Defective Endoplasmic Reticulum–Mitochondria Connection Is a Hallmark of Wolfram Syndrome

Abstract

Interactions between endoplasmic reticulum (ER) and mitochondria are key components of essential cellular functions. Indeed, these membrane appositions are necessary for proper Ca²⁺ transfer from ER to mitochondria, to regulate lipid metabolism, apoptosis, and inflammation. We report that the ER protein WFS1 interacts with the neuronal calcium sensor protein NCS1 to regulate mitochondria associated-ER membrane formation. Mutations in the WFS1 gene are associated with Wolfram syndrome, a rare neurodegenerative disease. We demonstrated that human WFS1-deficient cells lack NCS1 and fail to tether ER and mitochondria, resulting in a decrease in Ca²⁺ transfer and mitochondrial respiration. Interestingly, we showed that NCS1 overexpression in WFS1-deficient cells restored ER–mitochondria interactions and calcium exchange. Our results suggest that WFS1 regulates ER tethering to mitochondria through NCS1 and that restoration of NCS1 expression could be a therapeutic tool for restoring calcium signaling at the mitochondria associated-ER membrane interface and mitochondrial function in Wolfram syndrome.

Wolfram Syndrome

Wolfram syndrome (WS) is a very rare neurodegenerative disorder characterized by diabetes mellitus, diabetes insipidus, optic atrophy, and sensorineural deafness (Barrett, Bundey, & Macleod, 1995). The gene, WFS1, codes for an endoplasmic reticulum (ER) glycosylated transmembrane protein of 890 amino acids that plays a role in ER stress signaling (Morikawa, Tajima, Nakamura, Ishizu, & Ariga, 2017) or Ca²⁺ homeostasis (Cagalinec et al., 2016). More recently, Amr et al. (2007) identified ERIS (ER intermembrane small protein, now named WFS2) as a novel protein mutated in WS patients. Recently, Willey et al. (2013) detected the protein mostly in the ER fraction and also in the fraction of contact between ER and mitochondria, the so-called mitochondria-associated ER membranes (MAMs). MAMs are highly functionalized subcellular domains of interaction between ER and mitochondria, stabilized by protein bridges and sequestering numerous protein assemblies. Focused Ca²⁺ exchanges are driven by IP3 receptors (IP3Rs) on ER and voltage-dependent anion channel-1 on mitochondrial membranes (Giorgi et al., 2015). MAMs are involved in a plethora of function from Ca²⁺ homeostasis, lipid synthesis, mitochondrial function and dynamics, and apoptosis to inflammation (Pinton, 2018). Interestingly, MAMs defects are a hallmark of some neurodegenerative diseases (Delprat, Maurice, & Delettre, 2018).

Even if WFS1 plays an important role in ER stress, a putative role in Ca²⁺ signaling has been suggested. The first evidence of a role of WFS1 in Ca²⁺ homeostasis was shown by Osman et al. (2003). In their work, they reconstituted WFS1 from Xenopus oocyte membrane into planar lipid bilayers and were able to record a large cation-selective ion channel. Moreover, the addition of IP3 activated channel properties, suggesting that WFS1 may be an IP3R or a regulatory subunit of another IP3R. In another work, Takei et al. (2006) demonstrated that WFS1 was necessary for maintaining [Ca²⁺]_ER in HEK293 cells. Zatyka et al. (2015) confirmed this observation by showing the interaction and direct regulation of Serca2b by WFS1. In their study, they showed that WFS1 deficiency leads to reduced increases in cytosolic Ca²⁺ in response to elevated glucose concentrations in MIN6 cells. Finally, Cagalinec et al. (2016) demonstrated that WFS1 deficiency in mice affects the proper functioning of IP3R, leading to altered cytosolic Ca²⁺ homeostasis and mitochondrial dynamics. Even if these results are important for the understanding of the role of WFS1 in Ca²⁺ homeostasis, the exact role of WFS1 on IP3R physiology remains to be deciphered. Therefore, we analyzed Ca²⁺ homeostasis in human fibroblasts from WS patients (Angebault et al., 2018).

WFS1 Is Required for Structure and Function of MAMs

Our hypothesis was that WFS1 deficiency may affect Ca²⁺ flux between ER and mitochondria through regulation of IP3R. Indeed, we demonstrated by co-immunoprecipitation experiments that mouse WFS1 interacts with IP3R (Angebault et al., 2018). To elucidate the effects of loss of function mutations in the ER protein WFS1, we used fibroblasts from WS patients or normal individuals and we measured the effects of WFS1 loss of function on Ca²⁺ flux. We showed that there was no significant difference in steady-state ER Ca²⁺ concentration ([Ca²⁺]_ER) between control and WFS1 patient’s fibroblasts, but we found that mutated fibroblasts released less ER Ca²⁺. This result is in contradiction with those of Takei et al. (2006) that described a decrease in the ER Ca²⁺ content. This discrepancy may be explained by the difference in the cell type. Therefore, it is essential to analyze the [Ca²⁺]_ER in neurons deficient for WFS1 to have a clear view of what is going on in neurons. In addition, Ca²⁺ uptake by mitochondria was reduced in patients’ fibroblasts and was due to decreased ER Ca²⁺ release through IP3R (Figure 1). In addition, we evidenced that the number of ER–mitochondria contacts was reduced in patients’ cells (Figure 1). This reduced organelle tethering is expected to decrease Ca²⁺ transfer from ER to mitochondria in patients’ cells. We then evaluated mitochondrial functionality by analyzing mitochondrial respiration in patients’ fibroblasts and showed that WFS1 patient cells exhibited a significant decrease in Complex II-driven respiration, suggesting that WFS1 deficiency impairs mitochondrial functionality. To elucidate the molecular mechanisms underlying the WFS1 cell phenotype, we searched for binding proteins for WFS1 and identified a Neuronal Calcium Sensor, NCS1, preferentially expressed in neurons (Martone, Edelmann, Ellisman, & Nef, 1999). NCS1 regulates several neuronal functions including exocytosis, neurite outgrowth, neuroprotection, and axonal regeneration (Boeckel & Ehrlich, 2018). Notably, NCS1 is directly linked to neurological disorders (Bandura & Feng, 2019). Interestingly, NCS1 protein levels were decreased by almost 50% in WFS1-deficient fibroblasts (Figure 1). When we knocked down NCS1 in controls’ fibroblasts, IP3R-dependent mitochondrial Ca²⁺ uptake was decreased as well as the number of organelle contacts. This decrease in the number of ER–mitochondria contacts as well as the decrease in the length of the ER–mitochondria membrane apposition was unexpected (Figure 1). As we did not observe any alteration in the protein levels of MFN1-MFN2 or PTPIP51/VAPB, the two well-known complexes involved in MAM tethering, the decreased protein levels of NCS1 may perturb another not-yet-identified structural complex or signaling pathway. Interestingly, NCS1-depleted cells also displayed a decrease in both Complex I- and Complex II-driven respiration, similar to that observed in patient fibroblasts. From these findings, NCS1 is likely to mediate mitochondrial Ca²⁺ uptake and could be a key determinant to maintain mitochondrial function and MAM integrity. Therefore, we reasoned that NCS1 overexpression would rescue the defective mitochondrial phenotype of WFS1-deficient cells. To investigate this, we overexpressed NCS1 in WFS1-deficient cells and measured mitochondrial parameters such as Ca²⁺ uptake, respiration, and ER–mitochondria contacts. Indeed, NCS1 overexpression in patient cells restored ER–mitochondria interactions and Ca²⁺ exchange and increased Complex I and Complex II-driven respiration. Therefore, our results suggest that NCS1 rescue could protect WFS1-deficient cells and could be used as a tool to restore Ca²⁺ signaling at MAM interface and mitochondrial function.

MAMs Defect: Common Features in Neurodegenerative and Metabolic Diseases

Emerging evidence suggests a close relationship between ER–mitochondria miscommunication and neurodegenerative diseases, including WS, Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. Whereas these different neurodegenerative diseases affect different regions of the nervous system and involve distinct proteins, all display some common features such as altered Ca²⁺, cholesterol and phospholipid metabolism, altered mitochondrial dynamics, and reduced bioenergetic function and ER stress. As MAMs are a critical hub regulating all of these processes, they rapidly emerged as a common dysfunction. However, MAMs are either reinforced or disrupted depending of the neurodegenerative disease, or even within the same disease, suggesting that both abnormal increases and decreases of MAMs are likely to be detrimental to cells. Therefore, further studies are required to clarify the exact role of ER–mitochondria communication in neurodegenerative diseases.

The clinical hallmark of WS is the association of diabetes mellitus, optic atrophy, and deafness. The natural history of WS shows diabetes mellitus during the first decade of life together with progressive optic atrophy, whereas deafness appears during the second decade. The diabetic phenotype is associated with degeneration of pancreatic beta cells, a cell type in which NCS1 is abundant. Interestingly, ER–mitochondria miscommunication has been associated with beta cell dysfunction in type 2 diabetes (Thivolet, Vial, Cassel, Rieusset, & Madec, 2017). However, it is currently unknown whether NCS1 or WFS1 plays a role in these alterations.

In the context of type 2 diabetes, beta cell dysfunction is also associated with insulin resistance of peripheral tissues, such as the liver, skeletal muscle, and adipose tissue. Interestingly, ER–mitochondria miscommunication was recently associated with insulin resistance. Whereas conflicting results are observed in the insulin resistant liver, with either a disruption (Tubbs et al., 2014) or a reinforcement of MAMs (Arruda et al., 2014), disrupted ER–mitochondria interactions are found in insulin-resistant skeletal muscle in both mice and humans (Tubbs, Chanon, et al., 2018). In addition, loss of WFS2 induced a reduction of ER–mitochondria interactions as well as a reduction of insulin-mediated glucose uptake (Wang et al., 2014), pointing once again toward a link between organelle miscommunication and insulin resistance. Altogether, these data argue for ER–mitochondria miscommunication as a common physio-pathological mechanism in both neurodegenerative and metabolic diseases. In agreement, antidiabetic drugs such as metformin can reduce neurological symptoms in some patients and reduce disease phenotypes in animal and cell models (Rotermund, Machetanz, & Fitzgerald, 2018). Interestingly, metformin (Rieusset et al., 2016) and another potential antidiabetic drug, Sulforaphane, (Tubbs, Axelsson, et al., 2018) were shown to improve ER–mitochondria interactions in the liver. As there are still no cures for WS, Alzheimer’s disease, Parkinson’s disease, or amyotrophic lateral sclerosis, mechanistic insights into the role of MAMs in metabolic diseases will help to identify new therapeutic strategies.

Figure 1.

MAM alteration in Wolfram syndrome (WS) type 1. In healthy cells (left), WFS1 binds to NCS1 to form a complex with IP3R1 whose function is to maintain a physiological number and size of MAMs. In WS (right), the loss of function of WFS1 leads to NCS1 degradation. This results in a decrease in the size and the number (not shown in the figure) of ER–mitochondria contacts and a decrease in Ca²⁺ transfer from ER to mitochondrial matrix. MAM = mitochondria-associated ER membrane; IP3R = IP3 receptor; VDAC1 = voltage-dependent anion channel-1; NCS1 = neuronal calcium sensor 1; WFS1 = Wolfram syndrome 1; GRP75 = 75 kDa glucose-regulated protein.