Atp1a2 and Kcnj9 are candidate genes underlying oxycodone behavioral sensitivity and withdrawal in C57BL/6 substrains.

Opioid use disorder is heritable, yet its genetic etiology is largely unknown. Analysis of addiction model traits in rodents (e.g., opioid behavioral sensitivity and withdrawal) can facilitate genetic and mechanistic discovery. C57BL/6J and C57BL/6NJ substrains have extremely limited genetic diversity, yet can show reliable phenotypic diversity which together, can facilitate gene discovery. The C57BL/6NJ substrain was less sensitive to oxycodone (OXY)-induced locomotor activity compared to the C57BL/6J substrain. Quantitative trait locus (QTL) mapping in an F2 cross identified a distal chromosome 1 QTL explaining 7-12% of the variance in OXY locomotor sensitivity and anxiety-like withdrawal in the elevated plus maze. We identified a second QTL for withdrawal on chromosome 5 near the candidate gene Gabra2 (alpha-2 subunit of GABA-A receptor) explaining 9% of the variance. Next, we generated recombinant lines from an F2 founder spanning the distal chromosome 1 locus (163-181 Mb), captured the QTL for OXY sensitivity and withdrawal, and fine-mapped a 2.45-Mb region (170.16-172.61 Mb). There were five striatal cis-eQTL transcripts in this region (Pcp4l1, Ncstn, Atp1a2, Kcnj9, Igsf9), two of which were confirmed at the protein level (KCNJ9, ATP1A2). Kcnj9, a.k.a., GIRK3, codes for a potassium channel that is a major effector of mu opioid receptor signaling. Atp1a2 codes for a subunit of a Na+/K+ ATPase enzyme that regulates neuronal excitability and shows adaptations following chronic opioid administration. To summarize, we identified genetic sources of opioid behavioral differences in C57BL/6 substrains, two of the most widely and often interchangeably used substrains in opioid addiction research.

Longitudinal single-cell transcriptional dynamics throughout neurodegeneration in SCA1.

Neurodegeneration is a protracted process involving progressive changes in myriad cell types that ultimately results in the death of vulnerable neuronal populations. To dissect how individual cell types within a heterogeneous tissue contribute to the pathogenesis and progression of a neurodegenerative disorder, we performed longitudinal single-nucleus RNA sequencing of mouse and human spinocerebellar ataxia type 1 (SCA1) cerebellar tissue, establishing continuous dynamic trajectories of each cell population. Importantly, we defined the precise transcriptional changes that precede loss of Purkinje cells and, for the first time, identified robust early transcriptional dysregulation in unipolar brush cells and oligodendroglia. Finally, we applied a deep learning method to predict disease state accurately and identified specific features that enable accurate distinction of wild-type and SCA1 cells. Together, this work reveals new roles for diverse cerebellar cell types in SCA1 and provides a generalizable analysis framework for studying neurodegeneration.

Dysregulation of alternative splicing in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 is caused by an expansion of the polyglutamine tract in ATAXIN-1. Ataxin-1 is broadly expressed throughout the brain and is involved in regulating gene expression. However, it is not yet known if mutant ataxin-1 can impact the regulation of alternative splicing events. We performed RNA sequencing in mouse models of spinocerebellar ataxia type 1 and identified that mutant ataxin-1 expression abnormally leads to diverse splicing events in the mouse cerebellum of spinocerebellar ataxia type 1. We found that the diverse splicing events occurred in a predominantly cell autonomous manner. A majority of the transcripts with misregulated alternative splicing events were previously unknown, thus allowing us to identify overall new biological pathways that are distinctive to those affected by differential gene expression in spinocerebellar ataxia type 1. We also provide evidence that the splicing factor Rbfox1 mediates the effect of mutant ataxin-1 on misregulated alternative splicing and that genetic manipulation of Rbfox1 expression modifies neurodegenerative phenotypes in a Drosophila model of spinocerebellar ataxia type 1 in vivo. Together, this study provides novel molecular mechanistic insight into the pathogenesis of spinocerebellar ataxia type 1 and identifies potential therapeutic strategies for spinocerebellar ataxia type 1.

Reduction of nemo-like kinase increases lysosome biogenesis and ameliorates TDP-43-related neurodegeneration.

Protein aggregation is a hallmark of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although mutations in TARDBP, encoding transactive response DNA-binding protein 43 kDa (TDP-43), account for less than 1% of all ALS cases, TDP-43-positive aggregates are present in nearly all ALS patients, including patients with sporadic ALS (sALS) or carrying other familial ALS-causing (fALS-causing) mutations. Interestingly, TDP-43 inclusions are also present in subsets of patients with frontotemporal dementia, Alzheimer's disease, and Parkinson's disease; therefore, methods of activating intracellular protein quality control machinery capable of clearing toxic cytoplasmic TDP-43 species may alleviate disease-related phenotypes. Here, we identify a function of nemo-like kinase (Nlk) as a negative regulator of lysosome biogenesis. Genetic or pharmacological reduction of Nlk increased lysosome formation and improved clearance of aggregated TDP-43. Furthermore, Nlk reduction ameliorated pathological, behavioral, and life span deficits in 2 distinct mouse models of TDP-43 proteinopathy. Because many toxic proteins can be cleared through the autophagy/lysosome pathway, targeted reduction of Nlk represents a potential approach to therapy development for multiple neurodegenerative disorders.

Identifying Disease Signatures in the Spinocerebellar Ataxia Type 1 Mouse Cortex.

The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is known to lead to the progressive degeneration of specific neuronal populations, including cerebellar Purkinje cells (PCs), brainstem cranial nerve nuclei and inferior olive nuclei, and spinocerebellar tracts. The disease-causing protein ataxin-1 is fairly ubiquitously expressed throughout the brain and spinal cord, but most studies have primarily focused on the role of ataxin-1 in the cerebellum and brainstem. Therefore, the functions of ataxin-1 and the effects of SCA1 mutations in other brain regions including the cortex are not well-known. Here, we characterized pathology in the motor cortex of a SCA1 mouse model and performed RNA sequencing in this brain region to investigate the impact of mutant ataxin-1 towards transcriptomic alterations. We identified progressive cortical pathology and significant transcriptomic changes in the motor cortex of a SCA1 mouse model. We also identified progressive, region-specific, colocalization of p62 protein with mutant ataxin-1 aggregates in broad brain regions, but not the cerebellum or brainstem. A cross-regional comparison of the SCA1 cortical and cerebellar transcriptomic changes identified both common and unique gene expression changes between the two regions, including shared synaptic dysfunction and region-specific kinase regulation. These findings suggest that the cortex is progressively impacted via both shared and region-specific mechanisms in SCA1.

Differential effects of Wnt-ß-catenin signaling in Purkinje cells and Bergmann glia in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease characterized by progressive ataxia and degeneration of specific neuronal populations, including Purkinje cells (PCs) in the cerebellum. Previous studies have demonstrated a critical role for various evolutionarily conserved signaling pathways in cerebellar patterning, such as the Wnt-ß-catenin pathway; however, the roles of these pathways in adult cerebellar function and cerebellar neurodegeneration are largely unknown. In this study, we found that Wnt-ß-catenin signaling activity was progressively enhanced in multiple cell types in the adult SCA1 mouse cerebellum, and that activation of this signaling occurs in an ataxin-1 polyglutamine (polyQ) expansion-dependent manner. Genetic manipulation of the Wnt-ß-catenin signaling pathway in specific cerebellar cell populations revealed that activation of Wnt-ß-catenin signaling in PCs alone was not sufficient to induce SCA1-like phenotypes, while its activation in astrocytes, including Bergmann glia (BG), resulted in gliosis and disrupted BG localization, which was replicated in SCA1 mouse models. Our studies identify a mechanism in which polyQ-expanded ataxin-1 positively regulates Wnt-ß-catenin signaling and demonstrate that different cell types have distinct responses to the enhanced Wnt-ß-catenin signaling in the SCA1 cerebellum, underscoring an important role of BG in SCA1 pathogenesis.

The extra-cerebellar effects of spinocerebellar ataxia type 1 (SCA1): looking beyond the cerebellum.

Spinocerebellar ataxia type 1 (SCA1) is one of nine polyglutamine (polyQ) diseases and is characterized as an adult late-onset, progressive, dominantly inherited genetic disease. SCA1 is caused by an increase in the number of CAG repeats in the ATXN1 gene leading to an expanded polyQ tract in the ATAXIN-1 protein. ATAXIN-1 is broadly expressed throughout the brain. However, until recently, SCA1 research has primarily centered on the cerebellum, given the characteristic cerebellar Purkinje cell loss observed in patients, as well as the progressive motor deficits, including gait and limb incoordination, that SCA1 patients present with. There are, however, also other symptoms such as respiratory problems, cognitive defects and memory impairment, anxiety, and depression observed in SCA1 patients and mouse models, which indicate that there are extra-cerebellar effects of SCA1 that cannot be explained solely through changes in the cerebellar region of the brain alone. The existing gap between human and mouse model studies of extra-cerebellar regions in SCA1 makes it difficult to answer many important questions in the field. This review will cover both the cerebellar and extra-cerebellar effects of SCA1 and highlight the need for further investigations into the impact of mutant ATXN1 expression in these regions. This review will also discuss implications of extra-cerebellar effects not only for SCA1 but other neurodegenerative diseases showing diverse pathology as well.

Exploring the Role of Posttranslational Modifications in Spinal and Bulbar Muscular Atrophy.

Spinal and Bulbar Muscular Atrophy (SBMA) is an X-linked adult-onset progressive neuromuscular disease that affects the spinal and bulbar motor neurons and skeletal muscles. SBMA is caused by expansion of polymorphic CAG trinucleotide repeats in the Androgen Receptor (AR) gene, resulting in expanded glutamine tract in the AR protein. Polyglutamine (polyQ) expansion renders the mutant AR protein toxic, resulting in the formation of mutant protein aggregates and cell death. This classifies SBMA as one of the nine known polyQ diseases. Like other polyQ disorders, the expansion of the polyQ tract in the AR protein is the main genetic cause of the disease; however, multiple other mechanisms besides the polyQ tract expansion also contribute to the SBMA disease pathophysiology. Posttranslational modifications (PTMs), including phosphorylation, acetylation, methylation, ubiquitination, and SUMOylation are a category of mechanisms by which the functionality of AR has been found to be significantly modulated and can alter the neurotoxicity of SBMA. This review summarizes the different PTMs and their effects in regulating the AR function and discusses their pathogenic or protective roles in context of SBMA. This review also includes the therapeutic approaches that target the PTMs of AR in an effort to reduce the mutant AR-mediated toxicity in SBMA.

Microglia regulate brain progranulin levels through the endocytosis/lysosomal pathway.

Genetic variants in Granulin (GRN), which encodes the secreted glycoprotein progranulin (PGRN), are associated with several neurodegenerative diseases, including frontotemporal lobar degeneration, neuronal ceroid lipofuscinosis, and Alzheimer's disease. These genetic alterations manifest in pathological changes due to a reduction of PGRN expression; therefore, identifying factors that can modulate PGRN levels in vivo would enhance our understanding of PGRN in neurodegeneration and could reveal novel potential therapeutic targets. Here, we report that modulation of the endocytosis/lysosomal pathway via reduction of Nemo-like kinase (Nlk) in microglia, but not in neurons, can alter total brain Pgrn levels in mice. We demonstrate that Nlk reduction promotes Pgrn degradation by enhancing its trafficking through the endocytosis/lysosomal pathway, specifically in microglia. Furthermore, genetic interaction studies in mice showed that Nlk heterozygosity in Grn haploinsufficient mice further reduces Pgrn levels and induces neuropathological phenotypes associated with PGRN deficiency. Our results reveal a mechanism for Pgrn level regulation in the brain through the active catabolism by microglia and provide insights into the pathophysiology of PGRN-associated diseases.

Systems genetic analysis of binge-like eating in a C57BL/6J x DBA/2J-F2 cross.

Binge eating is a heritable trait associated with eating disorders and refers to the rapid consumption of a large quantity of energy-dense food that is, associated with loss of control and negative affect. Binge eating disorder is the most common eating disorder in the United States; however, the genetic basis is unknown. We previously identified robust mouse inbred strain differences between C57BL/6J and DBA/2J in binge-like eating of sweetened palatable food in an intermittent access, conditioned place preference paradigm. To map the genetic basis of changes in body weight and binge-like eating (BLE) and to identify candidate genes, we conducted quantitative trait locus (QTL) analysis in 128 C57BL/6J x DBA/2J-F2 mice combined with PheQTL and trait covariance analysis in GeneNetwork2 using legacy BXD-RI trait datasets. We identified a QTL on Chromosome 18 influencing changes in body weight across days in females (log of the odds [LOD] = 6.3; 1.5-LOD: 3-12 cM) that contains the candidate gene Zeb1. We also identified a sex-combined QTL influencing initial palatable food intake on Chromosome 5 (LOD = 5.8; 1.5-LOD: 21-28 cM) that contains the candidate gene Lcorl and a second QTL influencing escalated palatable food intake on Chromosome 6 in males (LOD = 5.4; 1.5-LOD: 50-59 cM) that contains the candidate genes Adipor2 and Plxnd1. Finally, we identified a suggestive QTL in females for slope of BLE on distal Chromosome 18 (LOD = 4.1; p = 0.055; 1.5-LOD: 23-35 cM). Future studies will use BXD-RI strains to fine map loci and support candidate gene nomination for gene editing.

Sex-dependent effects of an Hnrnph1 mutation on fentanyl addiction-relevant behaviors but not antinociception in mice.

Opioid Use Disorder (OUD) and opioid-related deaths remain a major public health concern in the United States. Both environmental and genetic factors influence risk for OUD. We previously identified Hnrnph1 as a quantitative trait gene underlying the stimulant, rewarding, and reinforcing properties of methamphetamine. Prior work shows that hnRNP H1, the RNA-binding protein encoded by Hnrnph1, post-transcriptionally regulates Oprm1 (mu opioid receptor gene)-the primary molecular target for the therapeutic and addictive properties of opioids. Because genetic variants can exert pleiotropic effects on behaviors induced by multiple drugs of abuse, in the current study, we tested the hypothesis that Hnrnph1 mutants would show reduced behavioral sensitivity to the mu opioid receptor agonist fentanyl. Hnrnph1 mutants showed reduced sensitivity to fentanyl-induced locomotor activity, along with a female-specific reduction in, and a male-specific induction of, locomotor sensitization following three, daily injections (0.2 mg/kg, i.p.). Hnrnph1 mutants also required a higher dose of fentanyl to exhibit opioid reward as measured via conditioned place preference (CPP). Male Hnrnph1 mutants showed reduced fentanyl reinforcement. Hnrnph1 mutants also showed reduced sucrose motivation, suggesting a reward deficit. No genotypic differences were observed in baseline thermal nociception, fentanyl-induced antinociception, physical or negative affective signs of opioid dependence, or in sensorimotor gating. In the context of our prior work, these findings suggest that Hnrnph1 dysfunction exerts a selective role in reducing the addiction liability to drugs of abuse (opioids and psychostimulants), which could provide new biological pathways to improve their therapeutic profiles.

5' UTR variants in the quantitative trait gene Hnrnph1 support reduced 5' UTR usage and hnRNP H protein as a molecular mechanism underlying reduced methamphetamine sensitivity.

We previously identified a 210 kb region on chromosome 11 (50.37-50.58 Mb, mm10) containing two protein-coding genes (Hnrnph1, Rufy1) that was necessary for reduced methamphetamine-induced locomotor activity in C57BL/6J congenic mice harboring DBA/2J polymorphisms. Gene editing of a small deletion in the first coding exon supported Hnrnph1 as a quantitative trait gene. We have since shown that Hnrnph1 mutants also exhibit reduced methamphetamine-induced reward, reinforcement, and dopamine release. However, the quantitative trait variants (QTVs) that modulate Hnrnph1 function at the molecular level are not known. Nine single nucleotide polymorphisms and seven indels distinguish C57BL/6J from DBA/2J within Hnrnph1, including four variants within the 5' untranslated region (UTR). Here, we show that a 114 kb introgressed region containing Hnrnph1 and Rufy1 was sufficient to cause a decrease in MA-induced locomotor activity. Gene-level transcriptome analysis of striatal tissue from 114 kb congenics vs Hnrnph1 mutants identified a nearly perfect correlation of fold-change in expression for those differentially expressed genes that were common to both mouse lines, indicating functionally similar effects on the transcriptome and behavior. Exon-level analysis (including noncoding exons) revealed decreased 5' UTR usage of Hnrnph1 and immunoblot analysis identified a corresponding decrease in hnRNP H protein in 114 kb congenic mice. Molecular cloning of the Hnrnph1 5' UTR containing all four variants (but none of them individually) upstream of a reporter induced a decrease in reporter signal in both HEK293 and N2a cells, thus, identifying a set of QTVs underlying molecular regulation of Hnrnph1.

A Mutation in Hnrnph1 That Decreases Methamphetamine-Induced Reinforcement, Reward, and Dopamine Release and Increases Synaptosomal hnRNP H and Mitochondrial Proteins.

Individual variation in the addiction liability of amphetamines has a heritable genetic component. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying decreased methamphetamine-induced locomotor activity in mice. Here, we showed that mice (both females and males) with a heterozygous mutation in the first coding exon of Hnrnph1 (H1+/-) showed reduced methamphetamine reinforcement and intake and dose-dependent changes in methamphetamine reward as measured via conditioned place preference. Furthermore, H1+/- mice showed a robust decrease in methamphetamine-induced dopamine release in the NAc with no change in baseline extracellular dopamine, striatal whole-tissue dopamine, dopamine transporter protein, dopamine uptake, or striatal methamphetamine and amphetamine metabolite levels. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for TH did not reveal any major changes in staining intensity, cell number, or forebrain puncta counts. Surprisingly, there was a twofold increase in hnRNP H protein in the striatal synaptosome of H1+/- mice with no change in whole-tissue levels. To gain insight into the mechanisms linking increased synaptic hnRNP H with decreased methamphetamine-induced dopamine release and behaviors, synaptosomal proteomic analysis identified an increased baseline abundance of several mitochondrial complex I and V proteins that rapidly decreased at 30 min after methamphetamine administration in H1+/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in WT mice showed a rapid increase. We conclude that H1+/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.SIGNIFICANCE STATEMENT Methamphetamine dependence is a significant public health concern with no FDA-approved treatment. We discovered a role for the RNA binding protein hnRNP H in methamphetamine reward and reinforcement. Hnrnph1 mutation also blunted methamphetamine-induced dopamine release in the NAc, a key neurochemical event contributing to methamphetamine addiction liability. Finally, Hnrnph1 mutants showed a marked increase in basal level of synaptosomal hnRNP H and mitochondrial proteins that decreased in response to methamphetamine, whereas WT mice showed a methamphetamine-induced increase in synaptosomal mitochondrial proteins. Thus, we identified a potential role for hnRNP H in basal and dynamic mitochondrial function that informs methamphetamine-induced cellular adaptations associated with reduced addiction liability.

Genetic differences in the behavioral organization of binge eating, conditioned food reward, and compulsive-like eating in C57BL/6J and DBA/2J strains.

Binge eating (BE) is a heritable symptom of eating disorders associated with anxiety, depression, malnutrition, and obesity. Genetic analysis of BE could facilitate therapeutic discovery. We used an intermittent, limited access BE paradigm involving sweetened palatable food (PF) to examine genetic differences in BE, conditioned food reward, and compulsive-like eating between C57BL/6J (B6J) and DBA/2J (D2J) inbred mouse strains. D2J mice showed a robust escalation in intake and conditioned place preference for the PF-paired side. D2J mice also showed a unique style of compulsive-like eating in the light/dark conflict test where they rapidly hoarded and consumed PF in the preferred unlit environment. BE and compulsive-like eating exhibited narrow-sense heritability estimates between 56 and 73%. To gain insight into the genetic basis, we phenotyped and genotyped a small cohort of 133 B6J × D2J-F2 mice at the peak location of three quantitative trait loci (QTL) previously identified in F2 mice for sweet taste (chromosome 4: 156 Mb), bitter taste (chromosome 6: 133 Mb) and behavioral sensitivity to drugs of abuse (chromosome 11: 50 Mb). The D2J allele on chromosome 6 was associated with greater PF intake on training days and greater compulsive-like PF intake, but only in males, suggesting that decreased bitter taste may increase BE in males. The D2J allele on chromosome 11 was associated with an increase in final PF intake and slope of escalation across days. Future studies employing larger crosses and genetic reference panels comprising B6J and D2J alleles will identify causal genes and neurobiological mechanisms.

Changes in neuronal immunofluorescence in the C- versus N-terminal domains of hnRNP H following D1 dopamine receptor activation.

RNA binding proteins are a diverse class of proteins that regulate all aspects of RNA metabolism. Accumulating studies indicate that heterogeneous nuclear ribonucleoproteins are associated with cellular adaptations in response to drugs of abuse. We recently mapped and validated heterogeneous nuclear ribonucleoprotein H1 (Hnrnph1) as a quantitative trait gene underlying differential behavioral sensitivity to methamphetamine. The molecular mechanisms by which hnRNP H1 alters methamphetamine behaviors are unknown but could involve pre- and/or post-synaptic changes in protein localization and function. Methamphetamine initiates post-synaptic D1 dopamine receptor signaling indirectly by binding to pre-synaptic dopamine transporters and vesicular monoamine transporters of midbrain dopaminergic neurons which triggers reverse transport and accumulation of dopamine at the synapse. Here, we examined changes in neuronal localization of hnRNP H in primary rat cortical neurons that express dopamine receptors that can be modulated by the D1 or D2 dopamine receptor agonists SKF38393 and (-)-Quinpirole HCl, respectively. Basal immunostaining of hnRNP H was localized primarily to the nucleus. D1 dopamine receptor activation induced an increase in hnRNP H nuclear immunostaining as detected by immunocytochemistry with a C-domain directed antibody containing epitope near the glycine-rich domain but not with an N-domain specific antibody. Although there was no change in hnRNP H protein in the nucleus or cytoplasm, there was a decrease in Hnrnph1 transcript following D1 receptor stimulation. Taken together, these results suggest that D1 receptor activation increases availability of the hnRNP H C-terminal epitope, which could potentially reflect changes in protein-protein interactions. Thus, D1 receptor signaling could represent a key molecular post-synaptic event linking Hnrnph1 polymorphisms to drug-induced behavior.

Cytoplasmic FMR1-Interacting Protein 2 Is a Major Genetic Factor Underlying Binge Eating.

BACKGROUND: Eating disorders are lethal and heritable; however, the underlying genetic factors are unknown. Binge eating is a highly heritable trait associated with eating disorders that is comorbid with mood and substance use disorders. Therefore, understanding its genetic basis will inform therapeutic development that could improve several comorbid neuropsychiatric conditions. METHODS: We assessed binge eating in closely related C57BL/6 mouse substrains and in an F2 cross to identify quantitative trait loci associated with binge eating. We used gene targeting to validate candidate genetic factors. Finally, we used transcriptome analysis of the striatum via messenger RNA sequencing to identify the premorbid transcriptome and the binge-induced transcriptome to inform molecular mechanisms mediating binge eating susceptibility and establishment. RESULTS: C57BL/6NJ but not C57BL/6J mice showed rapid and robust escalation in palatable food consumption. We mapped a single genome-wide significant quantitative trait locus on chromosome 11 (logarithm of the odds = 7.4) to a missense mutation in cytoplasmic FMR1-interacting protein 2 (Cyfip2). We validated Cyfip2 as a major genetic factor underlying binge eating in heterozygous knockout mice on a C57BL/6N background that showed reduced binge eating toward a wild-type C57BL/6J-like level. Transcriptome analysis of premorbid genetic risk identified the enrichment terms morphine addiction and retrograde endocannabinoid signaling, whereas binge eating resulted in the downregulation of a gene set enriched for decreased myelination, oligodendrocyte differentiation, and expression. CONCLUSIONS: We identified Cyfip2 as a major significant genetic factor underlying binge eating and provide a behavioral paradigm for future genome-wide association studies in populations with increased genetic complexity.