Circadian rhythm disruptions associated with opioid use disorder in synaptic proteomes of human dorsolateral prefrontal cortex and nucleus accumbens.

Opioid craving and relapse vulnerability is associated with severe and persistent sleep and circadian rhythm disruptions. Understanding the neurobiological underpinnings of circadian rhythms and opioid use disorder (OUD) may prove valuable for developing new treatments for opioid addiction. Previous work indicated molecular rhythm disruptions in the human brain associated with OUD, highlighting synaptic alterations in the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc)-key brain regions involved in cognition and reward, and heavily implicated in the pathophysiology of OUD. To provide further insights into the synaptic alterations in OUD, we used mass-spectrometry based proteomics to deeply profile protein expression alterations in bulk tissue and synaptosome preparations from DLPFC and NAc of unaffected and OUD subjects. We identified 55 differentially expressed (DE) proteins in DLPFC homogenates, and 44 DE proteins in NAc homogenates, between unaffected and OUD subjects. In synaptosomes, we identified 161 and 56 DE proteins in DLPFC and NAc, respectively, of OUD subjects. By comparing homogenate and synaptosome protein expression, we identified proteins enriched specifically in synapses that were significantly altered in both DLPFC and NAc of OUD subjects. Across brain regions, synaptic protein alterations in OUD subjects were primarily identified in glutamate, GABA, and circadian rhythm signaling. Using time-of-death (TOD) analyses, where the TOD of each subject is used as a time-point across a 24-h cycle, we were able to map circadian-related changes associated with OUD in synaptic proteomes associated with vesicle-mediated transport and membrane trafficking in the NAc and platelet-derived growth factor receptor beta signaling in DLPFC. Collectively, our findings lend further support for molecular rhythm disruptions in synaptic signaling in the human brain as a key factor in opioid addiction.

Circadian rhythm disruptions associated with opioid use disorder in the synaptic proteomes of the human dorsolateral prefrontal cortex and nucleus accumbens.

Opioid craving and relapse vulnerability is associated with severe and persistent sleep and circadian rhythm disruptions. Understanding the neurobiological underpinnings of circadian rhythms and opioid use disorder (OUD) may prove valuable for developing new treatments for opioid addiction. Previous work indicated molecular rhythm disruptions in the human brain associated with OUD, highlighting synaptic alterations in the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc)-key brain regions involved in cognition and reward, and heavily implicated in the pathophysiology of OUD. To provide further insights into the synaptic alterations in OUD, we used mass-spectrometry based proteomics to deeply profile protein expression alterations in bulk tissue and synaptosome preparations from DLPFC and NAc of unaffected and OUD subjects. We identified 55 differentially expressed (DE) proteins in DLPFC homogenates, and 44 DE proteins in NAc homogenates, between unaffected and OUD subjects. In synaptosomes, we identified 161 and 56 DE proteins in DLPFC and NAc, respectively, of OUD subjects. By comparing homogenate and synaptosome protein expression, we identified proteins enriched specifically in synapses that were significantly altered in both DLPFC and NAc of OUD subjects. Across brain regions, synaptic protein alterations in OUD subjects were primarily identified in glutamate, GABA, and circadian rhythm signaling. Using time-of-death (TOD) analyses, where the TOD of each subject is used as a time-point across a 24- hour cycle, we were able to map circadian-related changes associated with OUD in synaptic proteomes related to vesicle-mediated transport and membrane trafficking in the NAc and platelet derived growth factor receptor beta signaling in DLPFC. Collectively, our findings lend further support for molecular rhythm disruptions in synaptic signaling in the human brain as a key factor in opioid addiction.

Dynamic proteomic and phosphoproteomic atlas of corticostriatal axons in neurodevelopment.

Mammalian axonal development begins in embryonic stages and continues postnatally. After birth, axonal proteomic landscape changes rapidly, coordinated by transcription, protein turnover, and post-translational modifications. Comprehensive profiling of axonal proteomes across neurodevelopment is limited, with most studies lacking cell-type and neural circuit specificity, resulting in substantial information loss. We create a Cre-dependent APEX2 reporter mouse line and map cell-type-specific proteome of corticostriatal projections across postnatal development. We synthesize analysis frameworks to define temporal patterns of axonal proteome and phosphoproteome, identifying co-regulated proteins and phosphorylations associated with genetic risk for human brain disorders. We discover proline-directed kinases as major developmental regulators. APEX2 transgenic reporter proximity labeling offers flexible strategies for subcellular proteomics with cell type specificity in early neurodevelopment, a critical period for neuropsychiatric disease.

GPCR kinases generate an APH1A phosphorylation barcode to regulate amyloid-ß generation.

Emerging evidence suggests that G protein-coupled receptor (GPCR) kinases (GRKs) are associated with the pathophysiology of Alzheimer's disease (AD). However, GRKs have not been directly implicated in regulation of the amyloid-ß (Aß) pathogenic cascade in AD. Here, we determine that GRKs phosphorylate a non-canonical substrate, anterior pharynx-defective 1A (APH1A), an integral component of the γ-secretase complex. Significantly, we show that GRKs generate distinct phosphorylation barcodes in intracellular loop 2 (ICL2) and the C terminus of APH1A, which differentially regulate recruitment of the scaffolding protein ß-arrestin 2 (ßarr2) to APH1A and γ-secretase-mediated Aß generation. Further molecular dynamics simulation studies reveal an interaction between the ßarr2 finger loop domain and ICL2 and ICL3 of APH1A, similar to a GPCR-ß-arrestin complex, which regulates γ-secretase activity. Collectively, these studies provide insight into the molecular and structural determinants of the APH1A-ßarr2 interaction that critically regulate Aß generation.

Modeling oxidative injury response in human kidney organoids.

BACKGROUND: Hemolysis occurs in many injury settings and can trigger disease processes. In the kidney, extracellular hemoglobin can induce damage via several mechanisms. These include oxidative stress, mitochondrial dysfunction, and inflammation, which promote fibrosis and chronic kidney disease. Understanding the pathophysiology of these injury pathways offers opportunities to develop new therapeutic strategies. METHODS: To model hemolysis-induced kidney injury, human kidney organoids were treated with hemin, an iron-containing porphyrin, that generates reactive oxygen species. In addition, we developed an induced pluripotent stem cell line expressing the biosensor, CytochromeC-GFP (CytoC-GFP), which provides a real-time readout of mitochondrial morphology, health, and early apoptotic events. RESULTS: We found that hemin-treated kidney organoids show oxidative damage, increased expression of injury markers, impaired functionality of organic anion and cation transport and undergo fibrosis. Injury could be detected in live CytoC-GFP organoids by cytoplasmic localization of fluorescence. Finally, we show that 4-(phenylthio)butanoic acid, an HDAC inhibitor with anti-fibrotic effects in vivo, reduces hemin-induced human kidney organoid fibrosis. CONCLUSION: This work establishes a hemin-induced model of kidney organoid injury. This platform provides a new tool to study the injury and repair response pathways in human kidney tissue and will assist in the development of new therapeutics.

Synaptic Proteome Alterations in the Primary Auditory Cortex of Individuals With Schizophrenia.

Importance: Findings from unbiased genetic studies have consistently implicated synaptic protein networks in schizophrenia, but the molecular pathologic features within these networks and their contribution to the synaptic and circuit deficits thought to underlie disease symptoms remain unknown. Objective: To determine whether protein levels are altered within synapses from the primary auditory cortex (A1) of individuals with schizophrenia and, if so, whether these differences are restricted to the synapse or occur throughout the gray matter. Design, Setting, and Participants: This paired case-control study included tissue samples from individuals with schizophrenia obtained from the Allegheny County Office of the Medical Examiner. An independent panel of health care professionals made consensus DSM-IV diagnoses. Each tissue sample from an individual with schizophrenia was matched by sex, age, and postmortem interval with 1 sample from an unaffected control individual. Targeted mass spectrometry was used to measure protein levels in A1 gray matter homogenate and synaptosome preparations. All experimenters were blinded to diagnosis. Mass spectrometry data were collected from September 26 through November 4, 2016, and analyzed from November 3, 2016, to July 15, 2019. Main Outcomes and Measures: Primary measures were homogenate and synaptosome protein levels and their coregulation network features. Hypotheses generated before data collection were (1) that levels of canonical postsynaptic proteins in A1 synaptosome preparations would differ between individuals with schizophrenia and controls and (2) that these differences would not be explained by changes in total A1 homogenate protein levels. Results: Synaptosome and homogenate protein levels were investigated in 48 individuals with a schizophrenia diagnosis and 48 controls (mean age in both groups, 48 years [range, 17-83 years]); each group included 35 males (73%) and 13 females (27%). Robust alterations (statistical cutoff set at an adjusted Limma P < .05) were observed in synaptosome levels of canonical mitochondrial and postsynaptic proteins that were highly coregulated and not readily explained by postmortem interval, antipsychotic drug treatment, synaptosome yield, or underlying alterations in homogenate protein levels. Conclusions and Relevance: These findings suggest a robust and highly coordinated rearrangement of the synaptic proteome. In line with unbiased genetic findings, alterations in synaptic levels of postsynaptic proteins were identified, providing a road map to identify the specific cells and circuits that are impaired in individuals with schizophrenia A1.