Calcium mishandling in absence of primary mitochondrial dysfunction drives cellular pathology in Wolfram Syndrome

Abstract

Wolfram syndrome (WS) is a recessive multisystem disorder defined by the association of diabetes mellitus and optic atrophy, reminiscent of mitochondrial diseases. The role played by mitochondria remains elusive, with contradictory results on the occurrence of mitochondrial dysfunction. We evaluated 13 recessive WS patients by deep clinical phenotyping, including optical coherence tomography (OCT), serum lactic acid at rest and after standardized exercise, brain Magnetic Resonance Imaging, and brain and muscle Magnetic Resonance Spectroscopy (MRS). Finally, we investigated mitochondrial bioenergetics, network morphology, and calcium handling in patient-derived fibroblasts. Our results do not support a primary mitochondrial dysfunction in WS patients, as suggested by MRS studies, OCT pattern of retinal nerve fiber layer loss, and, in fibroblasts, by mitochondrial bioenergetics and network morphology results. However, we clearly found calcium mishandling between endoplasmic reticulum (ER) and mitochondria, which, under specific metabolic conditions of increased energy requirements and in selected tissue or cell types, may turn into a secondary mitochondrial dysfunction. Critically, we showed that Wolframin (WFS1) protein is enriched at mitochondrial-associated ER membranes and that in patient-derived fibroblasts WFS1 protein is completely absent. These findings support a loss-of-function pathogenic mechanism for missense mutations in WFS1, ultimately leading to defective calcium influx within mitochondria. Wolfram Syndrome (WS) is a rare genetic disorder also known as DIDMOAD, i.e. Diabetes Insipidus (DI), Diabetes Mellitus (DM), Optic Atrophy (OA) and Deafness 1. Additional neurological features include brainstem atrophy, cerebellar ataxia, peripheral neuropathy, cognitive deterioration and epilepsy 2. The onset of OA and DM, the minimal diagnostic criteria for WS, is typically within the second decade. The large majority of WS patients carry recessive mutations in the WFS1 gene encoding for the Wolframin (WFS1) protein 3. WFS1 is associated with the endoplasmic reticulum (ER) 4 and cellular and animal model studies involved Wolframin in the ER stress response, regulation of calcium homeostasis and Na/K ATPase function 5. Due to the clinical features resembling a mitochondrial disorder 6 and reports of mitochondrial DNA (mtDNA)

Lack of functional wolframin causes drop in plasmalemmal sodium-calcium exchanger type 1 expression at early stage in rat model of Wolfram syndrome

“…In line with the Wfs1 calcium modulation feature, our previously published data have shown that cytosolic calcium signal in response to glutamate or KCl stimulation was twice lower in Wfs1 deficient neurons (Cagalinec et al 2016). Analogical decrease of the cytosolic calcium release amplitude has been observed recently in WS patients' derived fibroblasts after stimulation with bradykinin (La Morgia et al 2020) or histamine (Angebault et al 2018). Moreover, Zatyka et al (2015) have demonstrated that the Wfs1 co-immunoprecipitates with sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) and that it negatively regulates SERCA expression.…”

Molecular Mechanisms behind Inherited Neurodegeneration of the Optic Nerve

“…However, a case of homozygous CISD2 mutation associated with “classical” WS1 has been reported [ 253 ]. Interestingly, a proteomic study evidenced the presence of Wfs1 in the MAMs fraction isolated from mouse brain tissues [ 237 ], and we recently confirmed that WFS1 is enriched in the MAMs also in human cell lines [ 19 ]. These findings suggest that Wolfram syndrome, and associated optic atrophy, may be due to alterations in MAMs-related processes, as in CMT2A.…”

“…These findings suggest that Wolfram syndrome, and associated optic atrophy, may be due to alterations in MAMs-related processes, as in CMT2A. According to this consideration, WS1 fibroblasts show reduced mitochondrial Ca 2+ uptake driven by altered Ca 2+ release from ER and altered ER-mitochondria contacts, whereas mitochondrial dynamics and morphology are not affected [ 18 , 19 ]. The increase in non-oxidative glycolysis, documented by high lactate levels in plasma of WS1 patients, in absence of defective mitochondrial respiration, and enhanced extracellular acidification rate (ECAR) in fibroblasts, may be related to a reduced activity of PDH, as regulated by mitochondrial Ca 2+ , and to diabetes [ 19 ].…”

“…According to this consideration, WS1 fibroblasts show reduced mitochondrial Ca 2+ uptake driven by altered Ca 2+ release from ER and altered ER-mitochondria contacts, whereas mitochondrial dynamics and morphology are not affected [ 18 , 19 ]. The increase in non-oxidative glycolysis, documented by high lactate levels in plasma of WS1 patients, in absence of defective mitochondrial respiration, and enhanced extracellular acidification rate (ECAR) in fibroblasts, may be related to a reduced activity of PDH, as regulated by mitochondrial Ca 2+ , and to diabetes [ 19 ]. The presence of a mitochondrial bioenergetics dysfunction is still unclear, since controversial results have been reported [ 18 , 19 ].…”

“…The most relevant forms of isolated or syndromic optic atrophy that apparently do not fit directly the mitochondrial paradigm are those related to Wolframin, with either dominant or recessive mutations, the latter typically causing Wolfram syndrome [ 17 ]. This protein is now established to be involved with the contact points between mitochondria and endoplasmic reticulum (ER) [ 18 , 19 ], currently defined as mitochondria-associated ER membranes or MAMs [ 20 ]. This qualifies a third major mechanistic area for optic nerve neurodegeneration, involving the complex inter-organellar crosstalk, which, in turn, affects mitochondrial dynamics, in particular fission [ 21 ], but also quality control, specifically autophagy [ 22 ], ultimately impacting cell survival or death [ 20 ].…”