Tale of mitochondria and mitochondria-associated ER membrane in patient-derived neuronal models of Wolfram syndrome
Mitochondria and mitochondria-associated endoplasmic reticulum membrane in neurodegenerative diseases: Mitochondria generate most of the chemical energy needed to power the biochemical reactions of cells, and thus are often referred to as the “powerhouse” of the cell. Nevertheless, this organelle is also involved in a plethora of different cellular functions such as calcium (Ca 2+ ) homeostasis, apoptosis, oxidative stress, and several metabolic pathways including oxidative phosphorylation, tricarboxylic acid cycle, and β-oxidation of fatty acids. Many of these functions require the contact between the mitochondria and the endoplasmic reticulum (ER), which is mediated by several tether proteins located on the respective organellar surfaces, enabling the formation of mitochondria-associated ER membrane (MAMs). Given that the brain is one of the high-energy-demanding organs in the body, neurons are uniquely vulnerable to reactive oxygen species, and that Ca 2+ homeostasis is crucial for neuronal functionality, there has been a long-standing interest in mitochondrial functions and their communications with the ER within the fields of neurology and neuropathology. Alterations in mitochondrial physical and functional tethers along with their biochemical dysfunction are now recognized as common hallmarks of different neurodegenerative and neurodevelopmental conditions including Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and autistic spectrum disorders, as well as rare, early-onset neurodegenerative diseases such as Wolfram syndrome 1 (WS) (Johri and Beal, 2012; Paillusson et al., 2016; Delprat et al., 2018; Mishra et al., 2021). Thus, the identification of effective treatments acting on biochemical pathways involving the mitochondria is of great biomedical interest.
Mutations in MAM-resident protein WFS1 causes Wolfram syndrome: WS is an ultra-rare, genetic, multi-systemic disorder with features of abnormal brain development and neurodegeneration including progressive optic nerve atrophy, ataxia, brain stem and cerebellum atrophy, and cognitive impairment (Barrett et al., 1995; Urano, 2016). It is also associated with diabetes and deafness. The optic nerve atrophy, caused by the loss of retinal ganglion cells, is a key early pathological feature of WS, while brain dysfunction is the most common cause of death in WS patients. To date, despite the scientific advances achieved in the past few years, there is no cure for WS, and the prognosis of this syndrome is poor as most patients die prematurely before 40 years (Barrett et al., 1995; Urano, 2016). WS is caused by loss of function mutations of the WFS1 gene that encodes a MAM-resident protein called wolframin (WFS1). Likewise, mutations in another MAM-associated protein, CISD2, cause Wolfram syndrome type 2 (Delprat et al., 2018). Explorations of the molecular and cellular pathological mechanisms in different cellular and animal models of WS have associated the protein WFS1 with a constitutive activation of the unfolded protein response under ER stress, as well as to Ca 2+ homeostasis defects (Mishra et al., 2021). Mitochondrial energy metabolism and dynamics, which are also critical for cell survival, are likely impaired due to the loss of wolframin. Interestingly, WFS1 is enriched at MAMs and is part of the MAM architecture (Delprat et al., 2018), so it is well-positioned to influence mitochondrial function. Indeed, some recent studies indicate that loss of function of WFS1 is associated with mitochondrial perturbations (Mishra et al., 2021). However, the nature of mitochondrial impairments in WFS1-deficient cells reported in various non-human or non-disease relevant models remains controversial, and studies in patient-derived, disease-affected neuronal models have been lacking. Thus, despite advances in our understanding of WS pathophysiology, the exact function of wolframin and the precise mechanisms leading to neuronal cell death in patients remain largely unknown, thereby preventing the development of an effective treatment for WS. Therefore, in our recent publication (Zatyka et al., 2023), we studied mitochondrial function in cortical neurons generated from WS patient-derived induced pluripotent stem cells (iPSCs). The patient iPSC-derived neuronal cells provide a clinically relevant disease-affected cellular platform of WS for biomedical exploitation.
Mitochondrial dysfunction in Wolfram syndrome patient-derived neuronal cells: In our study, we demonstrated a causal link between WFS1 depletion and mitochondrial dysfunction, which could be rescued by genetic or pharmacological interventions in WS patient iPSC-derived neural stem cells and cortical neurons (Figure 1; Zatyka et al., 2023). Transcriptomic analysis revealed significant dysregulation in the gene expression associated with the mitochondria. Notably, the patient iPSC-derived neural stem cells and neurons exhibited mitochondrial depolarization, oxidative stress, and reduction in mitochondrial respiration and adenosine triphosphate production (Zatyka et al., 2023). Whilst there are contradictory data on the functionality of mitochondria in cell models that are not human or disease relevant, our data in WS patient-derived neuronal cells are consistent with some of the phenotypes reported in earlier studies. These include mitochondrial depolarization in rat cortical neurons with Wfs1 knockdown (Cagalinec et al., 2016), and decreased mitochondrial respiration in patient fibroblasts (Angebault et al., 2018). Concomitant with mitochondrial dysfunction in WS patient-derived neuronal cells, increased cell death was observed at basal state that appeared to be more exacerbated in cortical neurons than in neural stem cells (Zatyka et al., 2023).
Genetic and chemical rescue of mitochondrial dysfunction in a patient-derived neuronal cell model of Wolfram syndrome.
In Wolfram syndrome associated with loss of WFS1 protein (left panel), WFS1–VDAC1 interaction is abrogated and MAMs are decreased, along with mitochondrial dysfunction involving mitochondrial depolarization, oxidative stress, decreased mitochondrial respiration, and lower ATP production that ultimately leads to neuronal cell death. Genetic rescue by WFS1 overexpression or chemical rescue by cyclosporin A and MnTBAP (right panel; as indicated by arrows) restores WFS1–VDAC1 interaction and increases MAMs, accompanied by rescue of the mitochondrial and cell death phenotypes that improve the survival of Wolfram syndrome patient iPSC-derived neurons. Adapted from our original research article (Zatyka et al., 2023). ATP: Adenosine triphosphate; ER: endoplasmic reticulum; iPSC: induced pluripotent stem cell; MAM: mitochondria-associated ER membrane; MnTBAP: Mn(III)tetrakis(4-benzoic acid)porphyrin chloride; VDAC1: voltage-dependent anion channel 1; WFS1: wolframin.
Genetic rescue studies highlight the role of WFS1 in influencing MAM and mitochondrial function: We identified a possible mechanism underlying a potential role of WSF1 in regulating mitochondrial function, and consequently, neuronal survival (Figure 1). This could be via the interaction of WFS1 with voltage-dependent anion channel 1 (VDAC1), identified by mass spectrometry that also revealed VDAC2, VDAC3, and GRP75 amongst the potential WFS1 interactors (Zatyka et al., 2023). It has been previously shown that VDAC1 forms a complex with the inositol triphosphate receptor (IP 3 R) on the ER through the molecular chaperone GRP75 (Delprat et al., 2018). Since both WFS1 and VDAC1 are known to associate with MAMs (Delprat et al., 2018), our data implied that the interaction between WFS1 and VDAC1 is likely a part of the IP 3 R-GRP75-VDAC1 multicomplex that tethers the ER and mitochondria. In support of this idea, we found that genetic rescue by restoration of the wild-type WFS1 protein in WS patient iPSC-derived cells restored WFS1-VDAC1 interaction along with an increase in the MAMs. Concomitant to this, reinstating WFS1 levels in WS patient iPSC-derived cells increased mitochondrial branch length accompanied by a reduction in the mitochondrial fission protein DRP1, improved mitochondrial membrane potential and adenosine triphosphate production, and lowered oxidative stress. Genetic rescue of MAMs and mitochondrial parameters was associated with improvement in the survival of WS patient iPSC-derived neurons. We thus speculate that increasing the number of MAMs and improving mitochondrial dynamics could positively impact on mitochondrial function and neuronal health in WS patient-derived neurons. In addition, WFS1 has been shown to interact with several MAM resident proteins such as Sigma-1 receptor (Crouzier et al., 2022) and neuronal calcium sensor 1 (Angebault et al., 2018), which have a role in MAM structure and function by stabilizing it through their interaction with IP 3 R and/or VDAC1. These studies further highlight a possible role of WFS1 not only in the function of MAMs but also in their structural integrity by interacting and modulating the tethering proteins.
Pharmacological modulation of mitochondrial function improves neuronal viability: Of biomedical interest, we have shown that pharmacologically modulating mitochondrial function could rescue mitochondrial dysfunction and cell death in WS patient-derived neurons (Zatyka et al., 2023). Specifically, cyclosporin A (inhibits mitochondrial permeability transition pore and increases resting membrane potential) and MnTBAP (a superoxide scavenger), which have been previously reported to exert beneficial effects in vivo for other conditions, exhibited significant cytoprotective effects in WS patient iPSC-derived neurons by improving the energy status and cell viability (Zatyka et al., 2023). Our findings highlight a potential therapeutic strategy for WS that warrants further investigation for generalizability in other neurodegenerative diseases accompanied by mitochondrial dysfunction.
MAM and mitochondrial defects as shared patho-mechanisms in neurodevelopmental and neurodegenerative disorders: Although WS is an ultra-rare genetic disorder, its main clinical manifestations (e.g., neurodegeneration) and the underlying mitochondria/ER pathophysiology are not as rare and have been reported in other neurological conditions. Indeed, the dysregulated ER Ca 2+ signaling, ER stress-induced unfolded protein response, and impaired mitochondrial function have also been found to play important roles in the pathogenesis of a range of neurodegenerative diseases (Johri and Beal, 2012; Paillusson et al., 2016). Of particular interest, some studies have demonstrated that WFS1 may be directly and/or indirectly involved in the pathogenesis of other diseases affecting neurons such as hereditary optic neuropathies and Alzheimer’s disease (Li et al., 2020; Rocatcher et al., 2023). Moreover, a major question concerning neurodegenerative disease is how a mutation existing since the origin of nervous system development can lead to neurodegeneration several years later. Neural network formation is a multi-step process starting at embryonic stage with cell differentiation and migration, involving synaptogenesis and axon guidance, and ending with synaptic maturation. This process is fundamental for proper connectivity and functionality of neurons in the adult central nervous systems. Thus, an emerging concept proposes that some neurodegenerative diseases may be caused, or at least become primed, by defects that arise during neurodevelopment, suggesting that deregulation of developmental processes could be compensated to a certain point and then leads to degeneration (Lin et al., 2009). Interestingly, neurodevelopmental and neurodegenerative disorders share many overlapping molecular mechanisms, such as MAM and mitochondrial defects, and some causative genes (Schor and Bianchi, 2021). In WS, in addition to the neurodegenerative processes described since the origin of the discovery of this disease, recent studies revealed that WS patients display an overall reduction of brain volume, a hallmark ascribed to neurodevelopmental defects (Hershey et al., 2012). Coherent with this observation, neuronal developmental alterations were also observed in animal models of the disease as well as in WS patient-derived neurons (Cagalinec et al., 2016; Pourtoy-Brasselet et al., 2021). Whether the neurodevelopmental defects identified in these studies may be associated after years with the observed neurodegenerative phenomena or whether the neurodevelopmental and neurodegenerative processes are due to independent pathological mechanisms requires further research. Thus, WS represents a paradigmatic disease that could help to improve our knowledge on other neurodegenerative disorders and their eventual developmental origins.
Concluding remarks: Novel treatments designed for this ultra-rare condition like WS may have broader implications for common disorders, especially those related to MAMs and mitochondrial dysfunction. In our study, we have demonstrated mitochondrial dysfunction in clinically relevant, patient-derived neurons wherein pharmacological agents modulating mitochondrial function improved neuronal survival (Zatyka et al., 2023). By leveraging the tools and therapeutic efforts targeting WS, we plan to identify novel therapeutic options that are generalizable to other rare and more prevalent neurodegenerative diseases in the future.
We thank M.E. Korsgen (University of Birmingham, UK) and H. Salmonowicz (International Institute of Molecular Mechanisms and Machine, Poland) for assistance with the figure.
This work was supported by LifeArc Philanthropic Fund (P2019-0004) and LifeArc Pathfinder Award, along with Wellcome Trust Seed Award (109626/Z/15/Z), FAPESP-UoB Strategic Collaboration Fund, and Birmingham Fellowship (to SS); grants from Laboratoire d’Excellence Revive (Investissement d’Avenir; ANR-10-LABX-73), and the Region Ile-de-France via doctoral school Innovation Thérapeutique, du Fondamental à l’Appliqué (ED 569) from Université Paris-Saclay (to LA); and Medical Research Council (MRC) Developmental Pathway Funding Scheme (DPFS) grant (MR/P007732/1) (to TB). Istem/CECS is supported by the Association Française contre les Myopathies (AFM-Téléthon).
TB is a National Institute for Health and Care Research (NIHR) Senior Investigator. SS is also Former Fellow for life at Hughes Hall, University of Cambridge, UK.
Additional file:Open peer review reports 1 (85.9KB, pdf) -3 (88.1KB, pdf) .
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