Biology subjects

Ash, P. E. A.

Publications and source records attributed to Ash, P. E. A..

5 recordsLinked to original sources

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↗

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↗

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↗