Ultrastructural insights into nerve pathology in patients with peripheral neuropathies by super-resolution imaging

Today's methods of super-resolution fluorescence microscopy make it possible to study structures at the nanometer level with molecular specificity. One structure that can only be studied in greater detail using these techniques is the node of Ranvier, which is the key element for fast saltatory conduction in myelinated nerve fibers. Previous studies have shown a periodic organization of 190 nm of the ringlike organized proteins (such as Caspr-1, Neurofascin - 155 and βII-spectrin) at the nodes of Ranvier and the internodal axon in rodent models. However, the ultrastructural organization of the paranodal region and the internodal axon of human peripheral nerves has not been visualized yet, nor is it known how these proteins are affected in peripheral neuropathies. In this work, the structure of the paranodal proteins and their cytoskeletal anchor molecules in mice samples and in nerve biopsies from 17 patients with peripheral neuropathies were studied using two-color direct stochastic optical reconstruction microscopy (dSTORM) and high-content confocal microscopy. In line with the previous studies, a periodicity of 190 nm was measured for both paranode and internode in mice samples. Furthermore, high colocalization was observed within the proteins at the paranodal region in mice samples. This 190 nm periodicity was observed in all patients for the internodal βII-spectrin and was also found at the paranode in patients with demyelinating neuropathies. However, for patients with axonal neuropathies, an increased periodicity was observed. A periodicity increase was also seen in all patients for the proteins Caspr-1 and Neurofascin-155, parts of the paranodal axoglial complex. In the colocalization analysis I was able to show that the axoglial complex pathologically detaches from its cytoskeletal anchor in peripheral neuropathies. In the high content imagining experiments patients with acute axonal neuropathies had the least nodes of Ranvier left. Here, the nodes of Ranvier were significantly longer than in other patient subgroups. This concludes that the nodes of Ranvier are an important target to study in peripheral neuropathies. In summary, the proteins at the node of Ranvier also form periodic structures with a periodicity of 190 nm in humans. However, this periodicity is pathologically altered by peripheral neuropathies. This work therefore provides new insights into the structure of the node of Ranvier in humans in physiological and pathological conditions. Thus, in the future, advanced fluorescence microscopy may help to understand and differentiate subtypes and stages of polyneuropathies for the better.