a murine model of HIE. HIE mice (unilateral common carotid artery occlusion to induce ischemia, followed by hypoxia with a FiO2 of 0.08 for 45 min) and control mice were tested on discrimination and reversal touchscreen tasks (using their noses) shown to be sensitive to loss of basal ganglia or cortical function, respectively. We hypothesized that the HIE injury would result in deficits in reversal learning, revealing complex cognitive and executive functioning impairments. Following HIE, mice had a mild discrimination impairment as measured by incorrect responses but were able to learn the paradigm to similar levels as controls. During reversal, HIE mice required significantly more total trials, errors and correction trials across the paradigm. Analysis of specific stages showed that reversal impairments in HIE were driven by significant increases in all measured parameters during the late learning, striatal-mediated portion of the task. Together, these results support the concept that HIE occurring during the neonatal period results in abnormal neurodevelopment that persists into adulthood, which can impact efficient associated learning. Further, these data show that utilization of an established model of HIE coupled with touchscreen learning provides valuable information for screening therapeutic interventions that could mitigate these deficits to improve the long-term outcomes of this vulnerable population.

A psychiatric disease-related circular RNA controls synaptic gene expression and cognition.

Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Here, we show that circHomer1a, a neuronal-enriched circRNA abundantly expressed in the frontal cortex, derived from Homer protein homolog 1 (HOMER1), is significantly reduced in both the prefrontal cortex (PFC) and induced pluripotent stem cell-derived neuronal cultures from patients with schizophrenia (SCZ) and bipolar disorder (BD). Moreover, alterations in circHomer1a were positively associated with the age of onset of SCZ in both the dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex (OFC). No correlations between the age of onset of SCZ and linear HOMER1 mRNA were observed, whose expression was mostly unaltered in BD and SCZ postmortem brain. Using in vivo circRNA-specific knockdown of circHomer1a in mouse PFC, we show that it modulates the expression of numerous alternative mRNA transcripts from genes involved in synaptic plasticity and psychiatric disease. Intriguingly, in vivo circHomer1a knockdown in mouse OFC resulted in specific deficits in OFC-mediated cognitive flexibility. Lastly, we demonstrate that the neuronal RNA-binding protein HuD binds to circHomer1a and can influence its synaptic expression in the frontal cortex. Collectively, our data uncover a novel psychiatric disease-associated circRNA that regulates synaptic gene expression and cognitive flexibility.

Increased Maternal Care Rescues Altered Reinstatement Responding Following Moderate Prenatal Alcohol Exposure.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) commonly include deficits in learning, memory, and executive control that can have a severe negative impact on quality of life across the life span. It is still unclear how prenatal alcohol exposure (PAE) affects executive control processes, such as control over reward seeking, that lead to inappropriate behavior later in life. Learning and reinstatement of a previously learned response after extinction is a simple, well-validated measure of both acquisition of a rewarded instrumental response and sensitivity to reward and reward-associated cues. We investigated the effects of PAE on learning, extinction, and reinstatement of a simple instrumental response for food reward. Next, we assessed the effectiveness of an early intervention, communal nest (CN) housing, on increased reinstatement of an extinguished response seen after PAE. METHODS: To assess the effects of PAE on control over reward seeking, we tested male and female PAE and saccharine (SAC) controls raised in a standard nest (SN) on the acquisition, extinction, and food reward-induced reinstatement of an instrumental response utilizing a touch screen-based paradigm. Next, in order to examine the effects of an early-life intervention on these behaviors, we tested PAE and SAC mice raised in a CN early-life environment on these behaviors. RESULTS: PAE mice readily acquired and extinguished a simple touch response to a white square stimulus. However, PAE mice showed significantly increased and persistent reinstatement compared to controls. Increased maternal care via rearing in CN slowed acquisition and sped extinction learning and rescued the significantly increased reinstatement responding in PAE mice. CONCLUSIONS: Together these results demonstrate that even moderate PAE is sufficient to alter control over reward seeking as measured by reinstatement. Importantly, an early-life intervention previously shown to improve cognitive outcomes in PAE mice was sufficient to ameliorate this effect.

Hippocampal to basal forebrain transport of Mn2+ is impaired by deletion of KLC1, a subunit of the conventional kinesin microtubule-based motor.

Microtubule-based motors carry cargo back and forth between the synaptic region and the cell body. Defects in axonal transport result in peripheral neuropathies, some of which are caused by mutations in KIF5A, a gene encoding one of the heavy chain isoforms of conventional kinesin-1. Some mutations in KIF5A also cause severe central nervous system defects in humans. While transport dynamics in the peripheral nervous system have been well characterized experimentally, transport in the central nervous system is less experimentally accessible and until now not well described. Here we apply manganese-enhanced magnetic resonance (MEMRI) to study transport dynamics within the central nervous system, focusing on the hippocampal-forebrain circuit, and comparing kinesin-1 light chain 1 knock-out (KLC-KO) mice with age-matched wild-type littermates. We injected Mn2+ into CA3 of the posterior hippocampus and imaged axonal transport in vivo by capturing whole-brain 3D magnetic resonance images (MRI) in living mice at discrete time-points after injection. Precise placement of the injection site was monitored in both MR images and in histologic sections. Mn2+-induced intensity progressed along fiber tracts (fimbria and fornix) in both genotypes to the medial septal nuclei (MSN), correlating in location with the traditional histologic tract tracer, rhodamine dextran. Pairwise statistical parametric mapping (SPM) comparing intensities at successive time-points within genotype revealed Mn2+-enhanced MR signal as it proceeded from the injection site into the forebrain, the expected projection from CA3. By region of interest (ROI) analysis of the MSN, wide variation between individuals in each genotype was found. Despite this statistically significant intensity increases in the MSN at 6h post-injection was found in both genotypes, albeit less so in the KLC-KO. While the average accumulation at 6h was less in the KLC-KO, the difference between genotypes did not reach significance. Projections of SPM T-maps for each genotype onto the same grayscale image revealed differences in the anatomical location of significant voxels. Although KLC-KO mice had smaller brains than wild-type, the gross anatomy was normal with no apparent loss of septal cholinergic neurons. Hence anatomy alone does not explain the differences in SPM maps. We conclude that kinesin-1 defects may have only a minor effect on the rate and distribution of transported Mn2+ within the living brain. This impairment is less than expected for this abundant microtubule-based motor, yet such defects could still be functionally significant, resulting in cognitive/emotional dysfunction due to decreased replenishments of synaptic vesicles or mitochondria during synaptic activity. This study demonstrates the power of MEMRI to observe and measure vesicular transport dynamics in the central nervous system that may result from or lead to brain pathology.