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Introduction. Neurons with long neurites are physiologically remarkable, as they need to transport newly synthesized proteins and organelles (e.g. mitochondria) from the soma over long distances to the synapses, which is essential for neuronal homeostasis. Via adaptor proteins, which walk on microtubules, organelles find their target place. Next to microtubules, the neuronal cytoskeleton consists of neurofilaments, and actin: neurofilaments regulate axonal caliber, and actin is involved in the neuronal shape (Eira et al. 2016, Hoffman et al. 1984). A misbalance or dysfunction of cytoskeletal components might contribute to neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS). Neurofilament light chain (NfL) is an established biomarker for ALS (Bacioglu et al. 2016). Here, we study the human cytoskeletal components during aging. As a model we chose sensory neurons which possess long processes and axon endings can be accessed easily by skin biopsies. In this study we focus on the expression levels of microtubules, neurofilaments, and actin in skin biopsies of healthy individuals. Furthermore, we analyze the axonal diameter. Methods. Skin biopsies (thigh and ankle) of 27 men (mean age 54.2 ± 18.9 a, from 24 a to 79 a) were PFA-fixed and cut. Fluorescent immunostaining for non-phosphorylated neurofilament heavy chain (npNfH), betaIII-tubulin, and actin was performed. Images of the dermis were acquired by confocal microscopy. At least 20 axons per healthy individual were analyzed for the mean gray values and diameter. Statistics were calculated using GraphPad Prism (V9). This study was approved by the local ethics committee. Participants gave informed written consent. Results. Cytoskeletal components (npNfH, betaIII-tubulin, actin) drastically increased with age. NpNfH increased 2.7-fold from 24- to 75-year-old individuals. Actin showed a change of 3.8-fold from the youngest to the 62-year-old controls (peak, maximum values), whereas betaIII-tubulin levels increased 3.2-fold from the 24- to the 75-year-old healthy individuals. Sensory axon caliber increased 2.2-fold from the 29- (1.35 µm) to the 78/79-year old (2.97 µm) controls which results in a 4.9-fold increase of the cross-sectional area of the axons. Statistical analysis (Mann-Whitney U test) showed highly significant results between each staining/caliber and age (p Discussion. Our results suggest an age-driven “ossification” of the axonal cytoskeleton in peripheral sensory axons, which might influence cytoskeletal functionality and axonal caliber. The age-dependent increase in caliber can be explained by the higher expression of neurofilaments, which mainly regulate the diameter of axons (Costa et al. 2018). These findings are in accordance with the increased susceptibility of aged individuals to neurodegenerative diseases. The results reported here will be validated in a second independent cohort (27 men, age 26 to 74). We further plan the analysis of length dependency as biopsies were obtained from thigh and ankle. |
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