InteractiveFly: GeneBrief

wolfram syndrome 1: Biological Overview | References

Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, this study aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. It was found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, a role is proposed for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations (Hao, 2023).

Sleep disruptions are common in individuals with psychiatric disorders, and sleep disturbances are risk factors for future onset of depression. However, the mechanism underlying sleep disruptions in psychiatric disorders are largely unclear. Wolfram Syndrome 1 (WS1) is an autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness and psychiatric abnormalities such as severe depression, psychosis and aggression. It is caused by homozygous (and compound heterozygous) mutation of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes wolframin, an endoplasmic reticulum (ER) resident protein highly expressed in the heart, brain, and pancreas. On the other hand, heterozygous mutation of WFS1 does not lead to WS1 but increase the risk of depression by 26 fold. A study in mice further confirmed that WFS1 mutation is causative for depression. Consistent with the comorbidity of psychiatric conditions and sleep abnormalities, WS1 patients also experience increased sleep problems compared to individuals with type I diabetes and healthy controls. It has been proposed that sleep symptoms can be used as a biomarker of the disease, especially during relatively early stages, but the mechanisms underlying these sleep disturbances are unclear. Considering that heterozygous WFS1 mutation is present in up to 1% of the population and may be a significant cause of psychiatric disorder in the general population, it was decided to investigate the role of wolframin in sleep regulation so as to probe the mechanism underlying sleep disruptions in psychiatric disorders (Hao, 2023).

Although the wolframin protein does not possess distinct functional domains, a number of ex vivo studies in cultured cells demonstrated a role for it in regulating cellular responses to ER stress and calcium homeostasis, as well as ER-mitochondria cross-talk. Mice that lack Wfs1 in pancreatic β cells develop glucose intolerance and insulin deficiency due to enhanced ER stress and apoptosis. Knocking out Wfs1 in layer 2/3 pyramidal neurons of the medial prefrontal cortex in mice results in increased depression-like behaviors in response to acute restraint stress. This is accompanied by hyperactivation of the hypothalamic-pituitary-adrenal axis and altered accumulation of growth and neurotrophic factors, possibly due to defective ER function. A more recent study in Drosophila found that knocking down wfs1 in the nervous system does not increase ER stress, but enhances the susceptibility to oxidative stress-, endotoxicity- and tauopathy-induced behavioral deficits and neurodegeneration (Sakakibara, 2018). Overall, the physiological function of wolframin in vivo, especially in the brain, remains elusive for the most part. This study identified a role for wolframin in regulating sleep and circadian rhythm in flies. Wfs1 deficiency in the dopamine 2-like receptor (Dop2R) neurons leads to reduced sleep, while inhibiting dopamine synthesis blocks the effect of wfs1 on sleep, implying that wfs1 influences sleep via dopaminergic signaling. It was further found that these Dop2R neurons function to promote wakefulness. Depletion of wfs1 alters neural activity, which leads to increased wakefulness and reduced sleep. Consistent with this, it was found that knocking down the ER calcium channel Ryanodine receptor (RyR) or 1,4,5-trisphosphate receptor (Itpr) rescues while knocking down the sarco(endoplasmic)reticulum ATPase SERCA synergistically enhances the short-sleep phenotype caused by wfs1 deficiency, indicating that wolframin regulates sleep by modulating calcium homeostasis. Taken together, these findings provide a potential mechanism for the sleep disruptions associated with WFS1 mutation, and deepen understanding of the functional role of wolframin in the brain (Hao, 2023).

Sleep problems have been reported in WS1 patients. Their scores on Pediatric Sleep Questionnaire are more than 3 times higher than healthy controls and doubled compared to individuals with type I diabetes, indicating that the sleep issues are not merely due to metabolic disruptions. Indeed, this study suggests that the sleep problems in human patients are of neural origin, specifically in the wake-promoting Dop2R neurons. Given that the rebound sleep is not significantly altered in wfs1 depleted flies, it is believed that lack of wfs1 does not shorten sleep duration by impairing the sleep homeostasis system. Instead, wfs1 deficiency leads to excessive wakefulness which in turn results in decreased sleep. Considering that heterozygous WFS1 mutation is present in up to 1% of the population, it would be interesting to examine whether these heterozygous mutations contribute to sleep disruptions in the general population (Hao, 2023).

In mouse, chick, quail and turtle, Wfs1 has been shown to be expressed in brain regions where dopamine receptor Drd1 is expressed. D1-like dopamine receptor binding is increased while striatal dopamine release is decreased in Wfs1-/- mice. The current results also implicate a role for wolframin in dopamine receptor neurons and that lack of wfs1 impacts dopaminergic signaling, as the effects of wfs1 deficiency on both sleep and mushroom body (MB) calcium concentration is blocked by the tyrosine hydroxylase inhibitor AMPT. Both Dop2RGAL4 and GoαGAL4 exhibit prominent expression in the MB, and to be more specific, in the α and β lobes of MB. Previous studies have shown that dopaminergic neurons innervate wake promoting MB neurons, and this study found Dop2R and Goα+ cells to be wake-promoting as well. Therefore, it is suspected that wolframin functions in MB Dop2R/Goα+ neurons to influence sleep. Taken together, these findings suggest an evolutionarily conserved role of wolframin within the dopaminergic system. As this system is also important for sleep/wake regulation in mammals, it is reasonable to suspect that wolframin functions in mammals to modulate sleep by influencing the dopaminergic tone as well (Hao, 2023).

MB neural activity appears to be enhanced in wfs1 deficient flies based on the results obtained using CaLexA and spH reporters. This elevated activity is consistent with behavioral data, as activation of Dop2R/Goα+ cells reduces sleep, similar to the effects of wfs1 deficiency. Moreover, silencing Dop2R neurons rescues the short-sleep phenotype of wfs1 mutants, while over-expressing wfs1 restores the decreased sleep induced by activation of Dop2R neurons. These findings suggest that wolframin functions to suppress the excitability of MB Dop2R neurons, which in turn reduces wakefulness and promotes sleep. Comparable cellular changes have been observed in SERCA mutant flies. Electric stimulation leads to an initial increase followed by prolonged decrease of calcium concentration in mutant motor nerve terminal compared to the control, while action potential firing is increased in the mutants. This series of results underpin the importance of ER calcium homeostasis in determining membrane excitability and thus neural function (Hao, 2023).

GCaMP6 monitoring reveals that wfs1 deficiency selectively reduces fluorescence signal in the MB both under baseline condition and after dopamine treatment, which should reflect a reduction of cytosolic calcium level that is usually associated with decreased excitability. Previous studies have shown that lack of wolframin leads to increased basal calcium level in neural progenitor cells derived from induced pluripotent stem (iPS) cells of WS1 patients and primary rat cortical neurons, but after stimulation the rise of calcium concentration is smaller in Wfs1 deficient neurons, resulting in reduced calcium level compared to controls. Similarly, evoked calcium increase is also diminished in fibroblasts of WS1 patients and MIN6 insulinoma cells with WFS1 knocked down. Notably, wolframin has been shown to bind to calmodulin (CaM) in rat brain, and is capable of binding with calcium/CaM complex in vitro and in transfected cells. This may undermine the validity of using GCaMP to monitor calcium level in wfs1 deficient animals and cells, and could potentially account for the contradictory data acquired using CaLexA vs GCaMP (Hao, 2023).

It is intriguing that in this study wfs1 deficiency appears to selectively impair the function of Dop2R/Goα+ neurons. It has been shown that in the rodent brain Wfs1 is enriched in layer II/III of the cerebral cortex, CA1 field of the hippocampus, central extended amygdala, striatum, and various sensory and motor nuclei in the brainstem. Wfs1 expression starts to appear during late embryonic development in dorsal striatum and amygdala, and the expression quickly expands to other regions of the brain at birth. It is suspect that in flies wfs1 may be enriched in Dop2R/Goα+ cells during a critical developmental period, and that sufficient level of wolframin is required for their maturation and normal functioning in adults. Another possibility is that these cells are particularly susceptible to calcium dyshomeostasis induced by loss of wfs1. Indeed, this is believed to be an important cause of selective dopaminergic neuron loss in Parkinson's Disease, as dopaminergic neurons are unique in their autonomic excitability and selective dependence on calcium channel rather than sodium channel for action potential generation. It is reasoned that Dop2R/Goα+ neurons may also be more sensitive to abnormal intracellular calcium concentration, making them particularly vulnerable to wfs1 deficiency. The pathogenic mechanism underlying the neurodegeneration of WS1 is quite complex, possibly involving brain-wide neurodegenerative processes and neurodevelopmental dis-regulations. The findings of this study provide some evidence supporting a role for altered dopaminergic system during development. Obviously, much more needs to be done to test these hypotheses (Hao, 2023).

The precise role of wolframin in ER calcium handling is not yet clear. It has been shown in human embryonic kidney (HEK) 293 cells that knocking down WFS1 reduces while over-expressing WFS1 increases ER calcium level. The authors concluded that wolframin upregulates ER calcium concentration by increasing the rate of calcium uptake. Consistently, this study found by genetic interaction that knocking down RyR or Itpr (which act to reduce ER calcium level and thus knocking down either one will increase ER calcium level) rescues the short-sleep phenotype caused by wfs1 mutation, while knocking down SERCA (which acts to increase ER calcium level and thus knocking down this gene will reduce ER calcium level) synergistically enhances the short-sleep phenotype. Based on the results of these genetic interactions, it is proposed that lack of wfs1 increases cytosolic calcium while decreasing ER calcium, leading to hyperexcitability of Dop2R neurons and thus reduced sleep. Knocking down RyR or Itpr decreases cytosolic calcium and increases ER calcium, counteracting the influences of wfs1 deficiency and thus rescuing the short-sleep phenotype. On the other hand, knocking down SERCA further increases cytosolic calcium and decreases ER calcium, rendering an enhancement of the short-sleep phenotype. In line with this, study conducted in neural progenitor cells derived from iPS cells of WS1 patients demonstrated that pharmacological inhibition of RyR can prevent cell death caused by WFS1 mutation. In addition, inhibiting the function of IP3R may mitigate ER stress in wolframin deficient cells. One caveat is that SERCA protein level is increased in primary islets isolated from Wfs1 conditional knock-out mice, as well as in MIN6 cells and neuroblastoma cell line with WFS1 knocked down. It is reasoned that this may be a compensatory increase to make up for the reduced ER calcium level due to wolframin deficiency. It is acknowledged that the hypothesis proposed in in the papert is not supported by GCaMP data, which indicates decreased cytosolic calcium level in Dop2R neurons of wfs1 deficient flies. It is suspected that since the sleep phenotype associated with lack of wfs1 is of developmental origin, it is possible there is an initial increase of cytosolic calcium during critical developmental period in wfs1 deficient flies and this influences the function of Dop2R neurons in adults. Clearly, further characterizations need to be done to fully elucidate this issue, and preferably another calcium indicator independent of the GCaMP system should be employed (Hao, 2023).

In conclusion, this study identified a role for wolframin in the wake-promoting Dop2R neurons. wfs1 depletion in these cells lead to impaired calcium homeostasis and altered neural activity, which in turn leads to enhanced wakefulness and reduced sleep. This study may provide some insights for the mechanisms underlying the sleep disruptions in individuals with WFS1 mutation, as well as for the pathogenesis of WS1 (Hao, 2023).

Knockdown of wfs1, a fly homolog of Wolfram syndrome 1, in the nervous system increases susceptibility to age- and stress-induced neuronal dysfunction and degeneration in Drosophila

Wolfram syndrome (WS), caused by loss-of-function mutations in the Wolfram syndrome 1 gene (WFS1), is characterized by juvenile-onset diabetes mellitus, bilateral optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. WFS1 encodes an endoplasmic reticulum (ER)-resident transmembrane protein, and mutations in this gene lead to pancreatic beta-cell death induced by high levels of ER stress. However, the mechanisms underlying neurodegeneration caused by WFS1 deficiency remain elusive. This study investigated the role of WFS1 in the maintenance of neuronal integrity in vivo by knocking down the expression of wfs1, the Drosophila homolog of WFS1, in the central nervous system. Neuronal knockdown of wfs1 caused age-dependent behavioral deficits and neurodegeneration in the fly brain. Knockdown of wfs1 in neurons and glial cells resulted in premature death and significantly exacerbated behavioral deficits in flies, suggesting that wfs1 has important functions in both cell types. Although wfs1 knockdown alone did not promote ER stress, it increased the susceptibility to oxidative stress-, excitotoxicity- or tauopathy-induced behavioral deficits, and neurodegeneration. The glutamate release inhibitor riluzole significantly suppressed premature death phenotypes induced by neuronal and glial knockdown of wfs1. This study highlights the protective role of wfs1 against age-associated neurodegeneration and furthers understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases (Sakakibara, 2018).

Most WS patients die prematurely in association with severe neurological disabilities as a result of brain atrophy; however, the mechanisms underlying neurodegeneration caused by WFS1 deficiency remain elusive. This study investigated the effects of knockdown of wfs1, a fly homolog of WFS1, in the nervous system on neuronal integrity during aging. The results demonstrated that wfs1 expression was induced upon aging, and neuronal knockdown of wfs1 caused age-dependent behavioral deficits and neurodegeneration (Sakakibara, 2018).

The process by which wfs1 deficiency induced behavioral deficits and neurodegeneration in the Drosophila model remained unclear. In pancreatic β cells, WFS1 deficiency causes chronic ER stress and subsequent cell death through the dysregulation of intracellular Ca2+ levels and disruption of ER homeostasis. In addition, a recent report using primary cultured neurons demonstrated that WFS1 deficiency alters Ca2+ homeostasis in the ER and promotes mitophagy, leading to alterations in mitochondrial distribution and dynamics. This study found that knockdown of wfs1 in the nervous system did not induce ER stress or mitochondrial dysfunction even in aged fly brains. Moreover, autophagy flux was not significantly affected in these flies. These results suggest that reductions in wfs1 function alone do not severely disrupt cellular functions, which directly causes neurodegeneration. Rather, wfs1 may play a protective role against various stressors associated with aging, and its deficiency increased the susceptibility to neurodegeneration in aged flies (Sakakibara, 2018).

In support of this hypothesis, wfs1 expression was induced by aging and stressors, and neuronal knockdown of wfs1 rendered neurons vulnerable to oxidative stress-, altered neuronal excitability- or Alzheimer's associated tauopathy-induced behavioral deficits and neurodegeneration. Moreover, although knockdown of wfs1 in neurons did not induce obvious stress responses (Figs 4 and 5C), wfs1 deficiency significantly augmented oxidative stress and ER stress responses under stressed conditions (Fig 6F and 6G). These results suggest that wfs1 deficiency modifies the onset, progression and severity of neurological and neurodegenerative phenotypes caused by a combination of complex environmental, genetic, and age-associated factors (Sakakibara, 2018).

In this study, knockdown of wfs1 in neurons and glial cells significantly exaggerated behavioral deficits and premature death, suggesting that neuronal and glial wfs1 work in concert to maintain neuronal integrity in flies. The mRNA expression levels of Eaat1 and Grd, which are fly orthologs of glial glutamate transporters (SLC1A3) and GABA-A receptor α subunit (GABRA6), respectively, were significantly altered in these fly brains. Moreover, loss of one copy of Eaat1 increased locomotor deficits induced by wfs1 deficiency, whereas feeding of riluzole, which is thought to inhibit glutamate release as well as GABA uptake, significantly suppressed premature death phenotypes in these flies. These results suggest that stresses caused by altered neuronal excitability, possibly due to an imbalance between glutamatergic and GABAergic tones, may underlie the observed behavioral deficits in flies with wfs1 deficiency (Sakakibara, 2018).

Recent reports suggest that the neurological manifestations of WFS1 deficiency are associated with endocrine problems. In addition to the ER, the WFS1 protein localizes to secretory granules and regulates intragranular acidification, proinsulin processing, and exocytosis in pancreatic β cells, in part by interacting with the V1A subunit of H+ V-ATPase. Consistent with this, vasopressin processing is defective in some hypothalamic nuclei in the brain of WS patients, and the processing and secretion of growth and trophic factors is impaired in Wfs1-deficient mice. This study examined whether knockdown of wfs1 in neurons and glial cells alters the levels of a fly ortholog of Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) in fly brains. Western blot analyses revealed that the level of MANF was not significantly altered in fly brains. Further analysis is required to clarify the mechanism by which wfs1 functions in neurons and glial cells to maintain neuronal function and integrity in fly brains (Sakakibara, 2018).

The wfs1 mutant and RNAi lines used in this study did not completely abolish wfs1 expression and function in flies. This indicates that some of the phenotypes induced by knockdown of wfs1 may be different from those caused by complete null mutation of wfs1. Nevertheless, this study demonstrated that wfs1 deficiency in the nervous system is sufficient to render neurons vulnerable to age-associated neurodegeneration in Drosophila. The results also highlight the importance of the functions of wfs1 in both neuronal and glial cells. Moreover, these data revealed a novel genetic interaction between wfs1 and Eaat1, and suggest that altered neural activity and vulnerability to oxidative stress underlie neurological phenotypes in wfs1 deficient flies (Sakakibara, 2018).

In conclusion, this study provides insight into the molecular mechanisms underlying neurodegeneration in WS and furthers our understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases such as Alzheimer's disease (Sakakibara, 2018).

Loss of Function of WFS1 Causes ER Stress-Mediated Inflammation in Pancreatic Beta-Cells

Wolfram syndrome is a rare genetic disorder characterized by juvenile-onset diabetes mellitus, optic nerve atrophy, hearing loss, diabetes insipidus, and progressive neurodegeneration. Pathogenic variants in the WFS1 gene are the main causes of Wolfram syndrome. WFS1 encodes a transmembrane protein localized to the endoplasmic reticulum (ER) and regulates the unfolded protein response (UPR). Loss of function of WFS1 leads to dysregulation of insulin production and secretion, ER calcium depletion, and cytosolic calpains activation, resulting in activation of apoptotic cascades. Although the terminal UPR has been shown to induce inflammation that accelerates pancreatic beta-cell dysfunction and death in diabetes, the contribution of pancreatic beta-cell inflammation to the development of diabetes in Wolfram syndrome has not been fully understood. This study shows that WFS1-deficiency enhances the gene expression of pro-inflammatory cytokines and chemokines, leading to cytokine-induced ER-stress and cell death in pancreatic beta-cells. PERK and IRE1alpha pathways mediate high glucose-induced inflammation in a beta-cell model of Wolfram syndrome. M1-macrophage infiltration and hypervascularization are seen in the pancreatic islets of Wfs1 whole-body knockout mice, demonstrating that WFS1 regulates anti-inflammatory responses in pancreatic beta-cells. These results indicate that inflammation plays an essential role in the progression of beta-cell death and diabetes in Wolfram syndrome. The pathways involved in ER stress-mediated inflammation provide potential therapeutic targets for the treatment of Wolfram syndrome (Morikawa, 2022).

Calpain inhibitor and ibudilast rescue beta cell functions in a cellular model of Wolfram syndrome

Wolfram syndrome is a rare multisystem disease characterized by childhood-onset diabetes mellitus and progressive neurodegeneration. Most cases are attributed to pathogenic variants in a single gene, Wolfram syndrome 1 (WFS1). There currently is no disease-modifying treatment for Wolfram syndrome, as the molecular consequences of the loss of WFS1 remain elusive. Because diabetes mellitus is the first diagnosed symptom of Wolfram syndrome, this study was aimed to further examine the functions of WFS1 in pancreatic beta cells in the context of hyperglycemia. Knockout (KO) of WFS1 in rat insulinoma (INS1) cells impaired calcium homeostasis and protein kinase B/Akt signaling and, subsequently, decreased cell viability and glucose-stimulated insulin secretion. Targeting calcium homeostasis with reexpression of WFS1, overexpression of WFS1's interacting partner neuronal calcium sensor-1 (NCS1), or treatment with calpain inhibitor and ibudilast reversed deficits observed in WFS1-KO cells. Collectively, these findings provide insight into the disease mechanism of Wolfram syndrome and highlight new targets and drug candidates to facilitate the development of a treatment for this disorder and similar diseases (Nguyen, 2020).

Wfs1 is expressed in dopaminoceptive regions of the amniote brain and modulates levels of D1-like receptors

During amniote evolution, the construction of the forebrain has diverged across different lineages, and accompanying the structural changes, functional diversification of the homologous brain regions has occurred. This can be assessed by studying the expression patterns of marker genes that are relevant in particular functional circuits. In all vertebrates, the dopaminergic system is responsible for the behavioral responses to environmental stimuli. This study shows that the brain regions that receive dopaminergic input through dopamine receptor D1 are relatively conserved, but with some important variations between three evolutionarily distant vertebrate lines-house mouse (Mus musculus), domestic chick (Gallus gallus domesticus) / common quail (Coturnix coturnix) and red-eared slider turtle (Trachemys scripta). Moreover, this study found that in almost all instances, those brain regions expressing D1-like dopamine receptor genes also express Wfs1. Wfs1 has been studied primarily in the pancreas, where it regulates the endoplasmic reticulum (ER) stress response, cellular Ca2+ homeostasis, and insulin production and secretion. Using radioligand binding assays in wild type and Wfs1-/- mouse brains, this study showed that the number of binding sites of D1-like dopamine receptors is increased in the hippocampus of the mutant mice. It is proposed that the functional link between Wfs1 and D1-like dopamine receptors is evolutionarily conserved and plays an important role in adjusting behavioral reactions to environmental stimuli (Takko, 2017).

Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression

Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca(2+) imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca(2+) concentration ([Ca(2+)]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. It was also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. This study demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis (Zatyka, 2015).

Initiation and developmental dynamics of Wfs1 expression in the context of neural differentiation and ER stress in mouse forebrain

Wolframin (Wfs1) is a membrane glycoprotein that resides in the endoplasmic reticulum (ER) and regulates cellular Ca(2+) homeostasis. In pancreas Wfs1 attenuates unfolded protein response (UPR) and protects cells from apoptosis. Loss of Wfs1 function results in Wolfram syndrome (OMIM 222300) characterized by early-onset diabetes mellitus, progressive optic atrophy, diabetes insipidus, deafness, and psychiatric disorders. Similarly, Wfs1-/- mice exhibit diabetes and increased basal anxiety. In the adult central nervous system Wfs1 is prominent in central extended amygdala, striatum and hippocampus, brain structures largely involved in behavioral adaptation of the organism. This study describes the initiation pattern of Wfs1 expression in mouse forebrain using mRNA in situ hybridization and compares it with Synaptophysin (Syp1), a gene encoding synaptic vesicle protein widely used as neuronal differentiation marker. The expression of Wfs1 was shown to start during late embryonic development in the dorsal striatum and amygdala, then expands broadly at birth, possessing several transitory regions during maturation. Syp1 expression precedes Wfs1 and it is remarkably upregulated during the period of Wfs1 expression initiation and maturation, suggesting relationship between neural activation and Wfs1 expression. Using in situ hybridization and quantitative real-time PCR it was shown that UPR-related genes (Grp78, Grp94, and Chop) display dynamic expression in the perinatal brain when Wfs1 is initiated and their expression pattern is not altered in the brain lacking functional Wfs1 (Tekko, 2014).

A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration and considered as an endoplasmic reticulum (ER) disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome and the identification of two causative genes, Wolfram syndrome 1 (WFS1) and Wolfram syndrome 2 (WFS2), a molecular mechanism linking the ER to death of neurons and beta cells has not been elucidated. This study implicate calpain 2 in the mechanism of cell death in Wolfram syndrome. Calpain 2 is negatively regulated by WFS2, and elevated activation of calpain 2 by WFS2-knockdown correlates with cell death. Calpain activation is also induced by high cytosolic calcium mediated by the loss of function of WFS1. Calpain hyperactivation is observed in the WFS1 knockout mouse as well as in neural progenitor cells derived from induced pluripotent stem (iPS) cells of Wolfram syndrome patients. A small-scale small-molecule screen targeting ER calcium homeostasis reveals that dantrolene can prevent cell death in neural progenitor cells derived from Wolfram syndrome iPS cells. These results demonstrate that calpain and the pathway leading its activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases (Lu, 2014).

Evidence for impaired function of dopaminergic system in Wfs1-deficient mice

Immunohistological studies suggest abundant expression of Wfs1 protein in neurons and nerve fibers that lie in the vicinity of dopaminergic (DA-ergic) fibers and neurons. Therefore, this study sought to characterize the function of DA-ergic system in Wfs1-deficient mice. In wild-type mice, amphetamine, an indirect agonist of DA, caused significant hyperlocomotion and increase in tissue DA levels in the dorsal and ventral striatum. Both effects of amphetamine were significantly blunted in homozygous Wfs1-deficient mice. Motor stimulation caused by apomorphine, a direct DA receptor agonist, was somewhat stronger in Wfs1-deficient mice compared to their wild-type littermates. However, apomorphine caused a similar reduction in levels of DA metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) in the dorsal and ventral striatum in all genotypes. Behavioral sensitization to repeated treatment with amphetamine (2.5 mg/kg) was observed in wild-type, but not in Wfs1-deficient mice. The expression of DA transporter gene (Dat) mRNA was significantly lower in the midbrain of male and female homozygous mice compared to wild-type littermates. Altogether, the blunted effects of amphetamine and the reduced gene expression of DA transporter are probably indicative of an impaired functioning of the DA-ergic system in Wfs1-deficient mice (Raud, 2013).

Impaired striatal dopamine output of homozygous Wfs1 mutant mice in response to [K+] challenge

Loss of function of the Wfs1 gene causes Wolfram syndrome, a rare multisystem degenerative disorder. Mutant mice with targeted Wfs1 gene disruption (Wfs1 KO) display morphological and behavioral impairments that are not well understood. This study aimed to investigate the striatal dopamine output of wild-type, heterozygous, and homozygous Wfs1 null-mutant mice using in vivo microdialysis technique. The baseline dopamine output in striatum was similar in all three animal groups. The application of 100 mM [K+]-rich modified Ringer solution caused in homozygous Wfs1 mutant mice an increase of dopamine output by 400%, while in wild-type and heterozygous animals, the increase of the dopamine output yielded up to 1,200%. In sum, the homozygous Wfs1 mutant mice (AUC(0)(-)(3) = 0.212 nM/mμl h) show significantly decreased striatal dopamine output in response to high-concentration [K+] challenge as compared with wild-type or heterozygous Wfs1 mutant conspecifics (AUC(0)(-)(3) = 0.427 and 0.505 nM/mul h, respectively). This could explain at least some of the behavioral alterations in Wfs1 mutant mice (Matto, 2011).

Wolfram syndrome 1 (Wfs1) mRNA expression in the normal mouse brain during postnatal development

Wolfram syndrome is a rare genetic disorder accompanying diabetes insipidus, sensorineural hearing loss, neurological complications, and psychiatric illness. This syndrome has been attributed to mutations in the WFS1 gene. In this study, a detailed histochemical analysis was made of the distribution of Wfs1 mRNA in the brain of developing mice. There were three patterns of change in the strength of Wfs1 mRNA signals from birth to early adulthood. In type 1, the signals were weak or absent in neonates but strong or moderate in young adults. This pattern was observed in the CA1 field, parasubiculum, and entorhinal cortex. In type 2, the signals were of a relatively constant strength during development. This pattern was seen in limbic structures (e.g. subiculum and central amygdaloid nucleus) and brainstem nuclei (e.g. facial and chochlear nuclei). In type 3, the signals peaked in the second week of age. This pattern was observed in the thalamic reticular nucleus. Thus, Wfs1 mRNA was widely distributed in the normal mouse brain during postnatal development. This evidence may provide clues as to the physiological role of the Wfs1 gene in the central nervous system, and help to explain endocrinological, otological, neurological, and psychiatric symptoms in Wolfram syndrome patients (Kawano, 2009).

Identification and characterization of wolframin, the product of the wolfram syndrome gene (WFS1), as a novel calmodulin-binding protein

To search for calmodulin (CaM) targets, affinity chromatography purification was performed of a rat brain extract using CaM fused with GST as the affinity ligand. Proteomic analysis was then carried out to identify CaM-binding proteins. In addition to identifying 36 known CaM-binding proteins, including CaM kinases, calcineurin, nNOS, the IP(3) receptor, and Ca(2+)-ATPase, an ER transmembrane protein, wolframin [the product of the Wolfram syndrome gene (WFS1)] was identified as interacting. A CaM overlay and an immunoprecipitation assay revealed that wolframin is capable of binding the Ca(2+)/CaM complex in vitro and in transfected cells. Surface plasmon resonance analysis and zero-length cross-linking showed that the N-terminal cytoplasmic domain (residues 2-285) of wolframin binds to an equimolar unit of CaM in a Ca(2+)-dependent manner with a K(D) for CaM of 0.15 muM. Various truncation and deletion mutants showed that the Ca(2+)/CaM binding region in wolframin is located from Glu90 to Trp186. Furthermore, it was demonstrated that three mutations (Ala127Thr, Ala134Thr, and Arg178Pro) associated with Wolfram syndrome completely abolished CaM binding of wolframin. This observation may indicate that CaM binding is important for wolframin function and that impairment of this interaction by mutation contributes to the pathology seen in Wolfram syndrome (Yurimoto, 2009).

Search PubMed for articles about Drosophila Wolfram syndrome 1

Baur M, Lin F, Morgen TO, Odenwald L, Mecking S. Polyethylene materials with in-chain ketones from nonalternating catalytic copolymerization. (2021) Science374(6567):604-607. PubMed ID: 34709904

Hao, H., Song, L. and Zhang, L. (2023). Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis. PLoS Genet 19(7): e1010827. PubMed ID: 37399203

Kawano J., Fujinaga, R., Yamamoto-Hanada, K., Oka, Y., Tanizawa, Y., Shinoda, K. (2009). Wolfram syndrome 1 (Wfs1) mRNA expression in the normal mouse brain during postnatal development. Neurosci Res64(2):213-230. PubMed ID: 19428703

Mahadevan J., Spears L. D., Oslowski C. M., Martinez R., Yamazaki-Inoue M., Toyoda M., Neilson A., Blanner P., Brown C. M., Semenkovich C. F., Marshall B. A., Hershey T., Umezawa A., Greer P. A., Urano F. A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome. (2014) Proc Natl Acad Sci U S A111(49):E5292-5301. PubMed ID: 25422446

Matto V., Terasmaa A., Vasar E., Koks S. Impaired striatal dopamine output of homozygous Wfs1 mutant mice in response to [K+] challenge. (2011) J Physiol Biochem67(1):53-60. PubMed ID: 20972658

Morikawa S., Blacher L., Onwumere C., Urano F. Loss of Function of WFS1 Causes ER Stress-Mediated Inflammation in Pancreatic Beta-Cells. (2022) Front Endocrinol (Lausanne)13:849204. PubMed ID: 35399956

Nguyen L. D., Fischer, T. T., Abreu, D., Arroyo, A., Urano, F., Ehrlich, B. E. (2020). Calpain inhibitor and ibudilast rescue beta cell functions in a cellular model of Wolfram syndrome. Proc Natl Acad Sci U S A117(29):17389-17398. PubMed ID: 32632005

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Zatyka M., Da Silva Xavier G., Bellomo E. A., Leadbeater W., Astuti D., Smith J., Michelangeli F., Rutter G. A., Barrett T. G. Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression. (2015) Hum Mol Genet24(3):814-827. PubMed ID: 25274773 Biological Overview

date revised: 11 November 2023

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