Identification of four novel mutations in VSP13A in Iranian patients with Chorea-acanthocytosis (ChAc).

Chorea-acanthocytosis (ChAc) is a rare autosomal recessive neurodegenerative disorder characterized by a variety of involuntary movements, predominantly chorea, and the presence of acanthocytosis in peripheral blood smears. ChAc is caused by mutations in the vacuolar protein sorting-associated protein 13A (VPS13A) gene. The aim of the present study was to conduct a clinical and genetic analysis of five patients with suspected ChAc in Iran. This study included five patients who were referred to the genetic department of the Endocrinology and Metabolism Research Institute between 2020 and 2022, with a suspicion of ChAc. Clinical features and the presence of characteristic MRI findings were evaluated in the patients. Whole-exome sequencing (WES) followed by Sanger sequencing was employed to identify the disease-causing variants. The functional effects of novel mutations were analyzed by specific bioinformatics prediction tools. WES and data analysis revealed the presence of five distinct VPS13A mutations in the patients, four of which were novel. These included one nonsense mutation (p.L984X), and three splice site mutations (c.755-1G>A, c.144+1 G>C, c.2512+1G>A). All mutations were validated by Sanger sequencing, and in silico analysis predicted that all mutations were pathogenic. This study provides the first molecular genetic characteristics of Iranian patients with ChAc, identifying four novel mutations in the VPS13A gene. These findings expand the VPS13A variants spectrum and confirm the clinical variability in ChAc patients.

A chorea-acanthocytosis patient with novel mutations in the VPS13A gene without acanthocyte.

Chorea-acanthocytosis (ChAc) is a rare clinical genetic disorder of the nervous system, which is characterized by choreiform movement disorder, cognitive decline, and psychiatric disorders. ChAc is mostly diagnosed based on its typical clinical manifestations and the increased number of acanthocytes in peripheral blood smears. Here, we report a patient, who has the characteristic clinical manifestations of ChAc with limb choreiform movements, involuntary lip and tongue bites, seizures, and emotional instability. However, her blood smear was negative for acanthocytes with scanning electron microscopy. We later identified two novel pathogenic mutations in the patient's vacuolar protein sorting homolog 13 A (VPS13A) on chromosome 9q21 by targeted gene sequencing, and she was definitively diagnosed with "ChAc." After treatment with carbamazepine, haloperidol, the patient's symptoms gradually improved. We consider that an acanthocyte negative blood smear cannot rule out ChAC diagnosis, and genetic testing is the "gold standard" for the diagnosis. Through a review of previous research, it is rare for a patient to have a clear diagnosis of ChAc by genetic testing, but whose blood smear is negative for acanthocytes with electron microscopy. In addition, in this report, we discovered two novel pathogenic mutations, which have not been reported previously, and extended the genetic characteristics of ChAc.

Multisystem pathology in McLeod syndrome.

We present a comprehensive characterization of clinical, neuropathological, and multisystem features of a man with genetically confirmed McLeod neuroacanthocytosis syndrome, including video and autopsy findings. A 61-year-old man presented with a movement disorder and behavioral change. Examination showed dystonic choreiform movements in all four limbs, reduced deep-tendon reflexes, and wide-based gait. He had oromandibular dyskinesia causing severe dysphagia. Elevated serum creatinine kinase (CK) was first noted in his thirties, but investigations, including muscle biopsy at that time, were inconclusive. Brain magnetic resonance imaging showed white matter volume loss, atrophic basal ganglia, and chronic small vessel ischemia. Despite raised CK, electromyography did not show myopathic changes. Exome gene panel testing was negative, but targeted genetic analysis revealed a hemizygous pathogenic variant in the XK gene c.895C > T p.(Gln299Ter), consistent with a diagnosis of McLeod syndrome. The patient died of sepsis, and autopsy showed astrocytic gliosis and atrophy of the basal ganglia, diffuse iron deposition in the putamen, and mild Alzheimer's pathology. Muscle pathology was indicative of mild chronic neurogenic atrophy without overt myopathic features. He had non-specific cardiomyopathy and splenomegaly. McLeod syndrome is an ultra-rare neurodegenerative disorder caused by X-linked recessive mutations in the XK gene. Diagnosis has management implications since patients are at risk of severe transfusion reactions and cardiac complications. When a clinical diagnosis is suspected, candidate genes should be interrogated rather than solely relying on exome panels.

Exome sequencing of choreoacanthocytosis reveals novel mutations in VPS13A and co-mutation in modifier gene(s).

Choreoacanthocytosis, one of the forms of neuroacanthocytosis, is caused by mutations in vacuolar protein sorting-associated protein A (VPS13A), and is often misdiagnosed with other form of neuroacanthocytosis with discrete genetic defects. The phenotypic variations among the patients with VPS13A mutations significantly obfuscates the understanding of the disease and treatment strategies. In this study, two unrelated cases were identified, exhibiting the core phenotype of neuroacanthocytosis but with considerable clinical heterogeneity. Case 1 presented with an additional Parkinsonism phenotype, whereas seizures were evident in case 2. To decipher the genetic basis, whole exome sequencing followed by validation with Sanger sequencing was performed. A known homozygous pathogenic nonsense mutation (c.799C > T; p.R267X) in exon 11 of the VPS13A gene was identified in case 1 that resulted in a truncated protein. A novel missense mutation (c.9263T > G; p.M3088R) in exon 69 of VPS13A identified in case 2 was predicted as pathogenic. In silico analysis of the p.M3088R mutation at the C-terminus of VPS13A suggests a loss of interaction with TOMM40 and may disrupt mitochondrial localization. We also observed an increase in mitochondrial DNA copy numbers in case 2. Mutation analysis revealed benign heterozygous variants in interacting partners of VPS13A such as VAPA in case 1. Our study confirmed the cases as ChAc and identified the novel homozygous variant of VPS13A (c.9263T > G; p.M3088R) within the mutation spectrum of VPS13A-associated ChAc. Furthermore, mutations in VPS13A and co-mutations in its potential interacting partner(s) might contribute to the diverse clinical manifestations of ChAc, which requires further study.

An Autopsy Series of Seven Cases of VPS13A Disease (Chorea-Acanthocytosis).

BACKGROUND: Vacuolar protein sorting 13 homolog A (VPS13A) disease, historically known as chorea-acanthocytosis, is a rare neurodegenerative disorder caused by biallelic mutations in VPS13A, usually resulting in reduced or absent levels of its protein product, VPS13A. VPS13A localizes to contact sites between subcellular organelles, consistent with its recently identified role in lipid transfer between membranes. Mutations are associated with neuronal loss in the striatum, most prominently in the caudate nucleus, and associated marked astrogliosis. There are no other known disease-specific cellular changes (eg, protein aggregation), but autopsy reports to date have been limited, often lacking genetic or biochemical diagnostic confirmation. OBJECTIVE: The goal of this study was to characterize neuropathological findings in the brains of seven patients with VPS13A disease (chorea-acanthocytosis). METHODS: In this study, we collected brain tissues and clinical data from seven cases of VPS13A for neuropathological analysis. The clinical diagnosis was confirmed by the presence of VPS13A mutations and/or immunoblot showing the loss or reduction of VPS13A protein. Tissues underwent routine, special, and immunohistochemical staining focused on neurodegeneration. Electron microscopy was performed in one case. RESULTS: Gross examination showed severe striatal atrophy. Microscopically, there was neuronal loss and astrogliosis in affected regions. Luxol fast blue staining showed variable lipid accumulation with diverse morphology, which was further characterized by electron microscopy. In some cases, rare degenerating p62- and ubiquitin-positive cells were present in affected regions. Calcifications were present in four cases, being extensive in one. CONCLUSIONS: We present the largest autopsy series of biochemically and genetically confirmed VPS13A disease and identify novel histopathological findings implicating abnormal lipid accumulation. © 2023 International Parkinson and Movement Disorder Society.

Novel pathogenic VPS13A mutation in Moroccan family with Choreoacanthocytosis: a case report.

BACKGROUND: Choreoacanthocytosis (ChAc), is a rare neurodegenerative disease, characterized by movement disorders and acanthocytosis in the peripheral blood smears, and various neurological, neuropsychiatric and neuromuscular signs. It is caused by mutations in VPS13A gene with autosomal recessive pattern of inheritance. CASE PRESENTATION: Here we report two patients belonging to a consanguineous Moroccan family who present with movement disorder pathology. They were suspected to have choreoacanthocytosis according to biological, clinical and radiological finding. Thus, whole-exome sequencing was performed for precise diagnosis and identified a homozygous novel nonsense mutation c.337C > T (p.Gln113*) in exon 5 of VPS13A in the two affected siblings. CONCLUSION: Here, we report a novel nonsense p.Gln113* mutation in VPS13A identified by whole-exome sequencing, which caused ChAc in a Moroccan family. This is the first description of ChAc in Morocco with genetic confirmation, that expands the mutation diversity of VPS13A and provide clinical, neuroimaging and deep brain stimulation findings.

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), yet the biology of these proteins is still poorly understood. Studies in Saccharomyces cerevisiae, Dictyostelium discoideum, Tetrahymena thermophila and Drosophila melanogaster point to the involvement of Vps13 in cytoskeleton organization, vesicular trafficking, autophagy, phagocytosis, endocytosis, proteostasis, sporulation and mitochondrial functioning. Recent findings show that yeast Vps13 binds to phosphatidylinositol lipids via 4 different regions and functions at membrane contact sites, enlarging the list of Vps13 functions. This review describes the great potential of simple eukaryotes to decipher disease mechanisms in higher organisms and highlights novel insights into the pathological role of Vps13 towards ChAc.

Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate.

The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc.

Pathoarchitectonics of the cerebral cortex in chorea-acanthocytosis and Huntington's disease.

AIMS: Quantitative estimation of cortical neurone loss in cases with chorea-acanthocytosis (ChAc) and its impact on laminar composition. METHODS: We used unbiased stereological tools to estimate the degree of cortical pathology in serial gallocyanin-stained brain sections through the complete hemispheres of three subjects with genetically verified ChAc and a range of disease durations. We compared these results with our previous data of five Huntington's disease (HD) and five control cases. Pathoarchitectonic changes were exemplarily documented in TE1 of a 61-year-old female HD-, a 60-year-old female control case, and ChAc3. RESULTS: Macroscopically, the cortical volume of our ChAc cases (ChAc1-3) remained close to normal. However, the average number of neurones was reduced by 46% in ChAc and by 33% in HD (P = 0.03 for ChAc & HD vs. controls; P = 0.64 for ChAc vs. HD). Terminal HD cases featured selective laminar neurone loss with pallor of layers III, V and VIa, a high density of small, pale, closely packed radial fibres in deep cortical layers VI and V, shrinkage, and chromophilia of subcortical white matter. In ChAc, pronounced diffuse astrogliosis blurred the laminar borders, thus masking the complete and partial loss of pyramidal cells in layer IIIc and of neurones in layers III, V and VI. CONCLUSION: ChAc is a neurodegenerative disease with distinct cortical neurodegeneration. The hypertrophy of the peripheral neuropil space of minicolumns with coarse vertical striation was characteristic of ChAc. The role of astroglia in the pathogenesis of this disorder remains to be elucidated.

Defective mitochondrial and lysosomal trafficking in chorea-acanthocytosis is independent of Src-kinase signaling.

Mutations in the VPS13A gene leading to depletion of chorein protein are causative for Chorea Acanthocytosis (ChAc), a rare devastating disease, which is characterized by neurodegeneration mainly affecting the basal ganglia as well as deformation of erythrocytes. Studies on patient blood samples highlighted a dysregulation of Actin cytoskeleton caused by downregulation of the PI3K pathway and hyper-activation of Lyn-kinase, but to what extent these mechanisms are present and relevant in the affected neurons remains elusive. We studied the effects of the absence of chorein protein on the morphology and trafficking of lysosomal and mitochondrial compartments in ChAc patient-specific induced pluripotent stem cell-derived medium spiny neurons (MSNs). Numbers of both organelle types were reduced in ChAc MSNs. Mitochondrial length was shortened and their membrane potential showed significant hyperpolarization. In contrast to previous studies, showing Lyn kinase dependency of ChAc-associated pathological events in erythrocytes, pharmacological studies demonstrate that the impairment of mitochondria and lysosomes are independent of Lyn kinase activity. These data suggest that impairment in mitochondrial and lysosomal morphologies in MSNs is not mediated by a dysregulation of Lyn kinase and thus the pathological pathways in ChAc might be - at least in part - cell-type specific.

Neuroacanthocytosis: a case report of chorea-acanthocytosis.

Neuroacanthocytosis is a rare progressive neurodegenerative disease, including Chorea-acanthocytosis, McLeod syndrome, Huntington's disease-like 2, and pantothenate kinase-associated neurodegeneration, where Chorea-acanthocytosis occupies the main entity of this disease group. Here, a classic case of Chorea-acanthocytosis is reported that exhibited gradually deteriorating abnormal movements of limbs and face, swallowing difficulty, and lip and cheek biting for the past two years. Peripheral blood smears revealed that 35% of the red blood cells were acanthocytes and electron microcopy scans clearly showed the morphology of acanthocytes. VPS13A gene sequencing found a heterozygous novel VPS13A gene mutation (c.80dupT). Brain magnetic resonance imaging scans showed moderate anterior horn dilation of lateral ventricles and bilateral atrophy of the head of caudate nucleus. Several suggestive features are summarized to provide diagnostic clues for Chorea-acanthocytosis and facilitate future diagnosis and treatment.

A new molecular link between defective autophagy and erythroid abnormalities in chorea-acanthocytosis.

Chorea-acanthocytosis is one of the hereditary neurodegenerative disorders known as the neuroacanthocytoses. Chorea-acanthocytosis is characterized by circulating acanthocytes deficient in chorein, a protein of unknown function. We report here for the first time that chorea-acanthocytosis red cells are characterized by impaired autophagy, with cytoplasmic accumulation of active Lyn and of autophagy-related proteins Ulk1 and Atg7. In chorea-acanthocytosis erythrocytes, active Lyn is sequestered by HSP90-70 to form high-molecular-weight complexes that stabilize and protect Lyn from its proteasomal degradation, contributing to toxic Lyn accumulation. An interplay between accumulation of active Lyn and autophagy was found in chorea-acanthocytosis based on Lyn coimmunoprecipitation with Ulk1 and Atg7 and on the presence of Ulk1 in Lyn-containing high-molecular-weight complexes. In addition, chorein associated with Atg7 in healthy but not in chorea-acanthocytosis erythrocytes. Electron microscopy detected multivesicular bodies and membrane remnants only in circulating chorea-acanthocytosis red cells. In addition, reticulocyte-enriched chorea-acanthocytosis red cell fractions exhibited delayed clearance of mitochondria and lysosomes, further supporting the impairment of authophagic flux. Because autophagy is also important in erythropoiesis, we studied in vitro CD34+-derived erythroid precursors. In chorea-acanthocytosis, we found (1) dyserythropoiesis; (2) increased active Lyn; (3) accumulation of a marker of autophagic flux and autolysososme degradation; (4) accumlation of Lamp1, a lysosmal membrane protein, and LAMP1-positive aggregates; and (5) reduced clearance of lysosomes and mitochondria. Our results uncover in chorea-acanthocytosis erythroid cells an association between accumulation of active Lyn and impaired autophagy, suggesting a link between chorein and autophagic vesicle trafficking in erythroid maturation.

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. SIGNIFICANCE STATEMENT: Chorea-acanthocytosis (ChAc) is a fatal neurodegenerative disease without a known cure. To gain pathophysiological insight, we newly established a human in vitro model using skin biopsies from ChAc patients to generate disease-specific induced pluripotent stem cells (iPSCs) and developed an efficient iPSC differentiation protocol providing striatal medium spiny neurons. Using patch-clamp electrophysiology, we detected a pathologically enhanced synaptic activity in ChAc neurons. Healthy control levels of synaptic activity could be restored by treatment of ChAc neurons with the F-actin stabilizer phallacidin and the Src kinase inhibitor PP2. Because Src kinases are involved in bridging the membrane to the actin cytoskeleton by membrane protein phosphorylation, our data suggest an actin-dependent mechanism of this dysfunctional phenotype and potential treatment targets in ChAc.

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

BACKGROUND: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. METHODS: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. RESULTS: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. CONCLUSIONS: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.

An Interesting Case of a Movement Disorder.

Neuroacanthocytosis is a genetic neurodegenerative disorder with syndromes of variable inheritance. These hyperkinetic movement disorders are reported to be very rare. It is associated with choreiform movements, orofacial and lingual dyskinesias and acanthocytes on peripheral smear and normolipoproteinemia. Here we present a similar case.

Giant axon formation in mice lacking Kell, XK, or Kell and XK: animal models of McLeod neuroacanthocytosis syndrome.

McLeod neuroacanthocytosis syndrome (MLS) is a rare X-linked multisystem disease caused by XK gene mutations and characterized by hematological and neurological abnormalities. XK, a putative membrane transporter, is expressed ubiquitously and is covalently linked to Kell, an endothelin-3-converting enzyme (ECE-3). Absence of XK results in reduction of Kell at sites where both proteins are coexpressed. To elucidate the functional roles of XK, Kell, and the XK-Kell complex associated with pathogenesis in MLS, we studied the pathology of the spinal cord, anterior roots, sciatic nerve, and skeletal muscle from knockout mouse models, using Kel(-/-), Xk(-/-), Kel(-/-)Xk(-/-), and wild-type mice aged 6 to 18 months. A striking finding was that giant axons were frequently associated with paranodal demyelination. The pathology suggests probable anterograde progression from the spinal cord to the sciatic nerve. The neuropathological abnormalities were found in all three genotypes, but were more marked in the double-knockout Kel(-/-)Xk(-/-) mice than in either Kel(-/-) or Xk(-/-) mice. Skeletal muscles from Xk(-/-) and Kel(-/-)Xk(-/-) mice showed mild abnormalities, but those from Kel(-/-) mice were similar to the wild type. The more marked neuropathological abnormalities in Kel(-/-)Xk(-/-) mice suggest a possible functional association between XK and Kell in nonerythroid tissues.

Physiological and Pathogenesis Significance of Chorein in Health and Disease.

This comprehensive review explores the physiological and pathophysiological significance of VPS13A, a protein encoded by the VPS13A gene. The VPS13A gene is associated with Chorea-acanthocytosis (ChAc), a rare hereditary neurodegenerative disorder. The review covers essential aspects, beginning with the genetics of VPS13A, highlighting its role in the pathogenesis of ChAc, and addressing the spectrum of genetic variants involved. It delves into the structure and function of the VPS13A protein, emphasizing its presence in various tissues and its potential involvement in protein trafficking and lipid homeostasis. Molecular functions of VPS13A in the brain tissue and other cell types or tissues with respect to their role in cytoskeletal regulation and autophagy are explored. Finally, it explores the intriguing link between VPS13A mutations, lipid imbalances, and neurodegeneration, shedding light on future research directions. Overall, this review serves as a comprehensive resource for understanding the pivotal role of VPS13A in health and disease, particularly in the context of ChAc. Key words: Chorein , Tumor, Actin, Microfilament, Gene expression, Chorea-acanthocytosis.

Chorein, the protein responsible for chorea-acanthocytosis, interacts with ß-adducin and ß-actin.

Chorea-acanthocytosis (ChAc) is an autosomal, recessive hereditary disease characterized by striatal neurodegeneration and acanthocytosis, and caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene. VPS13A encodes chorein whose physiological function at the molecular level is poorly understood. In this study, we show that chorein interacts with ß-adducin and ß-actin. We first compare protein expression in human erythrocyte membranes using proteomic analysis. Protein levels of ß-adducin isoform 1 and ß-actin are markedly decreased in erythrocyte membranes from a ChAc patient. Subsequent co-immunoprecipitation (co-IP) and reverse co-IP assays using extracts from chorein-overexpressing human embryonic kidney 293 (HEK293) cells, shows that ß-adducin (isoforms 1 and 2) and ß-actin interact with chorein. Immunocytochemical analysis using chorein-overexpressing HEK293 cells demonstrates co-localization of chorein with ß-adducin and ß-actin. In addition, immunoreactivity of ß-adducin isoform 1 is significantly decreased in the striatum of gene-targeted ChAc-model mice. Adducin and actin are membrane cytoskeletal proteins, involved in synaptic function. Expression of ß-adducin is restricted to the brain and hematopoietic tissues, corresponding to the main pathological lesions of ChAc, and thereby implicating ß-adducin and ß-actin in ChAc pathogenesis.

Erythrocyte membrane changes of chorea-acanthocytosis are the result of altered Lyn kinase activity.

Acanthocytic RBCs are a peculiar diagnostic feature of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder. Although recent years have witnessed some progress in the molecular characterization of ChAc, the mechanism(s) responsible for generation of acanthocytes in ChAc is largely unknown. As the membrane protein composition of ChAc RBCs is similar to that of normal RBCs, we evaluated the tyrosine (Tyr)-phosphorylation profile of RBCs using comparative proteomics. Increased Tyr phosphorylation state of several membrane proteins, including band 3, ß-spectrin, and adducin, was noted in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs, band 3 Tyr phosphorylation by Lyn was independent of the canonical Syk-mediated pathway. The ChAc-associated alterations in RBC membrane protein organization appear to be the result of increased Tyr phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton as supported by coimmunoprecipitation of ß-adducin with band 3 only in ChAc RBC-membrane treated with the Lyn-inhibitor PP2. We propose this altered association between membrane skeleton and membrane proteins as novel mechanism in the generation of acanthocytes in ChAc.

Chorein-sensitive polymerization of cortical actin and suicidal cell death in chorea-acanthocytosis.

Chorea-acanthocytosis is an inevitably lethal genetic disease characterized by a progressive hyperkinetic movement disorder and cognitive and behavioral abnormalities as well as acanthocytosis. The disease is caused by loss-of-function mutations of the gene encoding vacuolar protein sorting-associated protein 13A (VPS13A) or chorein, a protein with unknown function expressed in various cell types. How chorein deficiency leads to the pathophysiology of chorea-acanthocytosis remains enigmatic. Here we show decreased phosphoinositide-3-kinase (PI3K)-p85-subunit phosphorylation, ras-related C3 botulinum toxin substrate 1 (Rac1) activity, and p21 protein-activated kinase 1 (PAK1) phosphorylation as well as depolymerized cortical actin in erythrocytes from patients with chorea-acanthocytosis and in K562-erythrocytic cells following chorein silencing. Pharmacological inhibition of PI3K, Rac1, or PAK1 similarly triggered actin depolymerization. Moreover, in K562 cells, both chorein silencing and PAK1 inhibition with IPA-3 decreased phosphorylation of Bad, a Bcl2-associated protein, promoting apoptosis by forming mitochondrial pores, followed by mitochondrial depolarization, DNA fragmentation, and phosphatidylserine exposure at the cell surface, all hallmarks of apoptosis. Our observations reveal chorein as a novel powerful regulator of cytoskeletal architecture and cell survival, thus explaining erythrocyte misshape and possibly neurodegeneration in chorea-acanthocytosis.