Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration and considered as an endoplasmic reticulum (ER) disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome and the identification of two causative genes, Wolfram syndrome 1 (WFS1) and Wolfram syndrome 2 (WFS2), a molecular mechanism linking the ER to death of neurons and β cells has not been elucidated. Here we implicate calpain 2 in the mechanism of cell death in Wolfram syndrome. Calpain 2 is negatively regulated by WFS2, and elevated activation of calpain 2 by WFS2-knockdown correlates with cell death. Calpain activation is also induced by high cytosolic calcium mediated by the loss of function of WFS1. Calpain hyperactivation is observed in the WFS1 knockout mouse as well as in neural progenitor cells derived from induced pluripotent stem (iPS) cells of Wolfram syndrome patients. A small-scale small-molecule screen targeting ER calcium homeostasis reveals that dantrolene can prevent cell death in neural progenitor cells derived from Wolfram syndrome iPS cells. Our results demonstrate that calpain and the pathway leading its activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.Wolfram syndrome | endoplasmic reticulum | diabetes | neurodegeneration | treatment

Background: Wolfram syndrome is a rare endoplasmic reticulum disorder characterized by insulin-dependent diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration.Although there is currently no treatment to delay, halt, or reverse the progression of Wolfram syndrome, preclinical studies in cell and rodent models suggest that therapeutic strategies targeting endoplasmic reticulum calcium homeostasis, including dantrolene sodium, may be beneficial. Methods:Based on the results from preclinical studies on dantrolene sodium and ongoing longitudinal studies, our group put together the first-ever clinical trial in pediatric and adult patients with Wolfram syndrome. An open-label phase 1b/2a trial design was chosen. The primary objective of the study was to assess the safety and tolerability of dantrolene sodium in adult and pediatric patients with Wolfram syndrome. Secondary objectives were to evaluate the efficacy of dantrolene sodium on residual pancreatic -cell functions, visual acuity, quality of life measures related to vision, and neurological functions. Results:The results indicate that dantrolene sodium is well tolerated by patients with Wolfram syndrome. Overall, -cell functions were not significantly improved by dantrolene, but there was a significant correlation between baseline -cell functions and the change in -cell responsiveness (R 2 , p=0.004) after 6 months of dantrolene therapy. Other outcome measures, including visual acuity and neurological functions, were not improved by dantrolene sodium treatment within 6 months. As previously reported, markers of inflammatory cytokines and oxidative stress, such as IFN, IL-1, TNF, and isoprostane, were elevated in subjects with Wolfram syndrome. Conclusion:This study justifies further investigation into using dantrolene sodium and other small molecules targeting the endoplasmic reticulum for the treatment of Wolfram syndrome. Abreu & Stone et. al. 3 Trial registration ClinicalTrials.gov Identifier NCT02829268 Conflict of interest statement F. Urano received research funding from Eli Lilly, Ono Pharmaceuticals, and Amarantus BioScience for the development of MANF-based regenerative therapy for Wolfram syndrome, optic nerve atrophy, and diabetes. F. Urano received chemical compounds from Amylyx Pharmaceuticals, Mitochon Pharmaceuticals, Aetas Pharma, and National Center for Advancing Translational Sciences for the development of small molecule-based therapies for ER stress-related disorders, including Wolfram syndrome. F. Urano is an inventor of two patents related to the treatment of Wolfram syndrome, US 9,891,231 SOLUBLE MANF IN PANCREATIC BETA CELL DISORDERS and US 10,441,574 and US 10,695,324 TREATMENT FOR WOLFRAM SYNDROME AND OTHER ER STRESS DISORDERS. F. Urano is a Founder and President of CURE4WOLFRAM, INC.

Selective neuronal vulnerability to protein aggregation is found in many neurodegenerative diseases including Alzheimer's disease (AD). Understanding the molecular origins of this selective vulnerability is therefore of fundamental importance. Tau protein aggregates have been found in manuscriptClick here to access/download;manuscript;Manuscript_Final submission.docx Click here to view linked References Wolframin (WFS1)-expressing excitatory neurons in the entorhinal cortex, one of the earliest affected regions in AD. The role of WFS1 in Tauopathies and its levels in tau pathology-associated neurodegeneration, however, is largely unknown. Here we report WFS1 deficiency is associated with increased tau pathology and neurodegeneration, whereas overexpression of WFS1 reduces those changes. We also find that WFS1 interacts with tau protein and controls the susceptibility to tau pathology. Furthermore, chronic ER stress-and autophagy-lysosome pathway (ALP)associated genes are enriched in WFS1-high excitatory neurons in human AD at early Braak stages.The protein levels of ER stress-and autophagy-lysosome pathway (ALP)-associated proteins are changed in tau transgenic mice with WFS1 deficiency, while overexpression of WFS1 reverses those changes. This work demonstrates a possible role for WFS1 in the regulation of tau pathology and neurodegeneration via chronic ER stress and the downstream ALP. Our findings provide insights into mechanisms that underpin selective neuronal vulnerability, and for developing new therapeutics to protect vulnerable neurons in AD.

Endoplasmic reticulum (ER) stress-mediated cell death is an emerging target for human chronic disorders, including neurodegeneration and diabetes. However, there is currently no treatment for preventing ER stress-mediated cell death. Here, we show that mesencephalic astrocyte-derived neurotrophic factor (MANF), a neurotrophic factor secreted from ER stressed cells, prevents ER stress-mediated β cell death and enhances β cell proliferation in cell and mouse models of Wolfram syndrome, a prototype of ER disorders. Our results indicate that molecular pathways regulated by MANF are promising therapeutic targets for regenerative therapy of ER stress-related disorders, including diabetes, retinal degeneration, neurodegeneration, and Wolfram syndrome.

Conflict of interest:FU is an inventor of 3 patents related to Wolfram syndrome treatment, US 9,891,231 "Soluble MANF in pancreatic beta-cell disorders, " and US 10,441,574 and US 10,695,324 "Treatment for Wolfram syndrome and other endoplasmic reticulum stress disorders. " JRM is an inventor on a patent and patent application relating to stem cell-derived pancreatic islets, US 10,030,229 "SC-β cells and compositions and methods for generating the same" and US application PCT/US2019/032643 "Methods and Compositions for Generating Cells of Endodermal Lineage and Beta Cells and Uses Thereof. " FU is a founder and president of CURE4WOLFRAM, Inc. JRM is a consultant for Sana Biotechnology. JC and JK are co-CEOs of and report personal fees from and equity holdings in Amylyx Pharmaceuticals Inc. JC and JK have 2 patents, "Compositions for improving cell viability and methods of use thereof" (US 9,872,865; US Patent 10,251,896; AU 2014242123; EP 14775675.3; and JP2016515575A) and "For improving the composition of cell viability and using the method for the composition" (CN105050593B), issued to Amylyx Pharmaceuticals Inc. JL reports personal fees from and equity holdings in Amylyx Pharmaceuticals Inc.