W. G. McLean

Glycation of rat sciatic nerve tubulin in experimental diabetes mellitus.

SummaryDiabetic neuropathy is associated with some early defects of axonal transport in experimental animals. Axonal transport is dependent on intact microtubules, and unsubstituted lysine residues of tubulin are essential for microtubule polymerization. As lysine residues are the major target for the non-enzymatic attachment of glucose, the effect of diabetes on the extent of glycation of tubulin was investigated. There was a more than four-fold increase in the extent of glycation of tubulin in the sciatic nerve of rats with streptozotocin-induced diabetes of 2 weeks duration compared with control rats. In contrast, no such increase in glycation was observed in brain microtubule protein from diabetic rats at that stage of diabetes. Incubation of brain microtubule protein with glucose prior to in vitro polymerization showed that the early stages of glycation were not associated with inhibition of microtubule assembly. The observed glycation of peripheral nerve tubulin in early experimental diabetes may nevertheless contribute to axonal transport abnormalities through an as yet undetermined impairment of microtubule function.

Protein kinase C isozyme expression in sciatic nerves and spinal cords of experimentally diabetic rats

Changes in the expression and activation of protein kinase C (PKC) have been implicated in the pathogenesis of diabetic neuropathy. Recent studies in liver, retina, and cardiovascular tissues from experimentally diabetic rats have demonstrated that diabetes has a selective effect on the expression and subcellular distribution of isozymes of PKC. In the light of this evidence, we investigated the expression of the PKC isozymes alpha, betaI, betaII, and gamma in sciatic nerves, spinal cords, and in the L4,5 dorsal root ganglia from streptozotocin-induced diabetic rats. Six weeks of diabetes had differential effects on the expression and distribution of PKC isozymes in sciatic nerves and spinal cords. In the sciatic nerves there was an apparent translocation of the alpha isoform from the cytosolic to the particulate fractions, the betaII isoform was reduced in the cytosolic fraction, and the betaI and gamma isoforms were unaffected. The changes in the isozyme immunoreactivities in the nerves were not a direct result of changes in either spinal cord or dorsal root ganglia alone, suggesting that diabetes has different effects on motor and sensory fibres and/or on Schwann cells. In nerves that had been crushed 14 days previously there was an increase in total PKC alpha immunoreactivity. This increase was potentiated in diabetic rats. On the other hand, PKC betaII immunoreactivity in crushed nerves was unaffected by diabetes. The data are consistent with diabetes-induced changes in expression of PKC betaII contributing to nerve damage, and changes in PKC alpha being a consequence of it.

Effects of nerve compression on fast axonal transport in streptozotocin-induced diabetes mellitus. An experimental study in the sciatic nerve of rats.

SummaryThe hypothesis that nerves in diabetes mellitus exhibit an increased susceptibility to compression was experimentally tested. Inhibition of fast axonal transport was induced by local compression in sciatic nerves of rats with streptozotocin-induced diabetes mellitus. Fast anterograde axonal transport was measured after application of3H-leucine to the motor neurone cell bodies in the spinal cord. The sciatic nerve as subjected to local, graded compression in vivo by a small compression chamber. The amount of accumulation of proteins was quantified by calculation of a transport block ratio. Compression at 30 mm Hg for 3 h induced a significantly greater (p<0.05) accumulation of axonally transported proteins at the site of compression in nerves of diabetic animals (transport block ratio: 1.01±0.35; n=7) than in nerves of controls (0.67±0.16;n=7). Accumulation was significantly higher in ligature experiments of both control (1.34±0.44;n=8;p< 0.01) and diabetic animals (1.45±0.30;n=8 ;p< 0.05), indicating that the block of transport in compressed nerves was incomplete. Neither sham compressed diabetic (0.50±0.09;n=6) nor control (0.49±0.11;n=6) nerves showed any block of axonal transport. The possible causes of the increased inhibition of fast axonal transport in diabetic rats are discussed. The results indicate that diabetes may lead to an increased susceptibility of peripheral nerves to compression.

Tricresyl phosphate inhibits the formation of axon-like processes and disrupts neurofilaments in cultured mouse N2a and rat PC12 cells

Tricresyl phosphate (1 microg/ml) inhibited the outgrowth of axon-like processes in mouse N2a neuroblastoma and rat PC12 pheochromocytoma cell lines induced to differentiate by serum withdrawal and nerve growth factor addition, respectively. By contrast, it had no effect on the outgrowth of processes by rat C6 glioma cells induced to differentiate with sodium butyrate. The effect on axon outgrowth in the two neuronal cell lines correlated with altered distribution of neurofilament proteins, as determined by indirect immunofluorescence with monoclonal antibody RMd09. Western blots of neuronal cell extracts probed with the same antibody revealed decreased cross-reactivity after exposure to tricresyl phosphate. The results suggest that tricresyl phosphate has a selective effect on neuronal cell differentiation, which involves impaired axon outgrowth, reduced levels of the neurofilament heavy chain and disruption of the neurofilament network.

The toxicity of organophosphate compounds towards cultured PC12 cells

The effects of three representative organophosphates (OPs), tricresyl phosphate (TCP), triphenyl phosphite (TPP) and paraoxon (POX) on the proliferation and viability of rat PC12 pheochromocytoma cells were studied. With respect to its IC50, TCP was at least an order of magnitude more potent in its antiproliferative activity than both TPP and POX. All test OPs were cytotoxic at concentrations inhibiting cell proliferation. No compound inhibited cell growth below 10 micrograms/ml. For TCP and TPP the estimated IC50 values from proliferation assays were lower than published LD50 values in vivo, whereas paraoxon was much less toxic in vitro than in vivo. Subcytotoxic levels of TCP (1 micrograms/ml) were found to inhibit the maintenance of neurites on cells grown in the presence of nerve growth factor.

Glycation of Brain Actin in Experimental Diabetes

Abstract: Actin is a neuronal protein involved in axonal transport and nerve regeneration, both of which are known to be impaired in experimental diabetes. To determine if actin is subject to glycation, we rendered rats diabetic by injection of streptozotocin. Two or 6 weeks later brains were removed and a preparation of cytoskeletal proteins was analyzed by two‐dimensional polyacrylamide gel electrophoresis. Brains from diabetic animals contained an extra polypeptide that migrated close to actin and reacted with monoclonal antibody C4 against actin. It was also found in a preparation of soluble synaptic proteins from diabetic rat brain, indicating that it was at least partly neuronal in origin. This polypeptide could be produced by incubation of cytoskeletal proteins from brains of nondiabetic rats with glucose‐6‐phosphate in vitro. The appearance of this glycated actin in diabetic animals was prevented by administration of insulin for a period of 6 weeks. We could not detect any effect of glycation in vitro on the ability of muscle G‐actin to form F‐actin filaments and its significance for the function of actin remains to be determined. The finding that glycation of platelet‐derived actin from diabetic patients was significantly increased implies that the abnormality may also occur in clinical diabetes.

Axonal transport and morphological changes following nerve compression An experimental study in the rabbit vagus nerve

Axonal transport and morphological changes were studied in the rabbit vagus nerve after the nerves had been subjected to compression at either 0, 50 or 200 mmHg for two hours. Slow axonally transported proteins, tubulin and actin, were radiolabelled with 35S-methionine two, seven or 14 days after the injury and the distribution of radiolabelled tubulin and actin within component b of slow transport was measured three days later by densitometric analysis of fluorographs of polyacrylamide gel. No significant differences were found in the distribution of tubulin two (50 and 200 mmHg) or seven (200 mmHg) days after injury, but at 14 days (200 mmHg) there was significantly increased radiolabelling of tubulin relative to actin in the nerve 60 to 70 mm from the nodose ganglion. Morphometric measurements of the nerve cell bodies two days after the compression injury at 200 mmHg revealed no significant changes. Previous work has shown that morphological changes, similar to those found after axotomy, were present in nerve cell bodies seven days after a compression injury. This, taken together with the present results, indicates that compression can induce both morphological and biochemical changes in the neurone. The altered axonal transport of tubulin associated with nerve injury follows a slower time course and does not precede the morphological changes. The findings may be of relevance when discussing the double crush syndrome.

Axonal transport of protein in motor fibres of experimentally diabetic rats — Fast anterograde transport

Fast axonal transport of radiolabelled proteins in motor fibres of rat sciatic nerves was studied after 14 days of streptozotocin-induced diabetes. The rate of fast transport as measured at two time intervals after application of [3H]leucine to the motor neurone cell bodies in the spinal cord was reduced by 21% in diabetic rats. There was no significant change in the time between injection of isotope and the start of fast transport. The amount of axonal transport of radiolabelled proteins as measured by accumulation of proteins proximal to a ligation on the sciatic nerve was also unchanged. The reduction in fast transport rate in the diabetic rats was eliminated by maintenance of normal blood glucose levels in twice daily insulin administration. The results are discussed with regard to the known effects of experimental diabetes on axonal transport in sensory fibres and to the role of fast axonal transport in peripheral neuropathies in general.

Treatment with an aldose reductase inhibitor can reduce the susceptibility of fast axonal transport following nerve compression in the streptozotocin-diabetic rat

SummaryThe effect of treatment with an aldose reductase inhibitor on the susceptibility of peripheral nerves to compression was studied in rats made diabetic by the injection of streptozotocin (50 mg·kg−1). The response to nerve compression was determined in untreated diabetic rats after 22 days of diabetes and compared with the response in two similar groups of diabetic rats which had been treated with the aldose reductase inhibitor ‘Statil’ (ICI 128436; 25 mg·kg−1· day−1 orally) either from the induction of diabetes or for 7 days prior to nerve compression. Two groups of non-diabetic rats were treated with ‘Statil’ for either 22 days or 7 days to act as controls. Inhibition of fast axonally transported proteins was induced by local compression of the sciatic nerves 4 h after application of 3H-leucine to the motor neurone cell bodies in the spinal cord. The inhibition of fast axonal transport was quantified by calculation of a transport block ratio.Compression at 30 mmHg for 3 h induced a significantly greater (p<0.05) inhibition of axonal transport at the site of compression in nerves of untreated diabetic rats (transport block ratio 0.96±0.24, n=8) than in nerves of control rats treated with the aldose reductase inhibitor for either the shorter time of 7 days (0.71±0.17, n=10) or the longer time of 22 days (0.69±0.08, n=5). In diabetic rats treated with the aldose reductase inhibitor for 22 days the inhibition (0.77±0.12, n=6) was significantly less than that in untreated diabetic rats; treatment for 7 days reduced the transport block ratio to 0.85±0.11 (n=8), but the effect was not significant. Treatment for 22 days prevented the marked increase in nerve sorbitol found in the diabetic rats but did not prevent a fall in nerve myo-inositol. The results indicate that treatment with an aldose reductase inhibitor for a sufficient period of time can reduce the increased susceptibility of peripheral nerves to compression in streptozotocin-induced diabetes mellitus in the rat by a mechanism which may be related to the prevention of increases in sorbitol in the nerves.

Altered GAP-43 Immunoreactivity in Regenerating Sciatic Nerve of Diabetic Rats

Experimental diabetes in the rat is associated with impaired axon regeneration. Successful regeneration depends on the construction of axonal growth cones and establishment of appropriate target connections. The growth-associated protein (GAP)–43 is a major component of the axonal growth cone, and its synthesis and axonal transport are markedly increased during regeneration. The purpose of this study was to determine the effect of experimental diabetes on the synthesis and axonal transport of GAP–43 in regenerating sciatic nerves. Rats were rendered diabetic with 50 mg/kg streptozotocin i.p. Four weeks later, the rats were anesthetized, and one sciatic nerve was crushed to induce regeneration. After 2 weeks, nerves were ligated, and 6 h later, nerve pieces proximal to the ligature and dorsal root ganglia were removed, and proteins were separated by PAGE. Western blots of gels were probed with antibody 10E8/E7 against GAP-43. The presence of GAP-43 was confirmed by immunohistochemistry of nerve sections. Densitometric analysis of the blots showed a 45% reduction in native GAP-43 immunoreactivity in nerve pieces proximal to the ligature (P < 0.05; n = 7). Northern blots of total RNA extracted from pooled dorsal root ganglia were probed with a 32P-radiolabeled cDNA probe for GAP-43. There was no significant difference in the amount of GAP-43 mRNA between diabetic and nondiabetic rats. Immunohistochemistry of sciatic nerve confirmed the reduction in GAP-43 immunoreactivity. We conclude that a defect in turnover or axonal transport of GAP-43 may contribute to the impaired peripheral nerve regeneration in diabetes.