Protein aggregation critically affects cell viability in neurodegenerative diseases, but whether this also occurs in ischemic brain injury remains elusive. Prior studies report the post-ischemic aggregation of ubiquitin, small ubiquitin-related modifier (SUMO) and ribosomes, however whether other proteins are also affected is unknown. Here we employed a proteomic approach to identify the insoluble, aggregated proteome after cerebral ischemia. Mice underwent transient middle cerebral artery occlusion or sham-surgery. After 1-hour reperfusion, prior to apparent brain injury, mice were sacrificed and detergent-insoluble proteins were obtained and identified by nanoLC-MS/MS. Naturally existing insoluble proteins were determined in sham controls and aggregated proteins after cerebral ischemia/reperfusion were identified. Selected aggregated proteins found by proteomics were biochemically verified and aggregation propensities were studied during ischemia with or without reperfusion. We found that ischemia/reperfusion induces the aggregation of RNA-binding and heat-shock proteins, ubiquitin, SUMO and other proteins involved in cell signalling. RNA-binding proteins constitute the largest group of aggregating proteins in ischemia. These include TDP43, FUS, hnRNPA1, PSF/SFPQ and p54/NONO, all of which have been linked to neurodegeneration associated with amyotrophic lateral sclerosis and frontotemporal dementia. The aggregation of neurodegeneration-related disease proteins in cerebral ischemia unveils a previously unappreciated molecular overlap between neurodegenerative diseases and ischemic stroke.

BackgroundCancer-associated neoantigens (neoAg) derived from tumor genomic sequencing and predictive algorithms for mutated peptides are a promising basis for therapeutic vaccines under investigation. Although these are generally designed to bind major histocompatibility complex class I and induce CD8 cytolytic T lymphocyte (CTL) activity, results from preclinical and clinical studies demonstrate that the majority of neoAg vaccines efficiently induce CD4 T helper (Th) responses but not CTL. Despite this, these vaccines have demonstrated clinical efficacy. Therefore, understanding the mechanisms of CD4 + T cell-mediated tumor protection is critical to optimizing this immunotherapeutic strategy.MethodsWe investigated this phenomenon in the mineral oil-induced plasmacytoma (MOPC).315.BM (MOPC315) mouse model of multiple myeloma, a malignancy of plasma cells. MOPC315 cells express in their lambda chain a unique tumor-specific neoAg, an idiotypic (Id) peptide. We generated a vaccine formulated with this Id peptide fused to a heat shock protein HSC70 binding (HSB) motif co-delivered with poly (I:C). The immunogenicity of the Id-vaccine was measured in splenocytes by ELISpot. Mice were challenged with MOPC315 cells and antitumor immunity was assessed by co-incubating splenocytes and bone marrow mononuclear cells derived from vaccinated mice and controls, with the Id antigen and irradiated MOPC315 cells. The frequency of activated CD4 and CD8 T cells and their phenotype were characterized by flow cytometry.ResultsId-vaccine efficiently induced antigen-specific CD4 Th activity and antitumor immunity, protecting mice from MOPC315 tumor growth. CD4 cytolytic activity was not detected under these conditions. Polyfunctional CD8 T cells homed to the bone marrow microenvironment of protected mice and preferentially expanded only when restimulated ex vivo with both Id peptide and MOPC315 cells. Protective activity was abrogated by depletion of either CD4 or CD8 lymphocytes.ConclusionThese results demonstrate that Id-HSB +poly (I:C) vaccine protects against MOPC315 growth by priming Id-specific CD4 Th cells that confer protection against tumor but are not directly cytotoxic. These data indicate that activation of CD8 CTL against MOPC315-associated antigens not present in the vaccine is one of the major mechanisms of tumor immunity.

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Conventional genomic DNA (gDNA) extraction methods can take hours to complete, may require fume hoods and represent the most time-consuming step in many gDNA-based molecular assays. We systematically optimized a bead bashing-based (BBB) approach for rapid gDNA extraction without the need for a fume hood. Human tissue specimens (n = 34) subjected to the 12-min BBB method yielded 0.40 ± 0.17 (mean ± SD) μg of gDNA per milligram of tissue, sufficient for many downstream applications, and 3- and 6-min extensions resulted in an additional 0.43 ± 0.23 μg and 0.48 ± 0.43 μg per milligram of tissue, respectively. The BBB method provides a simple and rapid method for gDNA extraction from mammalian tissue that is applicable to time-sensitive clinical applications.