Batten disease is the most common progressive neurodegenerative disorder in children, with an incidence as high as one in 12,500 live births. Batten disease results from mutation of CLN3, and is characterized pathologically by the accumulation of autofluorescent hydrophobic material in the lysosome of neurons and other cell types. However, the mechanism driving these cellular alterations and the manner in which they relate to the neurodegeneration in Batten disease is unknown. Individuals with Batten disease, and a cln3-knockout mouse model for Batten disease have a circulating autoantibody to glutamic acid decarboxylase (GAD65) that is inhibitory to this enzyme's ability to convert glutamic acid to 7-aminobutyric acid (GABA). The GAD65 autoantibody associates with the brain in cln3-knockout mice, inhibits the activity of GAD, and results in a subsequent elevation of glutamate and altered expression of genes involved in the regulation and utilization of glutamate. The major goal of this proposal is to establish and characterize the contribution of the GAD65 autoantibody to the pathogenesis of Batten disease. As the model for this study is a genetically defined I knockout mouse, we will establish whether the presence of a GAD65 autoantibody is simply an epl- phenomenon, and whether it contributes directly to Batten disease. We shall: (i) construct cln3-knockout mice that are unable to mount an immune response to GAD65 by crossing to MuMT and C-alpha knockout mice, which lack the ability to generate B-cells, and CD4+ and CD8+ T-cells, respectively. This will enable us to determine whether the autoantibody contributes to the pathology of Batten disease. (ii) test by passive transfer of GAD65 autoantibodies to normal mice the degree of pathogenicity on the CNS mediated by these autoantibodies. Also, by transfer of GAD65 autoantibodies to immune deficient mice we will determine if the cln3-defect allows for preferential deposition of IgG in the brain (iii) define the inflammatory response to the presence of autoantibodies and the participation of the innate immune system in the disease process in cln3- knockout mice by examining cytokines such as IL-1 and TNF, complement proteins and microglial activation, respectively. (iv) characterize cell type specific molecular effects that elevated glutamate and exposure to the autoantibody have on primary neuronal culture from cln3-knockout mice.
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