Wolframin is in the endoplasmic reticulum (ER) membrane, which plays a key role in the ability of cells to properly fold and post-translate secretory and the ER transmembrane proteins
[29][30][31]. Mutations in
WFS1 cause an accumulation of misfolded proteins in the ER and therefore ER stress. High levels of misfolded proteins stimulate the unfolded protein response (UPR), which induces transcriptional and translational events that restore ER homeostasis. When ER stress is chronically persistent due to physiological processes (biosynthesis post-prandial of insulin) or to pathological processes (cancer, inflammatory diseases, viral infection, gene mutations), the UPR stimulates cell apoptosis.
[17][29][31][32][33]. For this reason, high levels of ER stress found in WS1 cause apoptosis of pancreatic cells and alterations of neuronal cells
[33].
Under conditions of physiological stress, IRE1 oligomerization and autophosphorylation occurs
[37]. Subsequently, the RNase domain of IRE1 induces a splice of the X-binding protein 1 (XBP-1) mRNA because of which a transcriptionally active mRNA (sXBP-1) is formed. sXBP-1 is activated to XBP-1, a transcription factor. After translocation to the nucleus, XBP-1 upregulates some UPR target genes to re-establish protein homeostasis and activate cellular protection. Under pathological conditions, hyperactivation of IRE1 with consequent apoptosis is found. Moreover, IRE1 affects the biosynthesis of insulin: in hyperglycemia, it induces β cell homeostasis and thus an improvement in pro-insulin biosynthesis
[38].
The transmembrane PERK protein is also involved in intricate mechanism of ER stress. Indeed, it activates the phosphorylation of eIF2alfa, a eukaryotic initiation translation factor 2alfa. The role of eIF2alfa is to decrease ER biosynthetic activity and to enhance the translation of both ATF4 transcription factor and apoptosis-antagonizing transcription factor (AATF) mRNAs. ATF4 activates genes involved in amino acid transport and metabolism, glutathione biosynthesis, and antioxidant responses. Moreover, ATF4- ATF3-CHOP axis promotes apoptosis. Thus, under conditions of pathological ER stress, apoptosis is induced by the continuous activation of these factors. On the other hand, AATF promotes the survival of cells
[39].
The intricate pathway of UPR is negatively regulated by
WFS1. Under physiological ER stress,
WFS1 negatively regulates ATF6, reduces the activation of the ER stress response element of the ER (ERSE) promoted by ATF6, and induces the stabilization of E3 ubiquitin ligase HRD1 (HMG-CoA reductase degradation protein) and thus the suppression of stress signals.
[17]. Conversely, in WS1, the hyperactivation of ATF6 promotes both the expression of genes involved in apoptosis, such as CHOP, ATF4, BIP, and sXBP1, and the reduced gene expression of insulin. Moreover, wolframin regulates the calcium release and absorption mechanisms in the ER. It is a calmodulin (CaM) with several functions, including the interaction with many cellular proteins and the regulation of the Ca2 + signal transduction processes involved in apoptosis
[43]. High levels of ER stress have been shown to cause alterations in mitochondrial function, thus suggesting that WS1 could be a mitochondrial disease
[44]. Recently, a link between ER stress, increased cytosolic Ca2+levels, impairment of mitochondrial dynamics, and inhibition of neuronal development in
WFS1-deficient neurons has been described
[45][46]. In healthy cells,
WFS1 is linked to neuronal calcium sensor 1 (NCS1) and inositol 1,4,5-trisphosphate receptor (IP3R) to induce transfer of Ca2+ between ER and mitochondria. In
WFS1-deficient cells, a severe decrease of NCS1 levels was found, which causes a reduction of ER-mitochondria interactions and transfer of Ca2+
[47]. Therefore, there is a strong causal link between ER stress, alterations in cytosolic levels of Ca 2+ and in mitochondrial dynamics, and developmental delay in
WFS1-deficient neuronal cells
[45]. In this intricate pathogenic mechanism, the alterations of mitochondria-associated ER membranes (MAMs) play an important role
[48][49]. MAMs are dynamic domains of interaction between mitochondria and ER in which several proteins involved in UPR are located. The role of these proteins is to stabilize the structure of MAMs and to facilitate the functional dialogue between ER and mitochondria. Indeed, MAMs facilitate the transfer of Ca2 + between ER and mitochondria mainly through IP3R
[46][49]. According to these observations, Cagalinec et al. suggested that a “mitochondrial phenotype” in WS1 patients could be due to severe alterations of mitochondrial dynamics caused by even mild ER stress
[46]. Zmyslowska et al. studied a human WS cell model in which skin fibroblasts reprogrammed into induced pluripotent stem cells (iPS) and then into neural stem cells (NSCs) were subjected to induced ER stress. The analysis of the proteins involved in mitochondrial function showed a down-regulation of the subunits of the respiratory chain complexes, an upregulation of the proteins involved in the Krebs cycle, and mechanisms of glycolysis in WS NSC cells. These alterations were not found in the control cells. These data have shown that severe mitochondrial damage resulting in functional and morphological alterations of mitochondria plays a key role in the pathogenesis of WS1
[50].