Collins, Mahlon, A
(2016)
Identification of Amyotrophic Lateral Sclerosis Disease Mechanisms by Cerebrospinal Fluid Proteomic Profiling.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motor neuron disease. Heterogeneity in clinical, genetic, and pathological features of ALS suggest the disease is a spectrum of disorders each resulting in motor neuron degeneration. Molecular profiling of ALS patients is, therefore, a useful means of characterizing and stratifying the ALS population. To this end, mass spectrometric proteomic profiling was performed on cerebrospinal fluid (CSF) from ALS, healthy control (HC), and other neurological disease (OND) subjects. This resulted in the identification of 1,712 CSF proteins, 123 of which exhibited altered relative abundance in ALS CSF. Biological processes related to these 123 proteins included synaptic activity, extracellular matrix, and inflammation. The application of feature selection and machine learning methods to these CSF proteomic profiles resulted in a classifier that used relative levels of WDR63, APLP1, SPARCL1, and CADM3 to predict independent ALS, HC, and OND samples with 83% sensitivity and 100% specificity. To aid in the validation of selected CSF proteins, a Western blot loading control method was developed and validated using a reversible, iodine-based total protein stain. This method improves the accuracy and sensitivity of the relative quantification of CSF proteins via Western blot. As RNA binding protein (RBP) pathology/dysfunction is common to several forms of ALS, the largest CSF RBP alteration, that of RNA binding motif 45 (RBM45) protein, was validated externally. The results demonstrated that RBM45 pathology is common to several forms of ALS, frontotemporal lobar degeneration (FTLD), and Alzheimer’s disease. To further understand the biological functions of RBM45, immunoprecipitation coupled to mass spectrometry was performed to identify RBM45 protein-protein interactions (PPIs). RBM45 PPIs and associated pathways were most strongly associated with hnRNP proteins, RNA processing, and cytoplasmic translation. RBM45 also participates in the general cellular response to stress via association with nuclear stress bodies. This association is dependent on RNA binding, is upregulated in ALS/FTLD, and is sufficient to induce the aggregation of the protein. Collectively, these results illustrate the utility of CSF proteomic profiling for characterizing mechanisms of neurological disease and provide new insights into the contributions of RNA binding protein dysregulation to ALS/FTLD.
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Item Type: |
University of Pittsburgh ETD
|
Status: |
Unpublished |
Creators/Authors: |
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ETD Committee: |
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Date: |
23 May 2016 |
Date Type: |
Publication |
Defense Date: |
14 April 2016 |
Approval Date: |
23 May 2016 |
Submission Date: |
20 April 2016 |
Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
Number of Pages: |
294 |
Institution: |
University of Pittsburgh |
Schools and Programs: |
School of Medicine > Neurobiology |
Degree: |
PhD - Doctor of Philosophy |
Thesis Type: |
Doctoral Dissertation |
Refereed: |
Yes |
Uncontrolled Keywords: |
cerebrospinal fluid; amyotrophic lateral sclerosis; frontotemporal lobar degeneration; RNA binding proteins; proteomics; mass spectrometry |
Date Deposited: |
23 May 2016 14:13 |
Last Modified: |
15 Nov 2016 14:32 |
URI: |
http://d-scholarship.pitt.edu/id/eprint/27749 |
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