Amyotrophic lateral sclerosis (ALS) is a progressive, inevitably fatal neurodegenerative disorder. Pathologically, ALS is characterized by the death of motor neurons of the corticospinal tract, resulting in a numerous debilitating symptoms culminating with paralysis and death. Research into the molecular mechanisms of ALS neurodegeneration has implicated impaired RNA processing and intracytoplasmic aggregation of RNA binding proteins in cell death. We recently performed a cerebrospinal fluid (CSF)-based proteomics study of ALS patients and identified the novel RNA binding protein RBM45 as a putative disease biomarker. Further investigation of RBM45 via immunohistochemistry of ALS patient spinal cord tissue revealed RBM45-positive intracytoplasmic inclusions in motor neurons similar in appearance to those containing the other ALS-associated RNA binding proteins TDP-43 and FUS. We now seek to extend these preliminary observations by studying the normal function of RBM45 as well as its contribution(s) to neurodegenerative disease pathology. To do so, we will first assess how overexpression and knockdown of RBM45 influences the morphology, formation of inclusions, and viability of two cell lines. We will also overexpress a naturally-occurring isoform of the protein lacking a putative nuclear localization signal to determine the propensity of this variant to aggregate in vitro. The next goal of this proposal is t understand what role (if any) RBM45 has in the cellular response to stress. Cells will be stressed via treatment with arsenite and the morphology, number of RBM45-containing inclusions, and cell viability will again be measured. We will also examine the cells for changes in RBM45 post-translational modifications, colocalization with other RNA binding proteins, and incorporation into stress granules to address the function of RBM45 during stress. In the second phase of this proposal, RBM45 gene targets will be defined via UV cross-linking and immunoprecipitation in conjunction with RNA-Seq. We will next assess the contribution of RBM45 to the regulation of gene expression and alternative splicing. For these experiments, RBM45 will be depleted from cells via shRNA and the changes in gene expression and exon splicing will be determined using exon junction- sensitive microarrays. In the last phase of this proposal, we will attempt to link findings obtained in aims 1 and 2 to human neurodegenerative disease using human post-mortem tissue for immunohistochemistry and confocal microscopy. Tissues will be evaluated for brain region-specific and cell type-specific expression of RBM45, inclusions, and colocalization with other ALS- and stress response-associated proteins. Collectively, these experiments will provide new information about the role of RBM45 in normal cell function and disease.
ALS is the most common form of adult-onset motor neuron disease. The disorder is inevitably fatal, has a mean survival time of approximately 3 years, and only one FDA-approved treatment. Thus, there is a pressing need for new insights into the mechanisms of this illness.
