saxitoxin and Nerve-Degeneration

Toxins produced by cyanobacteria and dinoflagellates have increasingly become a public health concern due to their degenerative effects on mammalian tissue and cells. In particular, emerging evidence has called attention to the neurodegenerative effects of the cyanobacterial toxin β-N-methylamino-L-alanine (BMAA). Other toxins such as the neurotoxins saxitoxin and ciguatoxin, as well as the hepatotoxic microcystin, have been previously shown to have a range of effects upon the nervous system. However, the capacity of these toxins to cause neurodegeneration in human cells has not, to our knowledge, been previously investigated. This study aimed to examine the cytotoxic effects of BMAA, microcystin-LR (MC-LR), saxitoxin (STX) and ciguatoxin (CTX-1B) on primary adult human astrocytes. We also demonstrated that α-lipoate attenuated MC-LR toxicity in primary astrocytes and characterised changes in gene expression which could potentially be caused by these toxins in primary astrocytes. Herein, we are the first to show that all of these toxins are capable of causing physiological changes consistent with neurodegeneration in glial cells, via oxidative stress and excitotoxicity, leading to a reduction in cell proliferation culminating in cell death. In addition, MC-LR toxicity was reduced significantly in astrocytes-treated α-lipoic acid. While there were no significant changes in gene expression, many of the probes that were altered were associated with neurodegenerative disease pathogenesis. Overall, this is important in advancing our current understanding of the mechanism of toxicity of MC-LR on human brain function in vitro, particularly in the context of neurodegeneration.

Topics: Amino Acids, Diamino; Astrocytes; Calcium; Cell Proliferation; Ciguatoxins; Cyanobacteria Toxins; Gene Expression; Humans; L-Lactate Dehydrogenase; Marine Toxins; Microcystins; Nerve Degeneration; Primary Cell Culture; Reactive Oxygen Species; Saxitoxin; Thioctic Acid

Severance of a peripheral nerve leads to a characteristic series of events in the distal stump, including the dissolution of axons and myelin and the proliferation of Schwann cells within their basal lamina. This study examines the relationship between the spatial-temporal pattern of the induction of the Schwann cell S phase, loss of the structural and functional properties of axolemma, and the clearance of myelin debris in the cat tibial nerve. Nerve transection stimulated a monophasic increase in [3H]thymidine incorporation that peaked at 4 days post-transection throughout an 80-mm length of distal stump. Light microscope autoradiography revealed prominent incorporation into Schwann cells of myelinated fibres. Treatment of distal stumps with mitomycin C at the time of nerve transection greatly retarded thymidine incorporation and clearance of myelin debris, but not the time course of axonal degeneration, decline in the synthesis of the major myelin glycoprotein, P0, or the onset of ovoid formation. Nerve transection also greatly reduced the specific uptake of [3H]saxitoxin (STX), a ligand which binds to voltage-sensitive sodium channels. Binding in the distal stump fell precipitously to 20% of the normal at 4 days post-transection, concurrent with the peak of thymidine incorporation. This low level of binding was maintained for periods of up to 70 days, demonstrating that some STX binds to structures other than axons in denervated distal stumps. Prior treatment with mitomycin C delayed the loss of specific STX binding. In conclusion, these studies suggest that: Schwann cell DNA replication and/or mitosis regulates other events during Wallerian degeneration, including myelin degeneration, catabolism of P0 and the clearance of sodium channels from nodal axolemma; the decline in P0 synthesis and/or shift to synthesis of less extensively processed P0 is independent of the induction of Schwann cell S phase; and Schwann cells enveloping myelinated axons enter S phase within a 24-h period throughout the entire 80-mm length of distal stump.

Topics: Animals; Autoradiography; Binding Sites; Cats; Female; Interphase; Male; Mitomycin; Mitomycins; Nerve Degeneration; Nerve Fibers; Nerve Tissue Proteins; Phosphates; Saxitoxin; Schwann Cells; Thymidine; Tibial Nerve; Time Factors; Tryptophan

The long-term excitability and the ionic currents in the nodes of Ranvier were studied in the severed frog sciatic nerve and the unoperated contralateral control nerve. After unilateral nerve section, compound action potentials of the nerve bundles and action potentials of single myelinated nerve fibres remained normal in amplitude and duration in either sciatic nerve for more than 38 days when frogs were kept at 11 degrees C. During this period the resting potentials averaged about -75 mV. Under voltage-clamp conditions Na currents and K currents in transected single myelinated nerve fibres also appeared normal in their kinetics, and in their peak amplitudes. These results indicate that the Na- and K-channel densities are quantitatively unchanged after the nerve transection, up to several weeks. The excitability of the severed sciatic nerve, expectedly, depends strongly on the time course of Wallerian degeneration. When frogs were kept at room temperature, the nerve excitability remained normal for only about 8-10 days, due to the faster Wallerian degeneration; whereas at 4 degrees C it was maintained for more than 84 days, as long as the myelinated nerve fibres did not degenerate. Together, these findings demonstrate that Na channels, K channels, and Na-K pumps are continuously present for several weeks in the transected nerve before nerve degeneration. It is surmised that either, (a) these proteins are extremely stable in the transected myelinated nerve fibres, or (b) they are supplied locally by Schwann cells, an open question recently posed by Chiu, Schrager & Ritchie (1984). In either event, the myelinated nerve fibres do not require cell bodies to provide a significant amount of new channels and pumps in order to retain their long-term excitability.

Topics: Action Potentials; Animals; Axons; Denervation; Ion Channels; Nerve Degeneration; Potassium; Rana pipiens; Ranvier's Nodes; Saxitoxin; Sciatic Nerve; Sodium; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin; Time Factors; Veratridine

Glial and Schwann cells undergo marked biochemical and morphological alterations following axonal injury. In the present experiments, the extent of enzyme activity associated with anaerobic (LDH, lactic dehydrogenase) vs aerobic (SDH, succinic dehydrogenase) respiration was assessed distal to the site of nerve fiber injury. Studies were performed in rat central (optic) and peripheral (sciatic) nerves at 2, 7 and 14 days postoperatively (d.p.o.). In sciatic nerves, LDH activity rose 3-fold in traumatized (vs unoperated control) nerve tissue between 2 and 7 d.p.o. and remained elevated at 14 d.p.o. SDH activity in traumatized nerve was equal to that in unoperated nerve at 7 d.p.o., but decreased at 14 d.p.o. LDH activity in optic nerve at 2 d.p.o. was equivalent to that in control nerve, but rose approximately two-fold by 7 d.p.o. However, unlike peripheral nerve, activity in traumatized optic nerve decreased to control levels at 14 d.p.o. SDH activity in traumatized optic nerve remained unchanged at any timepoint examined. Taken in concert, these data are consistent with the hypothesis that there is an overall shift in CNS glial and Schwann cell metabolism from aerobic to anaerobic respiration following nerve injury. Additional studies were performed to determine if this shift requires prior Schwann or glial cell mitosis. Administration of mitotic inhibitor (AraC, cytosine arabinofuranoside) inhibited post-traumatic elevations in LDH activity in optic, but not peripheral nerve. No significant effect of the drug on axonal degeneration (as assessed by saxitoxin binding) was observed.

Topics: Animals; L-Lactate Dehydrogenase; Mitosis; Nerve Degeneration; Optic Nerve Injuries; Rabbits; Rats; Rats, Inbred Strains; Saxitoxin; Species Specificity; Spinal Nerves; Succinate Dehydrogenase; Thymidine

Section of a rabbit peripheral nerve leads to axonal degeneration and a proliferation of Schwann cells, and it is known to lead to a profound increase in the saxitoxin binding capacity of its distal portion, suggesting that Schwann cells may bind this marker for sodium channels. The present study shows, however, that crush with subsequent axonal degeneration of the central axons of the rabbit optic nerve leads to a slow monotonic fall in the saxitoxin binding capacity, which by 100 days after crush is not significantly different from zero. This suggests that central glial cells (astrocytes and oligodendrocytes) do not bind saxitoxin, and that the saturable binding of saxitoxin to this nerve is entirely to the axolemma. On this basis, the value for total saxitoxin binding capacity of the normal rabbit optic nerve, taken together with the morphometric data of D. I. Vaney & A. Hughes (J. comp. Neurol. 80, 241-252 (1976)), yields a sodium channel density of about 400-700 channels per square micrometre nodal axolemma.

Topics: Animals; Axons; Ion Channels; Nerve Degeneration; Neuroglia; Optic Nerve; Rabbits; Saxitoxin; Sodium

The changes in binding of 3H-labeled saxitoxin (STX) to rabbit sciatic nerve during axonal regeneration (after nerve crush) and during axonal degeneration (after nerve section) were measured and compared with the corresponding changes in the sciatic nerves of other mammals (rat, guinea pig, and cat). In the rabbit and rat, regeneration after nerve crush is associated with a 2- to 4-fold increase in STX binding capacity, consistent with the known corresponding increase in the number of nodes of Ranvier in regenerating nerve. Furthermore, consistent with the disappearance of nodes that occurs with Wallerian degeneration, nerve section leads to a disappearance of all, or most, of the STX binding in rat and guinea pig nerve, similar to that previously found for cat nerve. However, in the rabbit, nerve section leads to a large maintained increase in STX binding. Intraneural injection of diphtheria toxin, which is known to damage Schwann cells and which causes an increase in STX binding in intact nerves, abolishes the binding in cut nerves. It is suggested that the increased binding in cut nerves is to nonneuronal sites situated on the surface membrane of the Schwann cells, which have greatly proliferated in number as axonal degeneration has progressed. The reason for the difference between rabbits and other species and the possibility that the binding sites of rabbit Schwann cells represent functional sodium channels remain to be investigated.

Topics: Amphibian Proteins; Animals; Carrier Proteins; Diphtheria Toxin; Guinea Pigs; Nerve Degeneration; Nerve Fibers, Myelinated; Nerve Regeneration; Rabbits; Rats; Saxitoxin; Sciatic Nerve

The biochemistry of gliotic CNS tissue was assessed by monotoring changes in de novo protein synthesis distal to site of crush in the rat optic nerve between 3 and 20 days post-operatively. Radioactivity profiles on 12% polyacrylamide SDS gels showed reproducible peaks associated with protein(s) with dissociated molecular weights of 57K, 51K, 42K, 40K, 37K and 23K. Differences between crush and control nerves were observed with respect to the latter two peaks. De novo synthesis of 23K protein (comigrant with myelin proteolipid protein) was evident in control but not crushed nerves. Synthesis of 37K protein (identity unknown) was evident at 7, 10 and (to a lesser extent) 20 days post-operatively in crushed nerves, but not in crushed nerves at 3 days post-operatively or in unoperated nerves at any time point. The appearance of the synthesis of the 37K protein coincides with a drop in the level of functional axolemma (assessed by [3H]saxitoxin binding) in crushed nerves from 78% to 36% of control levels between 3 and 7 days post-operatively.

Topics: Animals; Female; Nerve Degeneration; Nerve Tissue Proteins; Neuroglia; Optic Nerve; Rats; Rats, Inbred Strains; Saxitoxin