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

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

Role of the OPA3 protein in the pathogenesis of neurodegenerative diseases

OPA3 è una proteina codificata dal genoma nucleare che, grazie a una sequenza di targeting mitocondriale, viene indirizzata ai mitocondri dopo la sua sintesi. Le mutazioni nel gene OPA3 sono associate a due patologie neurodegenerative: la Sindrome di Costeff, causata da mutazioni recessive, e una forma di atrofia ottica dominante che si manifesta con cataratta e spesso sordità. L’esatta funzione e regolazione della proteina non sono ancora state completamente chiarite, così come la sua localizzazione nella membrana mitocondriale esterna o interna. Lo scopo di questa tesi era quello di fare luce sulla funzione della proteina OPA3, con particolare interesse alla dinamica mitocondriale e all’autofagia, sulla sua localizzazione subcellulare ed infine di definire il meccanismo patogenetico nelle patologie neurodegenerative causate da mutazioni in questo gene. A questo scopo abbiamo utilizzato sia una linea di neuroblastoma silenziata stabilmente per OPA3 che linee cellulari primarie derivate da pazienti. I risultati del presente studio dimostrano che la riduzione di OPA3, indotta nelle cellule del neuroblastoma e presente nei fibroblasti derivati dai pazienti, produce alterazioni nel network mitocondriale con uno sbilanciamento a favore della fusione. Questo fenomeno è probabilmente dovuto all’aumento della forma long della proteina OPA1 che è stato riscontrato in entrambi i modelli cellulari. Inoltre, seppur con direzione apparentemente opposta, in entrambi i modelli abbiamo osservato un’alterata regolazione dell’autofagia. Infine, abbiamo confermato che OPA3 localizza nella membrana mitocondriale interna ed è esposta per gran parte nella matrice. Inoltre, un segnale della proteina è stato trovato anche nelle mitochondrial associated membranes, suggerendo un possibile ruolo di OPA3 nel trasferimento dei lipidi tra i mitocondri e il reticolo endoplasmatico. Abbiamo rilevato un’interazione della proteina OPA3 con l’acido fosfatidico che non era mai stata evidenziata fino ad oggi. Queste osservazioni sono compatibili con le alterazioni della dinamica mitocondriale e la disregolazione dell’autofagia documentate nei modelli studiati.OPA3 is a protein encoded by the nuclear genome and, after its synthesis, is directed to mitochondria by a mitochondrial targeting sequence. Mutations in the OPA3 gene are responsible for two neurodegenerative diseases: Costeff syndrome, caused by recessive mutations, and a form of dominant optic atrophy with cataracts and hearing loss. To date, the exact function and regulation of the protein have not yet been understood, as well as its localization in the outer or inner mitochondrial membrane. This thesis aimed to shed light on the subcellular localization and function of the OPA3 protein, with particular focus on mitochondrial dynamics and autophagy, and to define the pathogenic mechanism of OPA3 mutations causing neurodegenerative diseases. For this purpose, we used neuroblastoma cell line stably silenced for OPA3 and patient-derived primary cell lines. The results of the present study demonstrate that a reduction of OPA3 content, induced in neuroblastoma cells and naturally occurring in patient-derived fibroblasts, produces alterations in the mitochondrial network with an unbalance toward fusion. This phenomenon is probably due to the increase in the long form of the OPA1 protein in both cell models. Furthermore, we observed an altered regulation of autophagy. Finally, we confirmed that OPA3 localizes in the inner mitochondrial membrane and is largely exposed in the matrix. Furthermore, the presence of OPA3 was also found in mitochondrial associated membranes, suggesting a possible role of the protein in lipid transfer between mitochondria and the endoplasmic reticulum. We detected an interaction of the OPA3 protein with phosphatidic acid that had never been shown until now. These observations are compatible with the alterations in mitochondrial dynamics and autophagic dysregulation documented in the models examined

Neuro-ophthalmological and clinical findings in Wolfram syndrome: what is the real mitochondrial role?

Wolfram syndrome (WS) type 1, characterized by early-onset diabetes mellitus and optic atrophy, is due to mutations in WSF1 gene. A longstanding debate exists on the possible role of mitochondrial dysfunction

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper

DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism

ADCA-DN and HSN-IE are rare neurodegenerative syndromes caused by dominant mutations in the replication foci targeting sequence (RFTS) of the DNA methyltransferase 1 (DNMT1) gene. Both phenotypes resemble mitochondrial disorders and mitochondrial dysfunction was first observed in ADCA-DN. To explore mitochondrial involvement we studied the effects of DNMT1 mutations in fibroblasts from four ADCA-DN and two HSN-IE patients. We documented impaired activity of purified DNMT1 mutant proteins, which in fibroblasts results in increased DNMT1 amount. We demonstrated that DNMT1 is not localized within mitochondria but it is associated to the mitochondrial outer membrane. Concordantly, mitochondrial DNA failed to show meaningful CpG methylation. Strikingly, we found activated mitobiogenesis and OXPHOS with significant increase of H2O2, sharply contrasting with a reduced ATP content. Metabolomics profiling of mutant cells highlighted purine, arginine/urea cycle and glutamate metabolisms as the most consistently altered pathways, similar to primary mitochondrial diseases. The most severe mutations showed activation of energy shortage AMPK-dependent sensing, leading to mTORC1 inhibition. We propose that DNMT1 RFTS mutations deregulate metabolism lowering ATP levels, as the result of increased purine catabolism and urea cycle pathways. This is associated with a paradoxical mitochondrial hyper-function and increased oxidative stress, possibly resulting in neurodegeneration in non-dividing cells