Cell-Type-Dependent Recruitment Dynamics of FUS Protein at Laser-Induced DNA Damage Sites.

Increased signs of DNA damage have been associated to aging and neurodegenerative diseases. DNA damage repair mechanisms are tightly regulated and involve different pathways depending on cell types and proliferative vs. postmitotic states. Amongst them, fused in sarcoma (FUS) was reported to be involved in different pathways of single- and double-strand break repair, including an early recruitment to DNA damage. FUS is a ubiquitously expressed protein, but if mutated, leads to a more or less selective motor neurodegeneration, causing amyotrophic lateral sclerosis (ALS). Of note, ALS-causing mutation leads to impaired DNA damage repair. We thus asked whether FUS recruitment dynamics differ across different cell types putatively contributing to such cell-type-specific vulnerability. For this, we generated engineered human induced pluripotent stem cells carrying wild-type FUS-eGFP and analyzed different derivatives from these, combining a laser micro-irradiation technique and a workflow to analyze the real-time process of FUS at DNA damage sites. All cells showed FUS recruitment to DNA damage sites except for hiPSC, with only 70% of cells recruiting FUS. In-depth analysis of the kinetics of FUS recruitment at DNA damage sites revealed differences among cellular types in response to laser-irradiation-induced DNA damage. Our work suggests a cell-type-dependent recruitment behavior of FUS during the DNA damage response and repair procedure. The presented workflow might be a valuable tool for studying the proteins recruited at the DNA damage site in a real-time course.

Mitochondria-Endoplasmic Reticulum Contact Sites Dynamics and Calcium Homeostasis Are Differentially Disrupted in PINK1-PD or PRKN-PD Neurons.

BACKGROUND: It is generally believed that the pathogenesis of PINK1/parkin-related Parkinson's disease (PD) is due to a disturbance in mitochondrial quality control. However, recent studies have found that PINK1 and Parkin play a significant role in mitochondrial calcium homeostasis and are involved in the regulation of mitochondria-endoplasmic reticulum contact sites (MERCSs). OBJECTIVE: The aim of our study was to perform an in-depth analysis of the role of MERCSs and impaired calcium homeostasis in PINK1/Parkin-linked PD. METHODS: In our study, we used induced pluripotent stem cell-derived dopaminergic neurons from patients with PD with loss-of-function mutations in PINK1 or PRKN. We employed a split-GFP-based contact site sensor in combination with the calcium-sensitive dye Rhod-2 AM and applied Airyscan live-cell super-resolution microscopy to determine how MERCSs are involved in the regulation of mitochondrial calcium homeostasis. RESULTS: Our results showed that thapsigargin-induced calcium stress leads to an increase of the abundance of narrow MERCSs in wild-type neurons. Intriguingly, calcium levels at the MERCSs remained stable, whereas the increased net calcium influx resulted in elevated mitochondrial calcium levels. However, PINK1-PD or PRKN-PD neurons showed an increased abundance of MERCSs at baseline, accompanied by an inability to further increase MERCSs upon thapsigargin-induced calcium stress. Consequently, calcium distribution at MERCSs and within mitochondria was disrupted. CONCLUSIONS: Our results demonstrated how the endoplasmic reticulum and mitochondria work together to cope with calcium stress in wild-type neurons. In addition, our results suggests that PRKN deficiency affects the dynamics and composition of MERCSs differently from PINK1 deficiency, resulting in differentially affected calcium homeostasis. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

A serum microRNA sequence reveals fragile X protein pathology in amyotrophic lateral sclerosis.

Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS.

Functional and Molecular Properties of DYT-SGCE Myoclonus-Dystonia Patient-Derived Striatal Medium Spiny Neurons.

Myoclonus-dystonia (DYT-SGCE, formerly DYT11) is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the SGCE gene encoding ε-sarcoglycan leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the SGCE gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca2+ content and lower frequency of spontaneous Ca2+ signals in SGCE MSNs. Blocking of voltage-gated Ca2+ channels by verapamil was less efficient in suppressing KCl-induced Ca2+ peaks of SGCE MSNs. Ca2+ amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca2+ channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia.

Human Spinal Motor Neurons Are Particularly Vulnerable to Cerebrospinal Fluid of Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is the most common and devastating motor neuron (MN) disease. Its pathophysiological cascade is still enigmatic. More than 90% of ALS patients suffer from sporadic ALS, which makes it specifically demanding to generate appropriate model systems. One interesting aspect considering the seeding, spreading and further disease development of ALS is the cerebrospinal fluid (CSF). We therefore asked whether CSF from sporadic ALS patients is capable of causing disease typical changes in human patient-derived spinal MN cultures and thus could represent a novel model system for sporadic ALS. By using induced pluripotent stem cell (iPSC)-derived MNs from healthy controls and monogenetic forms of ALS we could demonstrate a harmful effect of ALS-CSF on healthy donor-derived human MNs. Golgi fragmentation-a typical finding in lower organism models and human postmortem tissue-was induced solely by addition of ALS-CSF, but not control-CSF. No other neurodegenerative hallmarks-including pathological protein aggregation-were found, underpinning Golgi fragmentation as early event in the neurodegenerative cascade. Of note, these changes occurred predominantly in MNs, the cell type primarily affected in ALS. We thus present a novel way to model early features of sporadic ALS.

Combined Dendritic and Axonal Deterioration Are Responsible for Motoneuronopathy in Patient-Derived Neuronal Cell Models of Chorea-Acanthocytosis.

Chorea acanthocytosis (ChAc), an ultra-rare devastating neurodegenerative disease, is caused by mutations in the VPS13A gene, which encodes for the protein chorein. Affected patients suffer from chorea, orofacial dyskinesia, epilepsy, parkinsonism as well as peripheral neuropathy. Although medium spinal neurons of the striatum are mainly affected, other regions are impaired as well over the course of the disease. Animal studies as well as studies on human erythrocytes suggest Lynkinase inhibition as valuable novel opportunity to treat ChAc. In order to investigate the peripheral neuropathy aspect, we analyzed induced pluripotent stem cell derived midbrain/hindbrain cell cultures from ChAc patients in vitro. We observed dendritic microtubule fragmentation. Furthermore, by using in vitro live cell imaging, we found a reduction in the number of lysosomes and mitochondria, shortened mitochondria, an increase in retrograde transport and hyperpolarization as measured with the fluorescent probe JC-1. Deep phenotyping pointed towards a proximal axonal deterioration as the primary axonal disease phenotype. Interestingly, pharmacological interventions, which proved to be successful in different models of ChAc, were ineffective in treating the observed axonal phenotypes. Our data suggests that treatment of this multifaceted disease might be cell type and/or neuronal subtype specific, and thus necessitates precision medicine in this ultra-rare disease.

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.

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.

Glycolic acid and D-lactate-putative products of DJ-1-restore neurodegeneration in FUS - and SOD1-ALS.

Amyotrophic lateral sclerosis (ALS) leads to death within 2-5 yr. Currently, available drugs only slightly prolong survival. We present novel insights into the pathophysiology of Superoxide Dismutase 1 (SOD1)- and in particular Fused In Sarcoma (FUS)-ALS by revealing a supposedly central role of glycolic acid (GA) and D-lactic acid (DL)-both putative products of the Parkinson's disease associated glyoxylase DJ-1. Combined, not single, treatment with GA/DL restored axonal organelle phenotypes of mitochondria and lysosomes in FUS- and SOD1-ALS patient-derived motoneurons (MNs). This was not only accompanied by restoration of mitochondrial membrane potential but even dependent on it. Despite presenting an axonal transport deficiency as well, TDP43 patient-derived MNs did not share mitochondrial depolarization and did not respond to GA/DL treatment. GA and DL also restored cytoplasmic mislocalization of FUS and FUS recruitment to DNA damage sites, recently reported being upstream of the mitochondrial phenotypes in FUS-ALS. Whereas these data point towards the necessity of individualized (gene-) specific therapy stratification, it also suggests common therapeutic targets across different neurodegenerative diseases characterized by mitochondrial depolarization.

Differentiation efficiency of induced pluripotent stem cells depends on the number of reprogramming factors.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by retroviral overexpression of the transcription factors Oct4, Sox2, Klf4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk for chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC, albeit at lower efficiency. To elucidate the influence of factor reduction on subsequent differentiation, we compared the efficiency of neuronal differentiation in iPSC generated from postnatal murine neural stem cells with either one (Oct4; iPSC(1F-NSC) ), two (Oct4, Klf4; iPSC(2F-NSC) ), or all four factors (iPSC(4F-NSC) ) with those of embryonic stem cells (ESCs) and iPSC produced from fibroblasts with all four factors (iPSC(4F-MEF) ). After 2 weeks of coculture with PA6 stromal cells, neuronal differentiation of iPSC(1F-NSC) and iPSC(2F-NSC) was less efficient compared with iPSC(4F-NSC) and ESC, yielding lower proportions of colonies that stained positive for early and late neuronal markers. Electrophysiological analyses after 4 weeks of differentiation identified functional maturity in neurons differentiated from ESC, iPSC(2F-NSC) , iPSC(4F-NSC) , and iPSC(4F-MEF) but not in those from iPSC(1F-NSC) . Similar results were obtained after hematoendothelial differentiation on OP9 bone marrow stromal cells, where factor-reduced iPSC generated lower proportions of colonies with hematoendothelial progenitors than colonies of ESC, iPSC(4F-NSC) , and iPSC(4F-MEF) . We conclude that a reduction of reprogramming factors does not only reduce reprogramming efficiency but may also worsen subsequent differentiation and hinder future application of iPSC in cell replacement therapies.

Corrosion Products from Metallic Implants Induce ROS and Cell Death in Human Motoneurons In Vitro.

Due to advances in surgical procedures and the biocompatibility of materials used in total joint replacement, more and younger patients are undergoing these procedures. Although state-of-the-art joint replacements can last 20 years or longer, wear and corrosion is still a major risk for implant failure, and patients with these implants are exposed for longer to these corrosive products. It is therefore important to investigate the potential effects on the whole organism. Released nanoparticles and ions derived from commonly used metal implants consist, among others, of cobalt, nickel, and chromium. The effect of these metallic products in the process of osteolysis and aseptic implant loosening has already been studied; however, the systemic effect on other cell types, including neurons, remains elusive. To this end, we used human iPSC-derived motoneurons to investigate the effects of metal ions on human neurons. We treated human motoneurons with ion concentrations regularly found in patients, stained them with MitoSOX and propidium iodide, and analyzed them with fluorescence-assisted cell sorting (FACS). We found that upon treatment human motoneurons suffered from the formation of ROS and subsequently died. These effects were most prominent in motoneurons treated with 500 µM of cobalt or nickel, in which we observed significant cell death, whereas chromium showed fewer ROS and no apparent impairment of motoneurons. Our results show that the wear and corrosive products of metal implants at concentrations readily available in peri-implant tissues induced ROS and subsequently cell death in an iPSC-derived motoneuron cell model. We therefore conclude that monitoring of neuronal impairment is important in patients undergoing total joint replacement.

Restoring Axonal Organelle Motility and Regeneration in Cultured FUS-ALS Motoneurons through Magnetic Field Stimulation Suggests an Alternative Therapeutic Approach.

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron disease characterized by sustained loss of neuromuscular junctions, degenerating corticospinal motoneurons and rapidly progressing muscle paralysis. Motoneurons have unique features, essentially a highly polarized, lengthy architecture of axons, posing a considerable challenge for maintaining long-range trafficking routes for organelles, cargo, mRNA and secretion with a high energy effort to serve crucial neuronal functions. Impaired intracellular pathways implicated in ALS pathology comprise RNA metabolism, cytoplasmic protein aggregation, cytoskeletal integrity for organelle trafficking and maintenance of mitochondrial morphology and function, cumulatively leading to neurodegeneration. Current drug treatments only have marginal effects on survival, thereby calling for alternative ALS therapies. Exposure to magnetic fields, e.g., transcranial magnetic stimulations (TMS) on the central nervous system (CNS), has been broadly explored over the past 20 years to investigate and improve physical and mental activities through stimulated excitability as well as neuronal plasticity. However, studies of magnetic treatments on the peripheral nervous system are still scarce. Thus, we investigated the therapeutic potential of low frequency alternating current magnetic fields on cultured spinal motoneurons derived from induced pluripotent stem cells of FUS-ALS patients and healthy persons. We report a remarkable restoration induced by magnetic stimulation on axonal trafficking of mitochondria and lysosomes and axonal regenerative sprouting after axotomy in FUS-ALS in vitro without obvious harmful effects on diseased and healthy neurons. These beneficial effects seem to derive from improved microtubule integrity. Thus, our study suggests the therapeutic potential of magnetic stimulations in ALS, which awaits further exploration and validation in future long-term in vivo studies.

Live Cell Imaging of ATP Levels Reveals Metabolic Compartmentalization within Motoneurons and Early Metabolic Changes in FUS ALS Motoneurons.

Motoneurons are one of the most energy-demanding cell types and a primary target in Amyotrophic lateral sclerosis (ALS), a debilitating and lethal neurodegenerative disorder without currently available effective treatments. Disruption of mitochondrial ultrastructure, transport, and metabolism is a commonly reported phenotype in ALS models and can critically affect survival and the proper function of motor neurons. However, how changes in metabolic rates contribute to ALS progression is not fully understood yet. Here, we utilize hiPCS-derived motoneuron cultures and live imaging quantitative techniques to evaluate metabolic rates in fused in sarcoma (FUS)-ALS model cells. We show that differentiation and maturation of motoneurons are accompanied by an overall upregulation of mitochondrial components and a significant increase in metabolic rates that correspond to their high energy-demanding state. Detailed compartment-specific live measurements using a fluorescent ATP sensor and FLIM imaging show significantly lower levels of ATP in the somas of cells carrying FUS-ALS mutations. These changes lead to the increased vulnerability of diseased motoneurons to further metabolic challenges with mitochondrial inhibitors and could be due to the disruption of mitochondrial inner membrane integrity and an increase in its proton leakage. Furthermore, our measurements demonstrate heterogeneity between axonal and somatic compartments, with lower relative levels of ATP in axons. Our observations strongly support the hypothesis that mutated FUS impacts the metabolic states of motoneurons and makes them more susceptible to further neurodegenerative mechanisms.

Applied Bayesian Approaches for Research in Motor Neuron Disease.

Statistical evaluation of empirical data is the basis of the modern scientific method. Available tools include various hypothesis tests for specific data structures, as well as methods that are used to quantify the uncertainty of an obtained result. Statistics are pivotal, but many misconceptions arise due to their complexity and difficult-to-acquire mathematical background. Even though most studies rely on a frequentist interpretation of statistical readouts, the application of Bayesian statistics has increased due to the availability of easy-to-use software suites and an increased outreach favouring this topic in the scientific community. Bayesian statistics take our prior knowledge together with the obtained data to express a degree of belief how likely a certain event is. Bayes factor hypothesis testing (BFHT) provides a straightforward method to evaluate multiple hypotheses at the same time and provides evidence that favors the null hypothesis or alternative hypothesis. In the present perspective, we show the merits of BFHT for three different use cases, including a clinical trial, basic research as well as a single case study. Here we show that Bayesian statistics is a viable addition of a scientist's statistical toolset, which can help to interpret data.

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.

Reduced Expression of GABA A Receptor Alpha2 Subunit Is Associated With Disinhibition of DYT-THAP1 Dystonia Patient-Derived Striatal Medium Spiny Neurons.

DYT-THAP1 dystonia (formerly DYT6) is an adolescent-onset dystonia characterized by involuntary muscle contractions usually involving the upper body. It is caused by mutations in the gene THAP1 encoding for the transcription factor Thanatos-associated protein (THAP) domain containing apoptosis-associated protein 1 and inherited in an autosomal-dominant manner with reduced penetrance. Alterations in the development of striatal neuronal projections and synaptic function are known from transgenic mice models. To investigate pathogenetic mechanisms, human induced pluripotent stem cell (iPSC)-derived medium spiny neurons (MSNs) from two patients and one family member with reduced penetrance carrying a mutation in the gene THAP1 (c.474delA and c.38G > A) were functionally characterized in comparison to healthy controls. Calcium imaging and quantitative PCR analysis revealed significantly lower Ca2+ amplitudes upon GABA applications and a marked downregulation of the gene encoding the GABA A receptor alpha2 subunit in THAP1 MSNs indicating a decreased GABAergic transmission. Whole-cell patch-clamp recordings showed a significantly lower frequency of miniature postsynaptic currents (mPSCs), whereas the frequency of spontaneous action potentials (APs) was elevated in THAP1 MSNs suggesting that decreased synaptic activity might have resulted in enhanced generation of APs. Our molecular and functional data indicate that a reduced expression of GABA A receptor alpha2 subunit could eventually lead to limited GABAergic synaptic transmission, neuronal disinhibition, and hyperexcitability of THAP1 MSNs. These data give pathophysiological insight and may contribute to the development of novel treatment strategies for DYT-THAP1 dystonia.

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.

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.

Concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis.

Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis, a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOFs) through DPRs versus loss of C9ORF72 functions (LOFs). Through multiparametric high-content (HC) live profiling in spinal MNs from induced pluripotent stem cells and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA double-strand breaks (DSBs), RNA foci, DPRs, and apoptosis. We show that both GOFs and LOFs were necessary to yield the overall C9ORF72 pathology. Increased RNA foci and DPRs concurred with onset of axon trafficking defects, DSBs, and cell death, although DSB induction itself did not phenocopy C9ORF72 mutants. Interestingly, the majority of LOF-specific DEGs were shared with HRE-mediated GOF DEGs. Finally, C9ORF72 LOF was sufficient-albeit to a smaller extent-to induce premature distal axonal trafficking deficits and increased DSBs.

Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation.

Mutations in RNA binding proteins (RBPs) and in genes regulating autophagy are frequent causes of familial amyotrophic lateral sclerosis (fALS). The P56S mutation in vesicle-associated membrane protein-associated protein B (VAPB) leads to fALS (ALS8) and spinal muscular atrophy (SMA). While VAPB is primarily involved in the unfolded protein response (UPR), vesicular trafficking and in initial steps of the autophagy pathway, the effect of mutant P56S-VAPB on autophagy regulation in connection with RBP homeostasis has not been explored yet. Examining the muscle biopsy of our index ALS8 patient of European origin revealed globular accumulations of VAPB aggregates co-localised with autophagy markers LC3 and p62 in partially atrophic and atrophic muscle fibres. In line with this skin fibroblasts obtained from the same patient showed accumulation of P56S-VAPB aggregates together with LC3 and p62. Detailed investigations of autophagic flux in cell culture models revealed that P56S-VAPB alters both initial and late steps of the autophagy pathway. Accordingly, electron microscopy complemented with live cell imaging highlighted the impaired fusion of accumulated autophagosomes with lysosomes in cells expressing P56S-VAPB. Consistent with these observations, neuropathological studies of brain and spinal cord of P56S-VAPB transgenic mice revealed signs of neurodegeneration associated with altered protein quality control and defective autophagy. Autophagy and RBP homeostasis are interdependent, as demonstrated by the cytoplasmic mis-localisation of several RBPs including pTDP-43, FUS, Matrin 3 which often sequestered with P56S-VAPB aggregates both in cell culture and in the muscle biopsy of the ALS8 patient. Further confirming the notion that aggregation of the RBPs proceeds through the stress granule (SG) pathway, we found persistent G3BP- and TIAR1-positive SGs in P56S-VAPB expressing cells as well as in the ALS8 patient muscle biopsy. We conclude that P56S-VAPB-ALS8 involves a cohesive pathomechanism of aberrant RBP homeostasis together with dysfunctional autophagy.