Biology subjects

Tischfield, J. A.

Publications and source records attributed to Tischfield, J. A..

8 recordsLinked to original sources

bioRxiv2026

Identification of alcohol use disorder-associated and ethanol-responsive proteins and candidate biomarkers using human cortical organoids

Genome-wide association studies of alcohol use disorder (AUD) have identified >100 loci, yet the functional impact and the downstream molecular mechanisms are not well understood. As functional effectors of genes, proteins provide direct insight into disease mechanisms. However, most proteomic alterations in postmortem human brains of AUD likely reflect a combination of inherited susceptibility (AUD-associated), the consequences of alcohol exposure (ethanol-responsive), other environmental and lifestyle factors, as well as sample preparation. Here, we profiled the proteome of human microglia-containing cortical organoids derived from human induced pluripotent stem cells from individuals with AUD (n=11) and without (n=5), paired with or without intermittent ethanol exposure. To maximize statistical power, donors were selected based on high or low genetic liability for AUD as measured by polygenic scores. Among 8,952 proteins that passed quality control, 1,038 were AUD-associated and 718 were ethanol-responsive. AUD-associated proteins were enriched for neuronal signaling, immune, mitochondrial, and extracellular matrix pathways, whereas ethanol-responsive proteins were enriched for RNA processing, ribosome biogenesis, protein translation, vesicle trafficking, and membrane transport pathways. Twenty ethanol-responsive proteins were replicated in a postmortem human prefrontal cortex proteomic dataset. Using UK Biobank plasma proteomics data, we identified 26 candidate AUD-associated biomarkers and 9 candidate ethanol-responsive biomarkers. We also found that major proteomic variation was significantly correlated with AUD polygenic scores. Together, these findings reveal distinct proteomic signatures of inherited susceptibility to AUD and ethanol exposure, providing new insights into mechanisms underlying AUD and identifying candidate circulating biomarkers for further investigation.

cell biology↗

bioRxiv2024

Integrated Single-Cell Multiomic Profiling of Caudate Nucleus Suggests Key Mechanisms in Alcohol Use Disorder

Alcohol use disorder (AUD) induces complex transcriptional and regulatory changes across multiple brain regions including the caudate nucleus, which remains understudied. Using paired single-nucleus RNA-seq and ATAC-seq on caudate samples from 143 human postmortem brains, including 74 with AUD, we identified 17 distinct cell types. We found that a significant portion of the alcohol-induced changes in gene expression occurred through altered chromatin accessibility. Notably, we identified novel transcriptional and chromatin accessibility differences in medium spiny neurons, impacting pathways such as RNA metabolism and immune response. A small cluster of D1/D2 hybrid neurons showed distinct differences, suggesting a unique role in AUD. Microglia exhibited distinct activation states deviating from classical M1/M2 designations, and astrocytes entered a reactive state partially regulated by JUND, affecting glutamatergic synapse pathways. Oligodendrocyte dysregulation, driven in part by OLIG2, was linked to demyelination and increased TGF-{beta}1 signaling from microglia and astrocytes. We also observed increased microglia-astrocyte communication via the IL-1{beta} pathway. Leveraging our multiomic data, we performed cell type-specific expression quantitative trait loci analysis, integrating that with public genome-wide association studies to identify AUD risk genes such as ADAL and PPP2R3C, providing a direct link between genetic variants, chromatin accessibility, and gene expression in AUD. These findings not only provide new insights into the genetic and cellular mechanisms in the caudate related to AUD but also demonstrate the broader utility of large-scale multiomic studies in uncovering complex gene regulation across diverse cell types, which has implications beyond the substance use field.

neuroscience↗

bioRxiv2024

Functional 3'-UTR Variants Identify Regulatory Mechanisms Impacting Alcohol Use Disorder and Related Traits

Although genome-wide association studies (GWAS) have identified loci associated with alcohol consumption and alcohol use disorder (AUD), they do not identify which variants are functional. To approach this, we evaluated the impact of variants in 3 untranslated regions (3-UTRs) of genes in loci associated with substance use and neurological disorders using a massively parallel reporter assay (MPRA) in neuroblastoma and microglia cells. Functionally impactful variants explained a higher proportion of heritability of alcohol traits than non-functional variants. We identified genes whose 3-UTR activities are associated with AUD and alcohol consumption by combining variant effects from MPRA with GWAS results. We examined their effects by evaluating gene expression after CRISPR inhibition of neuronal cells and stratifying brain tissue samples by MPRA-derived 3-UTR activity. A pathway analysis of differentially expressed genes identified inflammation response pathways. These analyses suggest that variation in response to inflammation contributes to the propensity to increase alcohol consumption.

genomics↗

bioRxiv2023

Human mutations in high-confidence Tourette disorder genes affect sensorimotor behavior, reward learning, and striatal dopamine in mice

Tourette disorder (TD) is poorly understood, despite affecting 1/160 children. A lack of animal models possessing construct, face, and predictive validity hinders progress in the field. We used CRISPR/Cas9 genome editing to generate mice with mutations orthologous to human de novo variants in two high-confidence Tourette genes, CELSR3 and WWC1. Mice with human mutations in Celsr3 and Wwc1 exhibit cognitive and/or sensorimotor behavioral phenotypes consistent with TD. Sensorimotor gating deficits, as measured by acoustic prepulse inhibition, occur in both male and female Celsr3 TD models. Wwc1 mice show reduced prepulse inhibition only in females. Repetitive motor behaviors, common to Celsr3 mice and more pronounced in females, include vertical rearing and grooming. Sensorimotor gating deficits and rearing are attenuated by aripiprazole, a partial agonist at dopamine type II receptors. Unsupervised machine learning reveals numerous changes to spontaneous motor behavior and less predictable patterns of movement. Continuous fixed-ratio reinforcement shows Celsr3 TD mice have enhanced motor responding and reward learning. Electrically evoked striatal dopamine release, tested in one model, is greater. Brain development is otherwise grossly normal without signs of striatal interneuron loss. Altogether, mice expressing human mutations in high-confidence TD genes exhibit face and predictive validity. Reduced prepulse inhibition and repetitive motor behaviors are core behavioral phenotypes and are responsive to aripiprazole. Enhanced reward learning and motor responding occurs alongside greater evoked dopamine release. Phenotypes can also vary by sex and show stronger affection in females, an unexpected finding considering males are more frequently affected in TD. Significance StatementWe generated mouse models that express mutations in high-confidence genes linked to Tourette disorder (TD). These models show sensorimotor and cognitive behavioral phenotypes resembling TD-like behaviors. Sensorimotor gating deficits and repetitive motor behaviors are attenuated by drugs that act on dopamine. Reward learning and striatal dopamine is enhanced. Brain development is grossly normal, including cortical layering and patterning of major axon tracts. Further, no signs of striatal interneuron loss are detected. Interestingly, behavioral phenotypes in affected females can be more pronounced than in males, despite male sex bias in the diagnosis of TD. These novel mouse models with construct, face, and predictive validity provide a new resource to study neural substrates that cause tics and related behavioral phenotypes in TD.

neuroscience↗

bioRxiv2023

Upregulated GIRK2 counteracts ethanol-induced changes in excitability and respiration in human neurons

Genome-wide association analysis (GWAS) of electroencephalographic endophenotypes for alcohol use disorder (AUD) has identified non-coding polymorphisms within the KCNJ6 gene. KCNJ6 encodes GIRK2, a subunit of a G protein-coupled inwardly-rectifying potassium channel that regulates neuronal excitability. How changes in GIRK2 affect human neuronal excitability and the response to repeated ethanol exposure is poorly understood. Here, we studied the effect of upregulating KCNJ6 using an isogenic approach with human glutamatergic neurons derived from induced pluripotent stem cells (male and female donors). Using multi-electrode-arrays, population calcium imaging, single-cell patch-clamp electrophysiology, and mitochondrial stress tests, we find that elevated GIRK2 acts in concert with 7-21 days of ethanol exposure to inhibit neuronal activity, to counteract ethanol-induced increases in glutamate response, and to promote an increase intrinsic excitability. Furthermore, elevated GIRK2 prevented ethanol-dependent changes in basal and activity-dependent mitochondrial respiration. These data support a role for GIRK2 in mitigating the effects of ethanol and a previously unknown connection to mitochondrial function in human glutamatergic neurons. SIGNIFICANCE STATEMENTAlcohol use disorder (AUD) is a major health problem that has worsened since COVID, affecting over 100 million people worldwide. While it is known that heritability contributes to AUD, specific genes and their role in neuronal function remain poorly understood, especially in humans. In the current manuscript, we focused on the inwardly-rectifying potassium channel GIRK2, which has been identified in an AUD-endophenotype genome-wide association study. We used human excitatory neurons derived from healthy donors to study the impact of GIRK2 expression. Our results reveal that elevated GIRK2 counteracts ethanol-induced increases in glutamate response and intracellular calcium, as well as deficits in activity-dependent mitochondrial respiration. The role of GIRK2 in mitigating ethanol-induced hyper-glutamatergic and mitochondrial offers therapeutic promise for treating AUD.

neuroscience↗

bioRxiv2022

Alcohol reverses the effects of KCNJ6 (GIRK2) noncoding variants on excitability of human glutamatergic neurons

Synonymous and noncoding single nucleotide polymorphisms (SNPs) in the KCNJ6 gene, encoding G protein-gated inwardly rectifying potassium (GIRK2) channel subunit 2, have been linked with increased electroencephalographic frontal theta event-related oscillations (ERO) in subjects diagnosed with alcohol use disorder (AUD). To identify molecular and cellular mechanisms while retaining the appropriate genetic background, we generated induced excitatory glutamatergic neurons (iN) from iPSCs derived from four AUD-diagnosed subjects with KCNJ6 variants ( Affected: AF) and four control subjects without variants ( Unaffected: UN). Neurons were analyzed for changes in gene expression, morphology, excitability and physiological properties. Single cell RNA sequencing suggests that KCNJ6 AF variant neurons have altered patterns of synaptic transmission and cell projection morphogenesis. Results confirm that AF neurons express lower levels of GIRK2, have greater neurite area, and elevated excitability. Interestingly, exposure to intoxicating concentrations of ethanol induces GIRK2 expression and reverses functional effects in AF neurons. Ectopic overexpression of GIRK2 alone mimics the effect of ethanol to normalize induced excitability. We conclude that KCNJ6 variants decrease GIRK2 expression and increase excitability and that this effect can be minimized or reduced with ethanol.

neuroscience↗

bioRxiv2022

A cadherin mutation in Celsr3 linked to Tourette Disorder affects dendritic patterning and excitability of cholinergic interneurons

CELSR3 encodes an atypical protocadherin cell adhesion receptor that was recently identified as a high-risk gene for Tourette disorder. A putative damaging de novo variant was inserted into the mouse genome to generate an amino acid substitution within the fifth cadherin repeat. By contrast to Celsr3 constitutive null animals, mice homozygous for the R774H amino acid substitution are viable and have grossly normal forebrain development. The density of cortical and striatal interneuron subpopulations is normal, but 3D geometric analysis of cortical pyramidal neurons and striatal cholinergic interneurons revealed changes to dendritic patterning and types and distributions of spines. Furthermore, patch clamp recordings in cholinergic interneurons located within the sensorimotor striatum uncovered mild intrinsic hyperexcitability. Despite these changes, Celsr3R774H homozygous mice do not show obvious tic-like stereotypies at baseline nor motor learning impairments, but females exhibited perseverative digging behavior. Our findings show that a human mutation in CELSR3 linked to Tourette disorder is sufficient to alter dendritic patterning in the cortex and striatum and also the intrinsic excitability of cholinergic interneurons.

neuroscience↗

bioRxiv2018

Addiction associated N40D mu-opioid receptor variant modulates synaptic function in human neurons

BackgroundThe OPRM1 A118G gene variant (N40D) encoding the {micro}-opioid receptor (MOR) has been associated with dependence on opiates and other abused drugs but its mechanism is unknown. With opioid abuse-related deaths rising at unprecedented rates, understanding these mechanisms may provide a path to therapy.\n\nMethodsSeven human induced pluripotent stem (iPS) cell lines from homozygous N40D subjects (4 with N40 and 3 with D40 variants) were generated and human induced neuronal cells (iNs) were derived from these iPS cell lines. Morphological, gene expression as well as synaptic physiology analyses were conducted in human iN cells carrying N40D MOR variants; Two pairs of isogenic pluripotent stem cells carrying N40D were generated using CRISPR/Cas9 genome-editing and iN cells derived from them were analyzed.\n\nResultsInhibitory human neurons generated from subjects carrying N40D MOR gene variants show mature properties in morphological and functional analyses. Gene expression revealed that they express mature neuronal marker and MORs. Activation of MORs suppressed inhibitory synaptic transmission in human neurons carrying both N40 or D40 MOR variants but D40 show stronger effects. To mitigate the confounding effects of background genetic variation on neuronal function, the regulatory effects of MORs on synaptic transmission were validated in two sets of independently generated isogenic N40D iNs.\n\nConclusionsActivations of N40D MOR variants show different regulatory effects on synaptic transmission in inhibitory human neurons. This study identifies neurophysiological differences between human MOR variants that may predict altered opioid responsivity and/or dependence in this subset of individuals.

neuroscience↗