Investigating mitochondrial and ER protein profiles of cells expressing SPTLC1 mutations

Abstract

Axonal degeneration is the final common path in many neurological disorders. It is seen in its pure form in hereditary axonal neuropathies. The hereditary neuropathies are the most common group of diseases. Subsets of neuropathies involving the sensory neuron are known as hereditary sensory neuropathies (HSNs). Hereditary sensory neuropathy type I (HSN-I) (the most common subtype of HSNs) is an autosomal dominant inherited disorder, characterised by the progressive degeneration of the dorsal root ganglion and with onset of clinical symptoms occurring between the second or third decade of life. Heterozygous mutations in the serine palmitoyltransferase (SPT) long chain subunit 1 (SPTLC1) have been identified as the cause of HSN-I. In Paper I, we optimised an isolation method of mitochondria to allow the production of a full and in-depth proteomic profile to elucidate the molecular mechanisms underlying mitochondrial (dys) function in HSN-I. Paper II, detailed examinations of a small sub-set of proteins that were found to be altered in abundance within harvested mitochondria from HSN-I mutant SPTLC1 cells. Comparison of mitochondrial protein isolates from control and patient lymphoblasts, showed an increased abundance of Ubiquinol Cytochrome C Reductase Core Protein 1, an electron-transport chain protein, as well as the immunoglobulin, Ig Kappa Chain C. In, Paper III, endoplasmic reticulum (ER) protein lysates from HSN-I patient and control lymphoblasts, were examined leading to identification of changes in expression of five proteins; Hypoxia Up regulated Protein 1, Chloride Intracellular Channel Protein 1, Ubiqutin-40s Ribosomal Protein S27a, Coactosin and Ig Kappa chain C. Further investigations into mitochondrial and ER protein profiles were carried out in Paper IV, which showed a number of proteins that were altered in their relative abundance using membrane and soluble isolation techniques. Further analyses of these identified changes were carried out and replicated in Paper V, which revealed and confirmed the changes in protein expression and abundance of proteins earlier identified in Papers I and II. Changes were identified in V144D mutations, as well as C133W and C133Y mutations. All of which are implicated to be casual of HSN-I. Lipid droplets and alterations of lipid metabolism are hallmarks of a variety autosomal dominant neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Paper VI, revealed significant increases in the presence of lipid droplets in HSN-I patient-derived lymphoblasts, indicating a potential connection between lipid droplet formation and the molecular mechanisms of HSN-I. In conclusion, this study has shown alteration in mitochondrial and ER protein profiles in patient-derived lymphoblasts and in transfected neuronal cells expressing the mutations V144D, C133W and C133Y. This investigation has contributed to the field by identifying protein alterations which has yielded a more detailed and in-depth analysis of the cellular and molecular mechanisms involved in HSN-I.

Date of Award	2015
Original language	English