Axonal Transport Of Choline Acetyltransferase Research Articles

Mutant SOD1 impairs axonal transport of choline acetyltransferase and acetylcholine release by sequestering KAP3

Mutant SOD1 impairs axonal transport of choline acetyltransferase and acetylcholine release by sequestering KAP3

Mutant SOD1 impairs axonal transport of choline acetyltransferase and acetylcholine release by sequestering KAP3

Mutations in the superoxide dismutase 1 (sod1) gene cause familial amyotrophic lateral sclerosis (FALS), likely due to the toxic properties of misfolded mutant SOD1 protein. Here we demonstrated that, starting from the pre-onset stage of FALS, misfolded SOD1 species associates specifically with kinesin-associated protein 3 (KAP3) in the ventral white matter of SOD1G93A-transgenic mouse spinal cord. KAP3 is a kinesin-2 subunit responsible for binding to cargos including choline acetyltransferase (ChAT). Motor axons in SOD1G93A-Tg mice also showed a reduction in ChAT transport from the pre-onset stage. By employing a novel FALS modeling system using NG108-15 cells, we showed that microtubule-dependent release of acetylcholine was significantly impaired by misfolded SOD1 species. Furthermore, such impairment was able to be normalized by KAP3 overexpression. KAP3 was incorporated into SOD1 aggregates in human FALS cases as well. These results suggest that KAP3 sequestration by misfolded SOD1 species and the resultant inhibition of ChAT transport play a role in the dysfunction of ALS.

Follicle separation during Drosophila oogenesis requires the activity of the kinesin II-associated polypeptide Kap in germline cells.

Cellular localization of organelles, protein complexes and single mRNAs depends on the directed transport along microtubule tracks, a process mediated by ATP-driven molecular motor proteins of the dynein and kinesin superfamilies. Kinesin II is a heterotrimeric protein complex composed of two motor subunits and a unique nonmotor Kinesin-associated protein (Kap). Kap was shown to transport both particulate cargo, as axoneme components in rafts, and membrane-bounded organelles such as melanosomes. Drosophila Kinesin II was shown to be essential for the axonal transport of choline acetyltransferase in a specific set of neurons. We have generated Kap mutants and show that gene activity is not only required for neuronal function but also for separation of follicles during early oogenesis. The data suggest that Kap participates in the transport of signalling components required for instructive interactions between germline and soma cells.

Kinesin-II is required for axonal transport of choline acetyltransferase in Drosophila.

KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport.

Axonal transport of choline acetyltransferase and 6‐phosphofructokinase activities in genetically diabetic mice

This study examined the anterograde axonal transport of activities of the cytoplasmic enzymes choline acetyltransferase and 6-phosphofructokinase in genetically diabetic C57BL/Ks (db/db) mice and their nondiabetic (+/?) littermates. Diabetic mice exhibited marked reductions in the accumulation of both choline acetyltransferase and 6-phosphofructokinase activity against a constriction of the left sciatic nerve (38% and 51% of nondiabetic values, respectively). Enzyme activities per unit length of unconstricted nerve were not different from those of nondiabetic mice. The nerves of diabetic mice did not accumulate measurable amounts of sorbitol or fructose and showed no myo-inositol depletion. Thus this study concludes that, in diabetic mice, the deficits in anterograde axonal transport of these two enzymes do not arise from the accumulation of sorbitol and fructose nor from depletion of nerve free myo-inositol.

Studies on peripheral nerve and lens in long-term experimental diabetes: Effects of the aldose reductase inhibitor statil

This study determined the axonal transport of choline acetyltransferase (ChAT) activity and its content in several tissues in nondiabetic control rats and in four groups of rats with streptozotocin-induced diabetes of 6-months duration. Diabetic rats were either untreated, treated only with either the aldose reductase inhibitor Statil (Imperial Chemical Industries, Pharmaceuticals Division, Macclesfield, UK) or insulin, or were given the two in combination. Insulin treatment consisted of a single weekly injection of a long-acting insulin, a regime designed not to control the diabetes, but to provide regular respite from the catabolic dominance of uncontrolled diabetes. Elevated levels of sugars and polyols in the sciatic nerves of untreated diabetic animals were markedly attenuated by Statil. The reduced myo-inositol content and reduced axonal transport of ChAT activity also seen in these nerves were prevented by Statil, but a reduced motor nerve conduction velocity was attenuated only by Statil and insulin in combination. The presence of cataracts in all diabetic animals was associated with hyperhydration of the lens. The level of hydration and presence of cataracts were reduced by Statil, particularly in combination with insulin. ChAT activities of the iris, adrenal gland, and superior cervical ganglion were similar in all groups. Skeletal muscles showed wasting while the ileum showed an increased weight per unit length in diabetic rats. These tissues also displayed alterations in ChAT activities, particularly when referenced to unit weight of tissue, which may have been a consequence of the weight changes rather than diabetes per se. Thus, biochemical and functional defects of sciatic nerve previously reported in short-term experimental diabetes, preventable by aldose reductase inhibition, persist in rats with long-term experimental diabetes. Several of these defects were related to enhanced polyol pathway activity as demonstrated by their prevention or attenuation by Statil.

Studies of sorbinil on axonal transport in streptozotocin-diabetic rats

Deficits of axonal transport in short-term experimental diabetes may be a consequence of increased sorbitol pathway flux and may contribute to the development of degenerative neuropathies. Therefore, we studied the effect of the aldose reductase inhibitor sorbinil on the axonal transport of choline acetyltransferase (ChAT) in the cholinergic neurons of the sciatic nerve of rats with short-term streptozotocin diabetes. In addition, to examine the extent of axonal transport deficits, we studied the axonal transport of choline containing lipids in sensory neurons of the sciatic nerve of similarly diabetic rats and the effects of sorbinil thereon. In experimentally diabetic animals, sorbinil both prevented and reversed deficits of the axonal transport of ChAT and prevented a deficit in the axonal transport of choline containing lipids.

Prevention of Defects of Axonal Transport in Experimental Diabetes by Aldose Reductase Inhibitors

Experiments on streptozotocin-diabetic rats have indicated that axonal transport of choline acetyltransferase is reduced in sciatic nerve. Treatment with an aldose reductase inhibitor both prevented and reversed this defect which was related to marked accumulations of sorbitol and fructose. Concurrent with these accumulations the content of myo-inositol in diabetic peripheral nerve is depleted. Further experiments taking account of nerve water content showed that the depletion of myo-inositol was 'real' not apparent. When the level of myo-inositol was maintained, either by feeding myo-inositol or by the inhibition of aldose reductase, the development of defective axonal transport of choline acetyltransferase and choline-containing lipids was prevented.

Reversal of deficits in axonal transport and nerve conduction velocity by treatment of streptozotocin-diabetic rats with myo-inositol

This study examined the effects of myo-inositol treatment on the deficits of motor nerve conduction velocity and of axonal transport of choline acetyltransferase in rats with streptozotocin-induced diabetes. Motor nerve conduction velocity was measured in seven groups of male rats. Two groups formed nondiabetic controls; one survived for 3 and the other for 6 weeks, then motor nerve conduction velocity was again measured and the accumulation of choline acetyltransferase activity proximal to a 24-h sciatic nerve constriction was estimated. The other five groups were rendered diabetic and subjected to similar measurements. Two diabetic groups were untreated and survived 3 or 6 weeks. The other three groups received myo-inositol for 1, 2, and 3 weeks, respectively, commencing 21 days after induction of diabetes. These groups survived for 4, 5, or 6 weeks, respectively. In all groups surviving for longer than 3 weeks an interim measurement of motor nerve conduction velocity was made 21 days after the start of the experiment (immediately before onset of treatment in the treated groups). In these groups a third measurement was made on the day before death. At death, choline acetyltransferase accumulation was measured and the remainder of the sciatic nerves was assayed for sorbitol and myo-inositol. In the two untreated diabetic groups we found reduced motor nerve conduction velocity, a deficit in the accumulation of choline acetyltransferase proximal to the sciatic nerve constriction, and a marked build-up of nerve sorbitol and a depletion of nerve myo-inositol. All three treated groups showed a reversal of this myo-inositol depletion without a reduction in the nerve sorbitol content. One week of treatment was without effect on nerve conduction velocity or axonal transport. In the 2-week treatment group axonal transport of choline acetyltransferase was similar to controls but the nerve conduction deficit was not reversed. After 3 weeks of treatment choline acetyltransferase accumulation was slightly greater than controls and the motor nerve conduction velocity had increased significantly from the pretreatment value (after 3 weeks' diabetes) and was not significantly less than the prediabetic value.

Axonal transport of cholinergic transmitter enzymes in vagus and sciatic nerves of rats with acute experimental diabetes mellitus; correlation with motor nerve conduction velocity and effects of insulin

The accumulation of acetylcholinesterase and choline acetyltransferase proximal and distal to 24 h ligatures applied to the sciatic nerve was measured in rats. Additionally, the accumulation of choline acetyltransferase on both sides of 24 h ligatures applied to the left vagus was measured in the same animals. These procedures were performed in control rats and three groups of rats with streptozotocin (50 mg/kg i.p.)-induced diabetes. One group was diabetic for 1 week, the other 2 for 3 weeks. One of the latter groups received twice daily insulin to control their hyperglycaemia. The rats with 3 weeks' uncontrolled diabetes showed a reduction (approximately 50%) in the accumulation of choline acetyltransferase activity proximal to the ligature; there was also a reduction of the small accumulation of this enzyme distal to the ligature. The latter defect was also present in the rats with 1 week's diabetes, but in these animals the proximal choline acetyltransferase accumulation was not reduced. All these defects were absent in the insulin-treated diabetic group. Experimental diabetes did not alter the accumulations of acetyl-cholinesterase in the sciatic nerves nor the accumulation of choline acetyltransferase in the left vagi. The rats with 3 weeks' uncontrolled diabetes showed a significant reduction in motor nerve conduction velocity of about 9%. This deficit was not present in the insulin treated group. These findings indicate that experimental diabetes produces, at 3 weeks, a reduction in orthograde axonal transport of choline acetyltransferase in the sciatic nerve preceded by a possible defect of retrograde transport of the same enzyme at 1 week of diabetes. The development of the orthograde defect is contemporary with the development of a motor nerve conduction velocity defect. Both defects were obviated by insulin management.

Reversal, by treatment with an aldose reductase inhibitor, of impaired axonal transport and motor nerve conduction velocity in experimental diabetes mellitus

Streptozotocin-diabetic rats showed reduced motor nerve conduction velocity, impaired orthograde axonal transport of choline acetyltransferase, and accumulated sorbitol in the sciatic nerve compared with age-matched controls. Another age-matched group, with diabetes of similar intensity, but treated daily with an aldose reductase inhibitor, showed none of these neurological defects. These findings implicate aldose reductase activity in the development of acute conduction and axonal transport defects in experimental diabetes.

Some properties and the axonal transport of choline acetyltransferase in nervous tissue of the snail Helix pomatia

Some properties and the axonal transport of choline acetyltransferase in nervous tissue of the snail Helix pomatia

A developmental model for studying choline acetyltransferase transport in chick motoneurons.

The proximodistal axonal transport of choline acetyltransferase (ChAc) has been studied in the chick sciatic nerve in the absence of any experimental manipulation. The phenomenon utilized by us is a physiological transient fall in the activity of ChAc. This fall occurs in most areas of the chick central nervous system after hatching, and moves toward the periphery along the nerves. Having ruled out the presence of transient inhibitors, the movement of the fall towards skeletal muscles has been considered to correspond to the proximodistal transport of ChAc. The calculated average velocity is 7.86 mm/day, corresponding to the slow rate of transport. Nevertheless, the kinematics study suggests that the velocity varies along the nerve from a state of rest to an intermediate rate of transport (16 mm/day). The possible influence of variations in the resistance of the conductor on these changes in velocity is discussed.

Effect of ischemia on axonal transport of choline acetyltransferase and acetylcholinesterase and on ultrastructural changes of isolated segments of rabbit nerves in situ

Axonal transport of acetylcholinesterase (AChE) and choline acetyltransferase (ChAc) and ultrastructural degenerative changes were compared in isolated nerve segments of rabbit peroneal nerves kept in vivo for 22 h, either with preserved blood supply (control segments) or under conditions of ischemia (ischemic segments). Ischemia abolished the proximo-distal and disto-proximal axonal transport of AChE and the proximo-distal transport of ChAc which, in control segments, were revealed by accumulations of the enzymes at corresponding ends of the segments. Total activities of AChE and ChAc recovered in isolated segments with intact blood supply corresponded to the activities in normal nerves; in ischemic segments, 50% of ChAc activity was lost in 22 h, whereas all AChE activity was preserved. Ultrastructural changes were found in few fibres in control segments and in many fibres in ischemic segments 22 h after nerve interruption. The early changes in control segments correspond to those described in the literature for peripheral stump of severed nerves. The microtubules, neurofilaments and mitochondria were not affected. In ischemic segments, various stages of axoplasmic disintegration occurred in the myelinated and unmyelinated axons: flocculation and clumping of axoplasmic material, decomposition of neurofilaments and microtubules, swelling, formation of amorphous densities and breakdown of mitochondrial cristae. Swelling, amorphous densities, clumping of nuclear chromatin and necrotic mitochondrial changes appeared also in Schwann cells. It is concluded that ischemia blocks axonal transport and brings about, within 22 h, ultrastructural changes both in nerve fibres and in Schwann cells. Cytoplasmic ChAc is affected earlier by necrotic degeneration of the axons than membrane-bound AChE.

LEAD NEUROPATHY

Morphometric and pathologic studies along the length of the peripheral nervous system were obtained in groups of rats fed 4% lead carbonate for 3 and 6 months and in match-fed controls. The number and diameter histograms of L6 cytons of spinal ganglia and of myelinated fibers of proximal and distal portions of peroneal and sural nerve were not significantly different from the control groups. On the other hand, segmental demyelination occurred approximately as frequently in proximal as in distal parts of nerves. At 3 months approximately 1/3 of teased myelinated fibers showed changes of segmental demyelination (Condition C), or of remyelination after segmental demyelination (Condition F) or of both segmental demyelination and of remyelination (Condition D), while at 6 months more than 4/5ths of fibers showed these changes. As expected, regression lines of axonal area on number of lamellae of myelin, were less steep in nerves of rats fed on lead for 6 months as compared to controls. Axonal transport of choline acetyltransferase in lead neuropathy did not differ from that in control rats. As expected from the studies of others, conduction velocity of myelinated fibers of caudal nerve were low. A new finding was the often quite striking increase of transverse fascicular area of peripheral nerves. This was due to edema which appeared to develop at about the time of onset os segmental demyelination. Although the edema may be an epiphenomenon, it could be an important observation bearing on the development of lead neuropathy. It would be important to know next whether or not the blood nerve barrier is altered in lead neuropathy.

Effects of colchicine and vinblastine on axonal transport of choline acetyltransferase in rat sciatic nerve.

The effects of colchicine (0.5-10(-2) M) and vinblastine (10(-2)-10(-5) M) Upon axonal transport of choline acetyltranserase (CAT) and on nerve impulse conduction have been investigated in the rat sciatic nerve. High concentrations of colchicine (0.5 M) and vinblastine (10(-2) M) blocked completely both axonal transport of CAT and impulse conduction. 10(-3) M vinblastine did not affect impulse conduction until 20-22 h after injection, but this concentration of vinblastine did block CAT transport but not impulse conduction. 10(-2) M and 10(-1) M colchicine were without effect on impulse conduction, but did produce substantial, although incomplete, block of CAT transport. The results are discussed in relation to the possible involvement of microtubules in transport of CAT.

Transport of choline acetyltransferase and acetylcholinesterase in the central stump and isolated segments of a peripheral nerve

Axonal transport of choline acetyltransferase (ChAc, E.E.:2.3.1.6) and acetyl cholinesterase (AChE, E.C.:3.1.1.7) was studied in the peroneal fascicles of rabbit sciatic nerves. The accumulation of ChAc in the central nerve stump proceeded 5 times more slowly than that of AChE and occurred at a distanct of 2-4 mm proximally from the end, whereas AChE accumulated in the last 2 mm of the stump. In double-ligated segments of the nerve in situ the activity of ChAc decreased at the proximal and increased at the distal end; the activity of AChE rose at both ends, The increase of ChAc activity did not cease until 22 h, whereas that of AChE stopped before 10 h. The intensity of ChAc transport is considerably diminished in the part of axon separated from the nerve cell body. Differences between the behavior of ChAc and AChE are interpreted by the assumption that the axonal transport of ChAc is slow, unidirectional, concerns all of the enzyme in the nerve, and that most of the transported enzyme is not associated with intraaxonal organelles. In contrast to ChAc, the transport of AChE is fast, bidirectional, and concerns a minor proportion of enzyme in the nerve; the transported enzyme is associated with organelles. The rate of proximodistal transport of ChAc is estimated at 4 mm/day (based on the assumption that 100% of the enzyme moves proximo-distally) and that of AChE at 480 mm/day (based on the extimate that 5% of enzyme moved proximo-distally in the present experiments).

Choline acetyltransferase in identified neurons of abdominal ganglion of Aplysia californica.

Choline acetyltransferase in identified neurons of abdominal ganglion of Aplysia californica.