Regulatory role of sorting nexin 5 in protein stability and vesicular targeting of vesicular acetylcholine transporter to synaptic vesicle-like vesicles in PC12 cells

Accurate targeting of vesicular acetylcholine transporter (VAChT) to synaptic vesicles (SVs) is indispensable for efficient cholinergic transmission. Previous studies have suggested that the dileucine motif within the C-terminus of the transporter is sufficient for its targeting to SVs. However, the cytosolic machinery underlying specific regulation of VAChT trafficking and targeting to SVs is still unclear. Here we used the C-terminus of VAChT as a bait in a yeast two-hybrid screen to identify sorting nexin 5 (SNX5) as its novel interacting protein. SNX5 was detected in the SVs enriched LP2 subcellular fraction of rat brain homogenate and showed strong colocalization with VAChT in both brain sections and PC12 cells. Binding assays suggested that the C-terminal domain of VAChT can interact with both BAR and PX domain of SNX5. Depletion of SNX5 enhanced the degradation of VAChT and the process was mediated through the lysosomal pathway. More importantly, we found that, in PC12 cells, the depletion of SNX5 expression significantly decreased the synaptic vesicle-like vesicles (SVLVs) localization of VAChT. Therefore, the results suggest that SNX5 is a novel regulator for both stability and SV targeting of VAChT.     

The influence of supraspinal impulse activity on the intra-axonal transport of acetylcholine,choline acetyltransferase and acetylcholinesterase in rat motor neurons

The effect of supraspinal impulse activity upon the intra-axonal transport of acetylcholine (ACh), AChesterase (AChE) and cholineacetyltransferase (CAT) in rat sciatic nerve has been studied. A decreased impulse activity was obtained by spinal cord transsection (SCT) in the thoracic region 18 h, 6 days or 20 days before killing the rats. An increased neuronal activity was obtained by exercising the rats in a commercial rodent treadmill a couple of hours per day for 14 days. The amounts of substances which had accumulated in the sciatic nerve segments relative to a nerve crush performed 12 or 18 h. earlier were used to calculate the intra-axonal transport. The amounts of proximo-distally transported ACh decreased markedly with time after the SCT, while the proximo-distal transport of AChE-activity increased. Physical exercise appeared to increase ACh-transport. Thus, input to motor pericarya from supraspinal centres may regulate intra-axonal transport from the cell body of motor neurons into their axons.     

The axonal transport motor ‘kinesin’ is bound to anterogradely transported organelles: quantitative cytofluorimetric studies of fast axonal transport in the rat

Monoclonal antibodies to the axonal transport ATPase kinesin were used in an immunofluorescent study on mammalian nerves. Following crushing of the sciatic nerve and the ventral roots of adult rats, immunoreactive material was found to accumulate rapidly, mainly proximal to a crush but also, to some degree, distal to a crush. The strongest immunofluorescence was observed after incubation with the H2 antibody against the heavy subunit of kinesin. Using the cytofluorimetric scanning (CFS) procedure, the accumulated amounts were quantified and it was found that the retrogradely accumulating kinesin-like immunoreactivity (IR) was about 4–12% of the anterogradely transported kinesin-IR. The results were compared to the vesicle marker p38 (synaptophysin), which was found to accumulate to a significant extent on both sides of the crush. Cytofluorimetric scanning measurements indicated that nearly 50% of the anterogradely accumulated p38–IR was recycling to the cell body. The results demonstrate that kinesin in the living axon is affiliated with anterogradely transported organelles. Retrogradely transported organelles appeared to carry very little kinesin-IR, suggesting that kinesin may be subject to turnover, distinct from that of p38, in the distal regions of the axon.     

Changes in axonal protein transport produced by increased physiological activity of neurons

Department of Biology and Department of Pharmacology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 9, pp. 343–345, September, 1989.     

Quantitative autoradiographic evidence for axonal transport of imipramine receptors in the central nervous system of the rat

Interruption of the ascending serotonin axons of the medial forebrain bundle (MFB) in the rat brain produced a progressive time-dependent accumulation of imipramine receptors (labeled for autoradiography with [3H]imipramine). The largest accumulation of receptors occurred during the first 12 h at the caudal aspect of the lesion. An electrolytic lesion of the nucleus raphe dorsalis, administered 24 h prior to interruption of the medial forebrain bundle, markedly reduced the number of imipramine receptors on the caudal side of the lesion, while a significant accumulation was still evident on the rostral aspect. These results suggest that imipramine receptors are undergoing the process of orthograde axonal transport to terminals in the forebrain from the neuronal perikarya found in the nucleus raphe dorsalis. These receptors may also be undergoing retrograde transport back to their cell bodies of origin.     

Selective loss of alpha motor neurons with sparing of gamma motor neurons and spinal cord cholinergic neurons in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease characterised primarily by loss of lower motor neurons from the ventral grey horn of the spinal cord and proximal muscle atrophy. Recent experiments utilising mouse models of SMA have demonstrated that not all motor neurons are equally susceptible to the disease, revealing that other populations of neurons can also be affected. Here, we have extended investigations of selective vulnerability of neuronal populations in the spinal cord of SMA mice to include comparative assessments of alpha motor neuron (α‐MN) and gamma motor neuron (γ‐MN) pools, as well as other populations of cholinergic neurons. Immunohistochemical analyses of late‐symptomatic SMA mouse spinal cord revealed that numbers of α‐MNs were significantly reduced at all levels of the spinal cord compared with controls, whereas numbers of γ‐MNs remained stable. Likewise, the average size of α‐MN cell somata was decreased in SMA mice with no change occurring in γ‐MNs. Evaluation of other pools of spinal cord cholinergic neurons revealed that pre‐ganglionic sympathetic neurons, central canal cluster interneurons, partition interneurons and preganglionic autonomic dorsal commissural nucleus neuron numbers all remained unaffected in SMA mice. Taken together, these findings indicate that α‐MNs are uniquely vulnerable among cholinergic neuron populations in the SMA mouse spinal cord, with γ‐MNs and other cholinergic neuronal populations being largely spared.     

Localization and axonal transport of immunoreactive cholinergic organelles in rat motor neurons—An immunofluorescent study

Antisera produced in rabbits against pure fractions of cholinergic vesicles from Narcine brasiliensis were used to study cholinergic organelles in rat motor neurons. The indirect immunofluorescence method was used on perfusion-fixed material. The rats were surgically sympathectomized to remove sympathetic adrenergic and cholinergic nerves from the sciatic nerve. In the intact animal immunoreactive material, likely to represent cholinergic vesicles, was observed in motor endplates, identified by labelling with rhodamine-conjugated α-bungarotoxin or with subsequent acetylcholinesterase staining. The motor perikarya contained very little immunoreactive material. Non-terminal axons were virtually devoid of immunofluorescence in the intact animal. After crushing the sciatic nerve, immunoreactive material (likely to represent axonal cholinergic organelles) accumulated rapidly on both sides of the crush, indicating a rapid bidirectional transport. The transport was sensitive to local application of mitotic inhibitors.The axons which accumulated immunoreactive organelles were motor axons, as demonstrated by various procedures: (1) Cutting of ventral roots prevented accumulation of immunoreactive material in the nerve. (2) Deafferentation did not notably influence accumulations of immunoreactive material. (3) Ligated axons with immunoreactive material were acetylcholinesterase positive when identification was made on the same section; the intra-axonal distribution of immunoreactive material and acetylcholinesterase was not identical, however, and the Narcine antisera did not cross-react with bovine acetylcholinesterase in a solid phase immunoassay. (4) Most axons in ventral roots, but not in dorsal roots, accumulated strongly fluorescent immunoreactive material, while axons in dorsal roots contained weakly fluorescent material. On the other hand, substance P-like immune reactivity was present in many dorsal root axons, but only very rarely in ventral roots.It is suggested that the antisera against Narcine cholinergic vesicles can be used as a marker for cholinergic organelles in the motor neuron, and may be an important tool for studying the axonal cholinergic vesicles. It cannot, however, be used to identify cholinergic structures in unknown locations because it recognizes common antigenic determinants in transmitter organelles of other nerves e.g. adrenergic nerves. The axonal cholinergic organelles may carry important molecules, other than acetylcholine to the nerve endings.     

Axonal transport and subcellular distribution of dopamines-β-hydroxylase in the cod,Gadus morhua

The axonal transport of dopamine-β-hydroxylase (DBH; E.C. 1.14.17.1) was studied in the splanchnic nerve of the cod in vivo, and the subcellular localization of the same enzyme was studied in the chromaffin tissue from the cod head kidney. The mean rate of axonal transport for cod DBH was 18.6 mm/24 h at 10°C. The mobile fraction was estimated to 22 %, giving an absolute rate of transport of 85 mm/24 h at 10°C. Evidence for a retrograde transport of DBH was also obtained, with an accumulation distal to a ligature of 12% of the accumulation proximal to the ligature at 3 days. DBH from the chromaffin tissue appeared to be strongly bound to the adrenergic granules, with only a small amount (ca 4%) recovered in the soluble phase.     

KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons

Dense-core vesicles (DCVs) are responsible for transporting, processing, and secreting neuropeptide cargos that mediate a wide range of biological processes, including neuronal development, survival, and learning and memory. DCVs are synthesized in the cell body and are transported by kinesin motor proteins along microtubules to pre- and postsynaptic release sites. Due to the dependence on kinesin-based transport, we sought to determine if the kinesin-3 family member, KIF1A, transports DCVs in primary cultured hippocampal neurons, as has been described for invertebrate neurons. Two-color, live-cell imaging showed that the DCV markers, chromogranin A-RFP and BDNF-RFP, move together with KIF1A-GFP in both the anterograde and retrograde directions. To demonstrate a functional role for KIF1A in DCV transport, motor protein expression in neurons was reduced using RNA interference (shRNA). Fluorescently tagged DCV markers showed a significant reduction in organelle flux in cells expressing shRNA against KIF1A. The transport of cargo driven by motors other than KIF1A, including mitochondria and the transferrin receptor, was unaffected in KIF1A shRNA expressing cells. Taken together, these data support a primary role for KIF1A in the anterograde transport of DCVs in mammalian neurons, and also provide evidence that KIF1A remains associated with DCVs during retrograde DCV transport.     

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.     

Further evidence for the involvement of microtubules in the proximo-distal intra-axonal transport of acetylcholine and related enzymes in rat sciatic nerve

The two mitotic inhibitors colchicine (COL) and podofyllotoxin (POD) and their respective isomers, IumiCOL and picropodofyllin (picPOD) were tested for their effect on the intraaxonal transport (AXT) of acetylcholine (ACh) and the cholinergic enzymes in rat sciatic nerve. The mitotic inhibitors and their isomers were dissolved in saline + 10%, ethanol (COL and lumiCOL) or dimethylformamid (DMFA) (POD and picPOD) and injected (3–5 γl) subepineurally into the sciatic nerve. As controls the vehicle (saline+ 10% ethanol or DMFA) alone were injected into some rats. 2 h later a crush operation was performed 15 mm distal to the site of injection. The accumulation of ACh or the two enzymes, ACh-esterase (AChE) and cholineacetyltransferase (CAT), in the nerve segment proximal to the crush (12 h before death) was used as a measure of the AXT. COL and POD were very effective in inhibiting AXT of all 3 substances, while their isomers, lumiCOL and picPOD, were essentially without effect on AXT in equimolar concentrations (0.1 M). The effects on AXT of the 4 test substances thus appear related to their affinity to bind to tubulin, which is several orders of magnitude higher for COL and POD than for their isomers. The results further support the view that intact microtubules are essential for AXT of both membrane-bound (AChE) and soluble (CAT) enzymes, as well as of ACh in rat motor nerves.     

The use of retrograde transport of horseradish peroxidase for studying the dendritic trees and axonal courses of particular groups of tract cells in the spinal cord

Summary Modifications have been made in Mesulam's method for labelling neurons by retrograde transport of horseradish peroxidase, with tetramethylbenzidine as chromogen, with the object of increasing the extent of labelling of dendrites and axons. A procedure was devised specifically for studying spinomedullary and medullospinal tract systems, involving implanting easily-made HRP-agar pellets into areas of controlled damage in particular spinal fascicles, and sealing the site of implant with cyanoacrylate glue. Lesions of other fascicles were often made to limit transport to the implanted fascicle. Fourth-order dendrites were regularly labelled over long (30 cm or more) transport distances: axons were also labelled over this whole distance, often allowing exact study of the initial course of particular axons. Controls in both cat and rat showed that the uptake of HRP under these circumstances occurred almost wholly from the region of axonal damage at the site of implant which can be characterized histologically.     

Convergence on the same neurons in the feline ventrobasal thalamus of terminals from the dorsal column and the lateral cervical nuclei: an ultrastructural study combining orthograde degeneration and anterograde axonal transport of lectin conjugated horseradish peroxidase

After electrocoagulation of the dorsal column nuclei (DCN) and injections of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into the lateral cervical nucleus (LCN), two kinds of changes in fibers and boutons were observed in the dorsolateral part of the thalamic ventroposterolateral nucleus (VPL). The axons and boutons from neurons in the DCN demonstrated dark degeneration, while the fibers and synaptic terminals from the LCN-neurons contained the characteristic needle-shaped peroxidase reaction products following incubation with tetramethylbenzidine (TMB). Occasionally dark degenerating boutons and boutons with TMB-positive reaction products showed synaptic contact with the same dendritic profiles. After consideration of the possibility of an axoaxonal transfer of the tracer it is concluded that the findings demonstrate convergence of synaptic input both from the DCN and from the LCN on the same postsynaptic VPL neurons. The present results are in accordance with earlier light microscopic and neurophysiological findings. It is also concluded that the ultrastructural technique employed in this study is well suited to reveal convergence of different afferent systems on the same postsynaptic neurons.