Neuroacanthocytosis Syndromes: The Clinical Perspective.

The two very rare neurodegenerative diseases historically known as the "neuroacanthocytosis syndromes" are due to mutations of either VPS13A or XK. These are phenotypically similar disorders that affect primarily the basal ganglia and hence result in involuntary abnormal movements as well as neuropsychiatric and cognitive alterations. There are other shared features such as abnormalities of red cell membranes which result in acanthocytes, whose relationship to neurodegeneration is not yet known. Recent insights into the functions of these two proteins suggest dysfunction of lipid processing and trafficking at the subcellular level and may provide a mechanism for neuronal dysfunction and death, and potentially a target for therapeutic interventions.

VPS13 Forum Proceedings: XK, XK-Related and VPS13 Proteins in Membrane Lipid Dynamics.

In 2020, the pandemic interrupted the series of biannual International Neuroacanthocytosis Meetings that brought together clinicians, scientists, and patient groups to share research into a small group of devastating genetic diseases that combine both acanthocytosis (deformed red blood cells) and neurodegeneration with movement disorders. This Meeting Report describes talks at the 5th VPS13 Forum in January 2022, one of a series of online meetings held to fill the gap. The meeting addressed the basic biology of two key proteins implicated in chorea-acanthocytosis (mutations in VPS13A) and McLeod syndrome (mutations in XK). In a remarkable confluence of ideas, the speakers described different aspects of a single functional unit that comprises of VPS13A and XK proteins working together. Conditions caused by VPS13 (A-D) gene family mutations and related genes, such as XK, previously footnote knowledge, seem to turn central for a novel disease paradigm: bulk lipid transfer disorders.

Erysense, a Lab-on-a-Chip-Based Point-of-Care Device to Evaluate Red Blood Cell Flow Properties With Multiple Clinical Applications.

In many medical disciplines, red blood cells are discovered to be biomarkers since they "experience" various conditions in basically all organs of the body. Classical examples are diabetes and hypercholesterolemia. However, recently the red blood cell distribution width (RDW), is often referred to, as an unspecific parameter/marker (e.g., for cardiac events or in oncological studies). The measurement of RDW requires venous blood samples to perform the complete blood cell count (CBC). Here, we introduce Erysense, a lab-on-a-chip-based point-of-care device, to evaluate red blood cell flow properties. The capillary chip technology in combination with algorithms based on artificial neural networks allows the detection of very subtle changes in the red blood cell morphology. This flow-based method closely resembles in vivo conditions and blood sample volumes in the sub-microliter range are sufficient. We provide clinical examples for potential applications of Erysense as a diagnostic tool [here: neuroacanthocytosis syndromes (NAS)] and as cellular quality control for red blood cells [here: hemodiafiltration (HDF) and erythrocyte concentrate (EC) storage]. Due to the wide range of the applicable flow velocities (0.1-10 mm/s) different mechanical properties of the red blood cells can be addressed with Erysense providing the opportunity for differential diagnosis/judgments. Due to these versatile properties, we anticipate the value of Erysense for further diagnostic, prognostic, and theragnostic applications including but not limited to diabetes, iron deficiency, COVID-19, rheumatism, various red blood cell disorders and anemia, as well as inflammation-based diseases including sepsis.

Changes in Blood Cell Deformability in Chorea-Acanthocytosis and Effects of Treatment With Dasatinib or Lithium.

Misshaped red blood cells (RBCs), characterized by thorn-like protrusions known as acanthocytes, are a key diagnostic feature in Chorea-Acanthocytosis (ChAc), a rare neurodegenerative disorder. The altered RBC morphology likely influences their biomechanical properties which are crucial for the cells to pass the microvasculature. Here, we investigated blood cell deformability of five ChAc patients compared to healthy controls during up to 1-year individual off-label treatment with the tyrosine kinase inhibitor dasatinib or several weeks with lithium. Measurements with two microfluidic techniques allowed us to assess RBC deformability under different shear stresses. Furthermore, we characterized leukocyte stiffness at high shear stresses. The results showed that blood cell deformability-including both RBCs and leukocytes - in general was altered in ChAc patients compared to healthy donors. Therefore, this study shows for the first time an impairment of leukocyte properties in ChAc. During treatment with dasatinib or lithium, we observed alterations in RBC deformability and a stiffness increase for leukocytes. The hematological phenotype of ChAc patients hinted at a reorganization of the cytoskeleton in blood cells which partly explains the altered mechanical properties observed here. These findings highlight the need for a systematic assessment of the contribution of impaired blood cell mechanics to the clinical manifestation of ChAc.

XK-Associated McLeod Syndrome: Nonhematological Manifestations and Relation to VPS13A Disease.

BACKGROUND: McLeod syndrome (MLS) is an X-linked multisystemic progressive disorder caused by loss of function mutations in the XK gene. The rare blood group phenotype of MLS patients with absent Kx antigen requires the support of specialized transfusion institutions because of the risk of transfusion complications. Acanthocytosis of red blood cells occurs in almost all patients. Nonhematological manifestations of MLS are very similar to those of VPS13A disease (chorea-acanthocytosis), an autosomal-recessive condition. Their shared phenotype apart from acanthocytosis includes movement disorders such as chorea and dystonia, epilepsy, peripheral neuropathy, and muscle involvement, typically with creatine kinase (CK) elevation, cardiomyopathy included. SUMMARY: In this review, we describe the nonhematological manifestations of MLS in comparison with those of VPS13A disease. While there are many similarities, differences such as mode of inheritance, sex distribution, age at manifestation, severity of heart involvement, frequency of feeding dystonia or of involuntary head drops may help to distinguish these disorders in the clinic. Immunohematological demonstration of the McLeod-Kell phenotype or detection of pathogenic mutations of XK (or VPS13A, respectively) is the gold standard for distinction. "Neuroacanthocytosis" was often used as an overarching term, but is potentially misleading, as the term does not refer to a defined disease entity. Its use, if continued, must not prevent clinicians to seek a final diagnosis on the basis of molecular findings. The clinical similarity of MLS and VPS13A disease has long suggested some shared pathophysiology. Evidence for molecular interaction between XK, the McLeod protein, and chorein, the VPS13A gene product, has recently been put forward: XK forms a complex with chorein/VPS13A, a bulk lipid transporter located at various membrane contact sites. The exact role of XK in this complex needs to be further elucidated. Impairment of bulk lipid transport appears as the common denominator of both MLS and VPS13A disease. A variety of further conditions may in time be added to the "bulk lipid transport diseases," such as the recently recognized disorders caused by mutations in the VPS13B, VPS13C, and VPS13D genes. KEY MESSAGES: (1) Patients diagnosed with the rare red cell McLeod phenotype (McLeod syndrome, MLS) require interdisciplinary collaboration of transfusion medicine specialists, neurologists, and cardiologists for both their hematological and nonhematological disease manifestations. (2) The phenotypical similarity of MLS and VPS13A disease, often leading to either confusion or insufficient diagnostic depth (under the label of "neuroacanthocytosis"), is based on interaction of the respective proteins, XK and chorein, within the cellular machinery for bulk lipid transport. (3) Overall, the term "bulk lipid transport diseases" seems useful for further research on a group of conditions that may not only share pathophysiology, but may also share treatment approaches.

The Erythrocyte Sedimentation Rate and Its Relation to Cell Shape and Rigidity of Red Blood Cells from Chorea-Acanthocytosis Patients in an Off-Label Treatment with Dasatinib.

BACKGROUND: Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disease with deformed red blood cells (RBCs), so-called acanthocytes, as a typical marker of the disease. Erythrocyte sedimentation rate (ESR) was recently proposed as a diagnostic biomarker. To date, there is no treatment option for affected patients, but promising therapy candidates, such as dasatinib, a Lyn-kinase inhibitor, have been identified. METHODS: RBCs of two ChAc patients during and after dasatinib treatment were characterized by the ESR, clinical hematology parameters and the 3D shape classification in stasis based on an artificial neural network. Furthermore, mathematical modeling was performed to understand the contribution of cell morphology and cell rigidity to the ESR. Microfluidic measurements were used to compare the RBC rigidity between ChAc patients and healthy controls. RESULTS: The mechano-morphological characterization of RBCs from two ChAc patients in an off-label treatment with dasatinib revealed differences in the ESR and the acanthocyte count during and after the treatment period, which could not directly be related to each other. Clinical hematology parameters were in the normal range. Mathematical modeling indicated that RBC rigidity is more important for delayed ESR than cell shape. Microfluidic experiments confirmed a higher rigidity in the normocytes of ChAc patients compared to healthy controls. CONCLUSIONS: The results increase our understanding of the role of acanthocytes and their associated properties in the ESR, but the data are too sparse to answer the question of whether the ESR is a suitable biomarker for treatment success, whereas a correlation between hematological and neuronal phenotype is still subject to verification.

Targeting Lyn Kinase in Chorea-Acanthocytosis: A Translational Treatment Approach in a Rare Disease.

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 ChAc.

Cardiac manifestation is evident in chorea-acanthocytosis but different from McLeod syndrome.

INTRODUCTION: We aimed to study the various cardiac manifestations of the two core neuroacanthocytosis (NA) syndromes, namely chorea-acanthocytosis (ChAc) and McLeod syndrome (MLS). So far, cardiac involvement has been described as specific feature only for MLS. METHODS: We studied six patients with ChAc (mean age 44.5 years, five men, one woman) and six patients with MLS (mean age 57.1 years, all men). Cardiac evaluation included echocardiography and/or cardiac magnetic resonance imaging (cardiac MRI), 24-h ECG-recording and examination of cardiac biomarkers. RESULTS: Cardiac involvement of ChAc was found in four of six patients. Two patients showed mildly reduced left ventricular ejection fraction (LVEF), two other patients mild to moderate left ventricular (LV) dilatation. Neither an increase in ventricular ectopic beats nor ventricular tachycardia were evident in ChAc. Four of five MLS patients showed left ventricle dilatation and reduced left ventricular ejection fraction (LVEF). Two of these, in addition, had critical ventricular tachycardia. High sensitive troponin T was elevated in all patients, for whom data were available (n = 10). In contrast, elevation of high sensitive troponin I was found in one of six ChAc and one of two MLS patients. CONCLUSION: For the first time, we reveal cardiac involvement in a cohort of six ChAc patients, while the risk to develop heart failure seems lower than in MLS. Our study confirms the malignant nature of MLS in terms of ventricular arrhythmias and progression to advanced heart failure. Herein, we define disease-specific recommendations for cardiac assessment in both conditions.

Therapeutic targeting of Lyn kinase to treat chorea-acanthocytosis.

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a-/- mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a-/- basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a-/- Lyn-/- showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a-/- hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients.

Acanthocyte Sedimentation Rate as a Diagnostic Biomarker for Neuroacanthocytosis Syndromes: Experimental Evidence and Physical Justification.

(1) Background: Chorea-acanthocytosis and McLeod syndrome are the core diseases among the group of rare neurodegenerative disorders called neuroacanthocytosis syndromes (NASs). NAS patients have a variable number of irregularly spiky erythrocytes, so-called acanthocytes. Their detection is a crucial but error-prone parameter in the diagnosis of NASs, often leading to misdiagnoses. (2) Methods: We measured the standard Westergren erythrocyte sedimentation rate (ESR) of various blood samples from NAS patients and healthy controls. Furthermore, we manipulated the ESR by swapping the erythrocytes and plasma of different individuals, as well as replacing plasma with dextran. These measurements were complemented by clinical laboratory data and single-cell adhesion force measurements. Additionally, we followed theoretical modeling approaches. (3) Results: We show that the acanthocyte sedimentation rate (ASR) with a two-hour read-out is significantly prolonged in chorea-acanthocytosis and McLeod syndrome without overlap compared to the ESR of the controls. Mechanistically, through modern colloidal physics, we show that acanthocyte aggregation and plasma fibrinogen levels slow down the sedimentation. Moreover, the inverse of ASR correlates with the number of acanthocytes (R2=0.61, p=0.004). (4) Conclusions: The ASR/ESR is a clear, robust and easily obtainable diagnostic marker. Independently of NASs, we also regard this study as a hallmark of the physical view of erythrocyte sedimentation by describing anticoagulated blood in stasis as a percolating gel, allowing the application of colloidal physics theory.

Adaptative Up-Regulation of PRX2 and PRX5 Expression Characterizes Brain from a Mouse Model of Chorea-Acanthocytosis.

The peroxiredoxins (PRXs) constitute a ubiquitous antioxidant. Growing evidence in neurodegenerative disorders such as Parkinson's disease (PD) or Alzheimer's disease (AD) has highlighted a crucial role for PRXs against neuro-oxidation. Chorea-acanthocytosis/Vps13A disease (ChAc) is a devastating, life-shortening disorder characterized by acanthocytosis, neurodegeneration and abnormal proteostasis. We recently developed a Vps13a-/- ChAc-mouse model, showing acanthocytosis, neurodegeneration and neuroinflammation which could be restored by LYN inactivation. Here, we show in our Vps13a-/- mice protein oxidation, NRF2 activation and upregulation of downstream cytoprotective systems NQO1, SRXN1 and TRXR in basal ganglia. This was associated with upregulation of PRX2/5 expression compared to wild-type mice. PRX2 expression was age-dependent in both mouse strains, whereas only Vps13a-/- PRX5 expression was increased independent of age. LYN deficiency or nilotinib-mediated LYN inhibition improved autophagy in Vps13a-/- mice. In Vps13a-/-; Lyn-/- basal ganglia, absence of LYN resulted in reduced NRF2 activation and down-regulated expression of PRX2/5, SRXN1 and TRXR. Nilotinib treatment of Vps13a-/- mice reduced basal ganglia oxidation, and plasma PRX5 levels, suggesting plasma PRX5 as a possible ChAc biomarker. Our data support initiation of therapeutic Lyn inhibition as promptly as possible after ChAc diagnosis to minimize development of irreversible neuronal damage during otherwise inevitable ChAc progression.

Neurofilament light chain in serum is significantly increased in chorea-acanthocytosis.

INTRODUCTION: Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disease, characterized by hyper- and hypokinetic movement disorders, peripheral neuropathy and acanthocytosis. Biomarkers are not established; possible candidates include neurofilament reflecting neuroaxonal damage. METHODS: We studied serum neurofilament light chain (sNfL) of six ChAc patients compared to two healthy control cohorts (A, six age/sex matched and B, historical cohort of 59 healthy adult subjects) and in two patients with the very similar condition of McLeod syndrome (MLS), the second core syndrome of neuroacanthocytosis. sNfL was quantified using single-molecule array analysis. RESULTS: sNfL concentration was significantly higher in the ChAc cohort (18.73 pg/ml; IQR 15.65-27.70) compared to both healthy control cohorts (A, 7.37 pg/ml; IQR 5.60-9.05; B, 3.10 pg/ml; IQR 2.43-3.98). In MLS patients, a similar sNfL increase was observed. CONCLUSIONS: sNfL is significantly increased in ChAc and MLS and seems to reflect neuroaxonal damage in the peripheral as well as the central nervous system.

MDS criteria for the diagnosis of progressive supranuclear palsy overemphasize Richardson syndrome.

MDS-criteria for clinical diagnosis of progressive supranuclear palsy (PSP) were recently published, their usability in a classical clinical setting is yet unknown. We retrospectively applied the new criteria using PSP patients' case files. Assignment of PSP diagnosis according to the MDS-criteria was possible in 57/80 cases. The main difference to former specialist classification was a lower phenotype diversity and higher representation of PSP-RS. Furthermore, we examined those patients' brain MRIs. While neuroradiologists' reports were suggestive of PSP only in 11/62, the analysis of a blinded rater revealed pathological midbrain-to-pons-ratio in 40/62 implying this imaging feature is often missed.

Phenotypes and malignancy risk of different FUS mutations in genetic amyotrophic lateral sclerosis.

OBJECTIVE: Mutations in Fused in Sarcoma (FUS or TLS) are the fourth most prevalent in Western European familial amyotrophic lateral sclerosis (ALS) populations and have been associated with causing both early and very late disease onset. FUS aggregation, DNA repair deficiency, and genomic instability are contributors to the pathophysiology of FUS-ALS, but their clinical significance per se and their influence on the clinical variability have yet to be sufficiently investigated. The aim of this study was to analyze genotype-phenotype correlations and malignancy rates in a newly compiled FUS-ALS cohort. METHODS: We cross-sectionally reviewed FUS-ALS patient histories in a multicenter cohort with 36 novel cases and did a meta-analysis of published FUS-ALS cases reporting the largest genotype-phenotype correlation of FUS-ALS. RESULTS: The age of onset (median 39 years, range 11-80) was positively correlated with the disease duration. C-terminal domain mutations were found in 90%. Among all, P525L and truncating/ frameshift mutations most frequently caused juvenile onset, rapid disease progression, and atypical ALS often associated with negative family history while the R521 mutation site was associated with late disease onset and pure spinal phenotype. Malignancies were found in one of 40 patients. INTERPRETATION: We report the largest genotype-phenotype correlation of FUS-ALS, which enables a careful prediction of the clinical course in newly diagnosed patients. In this cohort, FUS-ALS patients did not have an increased risk for malignant diseases.

Off-Label Treatment of 4 Amyotrophic Lateral Sclerosis Patients With 4-Aminopyridine.

Amyotrophic lateral sclerosis (ALS) is a fatal disorder characterized by degeneration of the upper and lower motor neuron. Among the at least 25 known genes associated with familial (hereditary) and sporadic ALS, mutations in fused-in-sarcoma (FUS) and superoxide dismutase 1 (SOD1) have been extensively investigated in the past years, with emphasis on altered excitability of affected neurons. Recently, we reported on hypoexcitability and increased cell death in a FUS/SOD1-ALS-induced pluripotent stem cell-derived motor neuron model, which was partly reversible by a treatment with the potassium channel blocker 4-aminopyridine (4-AP). Based on this study, we aimed to examine this US Food and Drug Administration-approved drug as a potential individualized treatment for patients with ALS. We therefore retrospectively investigated 4 FUS/SOD1-ALS patients who were prescribed 4-AP. Two patients expressed an improved quality of life due to regain of facial muscle motor function and decreased disease progression rate, respectively. Together with recent pathophysiologic findings, this case series supports the need for clinical trials to examine the efficacy of this potential treatment in distinct ALS subgroups and disease stages.

Current state of knowledge in Chorea-Acanthocytosis as core Neuroacanthocytosis syndrome.

Neuroacanthocytosis (NA) syndromes are a group of rare diseases characterized by neurological disorders and misshaped spiky red blood cells (acanthocytes) including Chorea-Acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington disease-like 2 (HDL 2), pantothenate kinase-associated neurodegeneration (PKAN), abeta- and hypobetalipoproteinemia and aceruloplasminemia. This clinically and genetically heterogeneous group of diseases shares main clinical features presenting most often as a hyperkinetic movement disorder. Even though these are long noted disease conditions, we still know only little on the underlying disease mechanisms. The current review focuses upon ChAc as the core entity of NA syndromes caused by mutations in the VPS13A gene. The support of patient organizations and the ERA-NET initiative yielded to different multidisciplinary efforts with significant progress on our understanding of ChAc. Disturbances in two pathways are currently considered to be significantly involved in the pathophysiology of ChAc, namely elevated Lyn kinase phosphorylation and decreased signaling via Phosphoinositide 3-kinase (PI3K). These recent developments may reveal potential drugable targets for causative therapies of ChAc.

Sympathetic innervation of human muscle spindles.

The aim of the present study was to investigate the presence of sympathetic innervation in human muscle spindles, using antibodies against neuropeptide Y (NPY), NPY receptors and tyrosine hydroxylase (TH). A total of 232 muscle spindles were immunohistochemically examined. NPY and NPY receptors were found on the intrafusal fibers, on the blood vessels supplying muscle spindles and on free nerve endings in the periaxial space. TH-immunoreactivity was present mainly in the spindle nerve and vessel. This is, to our knowledge, the first morphological study concerning the sympathetic innervation of the human muscle spindles. The results provide anatomical evidence for direct sympathetic innervation of the intrafusal fibers and show that sympathetic innervation is not restricted to the blood vessels supplying spindles. Knowledge about direct sympathetic innervation of the muscle spindle might expand our understanding of motor and proprioceptive dysfunction under stress conditions, for example, chronic muscle pain syndromes.

The male bulbospongiosus muscle and its relation to the external anal sphincter.

PURPOSE: The bulbospongiosus muscle is part of the superficial muscular layer of the perineum and pelvic floor. Its morphology remains controversial in the literature. Therefore, we reinvestigated the fascial arrangement and fiber courses of the bulbospongiosus muscle and its topographical relation to the external anal sphincter. MATERIALS AND METHODS: The perineum was dissected in 9 male cadavers (mean ± SD age 78.3 ± 10.7 years). Select samples were obtained for histology and immunohistochemistry. In 43 patients (mean age 60.7 ± 12 years) the topographical relation between the bulbospongiosus muscle and the external anal sphincter was determined by magnetic resonance imaging. RESULTS: The perineum contains several fascial layers consisting of elastic and collagen fibers as well as bundles of smooth muscle cells. The bulbospongiosus muscle was subdivided into a ventral and dorsal portion, which developed in 4 variants. The ventral insertion formed a morphological unity with the ischiocavernous muscle while the dorsal origin had a variable relation to the external anal sphincter (5 variants). A muscle-like or connective tissue-like connection was frequently present between the muscles. However, in some cases the muscles were completely separated. CONCLUSIONS: We suggest a concept of variations of bulbospongiosus muscle morphology that unifies the conflicting literature. Its ventral fiber group and the ischiocavernosus muscle form a functional and morphological unity. While the bulbospongiosus muscle and the external anal sphincter remain independent muscles, their frequent connection might have clinical implications for perineal surgery and anogenital disorders.