Neurofilament Transport Defects And Corresponding Mechanisms In2,5-Hexanedione-Induced Neuropathy

ObjectiveThe organic solvent n-hexane belongs to one of the most important aliphatic glue industry, paints, varnishes, printing inks, shoe manufacturing, shoe repair, and the food industry. Occupational or experimental exposure to n-hexane induces a central-peripheral nervous system damage. Chronic exposure to n-hexane produces loss of sensory and motor function in arms and legs, which is the result of the nerve damages characterized as a central-peripheral distal axonopathy. Initial clinical manifestations include numbness and tingling sensation in the toes and fingers, followed by progressive weakness and areflexia, particularly in the distal limbs. Severely affected patients may develop sequelae of muscle wasting, foot drop, and spasticity, even paralysis. Electrophysiological studies demonstrate prominent prolongation of distal latencies, slowing of nerve conduction velocities, and conduction block with temporal dispersion particularly in severely intoxicated patients.Toxicokinetic studies have shown that2,5-Hexanedione (2,5-HD) is the active metabolite of n-hexane and mediates the neurotoxicity of the parent compound. However, the molecular mechanisms for2,5-HD-induced neuropathy remains unclear. The primary morphologic feature of2,5-HD-induced neuropathy is axon atrophy in peripheral and central nerves. Therefore, Lopachin and his partners proposed that axon atrophy in2,5-HD-induced neuropathy is mediated by a reduction in axonal NF contents. Axon atrophy involves disruption of the neurophysiological processes responsible for maintaining axon caliber in neurons. And it is affirmed that changes of axon caliber could impact the cable properties and nerve conduction velocity. And these descriptions are consistent with the electrophysiological alterations of n-hexane intoxicated patients. Axon is one important part of the neuron, its materials must be synthesizied and assembled in the neuronal cell body and transported along the axon toward the distal tip. This transportation is known as’axonal transport’. Growing evidence supports the idea that deficits in axonal transport contribute to pathogenesis in multiple neurodegenerative diseases, for example, Amyotrophic Lateral Sclerosis (ALS), Huntington’s disease, Alzheimer’s disease, and so on.In the present study, we investigated the effects of2,5-HD on slow axonal transport of GFP-tagged NF-M and partial factors involved in NF-M transport, as well as the cytoskeletal proteins in cultured DRG cells and subchronic poisoning rats, and studied the role of axonal transport played in2,5-HD-induced neuropathy.MethodsNeurons were dissociated from dorsal root ganglia(DRG) of postnatal2day old Sprague Dawley rats and cultured in vitro. After the cells were treated with0,4,8,16mM2,5-HD for24h, live-cell imaging experiment was performed. Under fluorescence microscope, PA-GFP-NFM was observed and pictured. The PA-GFP-NFM fluorescence intensity of each image was analyzed and compared. The amount of NF-M transported was measured by the decrease of the fluorescence intensity.The cultured DRG cells were harvested and crushed in lysis buffer (10mM Tris-HCl, pH7.5,150mM NaCl,1mM EDTA,1%Triton X-100,1%sodium deoxycholate,0.1%SDS, and10μl/ml proteinase inhibitor Cocktail) after24h of2,5-HD exposure. The lysate was sonicated and centrifuged at12,000g for10min at4℃. The supernatants were used for immunoblotting analysis and determination of ATP.Adult male Wistar rats were treated with2,5-HD by intraperitoneal injection at dosages of300mg/kg-bw for8weeks. The onset and development of neurotoxicity were determined by neurological testing (gait abnormality). The tissues of cerebrum, cerebellum, spinal cord and sciatic nerve, were homogenized in ice-cold homogenizing buffer containing1%X-100,50mM Tris (pH7.5),25mM KC1,2mM MgCl2,5mM EGTA,5mM dithiothreitol, Protease Inhibitor Cocktail (50ul/g tissue) and phosphatase inhibitors (5mM Na3VO4,10mM Na4P2O7and1mM iodoacetic acid) and then centrifuged at10,000xg for10min at4℃. The relative levels of NF-H, NF-M、NF-L、α-tubulin, β-tubulin, β-actin, kniesin heavy chain(KHC) and dynein in the supernatant of nerve tissues were determined by immunoblotting.Results1Cytotoxicity test2,5-HD inhibited the viability of cultured DRG cells, different concentration of2,5-HD shown different decrease of cell viability. There was an apparent concentration-response relationship. Finally,0,4,8,16mM2,5-HD were confirmed to be applied in the following experiments.22,5-HD decelerated NF-M transport in cultured DRG neuronsDuring the experiments, the density of fluorescence became weaker. Compared with the initial (set asl00%), the particular values were64.40±11.04%,71.52±11.64%,77.42±9.24%, and81.50±8.58%in0,4,8, and16mM2,5-HD-treated groups at120min, respectively. The decrease of the fluorescence density in2,5-HD-treated groups were all less than that of the control, indicating that the transport of NF-M was decelerated in2,5-HD groups.3Levels of cytoskeletal proteins in cultured DRG cells treated with2,5-HDThe prominent proteins in neurons (neurofilament subunits) were all reduced in2,5-HD groups. Among the three subunits, the decline of NF-M was the most evident. Compared with vehicle controls, a17%(P<0.05),38%(P<0.01) and41%(P<0.01) decrease of NF-M levels was observed in4,8,16mM2,5-HD-treated DRG cells, respectively, while the NF-H protein levels were decreased only by7%,21%(P<0.05) and33%(P<0.01), respectively. The decrease of NF-L was only observed in cells treated with16mM2,5-HD. In contrast to the obvious reduction of the NFs, no significant alteration of the levels of microtubules and microfilaments was observed.4Levels of motor proteins in cultured DRG cells treated with2,5-HDCompared with the control, kinesin heavy chain (KHC) protein levels in2,5-HD-treated DRG cells decreased by9.75%,24.02%(P<0.05) and36.06%(P<0.01) in4,8,16mM2,5-HD group, respectively. By contrast, the levels of dynactin p50were increased by43.37%(P<0.01),62.10%(P<0.01), and28.10%(P<0.05), respectively. However,2,5-HD intoxication failed to change the levels of dynein.5ATP concentration in cultured DRG cells treated with2,5-HD After24h intoxication, cells were collected and ATP content was determined. The ATP content in control cells was about15.9nmol/mg protein, and were10.7,8.3,7.8nmol/mg protein in cells treated with4,8,16mM2,5-HD, respectively.6Levels of μ-calpain and FK2in cultured DRG cells treated with2,5-HDIn cultured DRG cells, the levels of μ-calpain and FK2showed no significant changes after treated by2,5-HD (P>0.05)7The establishment of the animal modelAge-matched control rats had a starting mean body weight of272.3g, which increase steadily to483g at endpoint (8weeks). This represents a77.6%increase in body weight during the experiment period. Rats in the2,5-HD-treated groups had a similar starting weight, but gained only11.8%of their original weight, and, at the8weeks endpoint,2,5-HD-treated rats weighed63.1%of control.Intoxicated rats showed a progressive development of gait abnormalities. In the5th weekend, rats developed an unsteady walking pattern with a slight ataxia, and hindlimb weakness (2.05±0.23of mean gait score), and in the8th weekend, rats developed frank hindlimb weakness and an inability to rear (3.93±0.18of mean gait score).8Changes of cytoskeletal proteins in2,5-HD-intoxicated rats’nerve tissuesThe exposure to2,5-HD resulted in a significant decrease in NF-H, NF-M and NF-L in the homogenate of nerve tissues. In comparison with the control rats, the levels of NF-H significantly decreased (P<0.01) by65%,62%,66%and48%in cerebrum, cerebellum, spinal cord and sciatic nerve at the end of8weeks. Those of NF-L decreased by36%,66%, and45%in cerebrum, cerebellum and spinal cord respectively (P<0.01). The content of NF-M increased as the growth of rats, and compared with the age-matched control rats, those reductions were169%,73%,170%and60%in cerebrum, cerebellum, spinal cord and sciatic nerve at the end of8weeks (P<0.01).In cerebrum, β-tubulin showed no changes by2,5-HD-treated, α-tubulin and P-actin decreased slightly at the end of8weeks. In cerebellum, a-tubulin increased56%**, P-tubulin and β-actin reduced33%*and39%**, respectively. In sciatic nerve, a-tubulin showed a33%*reduction, but β-tubulin showed a78%**augment (*P <..05,**P<0.01) 9Changes of motor proteins in2,5-HD-intoxicated rats’ nerve tissuesThe motor protein, KHC declined by31%and57%, dynein dropped by35%and28%in spinal cord and sciatic nerve, respectively (P<0.01). There is no significant changes in cerebrum and cerebellum tissues.Conclusions1.2,5-HD significantly decelerated the axonal transport of NF-M in cultured DRG neurons.2. In cultured DRG cells, the levels of motor proteins and the content of ATP were decreased, as well as the levels of neurofilaments, after2,5-HD intoxication. This may be contributing to the deceleration of NF-M transport.3. The exposure to2,5-HD can lead to the reduction of KHC and dynein in rats’ cerebrum, cerebellum, spinal cord and sciatic nerve, and this reduction was more evident in the distal end of the axons. The alteration of neurofilaments had an similar tendency as KHC and dynein in the above nerve tissues. These may be involved in the onset and development of2,5-HD-induced neuropathy.