Identification of two compound heterozygous VPS13A large deletions in chorea-acanthocytosis only by protein and quantitative DNA analysis.

BACKGROUND: Chorea-acanthocytosis (ChAc; OMIM #200150) is a rare autosomal recessive condition with onset in early adulthood that is caused by mutations in the vacuolar protein sorting 13A (VPS13A) gene encoding chorein. Several diagnostic genomic DNA (gDNA) sequencing approaches are widely used. However, their limitations appear not to be acknowledged thoroughly enough. METHODS: Clinically, we deployed magnetic resonance imaging, blood smear analysis, and clinical chemistry for the index patient's characterization. The molecular analysis of the index patient next to his parents covered genomic DNA (gDNA) sequencing approaches, RNA/cDNA sequencing, and chorein specific Western blot. RESULTS: We report a 33-year-old male patient without functional protein due to compound heterozygosity for two VPS13A large deletions of 1168 and 1823 base pairs (bp) affecting, respectively, exons 8 and 9, and exon 13. To our knowledge, this represents the first ChAc case with two compound heterozygous large deletions identified so far. Of note, standard genomic DNA (gDNA) Sanger sequencing approaches alone yielded false negative findings. CONCLUSION: Our case demonstrates the need to carry out detection of chorein in patients suspected of having ChAc as a helpful and potentially decisive tool to establish diagnosis. Furthermore, the course of the molecular analysis in this case discloses diagnostic pitfalls in detecting some variations, such as deletions, using only standard genomic DNA (gDNA) Sanger sequencing approaches and exemplifies alternative methods, such as RNA/cDNA sequencing or qRT-PCR analysis, necessary to avoid false negative results.

Subcortical neurodegeneration in chorea: Similarities and differences between chorea-acanthocytosis and Huntington's disease.

INTRODUCTION: Chorea-acanthocytosis (ChAc) and Huntington's disease (HD) are neurodegenerative conditions that share clinical and neuropathological features, despite their distinct genetic etiologies. METHODS: In order to compare these neuropathologies, serial gallocyanin-stained brain sections from three subjects with ChAc were analyzed and compared with our previous studies of eight HD cases, in addition to three hemispheres from two male controls. RESULTS: Astrogliosis was much greater in the ChAc striatum, as compared to that found in HD, with dramatic increase in total striatal glia numbers and the number of glia per striatal neuron. Striatal astrocytes are most likely derived from the striatal subependymal layer in ChAc, which showed massive proliferation. The thalamic centromedian-parafascicular complex is reciprocally connected to the striatum and is more heavily affected in HD than in ChAc. CONCLUSION: The distinct patterns of selective vulnerability and gliosis observed in HD and ChAc challenge simplistic views on the pathogenesis of these two diseases with rather similar clinical signs. The particular roles played by astroglia in ChAc and in HD clearly need to be elucidated in more detail.

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.

The association of aphasia and right-sided motor impairment in corticobasal syndrome.

Corticobasal syndrome is defined clinically on the basis of symptoms and findings related to dysfunction of the cerebral cortex and the basal ganglia. Usually, marked asymmetry of motor findings is observed. Although aphasia has now been recognized as a frequent feature of corticobasal syndrome, it remains unclear whether it is usually associated with right-sided motor symptoms, pointing to the involvement of the left hemisphere. Hence, we set out to examine the relationship between the presence of language symptoms and the side affected by extrapyramidal symptoms. We analyzed the electronic care records of patients seen in the years 2003-2013 in the Neurology Department of the University Hospital of Munich. The diagnosis of corticobasal syndrome was discussed in ninety-two individuals. Of those, 38 cases fulfilled diagnostic criteria for corticobasal syndrome. Aphasia correlated highly with a predominant right-sided movement disorder (p = 0.002). In contrast, it was less common in patients with left-sided motor presentation. Dysarthria did not show a preferential correlation (p = 0.25). Our analysis suggests a characteristic presentation of corticobasal syndrome in which motor dysfunction of the right side of the body is associated with aphasia.

Neuroacanthocytosis in china: a review of published reports.

BACKGROUND: Neuroacanthocytosis (NA) syndromes are a group of rare diseases characterized by the presence of acanthocytes and neuronal multisystem pathology, including chorea-acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington's disease-like 2 (HDL-2), and pantothenate kinase-associated neurodegeneration (PKAN). China has the largest population in the world, which makes it a good location for investigating rare diseases like NA. METHODS: We searched Medline, ISI Proceedings, China National Knowledge Infrastructure, and Wanfang Data for literature published through December 31, 2013 for all the published Chinese NA case reports and extracted the clinical and laboratory findings. RESULTS: A total of 42 studies describing 66 cases were found to be eligible for inclusion. Age of symptom onset ranged from 5 to 74 years. The most common findings included hyperkinetic movements (88%), orofacial dyskinesia (80%), dystonia (67%), and dysarthria (68%), as well as caudate atrophy or enlarged lateral ventricles on neuroimaging (64%), and elevated creatine kinase (52%). Most cases were not confirmed by any specific molecular tests. Only two cases were genetically studied and diagnosed as ChAc or MLS. DISCUSSION: In view of the prevalence of NA syndromes in other countries, the number of patients in China appears to be underestimated. Chinese NA patients may benefit from the establishment of networks that offer specific diagnoses and care for rare diseases.

Modelling Ser129 phosphorylation inhibits membrane binding of pore-forming alpha-synuclein oligomers.

BACKGROUND: In several neurodegenerative diseases, hyperphosphorylation at position Ser129 is found in fibrillar deposits of alpha-synuclein (asyn), implying a pathophysiological role of asyn phosphorylation in neurodegeneration. However, recent animal models applying asyn phosphorylation mimics demonstrated a protective effect of phosphorylation. Since metal-ion induced asyn oligomers were identified as a potential neurotoxic aggregate species with membrane pore-forming abilities, the current study was undertaken to determine effects of asyn phosphorylation on oligomer membrane binding. METHODS: We investigated the influence of S129 phosphorylation on interactions of metal-ion induced asyn oligomers with small unilamellar lipid vesicles (SUV) composed of POPC and DPPC applying the phosphorylation mimic asyn129E. Confocal single-particle fluorescence techniques were used to monitor membrane binding at the single-particle level. RESULTS: Binding of asyn129E monomers to gel-state membranes (DPPC-SUV) is slightly reduced compared to wild-type asyn, while no interactions with membranes in the liquid-crystalline state (POPC-SUV) are seen for both asyn and asyn129E. Conversely, metal-ion induced oligomer formation is markedly increased in asyn129E. Surprisingly, membrane binding to POPC-SUV is nearly absent in Fe(3+) induced asyn129E oligomers and markedly reduced in Al(3+) induced oligomers. CONCLUSION: The protective effect of pseudophosphorylation seen in animal models may be due to impeded oligomer membrane binding. Phosphorylation at Ser129 may thus have a protective effect against neurotoxic asyn oligomers by preventing oligomer membrane binding and disruption of the cellular electrophysiological equilibrium. Importantly, these findings put a new complexion on experimental pharmaceutical interventions against POLO-2 kinase.

Short and long term outcome of bilateral pallidal stimulation in chorea-acanthocytosis.

BACKGROUND: Chorea-acanthocytosis (ChAc) is a neuroacanthocytosis syndrome presenting with severe movement disorders poorly responsive to drug therapy. Case reports suggest that bilateral deep brain stimulation (DBS) of the ventro-postero-lateral internal globus pallidus (GPi) may benefit these patients. To explore this issue, the present multicentre (n=12) retrospective study collected the short and long term outcome of 15 patients who underwent DBS. METHODS: Data were collected in a standardized way 2-6 months preoperatively, 1-5 months (early) and 6 months or more (late) after surgery at the last follow-up visit (mean follow-up: 29.5 months). RESULTS: Motor severity, assessed by the Unified Huntington's Disease Rating Scale-Motor Score, UHDRS-MS), was significantly reduced at both early and late post-surgery time points (mean improvement 54.3% and 44.1%, respectively). Functional capacity (UHDRS-Functional Capacity Score) was also significantly improved at both post-surgery time points (mean 75.5% and 73.3%, respectively), whereas incapacity (UHDRS-Independence Score) improvement reached significance at early post-surgery only (mean 37.3%). Long term significant improvement of motor symptom severity (≥ 20 % from baseline) was observed in 61.5 % of the patients. Chorea and dystonia improved, whereas effects on dysarthria and swallowing were variable. Parkinsonism did not improve. Linear regression analysis showed that preoperative motor severity predicted motor improvement at both post-surgery time points. The most serious adverse event was device infection and cerebral abscess, and one patient died suddenly of unclear cause, 4 years after surgery. CONCLUSION: This study shows that bilateral DBS of the GPi effectively reduces the severity of drug-resistant hyperkinetic movement disorders such as present in ChAc.

Alterations of red cell membrane properties in neuroacanthocytosis.

Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington's disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an "acanthocytic state" of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration.

Chorea-acanthocytosis: report of three cases from Iran.

Chorea-acanthocythosis (ChAc) is an inherited neurodegenerative disorder characterized by movement disorders, neuropsychiatric disturbances, neuropathy, myopathy, seizures and acanthocytosis accompanied by an elevated serum creatine kinase (CK) level. Its causative gene (VPS13A) produces chorein which is absent in ChAc patients as evaluated by Western blot assay. We report the first three Iranian patients whose disease has been confirmed by chorein Western blot. Our cases presented with heterogeneous courses of ChAc. A high sense of clinical awareness in approaching patients with deteriorating and/or multiple abnormal movements that are accompanied by other neurological signs such as neuropathy, myopathy, seizures and high serum CK level will support an early diagnosis of this disease. We also emphasize on the presence of axial flexion/extension spasms as a good clinical sign for narrowing differential diagnosis.

Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with α-synuclein at the single molecule level.

BACKGROUND: Fibrillar amyloid-like deposits and co-deposits of tau and α-synuclein are found in several common neurodegenerative diseases. Recent evidence indicates that small oligomers are the most relevant toxic aggregate species. While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and α-synuclein are not well understood. RESULTS: We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with α-synuclein at the level of individual oligomers. We show that Al3+ at physiologically relevant concentrations and tau phosphorylation by GSK-3ß exert synergistic effects on the formation of a distinct SDS-resistant tau oligomer species even at nanomolar protein concentration. Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of α-synuclein in pre-formed tau oligomers. CONCLUSIONS: Our findings provide a new perspective on interactions of tau phosphorylation, metal ions, and the formation of potentially toxic oligomer species, and elucidate molecular crosstalks between different aggregation pathways involved in neurodegeneration.

Computational identification of phospho-tyrosine sub-networks related to acanthocyte generation in neuroacanthocytosis.

Acanthocytes, abnormal thorny red blood cells (RBC), are one of the biological hallmarks of neuroacanthocytosis syndromes (NA), a group of rare hereditary neurodegenerative disorders. Since RBCs are easily accessible, the study of acanthocytes in NA may provide insights into potential mechanisms of neurodegeneration. Previous studies have shown that changes in RBC membrane protein phosphorylation state affect RBC membrane mechanical stability and morphology. Here, we coupled tyrosine-phosphoproteomic analysis to topological network analysis. We aimed to predict signaling sub-networks possibly involved in the generation of acanthocytes in patients affected by the two core NA disorders, namely McLeod syndrome (MLS, XK-related, Xk protein) and chorea-acanthocytosis (ChAc, VPS13A-related, chorein protein). The experimentally determined phosphoproteomic data-sets allowed us to relate the subsequent network analysis to the pathogenetic background. To reduce the network complexity, we combined several algorithms of topological network analysis including cluster determination by shortest path analysis, protein categorization based on centrality indexes, along with annotation-based node filtering. We first identified XK- and VPS13A-related protein-protein interaction networks by identifying all the interactomic shortest paths linking Xk and chorein to the corresponding set of proteins whose tyrosine phosphorylation was altered in patients. These networks include the most likely paths of functional influence of Xk and chorein on phosphorylated proteins. We further refined the analysis by extracting restricted sets of highly interacting signaling proteins representing a common molecular background bridging the generation of acanthocytes in MLS and ChAc. The final analysis pointed to a novel, very restricted, signaling module of 14 highly interconnected kinases, whose alteration is possibly involved in generation of acanthocytes in MLS and ChAc.

Chorea-acanthocytosis genotype in the original critchley kentucky neuroacanthocytosis kindred.

OBJECTIVE: To determine the molecular nature of the neurological disease in the seminal family reported by Critchley et al in the 1960s, characterized by a hyperkinetic movement disorder and the appearance of acanthocytosis on peripheral blood smear. The eponym Levine-Critchley syndrome, subsequently termed neuroacanthocytosis, has been applied to symptomatically similar, but genetically distinct, disorders, resulting in clinical and diagnostic confusion. DESIGN: DNA analysis. SETTING: Molecular biology research laboratories. PARTICIPANTS: First- and second-degree relatives of the original Critchley et al proband from Kentucky. MAIN OUTCOME MEASURES: Mutations in the VPS13A gene. RESULTS: A mutation was identified in the VPS13A gene, responsible for autosomal recessive chorea-acanthocytosis. Haplotype reconstruction suggested that this mutation was homozygous in the proband. CONCLUSION: These findings strongly support the diagnosis of chorea-acanthocytosis as the disorder described in the original report.

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.

Single particle analysis of tau oligomer formation induced by metal ions and organic solvents.

Pathological aggregates of tau protein are found in several neurodegenerative diseases termed 'tauopathies'. Increasing evidence indicates that tau oligomer species rather than the large amyloid cytoplasmic inclusions relevant for histopathological diagnosis might be crucial for cellular damage and neurodegeneration. Trivalent metal ions and polyanionic structures like heparin or arachidonic acid have been shown to induce tau aggregation. However, little is known about early processes of tau aggregation. In this study, we applied fluorescence correlation spectroscopy (FCS) and scanning for intensely fluorescent targets (SIFT) to investigate oligomer formation of tau protein at nanomolar protein concentrations at the single-particle level. Our results indicate that the formation of distinct tau oligomers is induced by the trivalent metal ions Fe(3+) and Al(3+) and by organic solvents like DMSO, respectively. In contrast, bivalent metal ions (Cu(2+), Zn(2+), Mn(2+), Ca(2+), Mg(2+)) had no effect. While DMSO-induced small tau oligomers are relatively stable in solution, dynamic remodeling can be initiated by non-ionic detergents. Moreover Al(3+) induces rapid formation of a different oligomer species of larger size. Our results provide further insights into early tau oligomerization and aggregation dynamics.

Clinical and genetic analysis of 29 Brazilian patients with Huntington's disease-like phenotype.

Huntington's disease (HD) is a neurodegenerative disorder characterized by chorea, behavioral disturbances and dementia, caused by a pathological expansion of the CAG trinucleotide in the HTT gene. Several patients have been recognized with the typical HD phenotype without the expected mutation. The objective of this study was to assess the occurrence of diseases such as Huntington's disease-like 2 (HDL2), spinocerebellar ataxia (SCA) 1, SCA2, SCA3, SCA7, dentatorubral-pallidoluysian atrophy (DRPLA) and chorea-acanthocytosis (ChAc) among 29 Brazilian patients with a HD-like phenotype. In the group analyzed, we found 3 patients with HDL2 and 2 patients with ChAc. The diagnosis was not reached in 79.3% of the patients. HDL2 was the main cause of the HD-like phenotype in the group analyzed, and is attributable to the African ancestry of this population. However, the etiology of the disease remains undetermined in the majority of the HD negative patients with HD-like phenotype.

Clinical and genetic analysis of 29 Brazilian patients with Huntington's disease-like phenotype / Análise clínica e genética em 29 pacientes Brasileiros com fenótipo doença de Huntington-símile

Huntington's disease (HD) is a neurodegenerative disorder characterized by chorea, behavioral disturbances and dementia, caused by a pathological expansion of the CAG trinucleotide in the HTT gene. Several patients have been recognized with the typical HD phenotype without the expected mutation. The objective of this study was to assess the occurrence of diseases such as Huntington's disease-like 2 (HDL2), spinocerebellar ataxia (SCA) 1, SCA2, SCA3, SCA7, dentatorubral-pallidoluysian atrophy (DRPLA) and chorea-acanthocytosis (ChAc) among 29 Brazilian patients with a HD-like phenotype. In the group analyzed, we found 3 patients with HDL2 and 2 patients with ChAc. The diagnosis was not reached in 79.3 percent of the patients. HDL2 was the main cause of the HD-like phenotype in the group analyzed, and is attributable to the African ancestry of this population. However, the etiology of the disease remains undetermined in the majority of the HD negative patients with HD-like phenotype.

A doença de Huntington (DH) é uma doença neurodegenerativa caracterizada por coréia, alterações comportamentais e demência, causada por uma expansão patológica do trinucleotídeo CAG no gene HTT. Vários pacientes têm sido descritos com o fenótipo típico para a DH porém sem a mutação esperada. O objetivo deste estudo foi avaliar a ocorrência de doenças como doença de Huntington-símile 2 (DHS-2), ataxias espinocerebelares tipo 1, 2, 3 e 17, atrofia dentatorubral-palidoluisiana e coreo-acantocitose (CAc) entre 29 pacientes brasileiros com fenótipo doença de Huntington-símile. No grupo analisado, encontramos 3 pacientes com DHS-2 e 2 pacientes com CAc. O diagnóstico permaneceu obscuro em 79,3 por cento dos pacientes. DHS-2 foi a principal causa do fenótipo DH-símile no grupo analisado, provavelmente devido a ancestralidade africana na população brasileira. Entretanto, a etiologia permaneceu indeterminada na maioria dos pacientes avaliados.