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Fast Axonal Transport: a Site of Acrylamide Neurotoxicity: a Rebuttal
NeuroToxicology 23 (2002) 265–270
Response to commentaries
Fast Axonal Transport: a Site of Acrylamide Neurotoxicity: a Rebuttal Dale W. Sickles1,*, Derek Stone1, Marvin Friedman2 1
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912-2000, USA Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2757, USA 2
Alternative viewpoints to our hypothesis are offered in the accompanying critiques. There appears to be a general consensus that acrylamide compromises fast axonal transport. This represents advancement in our thinking since the myriad of apparently contradictory experimental results have been resolved by identification of the differences in experimental designs that contributed to the variability. Both respondents recognize kinesin, the anterograde motor protein, as a site of action in producing fast anterograde axonal transport (faAXT) reductions. Sabri and Spencer present data supporting additional sites of acrylamide action. The most controversial component of our hypothesis is whether the faAXT reductions are related to the development of behavioral changes characteristic of acrylamide neurotoxicity. The void of information regarding this issue must be addressed to adequately assess the contribution of faAXT compromise to axon (including the axon terminals) functionality. Lopachin’s group has developed a new methodological approach for evaluating the relevance of acrylamide effects to the development of behavioral symptoms (neurotoxicity). Administration of acrylamide at 50 mg/kg per day for up to 11 days (subacute, short-term high-dose, intraperitoneal) or at 21 mg/kg per day for up to 38 days (subchronic, long-term lowdose, oral) results in similar neurotoxicity, but with differential outcome in other parameters (LoPachin et al., 1992; Lehning et al., 1994, 1998). A primary assumption is that pathophysiologically relevant changes should be expressed at both dosing regimens. Although it is feasible that different mechanisms are * Corresponding author. Tel.: þ1-706-721-7393; fax: þ1-706-721-6839. E-mail address: [email protected] (D.W. Sickles).
operative at the two dosing regimens, the probability of two mechanisms leading to similar symptoms is low, making the approach useful. The subchronic regimen produces the classic pathologies associated with acrylamide intoxication; atrophy, swellings and degeneration (LoPachin et al., 1992; Lehning et al., 1998). In addition, Na/K ATPase quantity (unpublished results) as well as axolemmal rubidium transport (measure of Na/K ATPase activity) are reduced (Lehning et al., 1994, 1998). Following subacute intoxication there is little degeneration and no changes in Na/K ATPase activity (Lehning et al., 1994, 1998). The correlation of reductions in Na/K ATPase activity with degeneration suggests a cause–effect relationship (Lehning et al., 1998; LoPachin and Lehning, 1997). However, the differential dosing rate dependent expression of degeneration and axonal ion homeostasis, despite similar neurotoxicity, led to the conclusion that these two characteristics are dissociated from the primary pathophysiologic event (Lehning et al., 1998). Therefore, these data support the conclusion that tibial nerve axonal degeneration is non-essential for neurotoxicity. However, these data are insufficient to conclude that degeneration and/or fast axonal transport compromises are unrelated to neurotoxicity. More inclusive morphological analyses (identified below) are required to resolve the relationship of degeneration to neurotoxicity. Furthermore, consideration of a broader spectrum of axonal functions and their associated proteins is necessary to evaluate the contribution of fast axonal transport compromise to axon or terminal functionality as well as neurotoxicity. Axonal degeneration is a well-defined outcome of faAXT blockade, and we have previously hypothesized degeneration as an endpoint of acrylamide toxicity. However, within the current forum article, we have
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clearly indicated that compromises in axonal transport by acrylamide are not complete and are, in addition, transient. Therefore, degeneration is anticipated to be less prominent in acrylamide toxicity than with agents such as microtubule poisons or metabolic inhibitors, whose effects are more potent and/or more prolonged (due to replenishment of these targets via slow axonal transport). The data demonstrating a lack of degeneration by subacute dosing rates are in distal nerves (LoPachin et al., 1992; Lehning et al., 1998). They do not include the sensory and motor terminals, the most distal elements that are predictably the most vulnerable to fast axonal transport deficits. Indeed, numerous studies have identified early pathologies of Pacinian corpuscles (emphasized in the accompanying critique by Sabri and Spencer); muscle spindle afferents and the motor end plates (Sumner and Asbury, 1975; Schaumburg et al., 1974; Lowndes et al., 1978; Lowndes and Baker, 1976; Goldstein and Lowndes, 1981). LoPachin (data presented in critique) apparently found CNS axonal degeneration primarily associated with low-rate, high-dose intoxication. However, they state that ‘‘regardless of the daily exposure rate (50 mg/ kg per day 5–11 days, i.p. or 21 mg/kg day 7–38 days, p.o.), ACR induced early nerve terminal degeneration’’. Therefore, there appears to be substantial terminal degeneration in the CNS and PNS, which can account for neurotoxicity. LoPachin appears to make a distinction between axonal versus terminal degeneration. However, in terms of evaluating the impact of fast axonal transport inhibition to pathological and neurotoxicological outcomes, the terminal is conclusively part of the axon and should not be semantically separated. We emphasize that degeneration is not required for inhibition of fast transport inhibition to be a primary neurotoxic mechanism. Fast transport compromise can lead to axonal and/or terminal dysfunction prior to, or in the absence of, degeneration. Earlier electrophysiological data identified dysfunction of axons and terminals prior to morphological changes (Anderson, 1981, 1982; Goldstein, 1985; Sumner and Asbury, 1975). Therefore, conclusions regarding relevance of a specific morphological effect to neurotoxicity are potentially overstated. LoPachin has concluded that distal axons of subacute acrylamide-intoxicated animals are not dysfunctional. This conclusion is based upon normal axolemmal Na/K ATPase and axoplasmic ion composition in tibial nerves of high ACR dosing rate rats (LoPachin et al., 1992; Lehning et al., 1994, 1998), despite the presence of neurotoxic symptoms. Addi-
tionally, intact mitochondrial transmembrane ion gradients and energy production were observed and axons were capable of regulating element composition. From these results, it is reasonable to conclude that homeostatic mechanisms, including total ion composition and energy metabolism, in tibial nerve are insufficiently changed by acrylamide to be a pathophysiological cause of neurotoxicity. However, these analyses are limited and do not include determination of axonal functions such as action potential propagation or terminal neurotransmission. As a consequence, they provide little information relevant to evaluating the outcome of faAXT compromise by acrylamide to neurotoxicity. Furthermore, these conclusions are contradictory to their previous observations of compromised ability of ACR exposed axons to recover from anoxia (Lehning et al., 1997). Several specific issues are of concern. The potential for an aerobic enzyme inhibition to contribute to neurotoxicity is considered below. However, we note here that reductions in axonal glycolytic enzyme activity may occur through direct inhibition or by changes in slow transport, not by reduced faAXT. Mitochondria are conveyed in the fast system but are capable of sustained longevity in the absence of translocation (fast transport). Therefore, aerobic metabolism and mitochondrial transmembrane ion gradients would be compromised only with direct toxicant action on this organelle. Since previous studies failed to identify an effect on mitochondrial respiration or ATP production (Medrano and LoPachin, 1989; Sickles et al., 1990) and therefore, are anticipated to remain active in the absence of translocation, the lack of effect of subacute doses on these functions is not surprising. Therefore, the absence of mitochondrial ion gradient and oxidative metabolism effects in the subacute animals are insufficient to discredit the fast axonal transport compromise as contributory. It is reasonable to anticipate compromise of fast axonal transport to cause dysfunction of all fast-transported, axolemmal proteins. However, differential protein turnover rates or differences in quantity of specific protein transport compared to functional requirements may result in differential sensitivity of various functions (as observed in kinesin knockout models discussed in greater detail below). Furthermore, differential efficacy of ACR inhibition of different kinesin family members, each with its own specific cargo, is possible. LoPachin indicates that similar sulfhydryl dependency of other kinesin motors has not been demonstrated and that differential sensitivity is doubtful. We have recently identified ACR inhibition of
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kinesin motors in two other kinesin subfamilies (Sperry and Sickles, 2001). Sequence differences within kinesin superfamily common domains, as well as higher variability within non-homologous domains (Cyr et al., 1995), are typically associated with differential sensitivity. We suggest that future studies assess the dose-response of acrylamide on the various kinesin superfamily members. We have concerns regarding the interpretation of electron probe microanalysis (EPMA) data, used to measure axon ion content, since it has some limitations. It measures total ion content (LoPachin and Saubermann, 1990). Therefore, functional differences in ionized species can go undetected under the large volume of total ion content. The toxicant-induced changes observed with this technique following acrylamide exposure are, primarily, increases in variability, rather than changes in means, and no spatial or temporal patterns were observed (LoPachin et al., 1992). These results are difficult to reconcile mechanistically. Changes in mean values in other experimental conditions do appear coincident with axonal degeneration (LoPachin and Lehning, 1997). Taken together, these observations suggest strong resistance to damage of these processes and/or insensitivity of the assay. Lastly, we emphasize that EPMA does not measure functional attributes specific to axonal or terminal function such as membrane excitability and communication, respectively. In contrast, acrylamide-intoxicated axons, using other parameters, have been shown to be dysfunctional. Changes in action potential characteristics are detected within 2–3 days (Anderson, 1981) following doses similar to the subacute dosing regimen of LoPachin. The author emphasized that the changes in relative area of the sural nerve action potential and in the relative refractory period were observed long before conduction velocity changes (due to neuropathy) would be expected. Therefore, at the higher dosing rates, the distal axons appear to be dysfunctional, yet morphologically intact. These distinctions should be tested systematically. The selective sural nerve electrophysiological dysfunction, in the absence of effects in the sciatic (Anderson, 1981), are consistent with axonal transport deficits as causative since the more distal axon segments are most vulnerable to intracellular transport defects. Kinesin knockout models provide additional justification to evaluate specific axon functional changes. Kinesin knockouts in Drosophila produce similar pathologies to ACR (Saxton et al., 1991; Hurd et al., 1996; Hurd and Saxton, 1996). Further analyses have identified resultant alterations in nerve
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conduction and neurotransmission (Hurd et al., 1996) and, similar to ACR, without any apparent changes in Na/K ATPase activity (Hurd et al., 1996). We are currently examining the effects of acrylamide on the voltage-gated ion channels of the nodes of Ranvier as a potential consequence of fast transport compromise and a mechanism of axonal dysfunction. Preliminary data demonstrate significant reductions in Na and K channels following either subacute or subchronic intoxication (Sickles, in press). The conclusion that ACR inhibition of fast transport has no pathophysiological significance ignores several reports of altered protein delivery following acrylamide intoxication. Fast transport of acetylcholinesterase was inhibited by ACR (Rasool and Bradley, 1978) and the quantity of a fast-transported form of acetylcholinesterase delivered to muscle was reduced by subacute ACR administration (Couraud et al., 1982). A systematic immunofluorescent study of synaptophysin content has identified a progressive decline in NMJ content with subacute ACR dosing and a correlation between NMJ synaptophysin content with neurotoxic subchronic dosing regimens (Sickles et al., 1998). Note that reductions in either (not both) axonal or terminal protein contents are necessary to support fast axonal transport reductions as a contributing factor in neurotoxicity. Since electrophysiological studies have identified dysfunctional axons or terminals by acrylamide, the most promising studies should be those that focus on proteins that are mechanistically related to these vital functions. Therefore, we conclude that insufficient data is available to alter our proposal of the covalent modification of kinesin and resultant compromise of fast axonal transport as a primary pathophysiological event leading to specific behavioral characteristics of acrylamide neurotoxicity. Neurobiologically-sound rationales, which are consistent with previous electrophysiological studies with acrylamide, can account for the lack of effects on some axonal functions. Future degeneration studies must include temporal studies on sensory and motor axon terminals. It must be determined if acrylamide produces differential effects on axonal protein content and if so, subsequent studies must identify the mechanisms by which these differences in acrylamide effects can lead to neurotoxicity. It must be determined whether critical physiological processes of axon excitability or terminal neurotransmission or mechanotransduction are altered. Dosing rate studies should be included, as they act as a discriminatory tool of relevant versus epiphenomenal events.
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Sabri and Spencer present evidence in support of alternative sites of action of acrylamide that could lead to neurotoxicity. They are convinced that altered energy metabolism is a contributing factor. We concluded that inhibition of energy production is one of the myriad of acrylamide effects, but a minor contributant. Inhibition of glycolytic enzyme such as glyceraldehyde-3-phosphate dehydrogenase by iodoacetate and of mitochondrial respiration by cyanide certainly block axonal transport (Ochs and Hollingsworth, 1971; Ochs and Smith, 1971). However, enzyme inhibition must be sufficient to produce a reduction in nerve ATP content to 1/2 normal before both axonal transport and axonal conduction were simultaneously blocked (Sabri and Ochs, 1972). The ACR data are not consistent with these requirements. ACR inhibition of glycolytic enzymes appears insufficient to inhibit the flux of metabolites through glycolysis (LoPachin et al., 1984) and there is no relationship between inhibition of glycolytic enzymes with the neurotoxicity of acrylamide analogues (Tanii and Hashimoto, 1985). Inhibition of oxidative enzymes does not compromise ATP production or oxygen consumption by mitochondria (Medrano and LoPachin, 1989; Sickles et al., 1990). Furthermore, several studies failed to identify significant changes in peripheral nerve ATP levels (Brimijoin and Hammond, 1985; Sickles and Pearson, 1987). The protective effect of pyruvate supplementation of diet is supportive of a significant effect of ACR on glycolysis (Sabri et al., 1989). However, the effect appears minor and certain aspects of the experimental design raise concerns. First, weekly time points of study were used. A single day difference in appearance of symptoms or degeneration, if it occurs near a sampling time, can exaggerate the pathophysiological significance. Secondly, the superimposition of caloric intake differences may have influenced the outcomes (Khanna et al., 1988, 1992). Lastly, the conclusions were based upon degeneration or b-glucuronidase activities, a characteristic of degeneration (Cavanagh and Nolan, 1982). Since LoPachin’s group demonstrated a lack of correlation of axonal degeneration with neurotoxicity (Lehning et al., 1998), mechanistic conclusions using these criteria are questionable. We endorse Sabri and Spencer’s suggestion to determine whether pyruvate administration alters the action of acrylamide on faAXT and would additionally suggest these studies to be conducted under different dosing rates in order to fully evaluate the contribution of compromised energy metabolism to neurotoxicity. We included in our hypothesis the possibility that acrylamide might act directly upon the retrograde
axonal transport motor dynein. Sabri and Spencer accurately indicate that there is no current evidence for such an action. We included this speculation based upon the observation of simultaneous reductions of retrograde vesicular traffic with anterograde transport whenever isolated axons are exposed to acrylamide. We cautioned that the same effects are observed with kinesin-specific antibodies and therefore, recommend a study of the direct effect on dynein to confirm or refute this aspect of the hypothesis. The additional unproven hypothesis of disrupted trophic signaling resulting in inappropriate protein synthesis was based upon reported differences in protein synthesis and transport composition (Miller and Spencer, 1985; Logan and McLean, 1988). It is surprising that Sabri and Spencer questioned this component since their group made the original speculation regarding the impact of altered retrograde transport (Miller and Spencer, 1985). Sabri and Spencer raised an interesting alternative outcome of faAXT compromise; namely, arrested organelles spilling their contents. Specifically they suggest accumulation of defective mitochondria spilling calcium, activating proteases and producing degeneration. This action would be accentuated over time at the paranodal and adjacent internodal regions, which are normal sites of Schwann cell phagocytosis of defective organelles. This suggestion is supported by acrylamide-induced reductions in calcium sequestering by microsomes (Xiwen et al., 1992). While mitochondria from ACR intoxicated animals do possess a darken matrix, ATP production and oxygen consumption were normal following acute exposures (Medrano and LoPachin, 1989; Sickles et al., 1990). Mitochondrial transmembrane ion gradients from subchronic exposed animals do change; but those from subacute studies were negative (LoPachin et al., 1992; Lehning et al., 1998). Since neurotoxicity is dose-independent and the altered mitochondrial function is associated with only subchronic exposure, mitochondrial dysfunction is an unlikely primary pathophysiological event. Furthermore, no changes in ATP levels of nerves are observed in chronically exposed animals (Brimijoin and Hammond, 1985), an anticipated outcome of mitochondrial damage. Furthermore, this hypothesis predicts degeneration as the endpoint precipitating neurotoxic symptoms. The dosing rate dependent production of axonal degeneration (Lehning et al., 1998) is non-supportive of this component of the energy hypothesis. It is possible that effects on energy production are below the threshold of detection, but that they are additive to kinesin (as well as other proteins)
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inhibitions. This may explain the subtle ameliorative effect of pyruvate on neurotoxicity. Sabri and Spencer emphasize the temporal difference in sensitivity of sensory and motor axons to acrylamide and that retrograde axonal transport may be affected earlier in motor than sensory axons. Numerous other factors, such as dosing rates (Lehning et al., 1998), axon firing rate (Satchell and Hersch, 1988), axonal caliber (Schaumburg et al., 1974), etc. contributes to variability in axonal sensitivity to acrylamide. Many of the studies used morphological studies of terminal degeneration. Since degeneration may not be necessary for development of symptoms (Lehning et al., 1998), we must look at functionality. Additionally, sensory axons or terminals may be more susceptible to axonal transport block. Therefore, differential effects of acrylamide on fast transport in different axons should be considered in future studies. Lastly, Sabri and Spencer speculate that actin or actin-associated membrane proteins may be a site of direct or indirect action resulting in the early alterations of subaxolemmal microfilaments, explaining the exquisite sensitivity of distal motor terminals and sensory receptors. We attempted to emphasize that axonal transport is not the only process that is compromised by acrylamide. Direct effects on microfilaments and/or their associated proteins are feasible and have received little consideration. Indirect effects on microfilaments via faAXT compromise would be limited to the membrane-associated proteins since actin is transported primarily in the slow transport system. We are in total agreement with the perspective that we need to focus upon functional aspects of the distal axons and their motor terminals or sensory endings since pathological changes are predictably later than functional losses. This is precisely our rationale for suggesting future studies that evaluate the concentrations of functionally important proteins in these distal sites coupled with both neurobehavioral and electrophysiological assays. It appears that the mechanism of acrylamide’s action in producing neurotoxicity remains unresolved, despite the substantial database. The interactions here have identified some consensus of effects and provide guidance for future investigations. Little argument exists that faAXT is significantly compromised by acrylamide. Kinesin appears to be a sensitive target that can lead to fast axonal transport compromise and the altered intra-axonal vesicular traffic changes are consistent with altered interaction of this motor with microtubules. The contribution of each protein inhibition to neurotoxicity must be assessed. One critical
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issue is whether the reductions in fast axonal transport have neurotoxicological relevance. Several critical questions must be addressed in future studies. Does the distal axon and/or terminals experience deficiencies due to compromised delivery? Are these deficiencies selective for certain proteins and therefore, certain functions? If so, what are the molecular mechanisms for such selectivity? If selectivity is demonstrated, which proteins and functions are most sensitive? Answers to these questions will not only validate or refute the faAXT hypothesis of acrylamide action but will be useful to furthering our understanding of neuronal intracellular transport and the consequences of toxic and disease associated compromises in this cellular process.
REFERENCES Anderson RJ. Selective effect on peripheral nerves after subchronic administration of acrylamide. Bull Environ Contam Toxicol 1981;27:888–93. Anderson RJ. Alterations in nerve and muscle compound action potentials after acute acrylamide administration. Environ Health Perspect 1982;44:153–7. Brimijoin WS, Hammond PI. Acrylamide neuropathy in the rat: effects on energy metabolism in sciatic nerve. Mayo Clin Proc 1985;60:3–8. Cavanagh JB, Nolan CC. The effects of acrylamide on bglucuronidase and acid phosphatase activities in rat sciatic nerve above and below a ligature. Neuropathol Appl Neurobiol 1982;8:465–76. Couraud JY, Giamberardino LD, Chretien M, Souyri F, Fardeau M. Acrylamide neuropathy and changes in the axonal transport and muscular content of the molecular forms of acetylcholinesterase. Muscle and Nerve 1982;5:302–12. Cyr JL, Sperry AO, Brady ST. The kinesin superfamily: variations on a theme. Adv Mol Cell Biol 1995;12:165–90. Goldstein BD. Acrylamide preferentially affects slowly adapting cutaneuous mechanoreceptors. Toxicol Appl Pharmacol 1985; 80:527–33. Goldstein BD, Lowndes HE. Group Ia primary afferent terminal defect in cats with acrylamide neuropathy. Neurotoxicology 1981;2:297–312. Hurd DD, Saxton WM. Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in drosophila. Genetics 1996;144:1075–85. Hurd DD, Stern M, Saxton WM. Mutation of the axonal transport motor kinesin enhances paralytic and suppresses shaker in drosophila. Genetics 1996;142:195–204. Khanna VK, Husain R, Seth PK. Low protein diet modifies acrylamide neurotoxicity. Toxicology 1988;49:395–401. Khanna VK, Husain R, Seth PK. Protein malnourishment: a predisposing factor in acrylamide toxicity in pregnant rats. J Toxicol Environ Health 1992;36:293–305. Lehning E, Gaughan CL, LoPachin Jr, RM. Acrylamide intoxication modifies in vitro responses of peripheral nerve axons to anoxia. J Peripher Nerv Syst 1997;2:165–74.
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Lehning EJ, LoPachin RM, Mathew J, Eichberg J. Changes in NaK ATpase and protein kinase C activities in peripheral nerve of acrylamide-treated rats. J Toxicol Environ Health 1994; 42:331–42. Lehning EJ, Persaud A, Dyer KR, Jortner BS, LoPachin RM. Biochemical and morphologic characterization of acrylamide peripheral neuropathy. Toxicol Appl Pharmacol 1998;151:211– 21. Logan MJ, McLean WG. A comparison of the effects of acrylamide and experimental diabetes on the retrograde axonal transport of proteins in the rat sciatic nerve: analysis by twodimensional polyacrylamide gel. J Neurochem 1988;50:183–9. LoPachin RM, Lehning E. Mechanism of calcium entry during axon injury and degeneration. Toxicol Appl Pharmacol 1997; 143:233–44. LoPachin RM, Saubermann AJ. Disruption of cellular elements and water in neurotoxicity: studies using electron probe X-ray microanalysis. Toxicol Appl Pharmacol 1990;106:355–74. LoPachin RM, Castiglia CM, Saubermann AJ. Acrylamide disrupts elemental composition. Toxicol Appl Pharmacol 1992;115:21–34. LoPachin RM, Moore RW, Menahan LA, Peterson RE. Glucosedependent lactate production by homogenates of neuronal tissues prepared from rats treated with 2,4-dithiobiuret, acrylamide, p-bromophenylacetylurea and 2,5-hexanedione. Neurotoxicology 1984;5:25–36. Lowndes HE, Baker T. Studies on drug-induced neuropathies. III. Motor nerve deficit in cats with experimental acrylamide neuropathy. Eur J Pharmacol 1976;35:177–84. Lowndes HE, Baker T, Cho E-S, Jortner BS. Position sensitivity of de-efferented muscle spindles in experimental acrylamide neuropathy. J Pharmacol Exp Ther 1978;205:40–8. Medrano CJ, LoPachin RM. Effects of acrylamide and 2,5hexanedione on brain mitochondrial respiration. Neurotoxicology 1989;10:249–56. Miller MS, Spencer PS. The mechanisms of acrylamide axonopathy. Annu Rev Pharmacol Toxicol 1985;25:643–66. Ochs S, Hollingsworth D. Dependence of fast axoplasmic transport in nerve on oxidative metabolism. J Neurochem 1971;18:107–14. Ochs S, Smith CB. Fast axoplasmic transport in mammalian nerve in vitro after block of glycolysis with iodoacetic acid. J Neurochem 1971;18:833–43.
Rasool CG, Bradley WG. Studies on axoplasmic transport of individual proteins: 1-acetylcholinesterase (AChE) in acrylamide neuropathy. J Neurochem 1978;31:419–25. Sabri MI, Ochs S. Relation of ATP and creatine phosphate to fast axoplasmic transport in mammalian nerve. J Neurochem 1972;19:2821–8. Sabri MI, Dairman W, Fenton M, Juhasz L, Ng T, Spencer PS. Effect of exogenous pyruvate on acrylamide neuropathy in rats. Brain Res 1989;483:1–11. Satchell PM, Hersch MI. Firing rate may be a determinant of nerve fibre vulnerability in axonopathies. J Neurol Sci 1988;87:289–97. Saxton WM, Hicks J, Goldstein LSB, Raff EC. Kinesin heavy chain is essential for viability and neuromuscular functions in drosophila, but mutants show no defects in mitosis. Cell 1991;64:1093–102. Schaumburg HH, Wisniewski HM, Spencer PS. Ultrastructural studies of the dying-back process. I. Peripheral nerve terminal and axon degeneration in systemic acrylamide intoxication. J Neuropathol Exp Neurol 1974;33:260–84. Sickles DW. Effects of acrylamide and 2,5-hexanedione on rat tibial nerve sodium and potassium channels. Toxicologist, in press. Sickles DW, Pearson JK. ATP, CP and axoplasmic transport in sciatic nerves of ACR, 2,5-HD and DMHD exposed rats. Toxicologist 1987;7:131. Sickles DW, Fowler SR, Testino AR. Effects of neurofilamentous axonopathy-producing neurotoxicants on in vitro production of ATP by brain mitochondria. Brain Res 1990;528:25–31. Sickles DW, Tsai N, Padilla S. Acrylamide produces a temporal and correlative decrease in fast anterogradely transported proteins in neuromuscular junctions. Toxicologist 1998;37:107. Sperry A, Sickles DW. Acrylamide inhibits kinesin related protein associated with the mitotic spindle. Toxicologist 2001;40:36–7. Sumner AJ, Asbury AK. Physiological studies of the dying-back phenomenon. Muscle stretch afferents in acrylamide neuropathy. Brain 1975;98:91–100. Tanii H, Hashimoto K. Effect of acrylamide and related compounds on glycolytic enzymes of rat brain. Toxicol Lett 1985;26:76–84. Xiwen H, Jing L, Tao C, Ke Y. Studies on biochemical mechanism of neurotoxicity induced by acrylamide in rats. Biomed Environ Sci 1992;5:276–81.
Response to commentaries
Fast Axonal Transport: a Site of Acrylamide Neurotoxicity: a Rebuttal Dale W. Sickles1,*, Derek Stone1, Marvin Friedman2 1
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912-2000, USA Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2757, USA 2
Alternative viewpoints to our hypothesis are offered in the accompanying critiques. There appears to be a general consensus that acrylamide compromises fast axonal transport. This represents advancement in our thinking since the myriad of apparently contradictory experimental results have been resolved by identification of the differences in experimental designs that contributed to the variability. Both respondents recognize kinesin, the anterograde motor protein, as a site of action in producing fast anterograde axonal transport (faAXT) reductions. Sabri and Spencer present data supporting additional sites of acrylamide action. The most controversial component of our hypothesis is whether the faAXT reductions are related to the development of behavioral changes characteristic of acrylamide neurotoxicity. The void of information regarding this issue must be addressed to adequately assess the contribution of faAXT compromise to axon (including the axon terminals) functionality. Lopachin’s group has developed a new methodological approach for evaluating the relevance of acrylamide effects to the development of behavioral symptoms (neurotoxicity). Administration of acrylamide at 50 mg/kg per day for up to 11 days (subacute, short-term high-dose, intraperitoneal) or at 21 mg/kg per day for up to 38 days (subchronic, long-term lowdose, oral) results in similar neurotoxicity, but with differential outcome in other parameters (LoPachin et al., 1992; Lehning et al., 1994, 1998). A primary assumption is that pathophysiologically relevant changes should be expressed at both dosing regimens. Although it is feasible that different mechanisms are * Corresponding author. Tel.: þ1-706-721-7393; fax: þ1-706-721-6839. E-mail address: [email protected] (D.W. Sickles).
operative at the two dosing regimens, the probability of two mechanisms leading to similar symptoms is low, making the approach useful. The subchronic regimen produces the classic pathologies associated with acrylamide intoxication; atrophy, swellings and degeneration (LoPachin et al., 1992; Lehning et al., 1998). In addition, Na/K ATPase quantity (unpublished results) as well as axolemmal rubidium transport (measure of Na/K ATPase activity) are reduced (Lehning et al., 1994, 1998). Following subacute intoxication there is little degeneration and no changes in Na/K ATPase activity (Lehning et al., 1994, 1998). The correlation of reductions in Na/K ATPase activity with degeneration suggests a cause–effect relationship (Lehning et al., 1998; LoPachin and Lehning, 1997). However, the differential dosing rate dependent expression of degeneration and axonal ion homeostasis, despite similar neurotoxicity, led to the conclusion that these two characteristics are dissociated from the primary pathophysiologic event (Lehning et al., 1998). Therefore, these data support the conclusion that tibial nerve axonal degeneration is non-essential for neurotoxicity. However, these data are insufficient to conclude that degeneration and/or fast axonal transport compromises are unrelated to neurotoxicity. More inclusive morphological analyses (identified below) are required to resolve the relationship of degeneration to neurotoxicity. Furthermore, consideration of a broader spectrum of axonal functions and their associated proteins is necessary to evaluate the contribution of fast axonal transport compromise to axon or terminal functionality as well as neurotoxicity. Axonal degeneration is a well-defined outcome of faAXT blockade, and we have previously hypothesized degeneration as an endpoint of acrylamide toxicity. However, within the current forum article, we have
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clearly indicated that compromises in axonal transport by acrylamide are not complete and are, in addition, transient. Therefore, degeneration is anticipated to be less prominent in acrylamide toxicity than with agents such as microtubule poisons or metabolic inhibitors, whose effects are more potent and/or more prolonged (due to replenishment of these targets via slow axonal transport). The data demonstrating a lack of degeneration by subacute dosing rates are in distal nerves (LoPachin et al., 1992; Lehning et al., 1998). They do not include the sensory and motor terminals, the most distal elements that are predictably the most vulnerable to fast axonal transport deficits. Indeed, numerous studies have identified early pathologies of Pacinian corpuscles (emphasized in the accompanying critique by Sabri and Spencer); muscle spindle afferents and the motor end plates (Sumner and Asbury, 1975; Schaumburg et al., 1974; Lowndes et al., 1978; Lowndes and Baker, 1976; Goldstein and Lowndes, 1981). LoPachin (data presented in critique) apparently found CNS axonal degeneration primarily associated with low-rate, high-dose intoxication. However, they state that ‘‘regardless of the daily exposure rate (50 mg/ kg per day 5–11 days, i.p. or 21 mg/kg day 7–38 days, p.o.), ACR induced early nerve terminal degeneration’’. Therefore, there appears to be substantial terminal degeneration in the CNS and PNS, which can account for neurotoxicity. LoPachin appears to make a distinction between axonal versus terminal degeneration. However, in terms of evaluating the impact of fast axonal transport inhibition to pathological and neurotoxicological outcomes, the terminal is conclusively part of the axon and should not be semantically separated. We emphasize that degeneration is not required for inhibition of fast transport inhibition to be a primary neurotoxic mechanism. Fast transport compromise can lead to axonal and/or terminal dysfunction prior to, or in the absence of, degeneration. Earlier electrophysiological data identified dysfunction of axons and terminals prior to morphological changes (Anderson, 1981, 1982; Goldstein, 1985; Sumner and Asbury, 1975). Therefore, conclusions regarding relevance of a specific morphological effect to neurotoxicity are potentially overstated. LoPachin has concluded that distal axons of subacute acrylamide-intoxicated animals are not dysfunctional. This conclusion is based upon normal axolemmal Na/K ATPase and axoplasmic ion composition in tibial nerves of high ACR dosing rate rats (LoPachin et al., 1992; Lehning et al., 1994, 1998), despite the presence of neurotoxic symptoms. Addi-
tionally, intact mitochondrial transmembrane ion gradients and energy production were observed and axons were capable of regulating element composition. From these results, it is reasonable to conclude that homeostatic mechanisms, including total ion composition and energy metabolism, in tibial nerve are insufficiently changed by acrylamide to be a pathophysiological cause of neurotoxicity. However, these analyses are limited and do not include determination of axonal functions such as action potential propagation or terminal neurotransmission. As a consequence, they provide little information relevant to evaluating the outcome of faAXT compromise by acrylamide to neurotoxicity. Furthermore, these conclusions are contradictory to their previous observations of compromised ability of ACR exposed axons to recover from anoxia (Lehning et al., 1997). Several specific issues are of concern. The potential for an aerobic enzyme inhibition to contribute to neurotoxicity is considered below. However, we note here that reductions in axonal glycolytic enzyme activity may occur through direct inhibition or by changes in slow transport, not by reduced faAXT. Mitochondria are conveyed in the fast system but are capable of sustained longevity in the absence of translocation (fast transport). Therefore, aerobic metabolism and mitochondrial transmembrane ion gradients would be compromised only with direct toxicant action on this organelle. Since previous studies failed to identify an effect on mitochondrial respiration or ATP production (Medrano and LoPachin, 1989; Sickles et al., 1990) and therefore, are anticipated to remain active in the absence of translocation, the lack of effect of subacute doses on these functions is not surprising. Therefore, the absence of mitochondrial ion gradient and oxidative metabolism effects in the subacute animals are insufficient to discredit the fast axonal transport compromise as contributory. It is reasonable to anticipate compromise of fast axonal transport to cause dysfunction of all fast-transported, axolemmal proteins. However, differential protein turnover rates or differences in quantity of specific protein transport compared to functional requirements may result in differential sensitivity of various functions (as observed in kinesin knockout models discussed in greater detail below). Furthermore, differential efficacy of ACR inhibition of different kinesin family members, each with its own specific cargo, is possible. LoPachin indicates that similar sulfhydryl dependency of other kinesin motors has not been demonstrated and that differential sensitivity is doubtful. We have recently identified ACR inhibition of
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kinesin motors in two other kinesin subfamilies (Sperry and Sickles, 2001). Sequence differences within kinesin superfamily common domains, as well as higher variability within non-homologous domains (Cyr et al., 1995), are typically associated with differential sensitivity. We suggest that future studies assess the dose-response of acrylamide on the various kinesin superfamily members. We have concerns regarding the interpretation of electron probe microanalysis (EPMA) data, used to measure axon ion content, since it has some limitations. It measures total ion content (LoPachin and Saubermann, 1990). Therefore, functional differences in ionized species can go undetected under the large volume of total ion content. The toxicant-induced changes observed with this technique following acrylamide exposure are, primarily, increases in variability, rather than changes in means, and no spatial or temporal patterns were observed (LoPachin et al., 1992). These results are difficult to reconcile mechanistically. Changes in mean values in other experimental conditions do appear coincident with axonal degeneration (LoPachin and Lehning, 1997). Taken together, these observations suggest strong resistance to damage of these processes and/or insensitivity of the assay. Lastly, we emphasize that EPMA does not measure functional attributes specific to axonal or terminal function such as membrane excitability and communication, respectively. In contrast, acrylamide-intoxicated axons, using other parameters, have been shown to be dysfunctional. Changes in action potential characteristics are detected within 2–3 days (Anderson, 1981) following doses similar to the subacute dosing regimen of LoPachin. The author emphasized that the changes in relative area of the sural nerve action potential and in the relative refractory period were observed long before conduction velocity changes (due to neuropathy) would be expected. Therefore, at the higher dosing rates, the distal axons appear to be dysfunctional, yet morphologically intact. These distinctions should be tested systematically. The selective sural nerve electrophysiological dysfunction, in the absence of effects in the sciatic (Anderson, 1981), are consistent with axonal transport deficits as causative since the more distal axon segments are most vulnerable to intracellular transport defects. Kinesin knockout models provide additional justification to evaluate specific axon functional changes. Kinesin knockouts in Drosophila produce similar pathologies to ACR (Saxton et al., 1991; Hurd et al., 1996; Hurd and Saxton, 1996). Further analyses have identified resultant alterations in nerve
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conduction and neurotransmission (Hurd et al., 1996) and, similar to ACR, without any apparent changes in Na/K ATPase activity (Hurd et al., 1996). We are currently examining the effects of acrylamide on the voltage-gated ion channels of the nodes of Ranvier as a potential consequence of fast transport compromise and a mechanism of axonal dysfunction. Preliminary data demonstrate significant reductions in Na and K channels following either subacute or subchronic intoxication (Sickles, in press). The conclusion that ACR inhibition of fast transport has no pathophysiological significance ignores several reports of altered protein delivery following acrylamide intoxication. Fast transport of acetylcholinesterase was inhibited by ACR (Rasool and Bradley, 1978) and the quantity of a fast-transported form of acetylcholinesterase delivered to muscle was reduced by subacute ACR administration (Couraud et al., 1982). A systematic immunofluorescent study of synaptophysin content has identified a progressive decline in NMJ content with subacute ACR dosing and a correlation between NMJ synaptophysin content with neurotoxic subchronic dosing regimens (Sickles et al., 1998). Note that reductions in either (not both) axonal or terminal protein contents are necessary to support fast axonal transport reductions as a contributing factor in neurotoxicity. Since electrophysiological studies have identified dysfunctional axons or terminals by acrylamide, the most promising studies should be those that focus on proteins that are mechanistically related to these vital functions. Therefore, we conclude that insufficient data is available to alter our proposal of the covalent modification of kinesin and resultant compromise of fast axonal transport as a primary pathophysiological event leading to specific behavioral characteristics of acrylamide neurotoxicity. Neurobiologically-sound rationales, which are consistent with previous electrophysiological studies with acrylamide, can account for the lack of effects on some axonal functions. Future degeneration studies must include temporal studies on sensory and motor axon terminals. It must be determined if acrylamide produces differential effects on axonal protein content and if so, subsequent studies must identify the mechanisms by which these differences in acrylamide effects can lead to neurotoxicity. It must be determined whether critical physiological processes of axon excitability or terminal neurotransmission or mechanotransduction are altered. Dosing rate studies should be included, as they act as a discriminatory tool of relevant versus epiphenomenal events.
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Sabri and Spencer present evidence in support of alternative sites of action of acrylamide that could lead to neurotoxicity. They are convinced that altered energy metabolism is a contributing factor. We concluded that inhibition of energy production is one of the myriad of acrylamide effects, but a minor contributant. Inhibition of glycolytic enzyme such as glyceraldehyde-3-phosphate dehydrogenase by iodoacetate and of mitochondrial respiration by cyanide certainly block axonal transport (Ochs and Hollingsworth, 1971; Ochs and Smith, 1971). However, enzyme inhibition must be sufficient to produce a reduction in nerve ATP content to 1/2 normal before both axonal transport and axonal conduction were simultaneously blocked (Sabri and Ochs, 1972). The ACR data are not consistent with these requirements. ACR inhibition of glycolytic enzymes appears insufficient to inhibit the flux of metabolites through glycolysis (LoPachin et al., 1984) and there is no relationship between inhibition of glycolytic enzymes with the neurotoxicity of acrylamide analogues (Tanii and Hashimoto, 1985). Inhibition of oxidative enzymes does not compromise ATP production or oxygen consumption by mitochondria (Medrano and LoPachin, 1989; Sickles et al., 1990). Furthermore, several studies failed to identify significant changes in peripheral nerve ATP levels (Brimijoin and Hammond, 1985; Sickles and Pearson, 1987). The protective effect of pyruvate supplementation of diet is supportive of a significant effect of ACR on glycolysis (Sabri et al., 1989). However, the effect appears minor and certain aspects of the experimental design raise concerns. First, weekly time points of study were used. A single day difference in appearance of symptoms or degeneration, if it occurs near a sampling time, can exaggerate the pathophysiological significance. Secondly, the superimposition of caloric intake differences may have influenced the outcomes (Khanna et al., 1988, 1992). Lastly, the conclusions were based upon degeneration or b-glucuronidase activities, a characteristic of degeneration (Cavanagh and Nolan, 1982). Since LoPachin’s group demonstrated a lack of correlation of axonal degeneration with neurotoxicity (Lehning et al., 1998), mechanistic conclusions using these criteria are questionable. We endorse Sabri and Spencer’s suggestion to determine whether pyruvate administration alters the action of acrylamide on faAXT and would additionally suggest these studies to be conducted under different dosing rates in order to fully evaluate the contribution of compromised energy metabolism to neurotoxicity. We included in our hypothesis the possibility that acrylamide might act directly upon the retrograde
axonal transport motor dynein. Sabri and Spencer accurately indicate that there is no current evidence for such an action. We included this speculation based upon the observation of simultaneous reductions of retrograde vesicular traffic with anterograde transport whenever isolated axons are exposed to acrylamide. We cautioned that the same effects are observed with kinesin-specific antibodies and therefore, recommend a study of the direct effect on dynein to confirm or refute this aspect of the hypothesis. The additional unproven hypothesis of disrupted trophic signaling resulting in inappropriate protein synthesis was based upon reported differences in protein synthesis and transport composition (Miller and Spencer, 1985; Logan and McLean, 1988). It is surprising that Sabri and Spencer questioned this component since their group made the original speculation regarding the impact of altered retrograde transport (Miller and Spencer, 1985). Sabri and Spencer raised an interesting alternative outcome of faAXT compromise; namely, arrested organelles spilling their contents. Specifically they suggest accumulation of defective mitochondria spilling calcium, activating proteases and producing degeneration. This action would be accentuated over time at the paranodal and adjacent internodal regions, which are normal sites of Schwann cell phagocytosis of defective organelles. This suggestion is supported by acrylamide-induced reductions in calcium sequestering by microsomes (Xiwen et al., 1992). While mitochondria from ACR intoxicated animals do possess a darken matrix, ATP production and oxygen consumption were normal following acute exposures (Medrano and LoPachin, 1989; Sickles et al., 1990). Mitochondrial transmembrane ion gradients from subchronic exposed animals do change; but those from subacute studies were negative (LoPachin et al., 1992; Lehning et al., 1998). Since neurotoxicity is dose-independent and the altered mitochondrial function is associated with only subchronic exposure, mitochondrial dysfunction is an unlikely primary pathophysiological event. Furthermore, no changes in ATP levels of nerves are observed in chronically exposed animals (Brimijoin and Hammond, 1985), an anticipated outcome of mitochondrial damage. Furthermore, this hypothesis predicts degeneration as the endpoint precipitating neurotoxic symptoms. The dosing rate dependent production of axonal degeneration (Lehning et al., 1998) is non-supportive of this component of the energy hypothesis. It is possible that effects on energy production are below the threshold of detection, but that they are additive to kinesin (as well as other proteins)
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inhibitions. This may explain the subtle ameliorative effect of pyruvate on neurotoxicity. Sabri and Spencer emphasize the temporal difference in sensitivity of sensory and motor axons to acrylamide and that retrograde axonal transport may be affected earlier in motor than sensory axons. Numerous other factors, such as dosing rates (Lehning et al., 1998), axon firing rate (Satchell and Hersch, 1988), axonal caliber (Schaumburg et al., 1974), etc. contributes to variability in axonal sensitivity to acrylamide. Many of the studies used morphological studies of terminal degeneration. Since degeneration may not be necessary for development of symptoms (Lehning et al., 1998), we must look at functionality. Additionally, sensory axons or terminals may be more susceptible to axonal transport block. Therefore, differential effects of acrylamide on fast transport in different axons should be considered in future studies. Lastly, Sabri and Spencer speculate that actin or actin-associated membrane proteins may be a site of direct or indirect action resulting in the early alterations of subaxolemmal microfilaments, explaining the exquisite sensitivity of distal motor terminals and sensory receptors. We attempted to emphasize that axonal transport is not the only process that is compromised by acrylamide. Direct effects on microfilaments and/or their associated proteins are feasible and have received little consideration. Indirect effects on microfilaments via faAXT compromise would be limited to the membrane-associated proteins since actin is transported primarily in the slow transport system. We are in total agreement with the perspective that we need to focus upon functional aspects of the distal axons and their motor terminals or sensory endings since pathological changes are predictably later than functional losses. This is precisely our rationale for suggesting future studies that evaluate the concentrations of functionally important proteins in these distal sites coupled with both neurobehavioral and electrophysiological assays. It appears that the mechanism of acrylamide’s action in producing neurotoxicity remains unresolved, despite the substantial database. The interactions here have identified some consensus of effects and provide guidance for future investigations. Little argument exists that faAXT is significantly compromised by acrylamide. Kinesin appears to be a sensitive target that can lead to fast axonal transport compromise and the altered intra-axonal vesicular traffic changes are consistent with altered interaction of this motor with microtubules. The contribution of each protein inhibition to neurotoxicity must be assessed. One critical
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issue is whether the reductions in fast axonal transport have neurotoxicological relevance. Several critical questions must be addressed in future studies. Does the distal axon and/or terminals experience deficiencies due to compromised delivery? Are these deficiencies selective for certain proteins and therefore, certain functions? If so, what are the molecular mechanisms for such selectivity? If selectivity is demonstrated, which proteins and functions are most sensitive? Answers to these questions will not only validate or refute the faAXT hypothesis of acrylamide action but will be useful to furthering our understanding of neuronal intracellular transport and the consequences of toxic and disease associated compromises in this cellular process.
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