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Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common condition of hereditary stroke and vascular dementia. CADASIL is caused by Notch3 mutation, leading to progressive degeneration of vascular smooth muscle cells (vSMCs) of the small arteries in the brain. However, the pathogenesis of CADASIL remains largely unknown, and treatment that can stop or delay the progression of CADASIL is not yet available. Using both wild type mice and transgenic mice carrying the human mutant Notch3 gene (CADASIL mice), we have recently characterized the pathological features of CADASIL and determined the therapeutic efficacy of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) in CADASIL. Our findings have revealed novel pathological changes in the endothelium of cerebral capillaries and in the neural stem cells (NSCs). We have also observed the impairment of cognitive function in CADASIL mice. Moreover, SCF+G-CSF treatment improves cognitive function, inhibits Notch3 mutation-induced vSMC degeneration, cerebral blood bed reduction, cerebral capillary damage, and NSC loss, and increases neurogenesis and angiogenesis. Here we compile an overview of our recently published studies, which provide new insights into understanding the pathogenesis of CADASIL and developing therapeutic strategies for this devastating neurological disease.

alphaB-crystallin is a member of the small heat shock proteins, which is abundantly expressed in various vertebrate tissues including the central nervous system. In our previous report, we showed alphaB-crystallin induction in activated astrocytes in the postischemic brain and in H2O2-treated primary astrocyte cultures. To investigate the functional significance of alphaB-crystallin induction in astrocytes, we generated a stable C6 astroglioma cell line overexpressing alphaB-crystallin. In these cells, hydrogen peroxide-induced apoptosis was reduced by 60% compared to parent cells. Furthermore, the repression of alphaB-crystallin expression by alphaB-crystallin siRNA transfection suppressed this protective effect, indicating that alphaB-crystallin is responsible for the protection against H2O2-induced apoptosis in C6 astroglioma cells. Similar level of aggravation in H2O2-induced apoptosis was observed in primary astrocyte cultures when alphaB-crystallin expression was suppressed by alphaB-crystallin siRNA transfection, confirming the importance of alphaB-crystallin. In addition, the induction of caspase-3 activity after H2O2 treatment was markedly suppressed in alphaB-crystallin-overexpressing cells, and immunoprecipitation proved binding between alphaB-crystallin and partially processed caspase-3 (a p24 intermediate). These results indicate that alphaB-crystallin confers protection against hydrogen peroxide-induced astrocytes apoptosis in part by inhibiting caspase-3 activation.

AlphaB-crystallin, known as a vertebrate lens protein, is a member of the small heat shock proteins (sHSP). AlphaB-crystallin is abundantly expressed in the vertebrate lens and striated muscles and it is also expressed constitutively in other tissues including the central nervous system (CNS). In our previous report, we showed alphaB-crystallin induction in activated astrocytes, which are enriched in the penumbra after transient focal cerebral ischemia. We also reported that alphaB-crystallin is significantly induced in astrocytes in the CA3 region of the hippocampus following KA-induced seizure. Here, we report that the expression of alphaB-crystallin is upregulated in H2O2-treated primary astrocyte cultures, which was prepared from newborn male Sprague-Dawley rats and that the proximal 408 bp of the alphaB-crystallin promoter harboring stress response element (STRE) is responsible for this induction. This effect of H2O2 was found to be virtually abolished by introducing mutations into STRE, and these mutations also impaired increased lens epithelial derived growth factor (LEDGF) binding to STRE after H2O2 treatment. Moreover, LEDGF was induced in primary astrocyte cultures after H2O2 treatment and alphaB-crystallin induction was significantly suppressed by transfecting small interfering RNA (siRNA) targeting LEDGF. Together these results indicate that the H2O2-induced upregulations of alphaB-crystallin in astrocytes are mediated by the LEDGF-STRE interaction on alphaB-crystallin promoter.

Cerebral ischemic injury proceeds with excitotoxicity-induced acute neuronal death in the ischemic core and with delayed damage processes in the penumbra. However, knowledge concerning the direct mediators that connect these two processes is limited. Here, we demonstrate that high-mobility group box 1 (HMGB1), a nonhistone DNA-binding protein, is massively released into the extracellular space immediately after ischemic insult and that it subsequently induces neuroinflammation in the postischemic brain. Short hairpin (sh)RNA-mediated HMGB1 downregulation in the postischemic brain suppressed infarct size, microglia activation, and proinflammatory marker induction, indicating that HMGB1 plays a crucial role in the inflammatory process. The proinflammatory cytokine-like function of extracellular HMGB1 was further verified in primary cortical cultures and microglial cultures. HMGB1 was found to accumulate in NMDA-treated primary cortical culture media, and supernatants collected from these cultures were found to trigger microglia activation, the hallmark of brain inflammation. Moreover, treatment with recombinant HMGB1 also induced microglial activation, but HMGB1-depleted supernatant produced by anti-HMGB1 antibody treatment or by HMGB1 shRNA expression did not, thus demonstrating the essential role of HMGB1 in microglial activation. Together, these results indicate that HMGB1 functions as a novel proinflammatory cytokine-like factor that connects excitotoxicity-induced acute damage processes and delayed inflammatory processes in the postischemic brain.

The p38 MAPK signaling pathway has been implicated in various pathological conditions of neuronal and non-neuronal cells. Here we report the differential induction of p38 MAPK isoforms, p38alpha and p38beta, in the adult gerbil brain following transient global ischemia. The p38alpha and p38beta kinase activities were gradually enhanced with the peak activity occurring around 2-4 days after ischemic insult. Immunohistochemical analysis revealed that p38alpha expression was increased as early as 4 h after ischemic insult and enhanced further reaching maximum induction around 4 days after ischemia. The induced p38alpha was concentrated in microglia in hippocampus as well as in frontal and parietal cortices of the brain, where significant neuronal damage was occurred. By contrast, immunostaining with anti-p38beta antibody indicated that p38beta was markedly induced in astrocytes in hippocampus around 4 days after ischemic insult, which lasted for the next several days. The differential induction of p38 MAPK isoforms following transient global ischemia, especially the induction of p38alpha and p38beta MAPKs in microglia and astrocytes, respectively, in different time points after ischemic insult suggest distinct roles of p38 MAPK isoforms in post-ischemic brain.