Summary Astrocytes are ubiquitous in the brain and are widely held to be largely identical. However, this view has not been fully tested and the possibility that astrocytes are neural circuit-specialized remains largely unexplored. Here, we used multiple, integrated approaches including RNA-Seq, mass spectrometry, electrophysiology, immunohistochemistry, serial block-face scanning electron microscopy, morphological reconstructions, pharmacogenetics, as well as diffusible dye, calcium and glutamate imaging, to directly compare adult striatal and hippocampal astrocytes under identical conditions. We found significant differences between striatal and hippocampal astrocytes in electrophysiological properties, Ca2+ signaling, morphology and astrocyte-synapse proximity. Unbiased evaluation of actively translated RNA and proteomic data confirmed significant astrocyte diversity between hippocampal and striatal circuits. We thus report core astrocyte properties, reveal evidence for specialized astrocytes within neural circuits and provide new, integrated database resources and approaches to explore astrocyte diversity and function throughout the adult brain.

Each of the 30 human amyloid diseases is associated with the aggregation of a particular precursor protein into amyloid fibrils. In transthyretin amyloidosis (ATTR), mutant or wild-type forms of the serum carrier protein transthyretin (TTR), synthesized and secreted by the liver, convert to amyloid fibrils deposited in the heart and other organs. The current standard of care for hereditary ATTR is liver transplantation, which replaces the mutant gene with the wild-type gene. However, the procedure is often followed by cardiac deposition of wild-type TTR secreted by the new liver. Here we find that amyloid fibrils extracted from autopsied and explanted hearts of ATTR patients robustly seed wild-type TTR into amyloid fibrils in vitro. Cardiac-derived ATTR seeds can accelerate fibril formation of wild-type and monomeric TTR at acidic pH and under physiological conditions, respectively. We show that this seeding is inhibited by peptides designed to complement structures of TTR fibrils. These inhibitors cap fibril growth, suggesting an approach for halting progression of ATTR.

Highlights d Collagen V deficiency increases scar size after acute heart injury d Mechanical properties of scars are altered with Col V deficiency d Altered mechanosensitive cues augment myofibroblast formation in scar d Inhibition of specific integrins rescues increased scarring in Col-V-deficient states

The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro. Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.

Objective Mitochondria are organelles primarily responsible for energy production, and recent evidence indicates that alterations in size, shape, location, and quantity occur in response to fluctuations in energy supply and demand. We tested the impact of acute and chronic exercise on mitochondrial dynamics signaling and determined the impact of the mitochondrial fission regulator Dynamin related protein (Drp)1 on exercise performance and muscle adaptations to training. Methods Wildtype and muscle-specific Drp1 heterozygote (mDrp1 +/− ) mice, as well as dysglycemic (DG) and healthy normoglycemic men (control) performed acute and chronic exercise. The Hybrid Mouse Diversity Panel, including 100 murine strains of recombinant inbred mice, was used to identify muscle Dnm1L (encodes Drp1)-gene relationships. Results Endurance exercise impacted all aspects of the mitochondrial life cycle, i.e. fission-fusion, biogenesis, and mitophagy. Dnm1L gene expression and Drp1 Ser616 phosphorylation were markedly increased by acute exercise and declined to baseline during post-exercise recovery. Dnm1L expression was strongly associated with transcripts known to regulate mitochondrial metabolism and adaptations to exercise. Exercise increased the expression of DNM1L in skeletal muscle of healthy control and DG subjects, despite a 15% ↓( P = 0.01) in muscle DNM1L expression in DG at baseline. To interrogate the role of Dnm1L further, we exercise trained male mDrp1 +/− mice and found that Drp1 deficiency reduced muscle endurance and running performance, and altered muscle adaptations in response to exercise training. Conclusion Our findings highlight the importance of mitochondrial dynamics, specifically Drp1 signaling, in the regulation of exercise performance and adaptations to endurance exercise training.

Alzheimer’s disease (AD) is the most common cause of dementia, yet there is no cure or diagnostics available prior to the onset of clinical symptoms. Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are released from almost all types of cell. Genome-wide association studies have linked multiple AD genetic risk factors to microglia-specific pathways. It is plausible that microglia-derived EVs may play a role in the progression of AD by contributing to the dissemination of insoluble pathogenic proteins, such as tau and Aβ. Despite the potential utility of EVs as a diagnostic tool, our knowledge of human brain EV subpopulations is limited. Here we present a method for isolating microglial CD11b-positive small EVs from cryopreserved human brain tissue, as well as an integrated multiomics analysis of microglial EVs enriched from the parietal cortex of four late-stage AD (Braak V-VI) and three age-matched normal/low pathology (NL) cases. This integrated analysis revealed 1,000 proteins, 594 lipids, and 105 miRNAs using shotgun proteomics, targeted lipidomics, and NanoString nCounter technology, respectively. The results showed a significant reduction in the abundance of homeostatic microglia markers P2RY12 and TMEM119, and increased levels of disease-associated microglia markers FTH1 and TREM2, in CD11b-positive EVs from AD brain compared to NL cases. Tau abundance was significantly higher in AD brain-derived microglial EVs. These changes were accompanied by the upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free cholesterol were elevated in microglial EVs from the AD brain. Lipidomic analysis also revealed a proinflammatory lipid profile, endolysosomal dysfunction, and a significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids, suggesting a potential defect in acyl-chain remodeling. Additionally, four miRNAs associated with immune and cellular senescence signaling pathways were significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may be accompanied by endolysosomal impairment and the release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. We suggest that the analysis of microglia-derived EVs has merit for identifying novel EV-associated biomarkers and providing a framework for future larger-scale multiomics studies on patient-derived cell-type-specific EVs.

IntroductionThe specific protein composition of stroke-causing emboli is unknown. Because ischemic stroke has a varied etiology, it is possible that the composition of the thrombus from which an embolus originated will have distinctive molecular characteristics reflective of the underlying pathophysiology. We used mass spectrometry to evaluate the protein composition of retrieved emboli from patients with differing stroke etiologies and correlated the protein levels to serum predictors of atherosclerosis.MethodsEmboli from 20 consecutive acute stroke patients were retrieved by thrombectomy during routine stroke care. Thrombus proteins were extracted, digested, and multidimensional fractionation of peptides was performed. Fractionated peptides underwent nano-liquid chromatography with tandem mass spectrometry. Spectra were searched using Mascot software in which results with p < 0.05 (95% confidence interval) were considered significant and indicating identity. The results were correlated to A1C, low-density lipoprotein (LDL), and erythrocyte sedimentation rate (ESR) taken on admission.ResultsEleven patients had atrial fibrillation, four had significant proximal vessel atherosclerosis, two were cryptogenic, and three had other identified stroke risk factors (left ventricular thrombus, dissection, endocarditis). Eighty-one common proteins (e.g., hemoglobin, fibrin, actin) were found in all 20 emboli. Serum LDL levels correlated with Septin-2 (rs = 0.78, p = 0.028), Phosphoglycerate Kinase 1 (rs = 0.75, p = 0.036), Integrin Alpha-M (rs = 0.68, p = 0.033) and Glucose-6-phosphate dehydrogenase (rs = 0.63, p = 0.05). Septin-7 levels inversely correlated to ESR (rs = −0.84, p = 0.01). No significant protein correlations to A1C or tPA use were found.ConclusionOur exploratory study presents mass spectrometry analysis of thrombi retrieved from acute stroke patients and correlates the thrombus proteome to clinical features of the patient. Notably, we found proteins associated with inflammation (e.g., Integrin Alpha-M) in emboli from patients with high LDL. Although these findings are tempered by a small sample size, we provide preliminary support for the feasibility of utilizing proteomic analysis of emboli to discover proteins that may be used as markers for stroke etiology.

Background & AimsHeavy alcohol drinking is associated with pancreatitis, whereas moderate intake lowers the risk. Mice fed ethanol long term show no pancreas damage unless adaptive/protective responses mediating proteostasis are disrupted. Pancreatic acini synthesize digestive enzymes (largely serine hydrolases) in the endoplasmic reticulum (ER), where perturbations (eg, alcohol consumption) activate adaptive unfolded protein responses orchestrated by spliced X-box binding protein 1 (XBP1). Here, we examined ethanol-induced early structural changes in pancreatic ER proteins.MethodsWild-type and Xbp1+/- mice were fed control and ethanol diets, then tissues were homogenized and fractionated. ER proteins were labeled with a cysteine-reactive probe, isotope-coded affinity tag to obtain a novel pancreatic redox ER proteome. Specific labeling of active serine hydrolases in ER with fluorophosphonate desthiobiotin also was characterized proteomically. Protein structural perturbation by redox changes was evaluated further in molecular dynamic simulations.ResultsEthanol feeding and Xbp1 genetic inhibition altered ER redox balance and destabilized key proteins. Proteomic data and molecular dynamic simulations of Carboxyl ester lipase (Cel), a unique serine hydrolase active within ER, showed an uncoupled disulfide bond involving Cel Cys266, Cel dimerization, ER retention, and complex formation in ethanol-fed, XBP1-deficient mice.ConclusionsResults documented in ethanol-fed mice lacking sufficient spliced XBP1 illustrate consequences of ER stress extended by preventing unfolded protein response from fully restoring pancreatic acinar cell proteostasis during ethanol-induced redox challenge. In this model, orderly protein folding and transport to the secretory pathway were disrupted, and abundant molecules including Cel with perturbed structures were retained in ER, promoting ER stress-related pancreas pathology.