tetracycline and lactacystin

Parkinson's disease (PD) is a common progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic neural cell death occurs remains unknown. Proteins encoded by two other genes in which mutations cause familial PD, parkin and UCH-L1, are involved in regulation of the ubiquitin-proteasome pathway, suggesting that dysregulation of the ubiquitin-proteasome pathway is involved in the mechanism by which these mutations cause PD. We established inducible PC12 cell lines in which wild-type or mutant alpha-synuclein can be de-repressed by removing doxycycline. Differentiated PC12 cell lines expressing mutant alpha-synuclein showed decreased activity of proteasomes without direct toxicity. Cells expressing mutant alpha-synuclein showed increased sensitivity to apoptotic cell death when treated with sub-toxic concentrations of an exogenous proteasome inhibitor. Apoptosis was accompanied by mitochondrial depolarization and elevation of caspase-3 and -9, and was blocked by cyclosporin A. These data suggest that expression of mutant alpha-synuclein results in sensitivity to impairment of proteasome activity, leading to mitochondrial abnormalities and neuronal cell death.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Caspases; Cyclosporine; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Gene Expression Regulation; Immunoenzyme Techniques; Membrane Potentials; Mitochondria; Multienzyme Complexes; Mutation; Nerve Tissue Proteins; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Rats; Synucleins; Tetracycline; Transfection

Neuronal intranuclear inclusions are a histopathological hallmark of Huntington's disease. Nevertheless, the precise mechanism by which they are formed and their relevance to neuronal cell death and/or dysfunction remains unclear. We recently generated a conditional mouse model of Huntington's disease (HD94) in which silencing expression of mutated huntingtin led to the disappearance of intranuclear aggregates and amelioration of the behavioral phenotype. Here, we analyze primary striatal neuronal cultures from HD94 mice to explore the dynamics of aggregate formation and reversal, the possible mechanisms involved, and the correlation between aggregates and neuronal death. In parallel, we examine symptomatic adult HD94 mice in similar studies and explored the relationship between aggregate clearance and behavioral reversal. We report that, in culture, aggregate formation and reversal were rapid processes, such that 2 d of transgene expression led to aggregate formation, and 5 d of transgene suppression led to aggregate disappearance. In mice, full reversal of aggregates and intranuclear mutant huntingtin was more rapid than reported previously and preceded the motor recovery by several weeks. Furthermore, the proteasome inhibitor lactacystin inhibited the aggregate clearance observed in culture, thus indicating that aggregate formation is a balance between the rate of huntingtin synthesis and its degradation by the proteasome. Finally, neither expression of the mutant huntingtin nor aggregates compromised the viability of HD94 cultures. This correlated with the lack of cell death in symptomatic HD94 mice, thus demonstrating that neuronal dysfunction, and not cell loss, triggered by mutant huntingtin underlies symptomatology.

Topics: Acetylcysteine; Animals; Behavior, Animal; Cell Death; Cell Survival; Cells, Cultured; Corpus Striatum; Cysteine Endopeptidases; Disease Models, Animal; Gene Silencing; Genes, Dominant; Huntingtin Protein; Huntington Disease; Locomotion; Macromolecular Substances; Mice; Mice, Neurologic Mutants; Multienzyme Complexes; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Phenotype; Proteasome Endopeptidase Complex; Remission Induction; Tetracycline; Transgenes; Ubiquitin

The proteasome is the principal provider of major histocompatibility complex (MHC) class I-presented peptides. Interferon (IFN)-gamma induces expression of three catalytically active proteasome subunits (LMP2, LMP7, and MECL-1) and the proteasome-associated activator PA28. These molecules are thought to optimize the generation of MHC class I-presented peptides. However, known information on their contribution in vivo is very limited. Here, we examined the antigen processing of two murine leukemia virus-encoded cytotoxic T lymphocyte (CTL) epitopes in murine cell lines equipped with a tetracycline-controlled, IFN-gamma-independent expression system. We thus were able to segregate the role of the immunosubunits from the role of PA28. The presence of either immunosubunits or PA28 did not alter the presentation of a subdominant murine leukemia virus (MuLV)-derived CTL epitope. However, the presentation of the immunodominant MuLV-derived epitope was markedly enhanced upon induction of each of these two sets of genes. Thus, the IFN-gamma-inducible proteasome subunits and PA28 can independently enhance antigen presentation of some CTL epitopes. Our data show that tetracycline-regulated expression of PA28 increases CTL epitope generation without affecting the 20S proteasome composition or half-life. The differential effect of these IFN-gamma-inducible proteins on MHC class I processing may have a decisive influence on the quality of the CTL immune response.

Topics: Acetylcysteine; Animals; Antigen Presentation; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; Autoantigens; Blotting, Western; Cell Cycle Proteins; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Epitopes, T-Lymphocyte; Histocompatibility Antigens Class I; Interferon-gamma; Leukemia Virus, Murine; Mice; Mice, Inbred C57BL; Multienzyme Complexes; Precipitin Tests; Proteasome Endopeptidase Complex; Proteins; Rats; Sulfones; T-Lymphocytes, Cytotoxic; Tetracycline