Abstract
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.
Highlights
Protein aggregation critically affects cell viability in neurodegenerative diseases, but whether this occurs in ischemic brain injury remains elusive
In Parkinson’s disease (PD), the aggregation of α-synuclein is thought to interfere with synaptic transmission, resulting in synaptic failure[3,4], whereas in Huntington’s disease (HD), huntingtin aggregation is involved in the transcriptional repression of BDNF and PGC-1α genes, mitochondrial dysfunction and defective axonal transport[5,6,7]
To identify aggregated proteins during I/R we subjected mice to middle cerebral artery occlusion (MCAO) or sham-surgery and harvested ipsilateral neocortical tissue after 1-hour reperfusion. This timepoint was chosen based on the prominent cortical aggregation of protein-modifiers ubiquitin and SUMO2/3 we had previously detected[12,13]
Summary
Protein aggregation critically affects cell viability in neurodegenerative diseases, but whether this occurs in ischemic brain injury remains elusive. 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. Protein aggregation is a pathological hallmark of neurodegenerative diseases (NDs), such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) In these diseases the aggregation of different proteins has been implicated in distinctive disease pathogeneses. Aggregation of RNA-binding proteins (RNABPs) TDP43 and FUS has been linked to ALS and FTD, where it may alter RNA processing[8,9,10] Taken together, these findings suggest that the formation of protein aggregates differentially affects distinct cellular processes in NDs, which may lead to neuronal dysfunction and death. Our results reveal an unexpected similarity between protein aggregates in chronic neurodegeneration and acute ischemic injury, which may suggest a formerly unrecognized link between the molecular pathology of neurodegenerative diseases and stroke
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