Our laboratory has previously shown that multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are characterized by severe oxidative stress and the accumulation of oxidized proteins in the CNS, which in turn reduces cell viability and leads to tissue damage. Build-up of oxidized/misfolded proteins often occurs as a consequence of deficiencies in the proteasome, the enzymatic machinery responsible for their proteolytic removal. We have also discovered that in both MS and chronic EAE there is a significant reduction in the peptidolytic activities of the proteasome, which may explain the accumulation of oxidized proteins observed in the neurodegenerative stages of these diseases. While very important, all of these studies were carried out in tissue homogenates using fluorescent peptide substrates, and a critical issue that remains to be established is whether these deficits are localized to specific cell types or affect all CNS cells o the same extent. Furthermore, the proteolytic activity of the standard (s) and immuno (i) proteasome core particles (i.e. s-20S and i-20S) are greatly affected by the binding of regulatory caps (19S and 11S) and inhibitors, which may be expressed differentially in the various CNS cells during the course of the disease. In this proposal, we hypothesize that as a result of disease activity neurons, oligodendrocytes, astrocytes and microglia express unique patterns of proteasome complexes, making some CNS cells less equipped than others to remove abnormal proteins and thereby more susceptible to stress. To test this idea, we will first determine by double-immunohistochemistry the temporal/spatial pattern of proteasome core (s-20S and i-20S) and proteasome cap (19S and 11S) expression and their relationship to the extent of inflammation and oxidative stress during the course of EAE. We will then isolate neurons, oligodendrocytes, astrocytes and microglial cells from the CNS of control and EAE mice and measure the levels of each complex (20S, 11S- 20S-11S, 19S-20S-19S and 19S-20S-11S) by blue native-polyacrylamide gel electrophoresis and quantitative western blot analysis. Finally, we will compare the ability of the various proteasome complexes isolated from control and EAE tissues to digest oxidized and ubiquitinated proteins. These exploratory studies are essential for understanding how the various CNS cells regulate the expression and assembly of functional proteasome complexes as EAE progresses from the inflammatory to the neurodegenerative stages, and for determining the molecular basis for proteasome impairment in chronic EAE. Identification of the cell(s) with altered proteasome function will ultimately serve to design a therapeutic approach geared to decrease the toxic consequence of oxidative burden in EAE and MS.
Multiple sclerosis (MS), the most common neurological disorder of young adults, is characterized by the accumulation of oxidized/damaged proteins in the nervous tissue, which seems to be due in part to their impaired degradation by the proteasome. The planned studies will characterize the alterations in the amount and structure of complex proteasomes of various nerve cells in an animal model of MS, and will determine the functional consequences of such changes. Identification of the cell(s) with altered proteasome function will ultimately serve to design a therapeutic approach geared to decrease the toxic consequence of oxidative burden in MS.