Yeast and other lower eukaryotic organisms for studies of Vps13 proteins in health and disease

Human Vps13 proteins are associated with several diseases, including the neurodegenerative disorder Chorea-acanthocytosis (ChAc). However, the biology of these proteins is relatively poorly understood. Studies in lower eukaryotic models, such as Saccharomyces cerevisiae and Dictyostelium discoideum, point to the involvement of Vps13 in many key cellular processes, the actin cytoskeleton organization, vesicular trafficking, regulation of membrane contact sites, mitochondrial functioning and autophagy. Recent findings revealed that yeast Vps13 binds phosphatidylinositol lipids via at least four different lipid binding regions. Modelling of ChAc-associated mutations in yeast has revealed that particular amino acid substitutions give rise to specific phenotypes. Noteworthy, a mutation in the APT1 domain of Vps13 results in diminished lipid binding and disturbances of all processes studied, including the actin cytoskeleton organization, vacuolar transport, endocytosis and sporulation. This review describes the great potential of simple eukaryotes to decipher disease mechanisms and highlights novel insights into the pathological role of Vps13 towards ChAc

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin

Background/Aims: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. Methods: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. Results: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). Conclusion: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition

Effect of Lithium treatment on SOCE components in Chorea Acanthocytosis

Chorea Acanthocytosis (ChAc) is an autosomal recessive neurodegenerative disease characterized by limb chorea, dystonia, epilepsy and acanthocytosis. The patients have a reduced life expectancy of only around 60 years. This disorder is caused by a loss-of-function mutation of the VPS13A (vacuolar protein sorting-associated protein A) gene, which is the encoding gene of chorein. Normally, chorein stimulates phosphoinositide 3-kinase (PI3K), which is involved in the regulation of Ca2+ influx. Oscillations of intracellular Ca2+ concentration regulate, among other cell components, proliferation, differentiation and apoptosis. Emptying of intracellular Ca2+ stores activates the Store-operated Ca2+ entry (SOCE), which leads to a conformational change and opening of the channel through the interaction between the Ca2+ concentration sensor protein, the Stromal Interacting Molecule 1 (STIM1), and the pore-forming channel, Orai1. The Ca2+ channel is known also as Ca2+ release-activated channel (CRAC). SOCE is increased by lithium via stimulation of serum & glucocorticoid-inducible kinase (SGK1), which is responsible in cells for stimulating the expression of Orai1 and STIM1 as well regulating the transcription factor: Nuclear Factor 'kappa-light-chain-enhancer' of activated B-cells (NFкB). Lithium is used in the treatment of bipolar disorders and can cross the blood-brain barrier (BBB). In this study, fibroblasts and neurons of ChAc patients have been shown to have decreased SOCE and an increased rate of apoptosis. Fibroblasts were isolated from six ChAc patients and six healthy donors. In addition, fibroblasts were obtained from three additional patients and healthy donors and induced pluripotent stem cells (iPSCs) were generated in order to differentiate them into neurons. Western blotting and calcium imaging showed a significant reduction in the amount of Orai1 protein and SOCE, respectively, in fibroblasts and iPSC-differentiated neurons of ChAc patients compared to healthy donors. RT-PCR showed a significant decrease in the mRNA levels of Orai1 and STIM1 in iPSC-differentiated neurons of ChAc patients. Apoptosis was detected by annexin-V / propidium iodide staining via flow cytometry and was in fibroblasts and iPSC-differentiated neurons of ChAc patients significantly higher than in those of healthy donors. In this study, it could be shown that the treatment of fibroblasts and neurons of ChAc patients with lithium positively influenced SOCE, an effect significantly reduced by the Orai1 blocker, 2-aminoethoxy diphenyl borate (2-APB). Lithium induced a significant decrease in apoptosis, an effect again abrogated by 2-APB. The mRNA and protein expression of Orai1 and STIM1 were increased by lithium, an effect reversed by inhibition of SGK1 and NFкB. In conclusion, the apoptotic effect of chorein deficiency in fibroblasts and neurons of ChAc patients is in part due to the decreased expression of Orai1, STIM1 and SOCE. Treatment with lithium could reverse this effect, via the SGK1/NFкB signaling pathway, which shows that lithium could be a new treatment for Chorea Acanthocytosis

Targeting lyn kinase in chorea-acanthocytosis: A translational treatment approach in a rare disease

Background: Chorea-acanthocytosis (ChAc) is a neurodegenerative disease caused by mutations in the VPS13A gene. It is characterized by several neurological symptoms and the appearance of acanthocytes. Elevated tyrosine kinase Lyn activity has been recently identified as one of the key pathophysiological mechanisms in this disease, and therefore represents a promising drug target. Methods: We evaluated an individual off-label treatment with the tyrosine kinase inhibitor dasatinib (100 mg/d, 25.8–50.4 weeks) of three ChAc patients. Alongside thorough safety monitoring, we assessed motor and non-motor scales (e.g., MDS-UPDRS, UHDRS, quality of life) as well as routine and experimental laboratory parameters (e.g., serum neurofilament, Lyn kinase activity, actin cytoskeleton in red blood cells). Results: Dasatinib appeared to be reasonably safe. The clinical parameters remained stable without significant improvement or deterioration. Regain of deep tendon reflexes was observed in one patient. Creatine kinase, serum neurofilament levels, and acanthocyte count did not reveal consistent effects. However, a reduction of initially elevated Lyn kinase activity and accumulated autophagy markers, as well as a partial restoration of the actin cytoskeleton, was found in red blood cells. Conclusions: We report on the first treatment approach with disease-modifying intention in ChAc. The experimental parameters indicate target engagement in red blood cells, while clinical effects on the central nervous system could not be proven within a rather short treatment time. Limited knowledge on the natural history of ChAc and the lack of appropriate biomarkers remain major barriers for “clinical trial readiness”. We suggest a panel of outcome parameters for future clinical trials in ChA

Neuronal Dysfunction in iPSC-Derived Medium Spiny Neurons from Chorea-Acanthocytosis Patients Is Reversed by Src Kinase Inhibition and F-Actin Stabilization

Chorea-acanthocytosis (ChAc) is a fatal neurological disorder characterized by red blood cell acanthocytes and striatal neurodegeneration. Recently, severe cell membrane disturbances based on depolymerized cortical actin and an elevated Lyn kinase activity in erythrocytes from ChAc patients were identified. How this contributes to the mechanism of neurodegeneration is still unknown. To gain insight into the pathophysiology, we established a ChAc patient-derived induced pluripotent stem cell model and an efficient differentiation protocol providing a large population of human striatal medium spiny neurons (MSNs), the main target of neurodegeneration in ChAc. Patient-derived MSNs displayed enhanced neurite outgrowth and ramification, whereas synaptic density was similar to controls. Electrophysiological analysis revealed a pathologically elevated synaptic activity in ChAc MSNs. Treatment with the F-actin stabilizer phallacidin or the Src kinase inhibitor PP2 resulted in the significant reduction of disinhibited synaptic currents to healthy control levels, suggesting a Src kinase- and actin-dependent mechanism. This was underlined by increased G/F-actin ratios and elevated Lyn kinase activity in patient-derived MSNs. These data indicate that F-actin stabilization and Src kinase inhibition represent potential therapeutic targets in ChAc that may restore neuronal function

The Parkinson’s Gene VPS13C Encodes an ER-Lysosome Lipid Transfer Protein Linking Lysosomal Lipid Homeostasis and cGAS/STING-Mediated Innate Immunity

Biallelic loss-of-function mutations in VPS13C cause early-onset Parkinson’s Disease (PD), and the VPS13C locus is a GWAS hit for sporadic PD risk. VPS13C is a member of the VPS13 family, which in humans contains three other proteins, VPS13A, VPS13B, and VPS13D, all with ties to neurological diseases. Mutations in VPS13A cause chorea-acanthocytosis a Huntington’s like syndrome with dysmorphic erythrocytes, mutations in VPS13B cause the neurodevelopmental disorder Cohen syndrome, and mutations in VPS13D cause spastic ataxia with varying presentation. The molecular function of these proteins, why their loss cause neurological diseases, and why they are each associated with distinct diseases despite their homology have all been open questions. In yeast, the single Vps13 protein localizes to contact sites between the mitochondria and vacuole, the yeast lysosome, and at the nuclear-vacuolar junction (NVJ), where multiple lines of indirect evidence has hinted that it may play a role in lipid transfer between these organelles. To understand whether human VPS13 proteins have diverged in their subcellular localization, we employed a combination of light and electron microscopy to demonstrate that VPS13A localizes to contact sites between the endoplasmic reticulum (ER) and mitochondria, while VPS13C localizes to contact sites between the ER and late endosomes/lysosomes. Both proteins also share a localization at ER-lipid droplet contact sites. We further show that the N-terminal portion of VPS13 forms a novel, tubular, hydrophobic cavity that can solubilize and transport glycerolipids between membranes. These findings identify VPS13 as a lipid transporter between the ER and other organelles, implicating defects in membrane lipid homeostasis in neurological disorders resulting from their mutations. Sequence and secondary structure similarity between the N-terminal portions of Vps13 and other proteins such as the autophagy protein ATG2 suggested lipid transport roles for these proteins as well, which has since been demonstrated. We next investigated the cellular phenotypes of VPS13C loss-of-function in an attempt to shed light on the pathophysiology of VPS13C-associated PD. We used CRISPR-Cas9 to generate VPS13C-knockout (VPS13CKO) HeLa cells. These cells have more lysosomes compared to WT, with accumulation of both membrane and luminal lysosomal proteins. These lysosomes have an altered lipid profile, including a substantial decrease in ether-linked phospholipids and an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS/STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation and place VPS13C in pathways relevant to PD pathogenesis. Further exploration of these pathways has the potential to yield new mechanistic understanding and novel therapeutic strategies for this debilitating illness