Ischemic brain injury is a common disorder linked to a variety of diseases. Significant progress has been made in our understanding of the underlying mechanisms. Previous studies show that protein misfolding, aggregation, and multiple organelle damage are major pathological events in postischemic neurons. The autophagy pathway is the chief route for bulk degradation of protein aggregates and damaged organelles. The latest studies suggest that impairment of autophagy contributes to abnormal protein aggregation and organelle damages after brain ischemia. This article reviews recent studies of protein misfolding, aggregation, and impairment of autophagy after brain ischemia.

Chaperone-mediated autophagy (CMA) and the ubiquitin-proteasomal system (UPS) are two major protein degradation systems responsible for maintaining cellular homeostasis, but how these two systems are regulated after traumatic brain injury (TBI) remains unknown. TBI produces primary mechanical damage that must be repaired to maintain neuronal homeostasis. The level of lysosomal-associated membrane protein type 2A (LAMP2A) is the hallmark of CMA activity. The level of polyubiquitinated proteins (ubi-proteins) reflects UPS activity. This study utilized a moderate fluid percussion injury model in rats to investigate the changes in CMA and the UPS after TBI. Induction of CMA was manifested by significant upregulation of LAMP2A and secondary lysosomes during the periods of 1-15 days of recovery after TBI. In comparison, the levels of ubiproteins were increased only moderately after TBI. The increases in the levels of LAMP2A and 70 kDa heat-shock protein for CMA after TBI were seen mainly in the secondary lysosome-containing fractions. Confocal and electron microscopy further showed that increased LAMP2A or lysosomes were found mainly in neurons and proliferated microglia. Because CMA and the UPS are two major routes for elimination of different types of cellular aberrant proteins, the consecutive activation of these two pathways may serve as a protective mechanism for maintaining cellular homeostasis after TBI.

Supplementary Figure I:Figure I. Western blot and quantitative analysis of NR1, NR2A, NR2B and phospho-NR2B Y1472 proteins in rat contralateral cortex after TBI. Neocortical tissues were obtained from the contralateral regions of sham-operated control rats and rats subjected to TBI followed by 0.5, 4 and 24 h recovery. Equal amounts of proteins from each sample in homogenate (Homo) and synaptosomal fractions (P2) were subjected to SDS-PAGE and immunoblotted with antibodies against NR1, NR2A, NR2B, phospho-NR2B Y1472, and β-actin respectively (A: NR1, B: NR2A, C: NR2B, D: phospho-NR2B Y1472). The changes of subunits NR1, NR2A, NR2B were normalized by β-actin, and phosphorylation level of NR2B Y1472 was normalized by total-NR2B. Data are

The microtubule-dependent GEF-H1 pathway controls synaptic re-networking and overall gene expression via regulating cytoskeleton dynamics. Understanding this pathway after ischemia is essential to developing new therapies for neuronal function recovery. However, how the GEF-H1 pathway is regulated following transient cerebral ischemia remains unknown. This study employed a rat model of transient forebrain ischemia to investigate alterations of the GEF-H1 pathway using Western blotting, confocal and electron microscopy, dephosphorylation analysis, and pull-down assay. The GEF-H1 activity was significantly upregulated by: (i) dephosphorylation and (ii) translocation to synaptic membrane and nuclear structures during the early phase of reperfusion. GEF-H1 protein was then downregulated in the brain regions where neurons were destined to undergo delayed neuronal death, but markedly upregulated in neurons that were resistant to the same episode of cerebral ischemia. Consistently, GTP-RhoA, a GEF-H1 substrate, was significantly upregulated after brain ischemia. Electron microscopy further showed that neuronal microtubules were persistently depolymerized in the brain region where GEF-H1 protein was downregulated after brain ischemia. The results demonstrate that the GEF-H1 activity is significantly upregulated in both vulnerable and resistant brain regions in the early phase of reperfusion. However, GEF-H1 protein is downregulated in the vulnerable neurons but upregulated in the ischemic resistant neurons during the recovery phase after ischemia. The initial upregulation of GEF-H1 activity may contribute to excitotoxicity, whereas the late upregulation of GEF-H1 protein may promote neuroplasticity after brain ischemia.