gms | German Medical Science

Many polyneuropathy-associated proteins are involved in processes that are regulated by protein post-translational modifications (PTMs), including protein synthesis, cell cycle regulation, cell proliferation, apoptosis, and cytoskeletal organization. Therefore, modulation of these processes by PTMs could play a role in the degeneration of peripheral nerves, rendering the systematic study of PTMs an important novel field in experimental neuropathology. Notably, it is already known that cytoskeletal components such as neurofilaments, that are synthesized within the cell bodies and transported into the axons, can be aberrantly hyper-phosphorylated in neuropathic disorders.

We established highly sensitive mass spectrometry-based protocols to study PTMs such as phosphorylation, ubiquitination and proteolytic cleavage and their role in the pathology and etiology of peripheral polyneuropathies using both cell culture models and nerve tissue. Thus, from as little as 60 µg of mouse sciatic nerve we identified 2053 high confidence phosphorylation sites from 990 proteins, and from 80 µg of MSC80 cells 4348 phosphorylation sites from 1884 proteins. Many of the identified phosphoproteins are involved in processes related to the ER, stress, cytoskeletal organization, the axon and myelin. Interestingly, we found phosphorylation sites in many polyneuropathy-associated proteins, such as Dnmt1, Fam134b, Wnk1, Kif1a, and Trp channels. A differential study between (i) severely demyelinated axons from sciatic nerve samples of an established demyelinating CMT neuropathy mouse model and (ii) nerves derived from respective control animals revealed clear differences in phosphorylation patterns.

Our results demonstrate that highly sensitive state-of-the-art methods for analyzing PTMs can be used to study their roles in neuropathology and neurodegeneration, not only in cell culture models, but more importantly in animal models and even biopsies, thus providing unprecedented insights into pathological processes of the peripheral nervous system.