Silencing of the WFS1 gene in HEK cells induces pathways related to neurodegeneration and mitochondrial damage

Kõks, S., Overall, R.W., Ivask, M., Soomets, U., Guha, M., Vasar, E., Fernandes, C. and Schalkwyk, L.C. (2013) Silencing of the WFS1 gene in HEK cells induces pathways related to neurodegeneration and mitochondrial damage. Physiological Genomics, 45 (5). pp. 182-190.

Abstract

The gene WFS1 encodes a protein with unknown function although its functional deficiency causes different neuropsychiatric and neuroendocrine syndromes. In the present study, we aimed to find the functional networks influenced by the time-dependent silencing of WFS1 in HEK cells. We performed whole genome gene expression profiling (Human Gene 1.0 ST Arrays) in HEK cells 24, 48, 72, and 96 h after transfection with three different WFS1 siRNAs. To verify silencing we performed quantitative RT-PCR and Western blot analysis. Analysis was conducted in two ways. First we analyzed the overall effect of the siRNA treatment on the gene expression profile. As a next step we performed time-course analysis separately for different siRNAs and combined for all siRNAs. Quantitative RT-PCR and Western blot analysis confirmed clear silencing of the expression of WFS1 after 48 h. Significant (FDR value <10%) changes in the expression of 11 genes was identified with most of these genes being related to the mitochondrial dysfunction and apoptosis. Time-course analysis confirmed significant correlations between WFS1 silencing and changes in the expression profiles of several genes. The pathways that were influenced significantly by WFS1 silencing were related to mitochondrial damage and neurodegenerative diseases. Our findings suggest a role of WFS1 gene in cell survival and its involvement in degenerative diseases.

wolfram syndrome (ws, mim222300) is an autosomal recessive disorder most frequently characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD), first described by Wolfram and Wagener as a juvenile diabetes mellitus with optic atrophy (40, 46). Only insulin-dependent diabetes mellitus and progressive optic atrophy are necessary to confirm WS, and both of these syndromes may be present in childhood or adolescence (3). WS can be characterized as polyendocrinopathy with significant pituitary deficiency and atrophy (13, 31). In addition to these diagnostic syndromes, most WS patients have highly variable clinical symptoms including several neurological abnormalities such as nystagmus, mental retardation, and seizures (3). Moreover, several studies have shown diffuse and widespread atrophy in the brain (32, 36). Central respiratory failure due to brain-stem atrophy has been described as a common cause of death, indicating the significance of neurodegeneration in WS (3, 36). In addition to the neurological manifestations, psychiatric illnesses have often been found in WS patients. The most prominent psychiatric manifestations in WS homozygous individuals are depression, violent or assaultive behavior, and organic brain syndromes (41).

WS is caused by mutations in the WFS1 gene, but the molecular function of WFS1 protein is not fully known. It is a transmembrane protein with 9–11 segments and is located in the endoplasmic reticulum (ER) (16). There is evidence that this protein plays a role in the regulation of ER calcium levels (30, 42). WFS1 is involved in the unfolded protein response (UPR), which is an adaptive response that counteracts ER stress (9). ER stress is defined as an imbalance between the actual folding capacity of the ER and the demand (24). Induction of ER stress with thapsigargin and tunicamycin causes significant upregulation of WFS1 expression (9). WFS1 seems to act as a survival factor; it is upregulated when ER stress is present, and its deficiency leads to more pronounced apoptosis (19).

WFS1 seems to be involved in the activation and secretion of bioactive peptides, including insulin. There is evidence that WFS1 is related to the processing of vasopressin in the hypothalamus (12). Increased insulin demand has been shown to promote apoptosis in WFS1-deficient mice (1). Our previous study indicated that WFS1 knockout (KO) mice exhibit impaired glucose tolerance and are significantly smaller than their wild-type littermates despite elevated growth hormone (GH) and IGF-1 levels (22, 29). These mice have lower plasma insulin and higher proinsulin levels, indicating potential insulin-processing problems. A recent study confirmed localization of WFS1 protein to the secretory granule and its role in the prohormone processing (15). Therefore, there is link between WFS1 gene function, peptide processing, and apoptosis. However, the available information is still fragmented and based on the diseased or gene targeted tissue samples, where compensatory changes may hide pathogenetic causal pathways. To add more causative information, time course-related effects of WFS1 silencing should be analyzed.

The aim of this study was to describe altered functional genetic networks in human embryonic kidney (HEK) cells after silencing the WFS1 gene and to follow the changes in the gene expression profile over time. This approach could give additional information on potential causative relations.

Item Type:	Journal Article
Publisher:	American Physiological Society
Copyright:	© 2013 the American Physiological Society
URI:	http://researchrepository.murdoch.edu.au/id/eprint/51773

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