Endoplasmic Reticulum Stress signalling - from basic mechanisms to clinical applications

The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling-centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes

Characterisation of novel gene mutations causing familiar amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a complex fatal degenerative disease selectively affecting motor neurones. The cause of the disease is still uncharacterised for the sporadic cases (SALS), whereas several disease-linked genes, such as SOD1 and VAPB, have been identified in families with ALS (FALS). However, since the pathological features are indistinguishable between FALS and SALS, it is generally believed that a common ALS-causing mechanism is responsible for both forms of disease. To gain a better understanding of ALS pathogenesis, which could also provide cues for therapeutic treatment, studies on the characterisation of FALS-linked mutations have been carried out. In this study, we screened for novel FALS-linked mutations and found three TDP-43 mutations in five unrelated FALS families, that caused marked changes at evolutional conserved amino acids and were absent in previously screened control populations. In addition, functional characterisation of novel ALS linked mutations in DAO (R199W) and VAPB (T46I) were undertaken whereby both mutations were shown to disrupt the physiological properties of wild type proteins. Whereas the T46I mutation in VAPB led to a sub-cellular redistribution and shift in solubility of VAPB protein, and an impairment in the unfolded protein response (UPR) and ubiquitin-proteasome system, R199W significantly abolished DAO enzyme activity. Cell culture-based functional studies showed that the expression of both mutant proteins triggered pathological features including ubiquitin-positive aggregates and cell death. Furthermore, an association study was conducted to investigate the cause of a dramatic VAPB down-regulation seen in SALS. An intronic SNP close to exon 5 of VAPB, rs6100067, was found to be significantly linked to the expression levels of VAPB in SALS. As the down-regulation of VAPB profoundly affects the activation of IRE1/XBP1, the most characterised UPR pathway, this SNP may present as a risk factor for ALS development. From these results, we propose an ALS pathogenesis mechanism, whereby VAPB plays a central role in sustaining multiple cellular events, such as UPR. Disruption or deprivation of VAPB causes impairments in these cellular functions and leads to motor neurone death.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

An investigation of genetic and epigenetic factors in the regulation of gene expression in schizophrenia : insights into pathways involved in pathogenesis

Schizophrenia is a common psychiatric disorder with a complex aetiology that has a strong genetic component as well as a major contribution from non-genetic factors. Whilst GWAS studies have not yielded substantial evidence of DNA variants linked to schizophrenia, microarray expression studies, which reflect both genetic and environmental factors, have provided evidence of candidate genes whose expression changes significantly in schizophrenia. The present thesis is based on a prospective microarray study carried out on two brain regions implicated in schizophrenia: frontal cortex and superior temporal cortex (Maycox et al., 2009; Barnes et al., 2011). SNP variants in six of these genes were genotyped and allele, genotype and haplotype analysis carried out initially to investigate associations with levels of expression found in these microarray studies. Subsequently SNP associations with disease were investigated in an extended case-control cohort from the East UK region. The selected candidates genes, highlighted from microarray studies, reflected two main functional categories: synaptic plasticity and the Wnt signalling pathway. Initial ANOVA revealed a significant interaction of disease status with genotype/haplotype in ZnT3 with respect to levels of expression. Significant effects of specific genotypes and haplotypes in CACNA1E and FRZB on expression were also observed. Furthermore, ZnT3 was the gene for which the strongest evidence of association with schizophrenia was obtained in the East UK cohort, with four SNPs associated at the allelic and genotypic level. Significant associations of a number of haplotypes of ZnT3 with disease status were also identified. In addition, modest evidence of statistical epistasis between genes within both functional groups was identified. Finally, a method for analysis of CpG island methylation was developed based on bisulphite sequencing and methylation specific PCR (MSP) using previously validated primers for DKK3 (Yue et al., 2008). Although DKK3 showed a low density of methylation, the application of this method may prove useful for other candidate genes.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Preclinical testing of potential therapeutics for Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurological disorder caused by the selective degeneration of upper and lower motor neurons, for which there are currently no effective treatments. 10% of ALS cases are familial, of which 15-20% are caused by mutations in the copper/zinc superoxide dismutase gene (SOD1). The primary triggers for motor neuron degeneration in ALS are unknown, but research in patients and SOD1 models has revealed several mechanisms which may contribute. These include: oxidative stress, mitochondrial abnormalities, inflammation and protein aggregation. This study focused on the pre-clinical testing of two-potential therapeutics for ALS in the SOD1G93A murine model of the disease; metformin, an anti type II diabetes drug which has been shown to have anti-inflammatory and anti-oxidant properties and the ability to bring about mitochondrial biogenesis, and trehalose, a chemical chaperone and enhancer of autophagy, which has been shown to reduce protein misfolding and aggregation. We performed an initial study in which oral metformin administration from 35 days increased the survival of functional motor units in the hindlimbs of male and female SOD1G93A mice at 100 days. Consequently we performed a dose-response survival study in SOD1G93A mice with longitudinal monitoring of weight and neurological score. Surprisingly, metformin had no effect in males and brought about a dose-dependent negative effect on the onset of neurological symptoms and on disease progression in females. We hypothesise this negative effect may have resulted from a metformin-induced reduction in oestrogen production. Oral trehalose administration was tested using the same survival study format. Although trehalose treatment brought about a dose-dependent delay in weight-loss in males, it had no effect on the onset or progression of neurological symptoms or on survival in male or female mice. We conclude that neither metformin nor trehalose represent strong candidates for clinical trial in ALS patients when administered orally.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Hsp27 overexpression in the R6/2 mouse model of Huntington’s disease: chronic neurodegeneration does not induce Hsp27 activation

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Hsp27 overexpression in the R6/2 mouse model o