Tariq Brown¹, Emily Petruccelli², John Hernandez³, Reza Azanchi³, Kristin Scaplen⁴, Erica Larschan⁵, Kate O’Connor-Giles² and Karla R. Kaun²

Tight regulation of transcription and RNA processing is required for appropriate cellular homeostasis, neurogenesis, and synaptic plasticity. This regulation can be dynamically and persistently perturbed by addiction-associated substances. Alcohol use has been associated with disrupted alternative splicing in humans, non-human primates, rats, mice, chickens, and flies, suggesting a fundamental mechanism through which alcohol affects the molecular processing and function of cells. However, the functional consequences of transcript switching, and how it can affect subsequent substance use are almost entirely unknown. We found that lasting memory for an odor associated with the rewarding properties of alcohol causes differential transcript usage in memory-encoding neurons. These switches included alternative start sites, exon skipping, and alternative use of splice donor/acceptor sites, and occurred in a variety of different genes including the Dopamine-2-like Receptor (Dop2R) and the Drosophila signal transducer and activator of transcription (Stat92E) genes. We hypothesized that alcohol-induced transcript switching causes functional changes to the plasticity of memory circuits and behavioral response to alcohol and other psychoactive drugs like nicotine and methamphetamine. Using a combination of functional in vivo imaging and high resolution behavior assays to test the rewarding properties of volatile substances, we demonstrate that alternative splicing contributes to memory circuit plasticity and behavioral decision-making. The conservation of drug-induced alternative splicing from flies to humans suggests it is likely a molecular mechanism through which addictive substances affect memory and consequent behavioral responses.

¹Neuroscience Graduate Program, Brown University; ²Department of Biological Sciences, Southern Illinois University; ³Department of Neuroscience, Brown University; ⁴Department of Psychology, Bryant University; ⁵Department of Molecular Biology, Cell Biology and Biochemistry, Brown University

K Czarnecki 1, K Krick 1, EA Heller 1

Alternative splicing differentiates identity and function of neuronal subtypes and is activity dependent. Defects in neuronal splicing are associated with neurological and psychiatric disorders. The hPTM, H3K36me3, is implicated in splicing and interacts with a variety of splicing-related proteins. The H3K36me3 writer, SETD2, directly interacts with splicing factors. We find that H3K36me3 is causally linked to alternative splicing in the context of cocaine reward behavior in mice. However, we still lack a mechanistic understanding of the interplay between hPTMs and alternative splicing. This project aims to define the regulation of alternative splicing by SETD2 and/or H3K36me3 by decoupling these putative splicing regulators. To accomplish this, we acutely depleted either H3K36me3 and SETD2 and then measured global splicing changes using RNA-sequencing in ex-vivo mouse neurons. We achieved depletion of H3K36me3 using a newly SETD2 inhibitor, EZM0414, and of SETD2 through a dTAG degron at the endogenous locus. In addition, we apply a CRISPR epigenetic editing tool to specifically deposit H3K36me3 and assess the sufficiency of H3K36me3 to drive splicing at target exons. This project will be the first to determine the relative contributions of SETD2 and H3K36me3 to alternative splicing to uncover how these factors might regulate diverse neuronal functions.

1 Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania USA

Funding Support: NIH/NIDA R01-DA052465

Luana Carvalho¹, Jonathas Almeida¹ and Amy W. Lasek¹

¹Department of Pharmacology and Toxicology, Virginia Commonwealth University

Alternative splicing is highly prevalent in the brain and tightly regulated by RNA-binding proteins (RBPs). Disruptions in this regulation have been implicated in psychiatric disorders, including alcohol use disorder (AUD). Poly(rC) binding protein 1 (PCBP1) is a multifunctional RBP that regulates exon retention and exclusion, influencing RNA splicing and protein function. Using the Lieber-DeCarli liquid diet model, we previously found that Pcbp1 expression is upregulated in the male rat hippocampus during alcohol withdrawal (AW), a period characterized by anxiety- and depressive-like behaviors. We demonstrated that PCBP1 modulates alternative splicing of Hapln2, a gene encoding an extracellular matrix (ECM) protein critical for nerve conduction velocity. PCBP1-driven splicing of Hapln2 leads to either nonsense-mediated decay or the production of a non-functional protein, potentially impairing neurotransmission and contributing to withdrawal-induced emotional and cognitive dysfunction. Beyond Hapln2, we hypothesized that PCBP1 regulates a broader network of genes involved in withdrawal-related behavioral deficits. To investigate this, we performed RNA Immunoprecipitation Sequencing (RIP-Seq) on hippocampal tissue from 12 males (6 control, 6 withdrawal) and 12 females (6 control, 6 withdrawal). Both IP and input samples were sequenced, and PCBP1-bound peaks were identified using MACS3. Differential enrichment analysis was performed with edgeR (FDR < 0.1). In males, we identified 73 highly enriched and 474 lowly enriched PCBP1 peaks, corresponding to 70 and 368 unique genes, respectively, compared to controls. These findings suggest a reduction in PCBP1 interaction with its RNA targets during AW. In contrast, no significant differences in PCBP1-enriched peaks were observed in females during withdrawal compared to female controls, suggesting potential sex-specific regulatory mechanisms. Notably, genes with high PCBP1 enrichment in males are involved in glycosaminoglycan metabolism, which plays a key role in ECM composition and cellular signaling. In contrast, genes with low PCBP1 enrichment are associated with synaptogenesis, glutamatergic receptor function, and myelination signaling, highlighting potential disruptions in neuronal connectivity and oligodendrocyte function during AW.

These findings provide new insights into PCBP1-mediated splicing regulation during alcohol withdrawal, linking altered RNA processing to ECM remodeling, synaptic plasticity, and myelination, while also revealing potential sex differences in PCBP1-RNA interactions. Understanding these mechanisms may reveal novel therapeutic targets for AUD-related neuroadaptations and withdrawal-induced behavioral deficits.

Rudong Li ^1,2, Jill L. Reiter ^1,2, Season K. Wyatt-Johnson ^3,4, Chuanpeng Dong ^1,2, Caine S. Smith ^5,6, Sheketha R. Hauser ^4,7, Manav Kapoor ^8,9, Julia Stevens ^5,6, R. Dayne Mayfield ^10,11, Alison M. Goate ^8,9, Yue Wang ², Howard J. Edenberg ^2,12, Richard L. Bell ⁷, Greg T. Sutherland ^5,6, Randy R. Brutkiewicz ^3,4_, Yunlong Liu ^1,2

Intron retention (IR) can be either a type of regulated alternative RNA splicing event or a consequence of dysregulated RNA splicing machinery. IR has been studied in neurological disease and cancer, particularly in the context of antiviral immune response, immune checkpoints, and spliceosome-targeted therapies. However, its involvement in complex neuropsychiatric disorders, such as alcohol use disorder (AUD), remains largely unexplored.

Here, we systematically identified IR events in post-mortem brain tissue of individuals with and without AUD. We analyzed the transcriptome of 320 samples from the superior frontal cortex, nucleus accumbens, central nucleus, and basolateral amygdala of 142 independent subjects (66 AUD and 76 controls). We observed an increase of total IR in the AUD samples, after adjusting for age, sex, sequencing cohort, brain regions and individuals. The same observation was confirmed in a well-characterized animal model of alcoholism. Significantly higher levels of total IR were found in the brains of selectively bred alcohol-preferring P rats consuming alcohol compared to water. Additionally, we found more IR events in the brains of individuals with AUD compared to controls. These AUD-associated introns exhibited lower splicing strength at the acceptor site compared to constitutively excised introns, suggesting that splicing of these introns were more susceptible to disease or environmental factors. Expression of the host genes of these IR events was enriched in several neuronal and glial cell types, including Purkinje neurons, visual cortex neurons, and oligodendrocytes. We also found a significantly higher proportion of long introns (>1 kilobase) among these IR events. Because longer introns could potentiate the formation of double-stranded RNA (dsRNA), we performed immunostaining of dsRNA in brain tissue sections from P rats. The dsRNA signal was co-localized with neurons and was significantly higher in multiple brain regions from P rats consuming alcohol compared to water. Moreover, in the human AUD samples, dsRNA-related response pathways were activated and neuronal counts were significantly decreased compared to non-AUD controls.

In conclusion, our findings indicate that chronic alcohol consumption is associated with elevated IR in the brain, particularly in key cell types such as GABAergic regulatory cells, excitatory neurons and oligodendrocytes. Our study provides evidence that IR may lead to the formation of dsRNA, which in turn can trigger immune signaling and apoptosis that contribute to neuroimmune activity and the loss of neurons observed in AUD. Thus, these findings support a potential dsRNA-mediated mechanism for the involvement of IR in AUD.

Rudong Li ^1,2, Jill L. Reiter ^1,2, Season K. Wyatt-Johnson ^3,4, Chuanpeng Dong ^1,2, Caine S. Smith ^5,6, Sheketha R. Hauser ^4,7, Manav Kapoor ^8,9, Julia Stevens ^5,6, R. Dayne Mayfield ^10,11, Alison M. Goate ^8,9, Yue Wang ², Howard J. Edenberg ^2,12, Richard L. Bell ⁷, Greg T. Sutherland ^5,6, Randy R. Brutkiewicz ^3,4_, Yunlong Liu ^1,2

¹ Center for Computational Biology and Bioinformatics (CCBB), Indiana University (IU) School of Medicine, IN, USA

² Department of Medical and Molecular Genetics (MMGE), IU School of Medicine, IN, USA

³ Department of Microbiology and Immunology, IU School of Medicine, IN, USA

⁴ Stark Neurosciences Research Institute, IU School of Medicine, IN, USA

⁵ New South Wales Brain Tissue Research Centre, University of Sydney, NSW, Australia

⁶ Charles Perkins Centre and School of Medical Sciences, University of Sydney, NSW, Australia

⁷ Department of Psychiatry, IU School of Medicine, IN, USA

⁸ Ronald M. Loeb Center for Alzheimer’s Disease, Department of Neuroscience, Icahn School of Medicine at Mount Sinai, NY, USA

⁹ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, NY, USA

¹⁰ Waggoner Center for Alcohol and Addiction Research (WCAAR), University of Texas at Austin, TX, USA

¹¹ Department of Neuroscience, University of Texas at Austin, TX, USA

¹² Department of Biochemistry and Molecular Biology, IU School of Medicine, IN