Posttranslational modifications of nerve cytoskeletal proteins in experimental diabetes

Abstract

Axonal transport is known to be impaired in peripheral nerve of experimentally diabetic rats. As axonal transport is dependent on the integrity of the neuronal cytoskeleton, we have studied the way in which rat brain and nerve cytoskeletal proteins are altered in experimental diabetes. Rats were made diabetic by injection of streptozotocin (STZ). Up to six weeks later, sciatic nerves, spinal cords, and brains were removed and used to prepare neurofilaments, microtubules, and a crude preparation of cytoskeletal proteins. The extent of nonenzymatic glycation of brain microtubule proteins and peripheral nerve tubulin was assessed by incubation with 3H-sodium borohydride followed by separation on two-dimensional polyacrylamide gels and affinity chromatography of the separated proteins. There was no difference in the nonenzymatic glycation of brain microtubule proteins from two-week diabetic and nondiabetic rats. Nor was the assembly of microtubule proteins into microtubules affected by the diabetic state. On the other hand, there was a significant increase in nonenzymatic glycation of sciatic nerve tubulin after 2 weeks of diabetes. We also identified an altered electrophoretic mobility of brain actin from a cytoskeletal protein preparation from brain of 2 week and 6 week diabetic rats. An additional novel polypeptide was demonstrated with a slightly more acidic isoelectric point than actin that could be immunostained with anti-actin antibodies. The same polypeptide could be produced by incubation of purified actin with glucose in vitro, thus identifying it as a product of nonenzymatic glycation. These results are discussed in relation to data from a clinical study of diabetic patients in which we identified increased glycation of platelet actin. STZ-diabetes also led to an increase in the phosphorylation of spinal cord neurofilament proteins in vivo during 6 weeks of diabetes. This hyperphosphorylation along with a reduced activity of a neurofilament-associated protein kinase led to a reduced incorporation of 32P into purified neurofilament proteins when they were incubated with 32P-ATP in vitro. Our combined data show a number of posttranslation modifications of neuronal cytoskeletal proteins that may contribute to the altered axonal transport and subsequent nerve dysfunction in experimental diabetes.

Effect of Pseudophosphorylation and Cross-linking by Lipid Peroxidation and Advanced Glycation End Product Precursors on Tau Aggregation and Filament Formation

“…Nearly a decade ago, it was suggested that carbonyl-derived post-translational modifications of neurofilaments may account for the biochemical properties of neurofibrillary tangles, possibly as a result of extensive cross-links (22,39). Because of their relatively long half-life, cytoskeletal proteins are preferred reactants with reactive carbonyl compounds, such as lipid peroxidation products or glucose (40,41). A first attempt to induce Tau glycation in vitro was made by short term incubation with glucose, but this did not result in the formation of PHFs (42).…”

How Is Proteinuric Diabetic Nephropathy Caused by Disturbed Proteostasis and Autophagy in Podocytes?

“…Thus, metabolically and genetically driven autophagy deficiency was associated with dysfunctional lysosomes accumulated in podocytes. The preservation of lysosome-and autophagy-mediated proteostasis may be critical for podocytes to cope with increased amounts of damaged proteins produced during diabetes, a condition favoring increased intensity of nonenzymatic modifications of amino acids (32) and high incidence of posttranslational modifications of proteins as reported in b-cells and nerves (33,34).…”

Axonal transport of cytoskeleton and cytoskeleton regulatory cargo proteins in neurodegenerative disorders

“…Pathological, post-translational changes of neurofilament structure, such as extensive phosphorylation [46][47][48] and nonenzymatic glycation 46,49,50 have also been shown to affect neurofilament transport, resulting in neurofilament accumulation and consequently altering axonal structure and function leading to nerve degeneration. Recent experiments focused on axonal transport impairment in the diabetic peripheral nerve revealed that neurofilament transport is indeed delayed and this delay coincides with the increased level of Carboxymethyllysine (CML), an advanced glycation end-product (AGE) indicative of high levels of glycation likely affecting neurofilament structure 19 .…”