Amyotrophic lateral sclerosis (ALS) is a severe progressive neurodegenerative disease characterized by degeneration of motor neurons in the brain and spinal cord, resulting in neurogenic muscle wasting, paralysis, and death. Nearly 95% of ALS cases have pathology featuring ubiquitinated and hyperphosphorylated inclusions of the TDP-43 protein in neurons and glial cells. Similar inclusions of TDP-43 are hallmarks of another neurodegenerative disease, frontotemporal lobar degeneration (FTLD-TDP), which is a major cause of mid- to late-life dementia. An understanding of the mechanisms controlling TDP-43 pathology in ALS and FTLD-TDP is critical to the design of neuroprotective strategies. TDP-43 is directly phosphorylated by the kinase CDC7, and this phosphorylation drives TDP-43 neurotoxicity and resultant neurodegeneration. This proposal describes experiments exploring the cellular and molecular changes that promote TDP-43 targeted CDC7 kinase activity, with a focus on the development of therapeutic interventions for the treatment of TDP-43 proteinopathies such as ALS and FTLD-TDP. Disease specific changes in CDC7 may promote TDP-43 phosphorylation. In fact, increased CDC7 protein levels and post-translational modifications of CDC7 are observed in parallel with pathological TDP-43 phosphorylation in a mammalian cell model of TDP-43 phosphorylation. CDC7 protein levels, localization, post-translational modifications, and gene expression will be evaluated in mouse primary neuron culture, mammalian cell culture, and transgenic C. elegans models exhibiting TDP-43 phosphorylation using molecular biology, biochemistry, and genetics techniques. Characterizing disease-relevant changes in CDC7 will further our understanding of the causes of TDP-43 pathology in ALS and FTLD-TDP. To identify pathway(s) controlling CDC7 kinase activation in neurons, CDC7 kinase regulators, pathway genes, and co-factors have been surveyed for effects on TDP-43 phosphorylation. The DNA damage response (DDR) pathway was identified by this screen as a key regulator of TDP-43 phosphorylation. Components of the DDR pathway will be evaluated in detail using mouse primary neurons, C. elegans and mammalian cell models of TDP-43 proteinopathy. The determination of the genes and gene pathways regulating TDP-43 targeted CDC7 kinase activity will provide insight into disease pathobiology, and potentially identify novel therapeutic targets for intervention. CDC7 is a promising therapeutic target for th inhibition of pathological TDP-43 phosphorylation that is observed in ALS and FTLD-TDP. However, small molecule inhibitors specific for CDC7 with appropriate pharmacokinetics in vivo are not commercially available. Therefore, a targeted library of small molecules structurally similar to CDC7 inhibitors will be assembled. These small molecules will be surveyed for specific inhibition of CDC7 kinase activity, and tested in vivo in C. elegans and in mammalian cell culture models for protection against TDP-43 driven neurodegeneration. This is a critical step towards developing viable treatments for ALS and FTLD-TDP.
The National Academy of Sciences Institute on Medicine has found an association between service in the U.S. military and ALS. American Veterans as a whole are at about 60% greater risk than U.S. civilians. Pathological TDP-43 occurs in the majority of ALS cases resulting in severe disability and premature death in American Veterans. Furthermore, ALS has been designated a service connected condition by the Department of Veterans Affairs. The burden placed on America Veterans afflicted with ALS is incalculable as this life ending neurologic condition causes rapid and severe disability ultimately leading to death. Here we propose to devise and test neuroprotective strategies targeting genes causing and contributing to disease using simple models of ALS. This in turn may ultimately lead to potential therapeutic interventions for the benefit of all those affected by ALS including Veterans.
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