Mononuclear phagocytes are a population of multi-phenotypic cells and have dual roles in brain destruction/reconstruction. The phenotype-specific roles of microglia/macrophages in traumatic brain injury (TBI) are, however, poorly characterized. In the present study, TBI was induced in mice by a controlled cortical impact (CCI) and animals were killed at 1 to 14 days post injury. Real-time polymerase chain reaction (RT-PCR) and immunofluorescence staining for M1 and M2 markers were performed to characterize phenotypic changes of microglia/macrophages in both gray and white matter. We found that the number of M1-like phagocytes increased in cortex, striatum and corpus callosum (CC) during the first week and remained elevated until at least 14 days after TBI. In contrast, M2-like microglia/macrophages peaked at 5 days, but decreased rapidly thereafter. Notably, the severity of white matter injury (WMI), manifested by immunohistochemical staining for neurofilament SMI-32, was strongly correlated with the number of M1-like phagocytes. In vitro experiments using a conditioned medium transfer system confirmed that M1 microglia-conditioned media exacerbated oxygen glucose deprivation-induced oligodendrocyte death. Our results indicate that microglia/macrophages respond dynamically to TBI, experiencing a transient M2 phenotype followed by a shift to the M1 phenotype. The M1 phenotypic shift may propel WMI progression and represents a rational target for TBI treatment.

Loss of mitochondrial membrane integrity and release of apoptogenic factors are a key step in the signaling cascade leading to neuronal cell death in various neurological disorders, including ischemic injury. Emerging evidence has suggested that the intramitochondrial protein apoptosis-inducing factor (AIF) translocates to the nucleus and promotes caspase-independent cell death induced by glutamate toxicity, oxidative stress, hypoxia, or ischemia. However, the mechanism by which AIF is released from mitochondria after neuronal injury is not fully understood. In this study, we identified calpain I as a direct activator of AIF release in neuronal cultures challenged with oxygen-glucose deprivation and in the rat model of transient global ischemia. Normally residing in both neuronal cytosol and mitochondrial intermembrane space, calpain I was found to be activated in neurons after ischemia and to cleave intramitochondrial AIF near its N terminus. The truncation of AIF by calpain activity appeared to be essential for its translocation from mitochondria to the nucleus, because neuronal transfection of the mutant AIF resistant to calpain cleavage was not released after oxygen-glucose deprivation. Adeno-associated virus-mediated overexpression of calpastatin, a specific calpain-inhibitory protein, or small interfering RNAmediated knockdown of calpain I expression in neurons prevented ischemia-induced AIF translocation. Moreover, overexpression of calpastatin or knockdown of AIF expression conferred neuroprotection against cell death in neuronal cultures and in hippocampal CA1 neurons after transient global ischemia. Together, these results define calpain I-dependent AIF release as a novel signaling pathway that mediates neuronal cell death after cerebral ischemia.

Emerging evidence describe heat shock proteins (HSPs) as critical regulators in normal neural physiological function as well as in cell stress responses. The functions of HSPs represent an enormous and diverse range of cellular activities, far beyond the originally identified role in protein folding and chaperoning. Now understood to be involved in processes such as synaptic transmission, autophagy, ER stress response, protein kinase and cell death signaling as well as protein chaperone and folding, manipulation of HSPs have robust effects on the fate of cells in neurological injury and disease states. The ongoing exploration of multiple HSP superfamilies has underscored the pluripotent nature of HSPs in the cellular context, and demanded the recent restructuring of the nomenclature referring to these families to reflect a re-organization based on structure and function. In keeping with this re-organization, we have first discussed the HSP superfamilies in terms of protein structure, regulation and expression and distribution in the brain. We then explore major cellular functions of HSPs that are relevant to neural physiological states, and from there discuss known and proposed HSP impact on major neurological disease states. This review article presents a three-part discussion on the array of HSPs families relevant to neuronal tissue, their cellular functions, and the exploration of therapeutic targets of these proteins in the context of neurological diseases.

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