Uncovering the interactome of peripheral myelin protein 22 kDa (PMP22) to understand its role in health and neuropathy
by Daniela Katharina Stausberg
Date of Examination:2025-05-09
Date of issue:2025-11-20
Advisor:Prof. Dr. Michael Werner Sereda
Referee:Prof. Dr. Michael Werner Sereda
Referee:Prof. Dr. Martin Göpfert
Persistent Address:
http://dx.doi.org/10.53846/goediss-11648
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Abstract
English
PMP22, a hydrophobic glycoprotein, is crucial for proper myelination in Schwann cells of the peripheral nervous system. Dosage alterations lead to the neuropathies Charcot-Marie-Tooth disease 1A (CMT1A), where PMP22 is overexpressed, resulting in hypomyelination of medium to large caliber axons, and hereditary neuropathy with liability to pressure palsies (HNPP) with focal hypermyelination due to PMP22 haploinsufficiency. While the pathological consequences of PMP22 mutations are well documented, their molecular effect, i.e. the mechanisms of PMP22 (dys)function, remain poorly understood. One possible explanation for this shortcoming is the limited knowledge about protein-protein interactions that may mediate the cellular function(s) of PMP22. In this study, I investigated PMP22’s protein-protein interactions across various cellular systems, including HEK293T, MDCKII, and primary Schwann cells, using highly stringent Co-immunoprecipitation coupled with mass spectrometric analysis. A comprehensive interactome was generated, validating existing proposed interactions in fundamental cellular processes with partners such as calnexin, LMAN2, and STIM1, as well as interactions that specifically occur in Schwann cells, such as myelin protein zero. In addition to the existing body of 124 direct or indirect interacting proteins of PMP22 I identified 577 novel candidates with proteins involved in cell adhesion, signaling, and lipid metabolism. A key focus of my study was the interaction between PMP22 and caveolin-1 (CAV1), the signature protein of caveolae, which in myelinating Schwann cells is found in non-compact myelin regions. I could validate their interaction biochemically and demonstrate their immediate proximity in the cell. In the peripheral nerve, the compact myelin protein PMP22 and CAV1 were shown via immune electron microscopy to coexist in plasma membrane caveolae and possibly caveolae at the outermost myelin layer. I also found that PMP22 overexpression had a major impact on CAV1/caveolae in Schwann cells, demonstrating a strong functional connection between these proteins. CAV1 protein expression initially increased upon PMP22 overexpression in CMT1A. However, protein levels normalized during development, while numbers of plasma-membrane-bound caveolae were substantially decreased in Schwann cells of CMT1A rats during developmental myelination and in adulthood. Intriguingly, animals showing a severe disease phenotype had less caveolae than mildly affected animals, indicating an involvement of caveolae in CMT1A pathogenesis. Cav1 knockout mice have been reported to suffer from neurological deficits, however, it is still unclear to what extent central or peripheral functions are affected. Electron microscopy showed that loss of Cav1 results in hypomyelination of peripheral axons in adult mice. At the same time, its overexpression in Schwann cells in co-cultures with neurons also resulted in a myelination defect, highlighting the need for correct CAV1 dosage for proper peripheral nerve myelination. Collectively, my data suggest that CAV1 is necessary for normal peripheral myelination and further provide evidence for an involvement of CAV1–through interaction with the disease-causing protein PMP22–in CMT1A pathogenesis. Finally, I investigated the actin cytoskeleton in CMT1A rats, where I hypothesized a functional role of PMP22 through its novel interaction with LIM domain kinase 2 (LIMK2), an important regulator of the actin-severing protein cofilin. CMT1A sciatic nerves showed a significant reduction in filamentous actin, in line with increased cofilin activation as evidenced by a reduction in protein phosphorylation. Schmidt-Lanterman incisures, which are highly enriched in actin filaments, are significantly affected by this loss. The work presented in this thesis identifies novel PMP22-interacting proteins that may contribute to PMP22’s role in peripheral nerve (dys)myelination, with a specific focus on CAV1.
Keywords: Schwann cell; Myelin; PMP22; Protein-protein interactions; CMT1A; Caveolin-1
