Definition of chaperones as disease modifiers of peripheral neuropathies

Keywords: Inherited peripheral neuropathy, induced neuronal progenitor cells, neurodegeneration

Abstract: Mutations in MPZ, which encodes myelin protein zero (P0), lead to different subtypes of Charcot-Marie-Tooth (CMT) disease, a neurodegenerative disorder affecting the peripheral nervous system (frequency is 1:2500). MPZ-mutations are a common cause of CMT and associated phenotypes range from demyelinating (CMT1) to axonal forms (CMT2). We have collected a large cohort of >500 patients with various forms of CMT in the North of England and determined epidemiology data of CMT for this region (Foley et al. 2012). We have identified 14 local patients carrying 10 different pathogenic mutations in the MPZ gene. For various mutations, expression of a (shortened) non-functional (proteotoxic) protein causing endoplasmic reticulum (ER) retention and thus impaired ER-function was demonstrated. Moreover, an important role of ER-chaperones such as CHOP, Calnexin and ERp57 was shown in the biology of peripheral myelin proteins including MPZ. Curcumin, a component of the spice turmeric, relieves ER stress and thus promotes maintenance of Schwann cell function in an Mpz mouse model (Khajavi et al. 2007). However, the precise molecular mechanisms are still elusive.
The aim of this study is to characterize relevant disease modifiers and thus suitable therapeutic targets for CMT neuropathies. Successful therapeutic targeting of specific chaperones such as SigR1 was already demonstrated in neurodegeneration (Prause et al. 2013). The first phase of the project will concentrate on the cellular effect of Curcumin on cells. The student will investigate the effect of MPZ mutations in vitro in induced Neuronal Progenitor Cells (iNPCs) of our patients and controls. We have established this method in our laboratory (Meyer et al. 2014). The study will focus on (i) the cellular fitness (proliferation, ELISA-based LDH and WST-1 assays and immunoblot studies of apoptosis and pro-survival related proteins), (ii) the stability and folding of the mutant protein (cyclohexamide-chase assays and native PAGEs) and on (iii) morphology of the ER along with UPR activation (immunofluorescence studies and immunoblot studies of ER proteins). We will compare the biochemical phenotype obtained in cell culture, with the severity of clinical presentations in order to establish genotype/phenotype correlations. We will explore the effect of Curcumin in these cells in vitro.
Proteomic studies on Curcumin-treated and non-treated patient iNPC cells will be carried out and the student will verify the altered expression of promising chaperones activated in treated iNPC cells with the help of immunoblotting and immunofluorescence. Afterwards, the beneficial effect of disease modifier candidates on pathogenesis of MPZ-related CMT will be tested by studying the effect of modifier (most likely chaperone) overexpression on the biochemical phenotype using the above mentioned techniques. In parallel, (iv) searching for new genetic modifiers in chaperone-encoding genes will be carried out by whole exome sequencing, and potentially by RNASeq analysis in a clinically well-defined cohort of 14 CMT patients carrying pathogenic mutations in the MPZ gene (Hermann et al. 2014; Bansagi et al. 2015a; Bansagi et al. 2015b). If genetic modifiers are detected in MPZ deficiency, we will also study their potential role as a modifier in other forms of CMT.
The results will improve the current understanding of chaperone function in the peripheral nervous system and on a more special note in the pathogenesis of neuropathies. The efficiency of the different chaperones will be carefully evaluated and will allow us to define the most striking candidates for the treatment of MPZ-associated CMT neuropathy. The long-term goal would be to select particular chaperones along with suitable targeted drugs like agonists with the aim to open new avenues for therapy of neuropathies.