MYELINATION DEFICIT IN NERVE OF SUCKLING RATS DUE TO CYCLOLEUCINE ‐INDUCED DEFICIENCY OF METHYL DONORS

Abstract

We used cycloleucine (CL) — which prevents methionine conversion to S‐adenosyl‐methionine (SAMe) by inhibiting ATP‐L‐methionine‐adenosyl‐transferase (MAT) — to characterize the lipid and protein changes induced by methyl donors deficit in peripheral nerve and brain myelin in rats during development. We have previously shown that CL (400 mg/kg ip) given to suckling rats at days 7, 8, 12, and 13 after birth reduced brain and sciatic nerve weight gain, brain myelin content, protein, phospholipid (PL), and galactolipid concentration in comparison to control. Among PLs, only sphingomyelin (SPH) significantly increased by 35–50%. SAMe p‐toluensulphonate (SAMe‐SD4) (100 mg/kg, ip) given daily from day 7, as with exogenous SAMe, partially prevented some lipid alterations induced by CL, particularly galactolipid and SPH. To test the ability of CL to affect PL metabolism we have measured de novo PL biosynthesis, ex vivo in nerve homogenates (in comparison with brain homogenates) from control and CL‐treated animals killed at day 18 after birth, starting from labelled substrates ([3H]‐choline, specific activity 20 mCi/mmol) in a Tris/HCl buffer, containing 5 mM MgCl2, 0.2 mM EDTA, 0.1 mM ATP, and 0.5 mM of the labelled substrates. After 60 min incubation, lipids were extracted, PL separated by TLC, and corresponding silica gel fractions scraped and counted in a liquid scintillator. Phosphatidylcholine enrichment in labelled choline resulted in slight increases in brain and sciatic nerve of CL‐treated rats, suggesting an increased synthesis rate via the Kennedy pathway, possibly due to the reduced availability of methyl donors. Interestingly, choline incorporation into SPH in brain and nerve myelin resulted in significant increases of 30–40%. In agreement with the observed decrease of galactolipid content and the relative increase in SPH, these data suggest an alteration in sphingolipid metabolism after CL.Among proteins, in sciatic nerves of CL‐treated pups the relative content of a number of polypeptides, namely the 116, 90, 66, 58, and 56 kDa bands, decreased, whereas others increased; the most abundant PNS protein, protein zero, remained unchanged. The analyses of myelin basic protein isoforms revealed a dramatic increase in the 14.0 and 18.5 forms, indicating early active myelination. SAMe‐SD4 treatment counteracted, and in some cases normalized, these changes.In summary, methyl donor deficiency induced by MAT inhibition produces myelin lipid and protein alterations, partly counteracted by SAMe‐SD4 administration.The financial support of Telethon‐Italy (grant No. D 51) is gratefully acknowledged.