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Impaired myelin production due to an intrinsic failure of oligodendrocytes in mTORpathies.

Neuropathology and applied neurobiology (2021-06-27)
Victoria-Elisabeth Gruber, Judith Lang, Verena Endmayr, Robert Diehm, Birgit Pimpel, Sarah Glatter, Jasper J Anink, Anika Bongaarts, Mark J Luinenburg, Roy J Reinten, Nicole van der Wel, Per Larsen, Johannes A Hainfellner, Karl Rössler, Eleonora Aronica, Theresa Scholl, Angelika Mühlebner, Martha Feucht

We aim to evaluate if the myelin pathology observed in epilepsy-associated focal cortical dysplasia type 2B (FCD2B) and-histologically indistinguishable-cortical tubers of tuberous sclerosis complex (TSC) is primarily related to the underlying malformation or constitutes a secondary phenomenon due to the toxic microenvironment created by epileptic seizures. We also aim to investigate the possible beneficial effect of the mTOR pathway regulator everolimus on white matter pathology. Primary mixed glial cell cultures derived from epilepsy surgery specimens of one TSC and seven FCD2B patients were grown on polycaprolactone fibre matrices and analysed using immunofluorescence and electron microscopy. Unaffected white matter from three age-matched epilepsy patients with mild malformations of cortical development (mMCD) and one with FCD3D served as controls. Additionally, TSC2 knock-out was performed using an oligodendroglial cell line. Myelination capacities of nanofibre grown cells in an inflammatory environment after mTOR-inhibitor treatment with everolimus were further investigated. Reduced oligodendroglial turnover, directly related to a lower myelin content was found in the patients' primary cells. In our culture model of myelination dynamics, primary cells grown under 'inflammatory condition' showed decreased myelination, that was repaired by treatment with everolimus. Results obtained in patient-derived primary oligodendroglial and TSC2 knock-out cells suggest that maturation of oligodendroglia and production of a proper myelin sheath seem to be impaired as a result of mTOR pathway disturbance. Hence, oligodendroglial pathology may reflect a more direct effect of the abnormal genetic programme rather than to be an inactive bystander of chronic epilepsy.

Product Number
Product Description

Anti-MAP2 Antibody, clone AP20, clone AP20, Chemicon®, from mouse
Anti-Myelin Basic Protein Antibody, a.a. 82-87, culture supernatant, clone 12, Chemicon®
Poly-L-lysine hydrobromide, mol wt 30,000-70,000
Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Phosphatase Inhibitor Cocktail 3, DMSO solution
Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody, Chemicon®, from rabbit
Anti-CNPase Antibody, clone 11-5B, clone 11-5B, Chemicon®, from mouse
RIPA Lysis Buffer, 10X, 100 mL RIPA Lysis Buffer, 10X for Immunoprecipitation & Western Blotting.
Anti-Glial Fibrillary Acidic Protein Antibody, clone GA5, clone GA5, Chemicon®, from mouse