Biology subjects

Wolozin, B.

Publications and source records attributed to Wolozin, B..

18 recordsLinked to original sources

bioRxiv2026

The loss of Tau in the adult brain triggers neuroplastic, epigenetic and behavioral deficits

INTRODUCTIONAccumulation of pathological Tau precipitates neuronal malfunction in Alzheimers disease (AD). However, the impact of loss of normal Tau function in the adult brain, independently of Tau aggregates, remains unclarified. METHODSWe used a mouse model with conditional mapt knocking-down in forebrain of 5-7 months old animals (cTau-KO), a virus-driven selective Tau knockdown in wild-types (WT) and Tau re-expression approaches, accompanied by neurostructural, epigenetic, proteomic, electrophysiological, neurochemical and behavioral analyses. RESULTSLoss of Tau in the adult brain of cTau-KOs triggers neuronal atrophy and malfunction, epigenetic as well cognitive and mood deficits. Importantly, these perturbations were confirmed by selective Tau loss in WT adult brain and reverted by Tau re-expression or pharmacologically-induced epigenetic correction in cTau-KOs. DISCUSSIONOur findings highlight the contribution of loss of normal Tau function in the adult brain malfunction that could be relevant in diverse brain pathologies beyond AD, associated to Tau and its dysfunction.

neuroscience↗

bioRxiv2025

Nanobodies targeting hnRNPA2/B1 and tau

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is an RNA-binding protein that mislocalizes to the cytoplasm and forms stress-induced granules in tauopathy and multisystem proteinopathy. It also preferentially interacts with oligomeric tau and is required for tau-mediated neurodegeneration in a mouse model of Alzheimers disease. To study endogenous hnRNPA2/B1 and tau, we generated nanobodies that specifically recognize these proteins. We screened yeast surface display nanobody libraries using an avidity-enhanced screening strategy that enabled selection of binders against short peptide ligands. This led to isolation of anti-hnRNPA2/B1 and anti-tau nanobodies with defined epitopes. Directed evolution of the anti-hnRNPA2/B1 nanobody improved binding affinity by over 20-fold but caused cytoplasmic aggregation, demonstrating a tradeoff between affinity and intracellular behavior. Although the final nanobodies retained modest affinities, they showed robust intracellular colocalization with their targets. Furthermore, fusion to ubiquitin ligase adaptor domains significantly decreased hnRNPA2/B1 and tau levels. Collectively, these nanobodies provide valuable tools for studying hnRNPA2/B1 and tau dynamics in their native cellular context.

bioengineering↗

bioRxiv2025

Progressive Remodeling of Global Protein Interaction Networks in a Mouse Model of Tauopathy

Neurodegenerative disease is marked not just by loss of proteins or cells, but by dynamic rewiring of macromolecular interaction networks that precede and drive pathology. Here, we present the first temporally resolved, systems-scale map of multi-protein complex remodeling in a tauopathy model, integrating co-fractionation mass spectrometry, quantitative phosphoproteomics, and machine learning to decode phosphorylation-dependent shifts in protein interactomes across disease progression. This interactomic atlas identifies functionally validated assemblies--including MAPT-Dpysl2 and Cyfip1-actin complexes--that modulate early disease phenotypes in vivo. By revealing how phosphorylation tunes macromolecular complex architecture and function, this work reframes tauopathy as a disease of dynamic network instability, and establishes a generalizable framework for early detection and mechanistic dissection of neurodegeneration.

neuroscience↗

bioRxiv2025

Distinct age- and pathology-dependent epitranscriptome and translational dysfunction in a tauopathy mouse model

Chronic neurodegenerative diseases, such as tauopathies, cause major metabolic changes in the brain affecting gene expression at both the transcriptional and translational levels. Our understanding of how regulation of translation changes with disease has focused on mRNA and its translational regulatory factors, RNA binding proteins, and microRNAs, despite clear evidence for translational and post-translational dysfunction in ADRD and tauopathies. The neurobiology of tRNA has only recently begun to be studied, but the impact of chronic neurodegenerative diseases on tRNA biology and translational dysfunction is largely unknown. We have previously shown that the tRNA pool and tRNA modifications behave as a system to regulate the cellular stress response by undergoing stress-specific reprogramming and causing selective translation of mRNAs from codon-biased stress response genes. Here we tested this stress-induced tRNA reprogramming and codon-biased translation system in the response to mutant tau expression by performing mass spectrometric quantification of [~]8500 proteins and 49 tRNA modifications, AQRNA-seq analysis of 222 cytosolic and mitochondrial tRNAs and other small RNAs, and informatic analysis of codon usage patterns in >23,000 protein-coding genes. Analysis of these datasets revealed that aging and tauopathy elicit major adaptation of the tRNA transcriptome and epitranscriptome as well as corresponding evidence of a program of translation of families of codon-biased genes for aging and disease responsive proteins. The mitochondrial tRNA transcriptome showed a strong response to aging and disease with 21 of the 22 mt-tRNAs showing age and disease-linked increases in expression, accompanied by mitochondria-specific modifications such as ms2i6A and f5C. Surprisingly, there were few significant changes in the 203 cytosolic tRNA isodecoders. However, a 10-fold increase tRNA isodecoder tRNA-Arg-TCT-5-1 was accompanied by increased translation of proteins encoded by genes highly enriched in its AGA cognate codon. These changes in tRNA biology are mirrored by strongly biased use of synonymous codons among the most highly upregulated and downregulated proteins in the P301S MAPT mice. Taken together these findings suggest the aging and disease brain produces an integrated response for translational control that is highly integrated with changes in tRNA biology.

neuroscience↗

bioRxiv2025

Loss of age-associated increase in m6A-modified RNA contributes to GABAergic dysregulation in Alzheimer's disease

Dysregulated RNA metabolism is a significant feature of Alzheimers disease (AD), yet how post-transcriptional RNA modifications like N6-methyladenosine (m6A) are altered in AD is unknown. Here, we performed deamination adjacent to RNA modification targets (DART-seq) on human dorsolateral prefrontal cortices to assess changes in m6A with nucleotide resolution. In non-AD brains, m6A sites increased with age, predominantly within the 3'UTR of transcripts encoding tripartite synapse proteins. In contrast, AD brains lost the age-associated m6A site increase and exhibited global hypomethylation of transcripts, including MAPT and APP. Hypomethylated genes involved with GABAergic signaling, glutamate transport, and ubiquitin-mediated proteolysis exhibited reduced expression, connecting m6A to synaptic excitotoxicity and disrupted proteostasis in AD. Site-specific m6A levels were linked with GABRA1 expression and protein levels, but this relationship was abolished in AD. Our findings provide insight into post-transcriptional mechanisms of dysregulated RNA metabolism in AD that are related to aging and GABAergic regulation. HIGHLIGHTSO_LIWith age, the number of m6A sites increases among Control cases (lacking AD pathology) but remains unchanged in AD cases. C_LIO_LITranscripts are globally hypomethylated in AD cases. C_LIO_LIHypomethylation is linked to decreased mRNA expression of transcripts related to synaptic and proteostatic function in AD. C_LIO_LI3UTR-localized m6A sites lack typical association with transcript metabolism of GABRA1 in AD. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/651974v3_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@995c55org.highwire.dtl.DTLVardef@7d0619org.highwire.dtl.DTLVardef@1b6a20org.highwire.dtl.DTLVardef@1d47dce_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

TIA1 Mediates Divergent Inflammatory Responses to Tauopathy in Microglia and Macrophages

The RNA binding protein TIA1 is known to regulate stress responses. Here we show that TIA1 plays a much broader role in inflammatory cells, being required for the microglial sensome. We crossed TIA1 cKO mice (using a CX3CR1 driven cre element) to PS19 MAPT P301S tauopathy mice. The peripheral macrophages of TIA1 cKO mice exhibited a hyper-inflammatory phenotype with increased cytokine signaling, as expected. Surprisingly, the brains of these mice showed striking reductions in inflammation, including decreases in microglial inflammatory cytokines (TNF and IL-1{beta}) and sensome markers (CLEC7A, TREM2, ITGAX); these reductions were accompanied by corresponding decreases in tau pathology. Analysis of the brain TIA1 protein interactome identified brain selective TIA1 protein mediated pathways, including strong interactions with the microglial protein C1q, which directs pruning of dystrophic neurons. These results uncover a previously unknown regulatory role for TIA1 in microglial activation in the context of neurodegenerative disease and highlights the divergent regulation of two mononuclear phagocytic lineages: microglia and macrophages.

neuroscience↗

bioRxiv2023

GSK3β phosphorylation catalyzes the aggregation of Tau into Alzheimer's disease-like amyloid strain

The pathological deposition of proteins is a hallmark of several devastating neurodegenerative diseases. These pathological deposits comprise aggregates of proteins that adopt distinct structures named strains. However, the molecular factors responsible for the formation of distinct aggregate strains are unknown. Here we show that the serine/threonine kinase GSK3{beta} catalyzes the aggregation of the protein tau into an Alzheimers disease-like amyloid strain. We demonstrate that phosphorylation by GSK3{beta}, but not by several other kinases, promotes the aggregation of full-length tau through enhanced phase separation into gel-like condensate structures. Cryo-electron microscopy further reveals that the amyloid fibrils formed by GSK3{beta}-phosphorylated tau adopt a fold comparable to that of paired helical filaments isolated from the brains of AD patients. Our results elucidate the intricate relationship between post-translational modification and the formation of tau strains in neurodegenerative diseases.

biophysics↗

bioRxiv2023

Proximity labeling reveals dynamic changes in the SQSTM1 protein network

Sequestosome1 (SQSTM1) is an autophagy receptor that mediates degradation of intracellular cargo, including protein aggregates, through multiple protein interactions. These interactions form the SQSTM1 protein network, and these interactions are mediated by SQSTM1 functional interaction domains, which include LIR, PB1, UBA and KIR. Technological advances in cell biology continue to expand our knowledge of the SQSTM1 protein network and of the relationship of the actions of the SQSTM1 protein network in cellular physiology and disease states. Here we apply proximity profile labeling to investigate the SQSTM1 protein interaction network by fusing TurboID with the human protein SQSTM1 (TurboID::SQSTM1). This chimeric protein displayed well-established SQSTM1 features including production of SQSTM1 intracellular bodies, binding to known SQSTM1 interacting partners, and capture of novel SQSTM1 protein interactors. Strikingly, aggregated tau protein altered the protein interaction network of SQSTM1 to include many stress-associated proteins. We demonstrate the importance of the PB1 and/or UBA domains for binding network members, including the K18 domain of tau. Overall, our work reveals the dynamic landscape of the SQSTM1 protein network and offers a resource to study SQSTM1 function in cellular physiology and disease state.

cell biology↗

bioRxiv2023

Human Herpesvirus 8 ORF57 protein is able to reduce TDP-43 pathology: Network analysis Identifies Interacting Pathways

Aggregation of TAR DNA-binding protein 43kDa (TDP-43) is thought to drive the pathophysiology of ALS and some Frontotemporal dementias. TDP-43 is normally a nuclear protein that in neurons translocates to the cytoplasm and forms insoluble aggregates upon activation of the integrated stress response (ISR). Viruses evolved to control the ISR. In the case of Herpesvirus 8, the protein ORF57 acts to bind protein kinase R, inhibit phosphorylation of eIF2 and reduce activation of the ISR. We hypothesized that ORF57 might also possess the ability to inhibit aggregation of TDP-43. ORF57 was expressed in the neuronal SH-SY5Y line and its effects on TDP-43 aggregation characterized. We report that ORF57 inhibits TDP-43 aggregation by 55% and elicits a 2.45-fold increase in the rate of dispersion of existing TDP-43 granules. These changes were associated with a 50% decrease in cell death. Proteomic studies were carried out to identify the protein interaction network of ORF57. We observed that ORF57 directly binds to TDP-43 as well as interacts with many components of the ISR, including elements of the proteostasis machinery known to reduce TDP-43 aggregation. We propose that viral proteins designed to inhibit a chronic ISR can be engineered to remove aggregated proteins and dampen a chronic ISR.

neuroscience↗

bioRxiv2023

Accumulation of m6A exhibits stronger correlation with MAPT than beta-amyloid pathology in an APP NL-G-F /MAPT P301S mouse model of Alzheimer's disease

The study for the pathophysiology study of Alzheimers disease (AD) has been hampered by lack animal models that recapitulate the major AD pathologies, including extracellular {beta}-amyloid (A{beta}) deposition, intracellular aggregation of microtubule associated protein tau (MAPT), inflammation and neurodegeneration. We now report on a double transgenic APPNL-G-F MAPTP301S mouse that at 6 months of age exhibits robust A{beta} plaque accumulation, intense MAPT pathology, strong inflammation and extensive neurodegeneration. The presence of A{beta} pathology potentiated the other major pathologies, including MAPT pathology, inflammation and neurodegeneration. However, MAPT pathology neither changed levels of amyloid precursor protein nor potentiated A{beta} accumulation. The APPNL-G-F/MAPTP301S mouse model also showed strong accumulation of N6-methyladenosine (m6A), which was recently shown to be elevated in the AD brain. M6A primarily accumulated in neuronal soma, but also co-localized with a subset of astrocytes and microglia. The accumulation of m6A corresponded with increases in METTL3 and decreases in ALKBH5, which are enzymes that add or remove m6A from mRNA, respectively. Thus, the APPNL- G-F/MAPTP301S mouse recapitulates many features of AD pathology beginning at 6 months of aging.

neuroscience↗

bioRxiv2022

Identification of circRNAs linked to Alzheimer's disease and related dementias

We conducted a circular-transcriptome-wide analysis and examined circRNA expression patterns associated with AD and clinical and neuropathological AD severity measures in human hippocampus and cortex brain regions. We validated prior studies of circRNA in AD cortex, and demonstrate novel patterns of expression in the AD hippocampus. We also examined circRNA expression across multiple types of dementia and show that circRNA expression differs by dementia subtype. We demonstrate robust circRNA expression in human neuronal precursor cells (NPCs). Then, using NPCs to study qPCR validated circRNA, we show that exposure to oligomeric tau elicits downregulation of circRNA similar to that observed in AD brain. These data identify circRNA that are changed with AD, and suggest that treating neuronal cells with oligomeric tau can recapitulate some of these changes.

molecular biology↗

bioRxiv2022

Single cell transcriptomic profiling of tauopathy in a novel 3D neuron-astrocyte coculture model

The use of iPSC derived brain organoid models to study neurodegenerative disease has been hampered by a lack of systems that accurately and expeditiously recapitulate pathogenesis in the context of neuron-glial interactions. Here we report development of a system, termed AstTau, which propagates toxic human tau oligomers in iPSC derived neuron-astrocyte spheroids. The AstTau system develops much of the neuronal and astrocytic pathology observed in tauopathies including misfolded, phosphorylated, oligomeric, and fibrillar tau, strong neurodegeneration, and reactive astrogliosis. Single cell transcriptomic profiling combined with immunochemistry characterizes a model system that can more closely recapitulate late-stage changes in adult neurodegeneration. The transcriptomic studies demonstrate striking changes in neuroinflammatory and heat shock protein (HSP) chaperone systems in the disease process. Treatment with the HSP90 inhibitor PU-H71 was used to address the putative dysfunctional HSP chaperone system and produced a strong reduction of pathology and neurodegeneration, highlighting the potential of AstTau as a rapid and reproducible tool for drug discovery.

neuroscience↗

bioRxiv2021

The methamphetamine-induced RNA targetome of hnRNP H in Hnrnph1 mutants showing reduced dopamine release and behavior

Methamphetamine addiction remains a major public health concern in the United States that has paralleled the opioid epidemic. Psychostimulant use disorders have a heritable genetic component that remains unexplained. Methamphetamine targets membrane and vesicular transporters to increase synaptic dopamine, norepinephrine, and serotonin. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene underlying methamphetamine behavioral sensitivity. Hnrnph1 encodes the RNA-binding protein hnRNP H1 that is ubiquitously expressed in neurons throughout the brain. Gene-edited mice with a heterozygous frameshift deletion in Hnrnph1s first coding exon of showed reduced methamphetamine-induced dopamine release and behaviors. To inform the mechanism linking hnRNP H with methamphetamine neurobehavioral effects, we surveyed the mRNA targetome of hnRNP H via cross-linking immunoprecipitation coupled with RNA-sequencing in striatal tissue at baseline and at 30 min post-methamphetamine in wild-type male and female C57BL/6J mice. Methamphetamine induced changes in RNA-binding targets of hnRNP H in mice, including differential binding to 3UTR targets and multiple enriched mRNAs involved in synaptic plasticity. Targetome, transcriptome, and spliceome analyses triangulated on Cacna2d2 as a suggestive target, with differences in hnRNP H binding, gene expression and splicing following methamphetamine treatment (2 mg/kg, i.p.). Furthermore, pre-treatment with pregabalin, an inhibitor of 2{delta}2 and 2{delta}1 voltage-gated calcium channel subunits, attenuated methamphetamine-induced locomotor activity in male and female mice, supporting a role for Cacna2d1/d2 in methamphetamine locomotor stimulant sensitivity. Our study identifies a dynamic hnRNP H RNA targetome that can rapidly and adaptively respond to methamphetamine to regulate gene expression and likely synaptic plasticity and behavior.

neuroscience↗

bioRxiv2021

Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer's disease pathology

BACKGROUNDC-reactive protein (CRP) in peripheral inflammation is associated with increased Alzheimers disease (AD) risk in Apolipoprotein E4 (ApoE4), but not ApoE3 or E2, humans. It remains unknown whether peripheral monomeric CRP (mCRP) induces AD pathogenesis through some receptor of blood-facing endothelia in the brain in an ApoE genotype dependent fashion. METHODSWe used human samples, ApoE knock-in and deficient mouse models, and primary brain endothelia. Different ApoE mice were intraperitoneally (i.p.) injected with mCRP. The characterizations by immunostaining, proximity ligation assay (PLA) and siRNA were conducted to identify the receptor for mCRP. Brain microvessel and endothelia were isolated for RNA sequencing to explore the molecular pathway. RESULTSWe demonstrate that CD31 (PECAM-1), a blood-facing endothelial receptor in brain, is a competitive target of both mCRP and ApoE protein. ApoE2 competes more strongly with mCRP for CD31 than ApoE4 does, and expressing ApoE4 or knocking out ApoE gene results in higher levels of mCRP-CD31 binding, leading to a decrease of CD31 expression but an increase in CD31 phosphorylation, along with greater cerebrovascular damage and AD pathology. This competitive binding mediates differential endothelial molecular responses depending on ApoE genotype, increasing cerebrovascular inflammation and mitochondria impairment in ApoE4 mice, while inducing vasculogenesis and protective changes in the presence of ApoE2. CONCLUSIONSOur study reveals a novel and dynamic endothelial ApoE-mCRP-CD31 pathway for AD pathogenesis during chronic inflammation and provides some insight into the opposing ApoE4-neurodegenerative and ApoE2-neuroprotective effects in AD. Clinical PerspectiveO_ST_ABSWHAT IS NEW?C_ST_ABSO_LICD31 is a competitive target of both mCRP and ApoE in brain endothelia in an ApoE-allele dependent pattern C_LIO_LImCRP increases CD31 phosphorylation in the brain endothelia and damages cerebrovasculature in ApoE4 carriers and AD brains C_LIO_LImCRP expression results in neuroprotective or neurodegenerative pathway activation in an ApoE-dependent manner C_LI WHAT ARE THE CLINICAL IMPLICATIONS?O_LIAlthough ApoE4 is a major genetic risk factor of AD, some ApoE4 carriers do not develop AD by the age of 90. C_LIO_LIElderly people often experience peripheral inflammatory attacks and develop chronic low-grade inflammation, which results in the formation and release of mCRP. Because CRP is routine clinical laboratory test, clinicians can use blood CRP level to predict AD risk in ApoE4 carriers. C_LIO_LIEvidence of Apoe4 genotype and chronic low-grade inflammation stages marked by elevated CRP levels should be targeted in personalized treatment and clinical trials for AD. C_LI

neuroscience↗

bioRxiv2020

A Complex Containing HNRNPA2B1 and N6-methyladenosine Modified Transcripts Mediates Actions of Toxic Tau Oligomers

The microtubule associated protein tau oligomerizes in response to stress and disease, but the function of oligomeric tau (oTau) and the ultimate mechanisms of toxicity are unknown. To gain insights, we have now used Cry2-based optogenetics to induce tau oligomers (oTau-c) in neuronal cultures. oTau-c can seed tau aggregation and biochemical fractionates in a manner similar to oTau. Optical induction of oTau elicits a translational stress response that includes cytoplasmic translocation of the TIA1, abundant stress granules (SGs) and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau. Imaging and immunoprecipitation verify the HNRNPA2B1 association with endogenous oTau in neurons, animal models and human Alzheimer brain tissue. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine modified transcripts (m6A). Knockdown of HNRNPA2B1 prevents oTau from associating with m6A, prevents oTau-induced reductions in protein synthesis and reduces oTau-induced toxicity. Finally, we show striking increases in m6A-oTau and -HNRNPA2B1 complexes in brains of Alzheimer subjects and P301S tau mice. These results reveal a novel complex containing oTau, HNRNPA2B1 and m6A that contributes to the integrated stress response of oTau. HighlightsO_LIDevelopment of a powerful method combining optical induction of tau oligomerization with precision mass spectrometry to obtain time resolved evolution of protein interaction networks. C_LIO_LIDemonstration of a tripartite complex that links tau oligomers with HNRNPA2B1 and N6-methyladenosine modified RNA in cytoplasmic stress granules. C_LIO_LIKnockdown of HNRNPA2B 1 abrogates the interactions of oTau with N6-methyladenosine modified RNA, as well as inhibits oTau-mediated neurodegeneration. C_LIO_LIDiscovery that N6-methyladenosine modified RNA is significantly increased in the brains of P301S tau transgenic mice and in patients with Alzheimers disease. C_LI

neuroscience↗

bioRxiv2020

Functional variants within the 5UTR support reduced exon usage as a molecular mechanism underlying the quantitative trait gene Hnrnph1 for 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 versus 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.

neuroscience↗

bioRxiv2019

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, mice (both male and female) 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 nucleus accumbens with no change in baseline extracellular dopamine, striatal whole tissue dopamine, dopamine transporter protein, or dopamine uptake. Immunohistochemical and immunoblot staining of midbrain dopaminergic neurons and their forebrain projections for tyrosine hydroxylase did not reveal any major changes in staining intensity, cell number, or in the number of forebrain puncta. Surprisingly, there was a two-fold increase in hnRNP H protein in the striatal synaptosome of H1+/- mice with no change in whole tissue levels. To gain insight into the molecular 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 post-methamphetamine administration in H1+/- mice. In contrast, the much lower level of basal synaptosomal mitochondrial proteins in wild-type mice showed a rapid increase in response to methamphetamine. We conclude that H1+/- decreases methamphetamine-induced dopamine release, reward, and reinforcement and induces dynamic changes in basal and methamphetamine-induced synaptic mitochondrial function.\n\nSIGNIFICANCE STATEMENTMethamphetamine 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 nucleus accumbens - 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 wild-type 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.

neuroscience↗

bioRxiv2018

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 preand/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 revers e 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.\n\nHighlightsO_LIWe investigated immunofluorescence and localization of hnRNP H following dopamine receptor activation in primary rat cortical neurons.\nC_LIO_LIActivation of D1 dopamine receptor increased nuclear immunofluorescence of the C-terminal domain but not the N-terminal domain of hnRNP H with no change in either nuclear or cytoplasmic levels of hnRNP H protein.\nC_LIO_LID1 dopamine signaling could alter interaction of hnRNP H with other proteins.\nC_LI

neuroscience↗