FV 3 Cytoskeletal analysis in sensory nerve fibre endings show a decrease of neurofilaments in high aggressive spinal Amyotrophic lateral sclerosis

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Introduction: Amyotrophic lateral sclerosis (ALS) is a multisystemic, neurodegenerative disease, which is characterized by distal axonopathy and motor neuron loss and thereby leads to muscular weakness and death during 3-5 years after symptom onset due to respiratory failure. Neurofilaments are well-established biomarkers in ALS as they show increased levels in CSF compared to healthy controls as an expression of axonal damage and predict disease aggressiveness (by using the D50 progression model) and survival in ALS [1]. Together with their posttranslational modifications, neurofilaments and microtubules are critical components in cytoskeletal pathology of motor neuron diseases. Also, there are several ALS-related genes known to directly affect cytoskeletal dynamics, like TUBA4A, KIF5A or DCTNI1, which can interfere axon stability and axonal transport. In this study, we investigate the cytoskeleton in sensory nerve fiber endings of ALS patients to get a closer look in axonal pathology in ALS. Material and Methods: We performed skin biopsies of the proximal and distal leg in ALS and healthy controls. Those were immunostained for microtubules (ßIII-tubulin), neurofilaments (phosphorylated/non-phosphorylated neurofilaments – pNfH/npNfH) and actin and the individual fibres were analyzed for mean grey value intensity and axonal caliber in confocal images of the dermis. Additionally, we added electron microscopy (EM) of the biopsies to evaluate accumulation of cytoskeleton components or disorders in organelle transport. By using the D50 progression modell, based on functional loss in ALSFRS-R score, we also included the individual disease course and evaluate disease aggressiveness and disease accumulation for each ALS patients. Results: Our preliminary results show that ALS patients have an increase of mean grey value intensities for neurofilaments, ßIII-tubulin and actin compared to healthy controls. In subgroup analysis of ALS, compared by disease onset and disease aggressiveness, we see a trend for decrease in high aggressive spinal and bulbar ALS vs. low aggressive spinal ALS. Also, EM shows organelle transport disorders like mitochondrial accumulation in sensory nerve fibre endings in some cases of ALS. Conclusion: Analysis of skin biopsies may provide a simple tool to study axonal pathology in ALS and may be useful to predict disease aggressiveness as neurofilaments in sensory nerve fibre endings show a decrease in high aggressive spinal ALS. Further, we want to take a closer look to posttranslational modifications of microtubules, investigate the length dependence (proximal vs. distal), add longitudinal analysis and also correlate the results with clinical data and biomarkers, eg. neurofilaments in serum and CSF.