The expression of major histocompatibility complex (MHC) class I and II antigens was studied in surgical and postmortem brain biopsy tissue using light and electron microscopic immunocytochemistry. In addition, monoclonal antibodies directed against human macrophages (EBM11) and alpha-smooth muscle actin were applied. It is shown that blood vessel-associated MHC class II immunoreactivity in histologically normal human brain can be localized to a distinct class of cells, termed perivascular cells, which share macrophage but not smooth muscle cell antigen. This immunophenotype, the location in the perivascular space as well as the morphology, frequency and tissue distribution distinguish perivascular cells from pericytes and intraparenchymal microglia. It is suggested that MHC class II positive perivascular cells are a normal constituent of the human cerebral microvasculature. The potential role of these cells in immunological reactions occurring at the blood-brain interface is discussed.

Mutation in CDC48 (cdc48 S565G ), a gene essential in the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, led to the discovery of apoptosis as a mechanism of cell death in the unicellular organism Saccharomyces cerevisiae. Elucidating Cdc48p-mediated apoptosis in yeast is of particular interest, because Cdc48p is the highly conserved yeast orthologue of human valosin-containing protein (VCP), a pathological effector for polyglutamine disorders and myopathies. Here we show distinct proteomic alterations in mitochondria in the cdc48 S565G yeast strain. These observed molecular alterations can be related to functional impairment of these organelles as suggested by respiratory deficiency of cdc48 S565G cells. Mitochondrial dysfunction in the cdc48 S565G strain is accompanied by structural damage of mitochondria indicated by the accumulation of cytochrome c in the cytosol and mitochondrial enlargement. We demonstrate accumulation of reactive oxygen species produced predominantly by the cytochrome bc 1 complex of the mitochondrial respiratory chain as suggested by the use of inhibitors of this complex. Concomitantly, emergence of caspase-like enzymatic activity occurs suggesting a role for caspases in the cell death process. These data strongly point for the first time to a mitochondrial involvement in Cdc48p/VCPdependent apoptosis.Fundamental cellular processes, such as the formation of organelles (ER,3 Golgi apparatus, and the nuclear envelope), or ubiquitin-dependent ER-associated protein degradation (ERAD) have been linked to the yeast protein Cdc48p and its highly conserved mammalian orthologue VCP (1-4). Mutations in VCP have been associated with "inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia" (IBMPFD), a dominant human disorder (5, 6). A genetic screening of a Drosophila model for human polyglutamine diseases, a class of inherited neurodegenerative disorders, identified the Drosophila homologue of Cdc48p/VCP as a modulator of apoptotic cell death (7), leading these authors to propose VCP as a pathological effector for polyglutamine-induced neurodegeneration. However, the cellular mechanisms underlying VCP-mediated cell death in these human disorders remain largely unknown. Apoptotic phenotypes in cells expressing mutated Cdc48p/ VCP have originally been described in budding yeast (8) and were thereafter confirmed in mammalian cell cultures (9, 10), in trypanosomes (11), and in zebrafish (12). Notably, Cdc48p was the first apoptotic mediator found in Saccharomyces cerevisiae (8). The expression of a point-mutated CDC48 gene (cdc48 S565G ) leads to a characteristic apoptotic phenotype: phosphatidylserine externalization, DNA fragmentation, chromatin condensation, nuclear fragmentation, and vacuolization (8, 13). These results obtained in the cdc48 S565G strain initiated the establishment of yeast as a model to study evolutionary conserved mechanisms of apoptotic regulation (14 -16).Mitochondria play a crucial role in many apoptotic pathways in ...

One major problem concerning the electrophoresis of mitochondria is the heterogeneity of mitochondrial appearance especially under pathological conditions. We show here the use of zone electrophoresis in a free flow electrophoresis device (ZE-FFE) as an analytical sensor to discriminate between different yeast mitochondrial populations. Impairment of the structural properties of the organelles by hyperosmotic stress resulted in broad separation profiles. Conversely untreated mitochondria gave rise to homogeneous populations reflected by sharp separation profiles. Yeast mitochondria with altered respiratory activity accompanied by a different outer membrane proteome composition could be discriminated based on electrophoretic deflection. Proteolysis of the mitochondrial surface proteome and the deletion of a single major protein species of the mitochondrial outer membrane altered the ZE-FFE deflection of these organelles. To demonstrate the usefulness of ZE-FFE for the analysis of mitochondria associated with pathological processes, we analyzed mitochondrial fractions from an apoptotic yeast strain. The cdc48 S565G strain carries a mutation in the CDC48 gene that is an essential participant in the endoplasmic reticulum-associated protein degradation pathway. Mutant cells accumulate polyubiquitinated proteins in microsomal and mitochondrial extracts. Subsequent ZE-FFE characterization could distinguish a mitochondrial subfraction specifically enriched with polyubiquitinated proteins from the majority of non-affected mitochondria. This result demonstrates that ZE-FFE may give important information on the specific properties of