Stressful events are a leading factor in the development of depression. The medial prefrontal cortex (mPFC) is strongly associated with depression etiology and exposure to uncontrollable stressors results in synaptic dysfunction and loss. Learned helplessness is a behavioral paradigm that measures effects of repeated exposure to uncontrollable, inescapable stress on later responses to escapable stress. We therefore performed a proteomic analysis of mPFC synaptosomes in a mouse learned helplessness model to identify molecular changes that could contribute to functional consequences of inescapable stress. Male and female mice were evaluated at baseline and following exposure to escapable or inescapable stress followed by an active avoidance test. Label-free mass spectrometry followed by pathway and protein-protein interaction network analyses identified alterations in signaling pathways involved in energy metabolism, neurotransmitter signaling, and protein shuttling. Furthermore, phosphoproteomics revealed alterations related to synaptic function, neurotransmitter signaling and protein internalization, as well as changes in activity of kinases previously identified as mediators of antidepressant efficacy (GSK3B) and receptor internalization (ADRBK1). We more deeply examined alterations in the acetylcholine receptor signaling pathway and identified muscarinic receptor proteins (Chrm1, Chrm2, Chrm4) and key proteins involved in their translocation to and from the membrane. These results identify substantial changes in the mPFC proteome following exposure to inescapable stressors. In addition, mPFC muscarinic cholinergic signaling is well placed to mediate responses to an inescapable stressor. This proteomic study will be useful in guiding studies of human mPFC relevant to depression. Data are available via ProteomeXchange with identifier PXD073765.

Dystrophic neurites (also termed axonal spheroids) are found around amyloid deposits in Alzheimer’s disease (AD), where they impair axonal electrical conduction, disrupt neural circuits and correlate with AD severity. Despite their importance, the mechanisms underlying spheroid formation remain incompletely understood. To address this, we developed a proximity labeling approach to uncover the proteome of spheroids in human postmortem and mouse brains. Additionally, we established a human induced pluripotent stem cell (iPSC)-derived AD model enabling mechanistic investigation and optical electrophysiology. These complementary approaches revealed the subcellular molecular architecture of spheroids and identified abnormalities in key biological processes, including protein turnover, cytoskeleton dynamics and lipid transport. Notably, the PI3K/AKT/mTOR pathway, which regulates these processes, was activated in spheroids. Furthermore, phosphorylated mTOR levels in spheroids correlated with AD severity in humans. Notably, mTOR inhibition in iPSC-derived neurons and mice ameliorated spheroid pathology. Altogether, our study provides a multidisciplinary toolkit for investigating mechanisms and therapeutic targets for axonal pathology in neurodegeneration. Axonal spheroids disrupt neural circuits in Alzheimer’s disease. In this study, using subcellular proximity labeling proteomics in human brain and iPSC modeling, the authors link spheroid formation to dysregulated mTOR, cytoskeletal and lipid transport signaling.

Background Accurate placement of the macaque within the Alzheimer's disease (AD) research framework is essential to discover early-stage predictive biomarkers. Objective To assess utility of the aging macaque in advancing translational biomarker development for preclinical AD, we evaluated relative signal strength of comparable neuropathologic phenomena in macaques and patients. Methods We compared pathology in patient and macaque formalin-fixed paraffin embedded (FFPE) tissues using identical criteria. We quantified expression of amyloid-β (Aβ), pTau, and inflammatory and senescence markers across species. Distribution of AD-relevant markers were compared in FFPE and perfused frozen macaque brain to assess expression of labile proteins that could inform in-life fluid biomarkers. Results Aβ pathology in macaques closely approximated patient pathology. Complex plaque composition in macaques implied significant disruption of synaptic connectivity. In FFPE tissue, pretangle pTau immunoreactivity placed the macaque in Braak Stage 1b. In perfused frozen tissue, soluble pTau distribution approximated Braak Stage III-IV. In macaque, Aβ, pTau, and acetylcholinesterase labeling co-localized to AD-vulnerable circuits. Significant association of glial fibrillary acidic protein with Aβ occurred in humans only. The senescence marker p16 correlated positively with pTau expression and negatively with Aβ in patients only. Macaques lacked neuropathologic co-morbidities. Conclusions AD-relevant neuropathologic signals in the macaque support biomarker discovery in the areas of Aβ plaque evolution and associated synaptic disruption as well as early-stage tau phosphorylation. Relative protection from accumulation of senescence markers, fibrillar tau and neuropathologic co-morbidities in macaque implicate species difference in rates of biological brain aging. We provide over 4000 digital slides for further study.

Actin polymerization and depolymerization are fundamental cellular processes required not only for the embryonic and postnatal development of the brain but also for the maintenance of neuronal plasticity and survival in the adult and aging brain. The orchestrated organization of actin filaments is controlled by various actin regulatory proteins. Wiskott‒Aldrich syndrome protein-family verprolin-homologous protein (WAVE) members are key activators of ARP2/3 complex-mediated actin polymerization. WAVE proteins exist as heteropentameric complexes together with regulatory proteins, including CYFIP, NCKAP, ABI and BRK1. The activity of the WAVE complex is tightly regulated by extracellular cues and intracellular signaling to execute its roles in specific intracellular events in brain cells. Notably, dysregulation of the WAVE complex and WAVE complex-mediated cellular processes confers vulnerability to a variety of brain disorders. De novo mutations in WAVE genes and other components of the WAVE complex have been identified in patients with developmental disorders such as intellectual disability, epileptic seizures, schizophrenia, and/or autism spectrum disorder. In addition, alterations in the WAVE complex are implicated in the pathophysiology of Alzheimer’s disease and Parkinson’s disease, as well as in behavioral adaptations to psychostimulants or maladaptive feeding. The WAVE complex is a group of proteins that work together to regulate actin, an essential component of the cell cytoskeleton. This article reviews how changes in the WAVE complex can affect brain function and contribute to various brain disorders. It summarizes how different parts of the WAVE complex interact with other proteins to regulate actin polymerization. This regulation is crucial for programming brain cell growth, migration, and synaptic formation and function. This article also explores recent studies on genetic changes in the WAVE complex and their links to developmental and adult brain disorders. Key findings suggest disruptions in the WAVE complex can lead to conditions like intellectual disabilities, autism, and neurodegenerative diseases such as Alzheimer’s. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

The differential diagnosis of Alzheimer’s disease (AD) and normal pressure hydrocephalus (NPH) is complicated by overlapping clinical manifestations. This challenges accurate clinical diagnosis and highlights the need for molecular level investigations to understand underlying pathologies. There have been few proteomic investigations into NPH, which were limited by low sample sizes and limited analytical depth. Here we applied machine learning to investigate the distinct and overlapping proteomic signatures associated with AD and NPH, compared to that of cognitively unimpaired controls (CU). Banked cerebrospinal fluid (CSF) samples were obtained from diagnostic lumbar punctures at an outpatient neurology clinic. Participants were classified based on clinical presentation, improvement after a high-volume LP, and CSF amyloid-b status as CU (N = 53), AD (N = 158), or NPH (N = 56). Proteomic quantification was done using data-independent acquisition mass spectrometry. Defining molecular signatures between classes was investigated with random forest models in two-way fashion and top features were assessed using Gini importance. Additionally, to determine functional changes, we examined enriched and depleted pathways with gene-set enrichment analysis (GSEA) using fold-change data between classes. Random forest models obtained high classification accuracy (>75%). Comparison of the top 30 proteins of each model indicated four proteins, MASP1, NRXN2, VCAN and LTBP2 as defining features in NPH compared to both CU and AD. Six proteins, SMOC1, PTPRN2, NPTX2, LUM, APLP1 and GFRA2 were defining features for both AD and NPH compared to CU. One protein, TTR, was a defining feature comparing AD to both CU and NPH. GSEA indicated 91 pathways to be differentially regulated in NPH compared to both CU and AD. Further analysis indicated specific enrichment of complement activation and immune response pathways in NPH. Pathways differentially regulated in AD compared to both CU and NPH included pathways related to glycolysis and metabolic processes. Our analyses validate previously suggested mechanisms and provide novel insights into the differential pathologies of AD and NPH. Hereby, we aim to contribute to the development of more refined and early diagnostic tools, facilitating targeted therapeutic approaches for these neurologically challenging disorders.

Many essential proteins require pyridoxal 5'-phosphate, the active form of vitamin B6, as a cofactor for their activity. These include enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian pyridoxal 5'-phosphate-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial pyridoxal 5'-phosphate levels in mammals remains unknown. In this study, we used a genome-wide CRISPR interference screen in erythroleukemia cells and organellar metabolomics to identify the mitochondrial inner membrane protein SLC25A38 as a regulator of mitochondrial pyridoxal 5'-phosphate. Loss of SLC25A38 causes depletion of mitochondrial, but not cellular, pyridoxal 5'-phosphate, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial pyridoxal 5'-phosphate-dependent enzymatic reactions, including serine to glycine conversion catalyzed by serine hydroxymethyltransferase-2 as well as ornithine aminotransferase. The proliferation defect of SLC25A38-null K562 cells in physiological and low vitamin B6 media can be explained by the loss of serine hydroxymethyltransferase-2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial pyridoxal 5'-phosphate accumulation and provides insights into the pathology of congenital sideroblastic anemia.

Alzheimer disease (AD) is characterized by plaques and tangles, including calcium dysregulation and glycated products produced by reactive carbonyl compounds. AD brains have increased glyoxalase I (GLO1), a major scavenger of inflammatory carbonyl compounds, at early, but not later, stages of disease. Calcium dysregulation includes calcium leak from phosphorylated ryanodine receptor 2 (pS2808-RyR2), seen in aged macaques and AD mouse models, but the downstream consequences of calcium leak remain unclear. Here, we show that chronic calcium leak is associated with increased GLO1 expression and activity. In macaques, we found age-related increases in GLO1 expression in the prefrontal cortex (PFC), correlating with pS2808-RyR2, and localized to dendrites and astrocytes. To examine the relationship between GLO1 and RyR2, we used S2808D-RyR2 mutant mice exhibiting chronic calcium leak through RyR2, and found increased GLO1 expression and activity in the PFC and hippocampus as early as 1 month and as late as 21 months of age, with a bell-shaped aging curve. These aged S2808D-RyR2 mice demonstrated impaired working memory. As with macaques, GLO1 was expressed in astrocytes and neurons. Proteomics data generated from S2808D-RyR2 synaptosomes confirmed GLO1 upregulation. Altogether, these data suggest potential association between GLO1 and chronic calcium leak, providing resilience in early stages of aging.

Post-traumatic stress disorder (PTSD) is a common and disabling psychiatric disorder. PTSD involves multiple brain regions and is often comorbid with other psychiatric disorders, such as major depressive disorder (MDD). Recent genome-wide association studies (GWASs) have identified many PTSD risk loci and transcriptomics studies of postmortem brain have found differentially expressed genes associated with PTSD cases. In this study, we integrated genome-wide measures across modalities to identify convergent molecular effects in the PTSD brain. We performed tandem mass spectrometry (MS/MS) on a large cohort of donors (N = 66) in two prefrontal cortical areas, dorsolateral prefrontal cortex (DLPFC), and subgenual prefrontal cortex (sgPFC). We also coupled the proteomics data with transcriptomics and microRNA (miRNA) profiling from RNA-seq and small-RNA sequencing, respectively for the same cohort. Additionally, we utilized published GWAS results of multiple psychiatric disorders for integrative analysis. We found differentially expressed proteins and co-expression protein modules disrupted by PTSD. Integrative analysis with transcriptomics and miRNA data from the same cohort pointed to hsa-mir-589 as a regulatory miRNA responsible for dysregulation of neuronal protein networks for PTSD, including the gamma-aminobutyric acid (GABA) vesicular transporter, SLC32A1. In addition, we identified significant enrichment of risk genes for other psychiatric disorders, such as autism spectrum disorder (ASD) and major depressive disorder (MDD) within PTSD protein co-expression modules, suggesting shared molecular pathology. We integrated genome-wide measures of mRNA and miRNA expression and proteomics profiling from PTSD, MDD, and control (CON) brains to identify convergent and divergent molecular processes across genomic modalities. We substantially expand the number of differentially expressed genes and proteins in PTSD and identify downregulation of GABAergic processes in the PTSD proteome. This provides a novel framework for future studies integrating proteomic profiling with transcriptomics and non-coding RNAs in the human brain studies.

Platelet production is a highly regulated process involving coordinated signaling and cytoskeletal remodeling. While thrombopoietin-driven pathways have been well characterized, the contribution of serine/threonine phosphatases remains poorly explored. In this study, we interrogated the specific function of the phosphatase PP1β in megakaryocyte development using a lineage-specific conditional knockout mouse model by crossing Ppp1cb-floxed mice with Pf4-Cre transgenic mice. Mice lacking PP1β in the megakaryocyte lineage exhibited severe macrothrombocytopenia, accompanied by hallmark features of bone marrow pathology, including myelofibrosis, osteosclerosis, and splenic enlargement. The depletion of PP1β let to an accumulation of smaller megakaryocytes in the bone marrow, which displayed abnormal ultrastructure and disorganized cytoplasm with impaired demarcation membrane system formation. In vitro assays confirmed elevated ploidy, changes in the expression of surface glycoproteins critical for platelet function defective proplatelet formation, and lower platelet lifespan. Notably, these main findings were also recapitulated in vivo using intravital imaging assays, further validating the physiological relevance of the observed defects. Mechanistically, we observed increased phosphorylation of myosin light chain 2 (Ser19), consistent with enhanced actomyosin contractility and stress fiber assembly. Surprisingly, canonical thrombopoietin signaling via Akt and STAT family members remained unaltered, while phosphorylation of Src family kinases at their activation loop was markedly diminished. These data suggest that PP1β modulates both cytoskeletal tension and tyrosine kinase signaling to support megakaryocyte maturation and platelet release. Together, our findings position PP1β as an essential regulator of megakaryopoiesis, acting through control of myosin II activity and Src kinase signaling, highlighting a distinct, non-redundant role for this phosphatase in hematopoiesis.

BACKGROUND AND OBJECTIVES:Idiopathic normal pressure hydrocephalus (iNPH) is a reversible neurologic disorder that remains poorly understood. Accurate differential diagnosis of iNPH and Alzheimer disease (AD) is complicated by overlapping clinical manifestations. Beyond neuroimaging, there are currently no biomarkers available for iNPH leading to frequent misdiagnosis, and proteomic studies into iNPH have been limited by low sample sizes and inadequate analytical depth. In this study, we report the results of a large-scale proteomic analysis of CSF from patients with iNPH to elucidate pathogenesis and identify potential disease biomarkers. METHODS:CSF samples were collected through lumbar puncture during diagnostic visits to the Mass General Brigham neurology clinic. Samples were analyzed using mass spectrometry. Differential expression of proteins was studied using linear regression models. Results were integrated with publicly available single-nucleus transcriptomic data to explore potential cellular origins. Biological process enrichment was analyzed using gene-set enrichment analyses. To identify potential diagnostic biomarkers, decision tree-based machine learning algorithms were applied. RESULTS:Participants were classified as cognitively unimpaired (N = 53, mean age: 66.5 years, 58.5% female), AD (N = 124, mean age: 71.2 years, 46.0% female), or iNPH (N = 44, mean age: 74.6 years, 34.1% female) based on clinical diagnosis and AD biomarker status. Gene Ontology analyses indicated upregulation of the immune system and coagulation processes and downregulation of neuronal signaling processes in iNPH. Differential expression analysis showed a general downregulation of proteins in iNPH. Integration of differentially expressed proteins with transcriptomic data indicated that changes likely originated from neuronal, endothelial, and glial origins. Using machine learning algorithms, a panel of 12 markers with high diagnostic potential for iNPH were identified, which were not all detected using univariate linear regression models. These markers spanned the various molecular processes found to be affected in iNPH, such as LTBP2, neuronal pentraxin receptor (NPTXR), and coagulation factor 5. DISCUSSION:Leveraging the etiologic insights from a typical neurologic clinical cohort, our results indicate that processes of immune response, coagulation, and neuronal signaling are affected in iNPH. We highlight specific markers of potential diagnostic interest. Together, our findings provide novel insights into the pathophysiology of iNPH and may facilitate improved diagnosis of this poorly understood disorder.

Protein phosphatase 1 (PP1) regulates synaptic plasticity and has been described as a molecular constraint on learning and memory. There are three neuronal isoforms, PP1α, PP1β, and PP1γ, but little is known about their individual functions. PP1α and PP1γ are assumed to mediate the effects of PP1 on learning and memory based on their enrichment at dendritic spines and their preferential binding to neurabin and spinophilin, major PP1 synaptic scaffolding proteins. However, it was recently discovered that human de novo PP1β mutations cause intellectual disability, suggesting an important but ill-defined role for PP1β. In this study, we investigated the functions of each PP1 isoform in hippocampal synaptic physiology using conditional CA1-specific knockout mice. In stark contrast to classic PP1 function, we found that PP1β promotes synaptic plasticity as well as spatial memory. These changes in synaptic plasticity and memory are accompanied by changes in GluA1 phosphorylation, GluN2A levels, and dendritic spine density and morphology, including silent synapse number. These functions of PP1β reveal a previously unidentified signaling pathway regulating spine maturation and plasticity, broadening our understanding of the complex role of PP1 in synaptic physiology.

BACKGROUND:Highly palatable food triggers behavioral alterations reminiscent of those induced by addictive drugs. These effects involve the reward system and dopamine neurons, which modulate neurons in the nucleus accumbens (NAc). The molecular mechanisms underlying the effects of highly palatable food on feeding behavior are poorly understood.METHODS:We studied the effects of 2-week operant conditioning of mice with standard or isocaloric highly palatable food. We investigated the behavioral effects and dendritic spine modifications in the NAc. We compared the translating mRNA in NAc neurons identified by the type of dopamine receptors they express, depending on the type of food and training. We tested the consequences of invalidation of an abundant downregulated gene, Ncdn (Neurochondrin).RESULTS:Operant conditioning for highly palatable food increases motivation for food even in well-fed mice. In control mice, free access to regular or highly palatable food results in increased weight as compared to regular food only. Highly palatable food increases spine density in the NAc. In animals trained for highly palatable food, translating mRNAs are modified in NAc dopamine D2-receptor-expressing neurons, mostly corresponding to striatal projection neurons, but not in those expressing D1-receptors. Knock-out of Ncdn, an abundant down-regulated gene, opposes the conditioning-induced changes in satiety-sensitive feeding behavior and apparent motivation for highly palatable food, suggesting down-regulation may be a compensatory mechanism.CONCLUSIONS:Our results emphasize the importance of mRNA alterations D2 striatal projection neurons in the NAc in the behavioral consequences of highly palatable food conditioning and suggest a modulatory contribution of Ncdn downregulation.

Liquid Chromatography Mass Spectrometry (LC-MS) is a powerful method for profiling complex biological samples. However, batch effects typically arise from differences in sample processing protocols, experimental conditions and data acquisition techniques, significantlyimpacting the interpretability of results. Correcting batch effects is crucial for the reproducibility of proteomics research, but current methods are not optimal for removal of batch effects without compressing the genuine biological variation under study. We propose a suite of Batch Effect Removal Neural Networks (BERNN) to remove batch effects in large LC-MS experiments, with the goal of maximizing sample classification performance between conditions. More importantly, these models must efficiently generalize in batches not seen during training. Comparison of batch effect correction methods across three diverse datasets demonstrated that BERNN models consistently showed the strongest sample classification performance. However, the model producing the greatest classification improvements did not always perform best in terms of batch effect removal. Finally, we show that overcorrection of batch effects resulted in the loss of some essential biological variability. These findings highlight the importance of balancing batch effect removal while preserving valuable biological diversity in large-scale LC-MS experiments.

To identify proteins by the bottom-up mass spectrometry workflow, enzymatic digestion is essential to break down proteins into smaller peptides amenable to both chromatographic separation and mass spectrometric analysis. Trypsin is the most extensively used protease due to its high cleavage specificity and generation of peptides with desirable positively charged N- and C-terminal amino acid residues that are amenable to reverse phase HPLC separation and MS/MS analyses. However, trypsin can yield variable digestion profiles and its protein cleavage activity is interdependent on trypsin source and quality, digestion time and temperature, pH, denaturant, trypsin and substrate concentrations, composition/complexity of the sample matrix, and other factors. There is therefore a need for a more standardized, general-purpose trypsin digestion protocol. Based on a review of the literature we delineate optimal conditions for carrying out trypsin digestions of complex proteomes from bulk samples to limiting amounts of protein extracts. Furthermore, we highlight recent developments and technological advances used in digestion protocols to quantify complex proteomes from single cells.SignificanceCurrently, bottom-up MS-based proteomics is the method of choice for global proteome analysis. Since trypsin is the most utilized protease in bottom-up MS proteomics, delineating optimal conditions for carrying out trypsin digestions of complex proteomes in samples ranging from tissues to single cells should positively impact a broad range of biomedical research.

Given the increasing use of cannabis in the US, there is an urgent need to better understand the drug’s effects on central signaling mechanisms. Extracellular vesicles (EVs) have been identified as intercellular signaling mediators that contain a variety of cargo, including proteins. Here, we examined whether the main psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), alters EV protein signaling dynamics in the brain. We first conducted in vitro studies, which found that THC activates signaling in choroid plexus epithelial cells, resulting in transcriptional upregulation of the cannabinoid 1 receptor and immediate early gene c-fos, in addition to the release of EVs containing RNA cargo. Next, male and female rats were examined for the effects of either acute or chronic exposure to aerosolized (‘vaped’) THC on circulating brain EVs. Cerebrospinal fluid was extracted from the brain, and EVs were isolated and processed with label-free quantitative proteomic analyses via high-resolution tandem mass spectrometry. Interestingly, circulating EV-localized proteins were differentially expressed based on acute or chronic THC exposure in a sex-specific manner. Taken together, these findings reveal that THC acts in the brain to modulate circulating EV signaling, thereby providing a novel understanding of how exogenous factors can regulate intercellular communication in the brain.

BACKGROUND:Identifying neurobiological targets predictive of the molecular neuropathophysiological signature of human opioid use disorder (OUD) could expedite new treatments. OUD is characterized by dysregulated cognition and goal-directed behavior mediated by the orbitofrontal cortex (OFC), and next-generation sequencing could provide insights regarding novel targets. METHODS:Here, we used machine learning to evaluate human postmortem OFC RNA sequencing datasets from heroin users and control participants to identify transcripts that were predictive of heroin use. To determine a causal link to OUD-related behaviors, we examined the effects of overexpressing the top target gene in a translational rat model of heroin seeking and behavioral updating. Additionally, we determined the effects of overexpression on the rat OFC transcriptome compared with that of human heroin users. Co-immunoprecipitation/mass spectrometry (co-IP/MS) from the rat OFC elucidated the protein complex of the novel target. RESULTS:Our machine learning approach identified SHISA7 as predictive of human heroin users. Shisa7 is understudied but appears to be an auxiliary protein of GABAA (gamma-aminobutyric acid A) or AMPA receptors. In rats, Shisa7 expression positively correlated with heroin-seeking behavior. Overexpressing Shisa7 in the OFC augmented heroin seeking and impaired behavioral updating for sucrose-based operant contingency. RNA sequencing of rat OFC revealed gene coexpression networks regulated by Shisa7 overexpression similar to human heroin users. Finally, co-IP/MS showed that heroin influenced Shisa7 binding to glutamatergic and GABAergic receptor subunits. Both gene expression signatures and Shisa7 protein complex emphasized perturbations of neurodegenerative and neuroimmune processes. CONCLUSIONS:Our findings suggest that OFC Shisa7 is a critical driver of neurobehavioral pathology related to drug-seeking behavior and behavioral updating, thus identifying a potential therapeutic target for OUD.

In addition to neuronal migration, brain development, and adult plasticity, the extracellular matrix protein Reelin has been extensively implicated in human psychiatric disorders such as schizophrenia, bipolar disorder, and autism spectrum disorder. Moreover, heterozygous reeler mice exhibit features reminiscent of these disorders, while overexpression of Reelin protects against its manifestation. However, how Reelin influences the structure and circuits of the striatal complex, a key region for the above-mentioned disorders, is far from being understood, especially when altered Reelin expression levels are found at adult stages. In the present study, we took advantage of complementary conditional gain- and loss-of-function mouse models to investigate how Reelin levels may modify adult brain striatal structure and neuronal composition. Using immunohistochemical techniques, we determined that Reelin does not seem to influence the striatal patch and matrix organization (studied by μ-opioid receptor immunohistochemistry) nor the density of medium spiny neurons (MSNs, studied with DARPP-32). We show that overexpression of Reelin leads to increased numbers of striatal parvalbumin- and cholinergic-interneurons, and to a slight increase in tyrosine hydroxylase-positive projections. We conclude that increased Reelin levels might modulate the numbers of striatal interneurons and the density of the nigrostriatal dopaminergic projections, suggesting that these changes may be involved in the protection of Reelin against neuropsychiatric disorders.

Substance use disorders are associated with disruptionsin sleepand circadian rhythms that persist during abstinence and may contributeto relapse risk. Repeated use of substances such as psychostimulantsand opioids may lead to significant alterations in molecular rhythmsin the nucleus accumbens (NAc), a brain region central to reward andmotivation. Previous studies have identified rhythm alterations inthe transcriptome of the NAc and other brain regions following theadministration of psychostimulants or opioids. However, little isknown about the impact of substance use on the diurnal rhythms ofthe proteome in the NAc. We used liquid chromatography coupled totandem mass spectrometry-based quantitative proteomics, along witha data-independent acquisition analysis pipeline, to investigate theeffects of cocaine or morphine administration on diurnal rhythms ofproteome in the mouse NAc. Overall, our data reveal cocaine and morphinedifferentially alter diurnal rhythms of the proteome in the NAc, withlargely independent differentially expressed proteins dependent ontime-of-day. Pathways enriched from cocaine altered protein rhythmswere primarily associated with glucocorticoid signaling and metabolism,whereas morphine was associated with neuroinflammation. Collectively,these findings are the first to characterize the diurnal regulationof the NAc proteome and demonstrate a novel relationship between thephase-dependent regulation of protein expression and the differentialeffects of cocaine and morphine on the NAc proteome. The proteomicsdata in this study are available via ProteomeXchange with identifierPXD042043.