The role of Wolframin in the pathogenesis of Wolfram or Didmoad syndrome

Abstract

The Wolfram Syndrome (WS) is an autosomal recessive neurodegenerative disorder characterised by insulin-dependent diabetes mellitus (IDDM) and bilateral progressive optic atrophy. Manifestations in neural and neuroendocrine tissues generally arise, but are not a requirement for diagnosis. A novel gene was identified in 1998 on chromosome 4p16.1 (WFS1 or Wolframin) that contains loss-of-function mutations in a majority of Wolfram patients. This gene encode for a putative hydrophobic transmembrane protein of 890aa and its predicted molecular weight is 100.29 kDa based on the amino acid sequence. This thesis was intended to study the localisation and function of this gene product in order to gain insight into its role in the pathogenesis of diabetes mellitus and neurodegeneration. In order to study the role of the Wolframin gene in diabetes, its mRNA expression was analysed during the development of diabetes in the Nonobese Diabetic (NOD) mouse model. This showed a decrease in Wolframin expression in the pancreas of NOD mice 9 days after induction for diabetes, indicating a possible involvement of Wolframin in the survival of β-cells. Fibroblast cells and blood samples from Wolfram patients were obtained and analysed for their mutations. One patient was compound heterozygotic for two separate mutations and one patient was homozygotic for a single insertion leading to an early stop codon. RT-PCR of these cells showed no significant difference in mRNA expression as compared to either fibroblasts from first-grade relatives or control cells. These cells were used in various functional assays. First of all, microarray analysis was used to identify genes that are affected when the Wolframin gene is non-functional. These results lead to the investigation of the role of Wolframin in senescence using markers of growth rate and radiation. No difference was found between fibroblasts from a Wolfram patient and her sibling, not supporting a role for Wolframin in senescence. Since the mouse homologue is 83% identical to the human variant on an amino acid level it is likely that there is also functional homology. Therefore, in order to study the pathomechanisms acting in human disease, a knock-out mouse construct was produced thus overcome the limitation to obtain human material. Two constructs were electroporated into embryonic stem cells and so far a total of 1080 colonies were screened, however, no positive clones were obtained. In order to investigate this gene on a protein level, antibodies were raised against the N- and C- terminus. The respective protein was found to be expressed in many tissues and cell lines with the highest expression in brain, pancreas, heart and insulinoma β-cell lines. Using sub-cellular fractionation techniques, the protein was found exclusively in microsomal fractions. Immunofluorescence on transfected cos-7 cells showed intracellular localisation to the endoplasmic reticulum. Incubating mouse pancreas slices with the Wolframin antibody showed exclusive staining in the β-cells and co-localisation to insulin. Immunofluorescence analysis also showed immunoreactivity to structures of the limbic system in the mouse brain. These results give the first clues towards a possible function in processing or trafficking of proteins involved in the survival of neuronal and endocrine cells