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Mutations in the human NOTCH3 gene cause cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), but the pathogenic mechanisms of the disorder remain unclear. We investigated the cytotoxic properties of mutant Notch3 using stable cell lines with inducible expression of either wild-type or two mutants p.R133C and p.C185R. We found that both mutants of Notch3 were prone to aggregation and retained in the endoplasmic reticulum (ER). The turnover rates of the mutated Notch3 proteins were strikingly slow, with half-lives greater than 6 days, whereas wild-type Notch3 was rapidly degraded, with a half-life of 0.7 days. The expression of mutant Notch3 also impaired cell proliferation compared with wild-type Notch3. In addition, cell lines expressing mutant Notch3 were more sensitive to proteasome inhibition resulting in cell death. These findings suggest that prolonged retention of mutant Notch3 aggregates in the ER decreases cell growth and increases sensitivity to other stresses. It is also possible that the aggregate-prone property of mutant Notch3 contributes to a pathogenic mechanism underlying CADASIL.

Abstract:Manganese is known to induce neurological disorders similar to parkinsonisms. A dopamine deficiency has been demonstrated in Parkinson's disease and in chronic manganese poisoning, suggesting that the mechanisms underlying the neurotoxic effects of the metal ion are related to a functional abnormality of the extrapyramidal system. However, the details have yet to be elucidated. Here we report that manganese causes characteristic internucleosomal DNA fragmentation, a biochemical hallmark of apoptosis, in PC12 cells. It was transcription dependent, relatively specific for manganese, and blocked in BcI-2-overexpressed PC12 cells.The results indicate that apoptosis may play a role in the dopaminergic neurotoxicity associated with manganese, the first metal to be reported to induce this form of cell death. The early biochemical events show the impairment of energy metabolism, and the process may require new synthesis of proteins such as c-Fos and c-Jun. In addition, manganese induces phosphorylation of c-Jun at Ser 63 and Ser73 and SEK1 /MKK4 (c-Jun N-terminal kinase kinase) at Thr258 and tyrosine phosphorylation of several proteins. These results indicate that manganese activates specific signal cascades including the c-Jun Nterminal kinase pathway. Key Words: Apoptosis-Manganese-DNA fragmentation-c-Jun N-terminal kinase/ stress-activated protein kinase-Tyrosine phosphorylation-Parkinson's disease.

We investigated the temporal and spatial expression patterns of the GDNF gene after subjecting rats to an acute contusion injury of the spinal cord using the weight drop technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that GDNF transcription in the spinal cord began to increase within 30 min after injury and peaked within 3 h. Immunohistochemical analysis showed GDNF immunoreactivity to be present mainly in microglia and macrophages 1 day after injury, but not in neurons or astrocytes. This immediate upregulation of GDNF gene expression may be a component of an inflammatory process and probably exerts a protective effect on neurons following spinal cord injury (SCI).

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Apolipoprotein E (apoE) ⑀4 and hyperhomocysteinemia are risk factors for Alzheimer disease (AD). The dimerization of apoE3 by disulfide bonds between cysteine residues enhances apoE3 function to generate HDL. Because homocysteine (Hcy) harbors a thiol group, we examined whether Hcy interferes with the dimerization of apoE3 and thereby impairs apoE3 function. We found that Hcy inhibits the dimerization of apoE3 and reduces apoE3-mediated HDL generation to a level similar to that by apoE4, whereas Hcy does not affect apoE4 function. Western blot analysis of cerebrospinal fluid showed that the ratio of apoE3 dimers was significantly lower in the samples from the patients with hyperhomocysteinemia than in those that from control subjects. Hyperhomocysteinemia induced by subcutaneous injection of Hcy to apoE3 knock-in mice decreased the level of the apoE3 dimer in the brain homogenate. Because apoE-HDL plays a role in amyloid ␤-protein clearance, these results suggest that two different risk factors, apoE4 and hyperhomocysteinemia, may share a common mechanism that accelerates the pathogenesis of AD in terms of reduced HDL generation.It has been shown that the possession of the apolipoprotein E (apoE) ⑀4 allele is a major risk factor for Alzheimer disease (AD) 2 (1). In the central nervous system, apoE is one of the major lipid acceptors to remove cholesterol from cells and generate HDL particles. Previous studies have shown that apoE isoforms do not affect apoE binding to ABCA1, that apoE-mediated ABCA1-dependent cholesterol efflux is not affected by apoE isoforms in fibroblasts (2), and that there is no apoE-isoform-dependence on apoE-mediated lipid efflux from mouse astrocytes (3). Other lines of evidence have shown that apoE induces lipid efflux from macrophages and neural cells in an isoform-dependent manner; apoE3 induces a greater lipid efflux than apoE4 (4 -9). It has been shown that two major factors cause this apoE-isoform-dependent generation of HDL. Namely, intramolecular domain interaction occurring in apoE4 attenuates apoE4 ability to generate HDL and intermolecular dimerization by disulfide bonds between cysteines in apoE3 enhances apoE3 ability to generate HDL in neural cells (10).Recent studies have shown other functions of apoE as well, including an intracellular function of apoE (11, 12) and a function of apoE in clearance and degradation of A␤. It has been demonstrated that apoE isoforms differentially regulate A␤ clearance from the brain (13), and that an increased level of lipidated apoE, namely, apoE-HDL, stimulates A␤ degradation (14). These lines of evidence suggest that the lower ability of apoE4 than apoE3 to generate HDL would result in an enhanced A␤ deposition in the brain owing to the lower A␤ degradation/clearance from the brain. Similarly, apoE-isoform-dependent HDL generation results in a lower HDL-cholesterol level in serum in those who possess apoE ⑀4 allele, which is a risk factor for atherosclerosis (15) and cerebral infarction (16).In light of these findings, it is interesting t...