γ-Diketone neuropathy: axon atrophy and the role of cytoskeletal protein adduction

https://doi.org/10.1016/j.taap.2004.03.008Get rights and content

Abstract

Multifocal giant neurofilamentous axonal swellings and secondary distal degeneration have been historically considered the hallmark features of γ-diketone neuropathy. Accordingly, research conducted over the past 25 years has been directed toward discerning mechanisms of axonal swelling. However, this neuropathological convention has been challenged by recent observations that swollen axons were an exclusive product of long-term 2,5-hexanedione (HD) intoxication at lower daily dose-rates (e.g., 175 mg/kg/day); that is, higher HD dose-rates (e.g., 400 mg/kg/day) produced neurological deficits in the absence of axonal swellings. The observation that neurological toxicity can be expressed without axonal swelling suggests that this lesion is not an important pathophysiological event. Instead, several research groups have now shown that axon atrophy is prevalent in nervous tissues of laboratory animals intoxicated over a wide range of HD dose-rates. The well-documented nerve conduction defects associated with axon atrophy, in conjunction with the temporal correspondence between this lesion and the onset of neurological deficits, strongly suggest that atrophy has pathophysiological significance. In this commentary, we present evidence that supports a pathognomonic role for axon atrophy in γ-diketone neuropathy and suggests that the functional consequences of this lesion mediate the corresponding neurological toxicity. Previous research has demonstrated that HD interacts with proteins via formation of pyrrole adducts. We therefore discuss the possibility that this chemical process is essential to the mechanism of atrophy. Evidence presented in this review suggests that “distal axonopathy” is an inaccurate classification and future nosological schemes should be based on the apparent primacy of axon atrophy.

Introduction

Giant neurofilamentous swelling of large myelinated axons in the PNS and CNS has been historically considered the hallmark lesion induced by the industrial hexacarbon chemicals, n-hexane and methyl n-butyl ketone (MBK), and by their common active γ-diketone metabolite, 2,5-hexanedione (HD; Couri and Milks, 1982, Krasavage et al., 1980, Spencer et al., 1980a, Spencer et al., 1980b). On the basis of an assumed neurotoxicological importance, giant axonal swellings have been the focus of most mechanistic research conducted over the past 30 years (reviewed in DeCaprio, 1987, DeCaprio, 2000, Graham et al., 1991, Graham et al., 1995, LoPachin and Lehning, 1997, Sayre et al., 1985, Spencer et al., 1980a, Spencer et al., 1980b). Axon atrophy, however, has also been identified as a morphological feature of γ-diketone neuropathy (e.g., see Lehning et al., 2000, Monaco et al., 1985, Yagi, 1994). Although it was assumed by early investigators that loss of axon caliber was secondary to proximal accumulation of neurofilaments (NFs) in giant swellings Brown et al., 1978, Spencer and Schaumburg, 1977a, Spencer et al., 1980a, more recent quantitative morphometric studies have demonstrated that these axonal lesions are independent phenomena Lehning et al., 1995, Lehning et al., 2000, LoPachin et al., 2003a. Quantitative studies have also shown that axonal atrophy was a prevalent effect that occurred during the early stages of γ-diketone intoxication over a wide range of daily dose-rates (100–400 mg/kg/day). In contrast, axonal swellings were scarce in both PNS and CNS, and their expression was restricted to intoxication at lower dose-rates (i.e., 100–250 mg/kg/day; Lehning et al., 1995, Lehning et al., 2000, LoPachin et al., 2003a). Together, these observations suggest that axon atrophy is a relevant component of the pathophysiological process that leads to neurological toxicity, whereas neurofilamentous swelling is of unclear neuropathogenic significance (LoPachin and Lehning, 1997).

The molecular mechanism by which HD causes either axon atrophy or swelling is not understood. However, it is known that NF triplet proteins are an important determinant of axon caliber Friede and Samorajski, 1970, Hoffman et al., 1988, Muma and Hoffman, 1993 and that HD reacts with these cytoskeletal proteins (e.g., DeCaprio et al., 1982, Graham et al., 1982). Indeed, research conducted over the past 20 years has demonstrated that HD interacts with ε-amine groups on lysine residues of NFs and other proteins to form N-substituted 2,5-dimethylpyrrole adducts (e.g., DeCaprio et al., 1982). This pyrrole-forming reaction is now considered to be the first step in γ-diketone neuropathy. Work by Graham et al., 1991, Graham et al., 1995 has suggested that once formed, pyrrole adducts can undergo secondary oxidative reactions that yield cross-linked proteins. This research has lead to the hypothesis that protein cross-linking is an important pathophysiological step in the neurotoxic mechanism of γ-diketone action. Whereas additional molecular details are needed (e.g., extent of cross-linking vs. pyrrole formation, location of adducted lysine residues), research to date suggests that adduction of NF proteins is involved in γ-diketone-induced alterations in axon diameter (atrophy or swelling).

The purpose of this commentary is to present evidence supporting axonal atrophy as the primary morphological lesion associated with γ-diketone neuropathy. The functional consequences of atrophy and how ensuing dysfunction might be related to the development of neurological deficits are also discussed. In this commentary, we will consider the possibility that HD adduction of cytoskeletal proteins plays a mechanistic role in axon atrophy. We begin with a background discussion of the classic morphological and neurological changes induced by γ-diketone exposure. This will form a basis for developing the thesis that axon atrophy and resulting neurophysiological dysfunction are necessary events in γ-diketone neurotoxicity.

Section snippets

Classical concepts of γ-diketone neurotoxicity: neurological deficits and morphological lesions

Long-term, low dose-rate exposure or short-term, high dose-rate intoxication of laboratory animals with HD produces decreases in body weight and changes in several neurological parameters including gait abnormalities (ataxia) and reductions in hindlimb skeletal muscle strength Jortner and Ehrich, 1993, LoPachin et al., 2002a, Shell et al., 1992, Spencer and Schaumburg, 1977a. Several lines of evidence suggest that induction of neurological toxicity by HD conforms in principle to Haber's Rule

Giant neurofilamentous axonal swellings

Neurofilamentous swelling is the presumed morphological manifestation of disrupted molecular processes in affected axons of γ-diketone-intoxicated animals or humans (reviewed in DeCaprio, 1985, DeCaprio, 1987, DeCaprio, 2000, Graham et al., 1991, Graham et al., 1995). It has been assumed that the focal increase in caliber associated with the swelling promotes axonal dysfunction and eventual degeneration Spencer et al., 1980a, Spencer et al., 1980b. Consequently, neurofilamentous swellings have

NF protein turnover as a determinant of axon caliber

In the mature neuron, axon caliber is correlated with cross-sectional neurofilament (NF) protein content, which is regulated by perikaryal gene expression/synthesis and by axonal triplet protein turnover Friede and Samorajski, 1970, Hoffman et al., 1988, Muma and Hoffman, 1993, Sakaguchi et al., 1993. NFs are composed of three subunits that assemble as obligate heteropolymers: light NF (NF-L, 68–70 kDa), midsize NF (NF-M; 140–160 kDa), and heavy NF (NF-H; 190–200 kDa; reviewed in Muma and

Mechanisms of γ-diketone axon atrophy

It is not known how HD adduction of NF subunits or possibly other cytoskeletal proteins might produce axon atrophy. Previous studies of PNS and CNS from HD-intoxicated animals have revealed significant reductions in NF subunit protein contents Carden et al., 1986, Chiu et al., 2000, DeCaprio and O'Neill, 1985, Karlsson et al., 1991, Lapadula et al., 1986, Lapadula et al., 1988, LoPachin et al., 2003a, LoPachin et al., 2003b, LoPachin et al., 2003c, Watson et al., 1991. These observations, in

Conclusions

The evidence presented in this review suggests that a redefinition of γ-diketone neuropathy is necessary. Clearly, the labels “giant neurofilamentous axonopathy” or “distal axonopathy” are misleading, and therefore, future nosological schemes should take into consideration the apparent primacy of axon atrophy. Determining the relative neurotoxic risk for different chemicals that have industrial, agricultural, or household application is, in part, dependent upon assessment of classic distal

Acknowledgements

This research was supported by NIH Grants from NIEHS to R.M.L. (RO1 ES07912-07) and to A.P.D. (RO1 ES05172).

References (142)

  • D.G Graham et al.

    Studies of the molecular pathogenesis of hexane neuropathy: II. Evidence that pyrrole derivitization of lysyl residues leads to protein crosslinking

    Toxicol. Appl. Pharmacol.

    (1982)
  • S Hisanaga et al.

    Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules

    J. Biol. Chem.

    (1991)
  • K.I Horan et al.

    Hexanedione effects on protein phosphorylation in rat peripheral nerve

    Brain Res.

    (1989)
  • C Jung et al.

    C-terminal phosphorylation of the high molecular weight neurofilament subunit correlates with decreased neurofilament axonal transport velocity

    Brain Res.

    (2000)
  • W.J Krasavage et al.

    The relative neurotoxicity of methyl n-butyl ketone, n-hexane and their metabolites

    Toxicol. Appl. Pharmacol.

    (1980)
  • S Kumar et al.

    Relating interactions between neurofilaments to the structure of axonal neurofilament distributions through polymer brush models

    Biophys. J.

    (2002)
  • D.M Lapadula et al.

    Evidence for multiple mechanisms responsible for 2,5-hexanedione-induced neuropathy

    Brain Res.

    (1988)
  • E.J Lehning et al.

    Axonal atrophy is a specific component of 2,5-hexanedione peripheral neuropathy

    Toxicol. Appl. Pharmacol.

    (1995)
  • E.J Lehning et al.

    γ-Diketone peripheral neuropathy: I. Quantitative morphometric analyses of axonal atrophy and swelling

    Toxicol. Appl. Pharmacol.

    (2000)
  • J.F Leterrier et al.

    Mechanical effects of neurofilament cross-bridges

    J. Biol. Chem.

    (1996)
  • R.M LoPachin et al.

    Rate of neurotoxicant exposure determines morphological manifestations

    Toxicol. Appl. Pharmacol.

    (2000)
  • R.M LoPachin et al.

    Neurological evaluation of toxic axonopathies in rats: acrylamide and 2,5-hexanedione

    Neurotoxicology

    (2002)
  • R.M LoPachin et al.

    Nerve terminals as the primary site of acrylamide action: a hypothesis

    Neurotoxicology

    (2002)
  • R.M LoPachin et al.

    γ-Diketone central neuropathy: quantitative morphometric analysis of axons in rat spinal cord white matter regions and nerve roots

    Toxicol. Appl. Pharmacol.

    (2003)
  • R.M LoPachin et al.

    Acrylamide axonopathy revisited

    Toxicol. Appl. Pharmacol.

    (2003)
  • H Miyasaka et al.

    Interaction of the tail domain of high molecular weight subunits of neurofilaments with the COOH-terminal region of tubulin and its regulation of τ protein kinase II

    J. Biol. Chem.

    (1993)
  • S Monaco et al.

    Axonal transport of neurofilament is accelerated in peripheral nerve during 2,5-hexanedione intoxication

    Brain Res.

    (1989)
  • S Monaco et al.

    Giant axonopathy characterized by intermediate location of axonal enlargements and acceleration of neurofilament transport

    Brain Res.

    (1990)
  • N.A Muma et al.

    Neurofilaments are intrinsic determinants of axonal caliber

    Micron

    (1993)
  • S Ochs et al.

    The origin and nature of beading: a reversible transformation of the shape of nerve fibers

    Prog. Neurobiol.

    (1997)
  • M Pappolla et al.

    Carbon disulfide axonopathy. Another experimental model characterized by acceleration of neurofilament transport and distinct changes of axonal size

    Brain Res.

    (1987)
  • R.J Anderson et al.

    Electrophysiological deficits in peripheral nerve as a discriminator of early hexacarbon neurotoxicity

    J. Toxicol. Environ. Health

    (1984)
  • D.C Anthony et al.

    The spatio-temporal pattern of the axonopathy associated with the neurotoxicity of 3,4-dimethyl-2,5-hexanedione in the rat

    J. Neuropathol. Exp. Neurol.

    (1983)
  • D.C Anthony et al.

    The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,5-hexanedione: I. Accelerated clinical neuropathy is accompanied by more proximal axonal swellings

    Toxicol. Appl. Pharmacol.

    (1983)
  • H Braendgaard et al.

    Anterograde components of axonal transport in motor and sensory nerves in experimental 2,5-hexanedione neuropathy

    J. Neurochem.

    (1986)
  • H.G Brown et al.

    Entropic exclusion by neurofilament sidearms: a mechanism for maintaining interfilament spacing

    Biochemistry

    (1997)
  • M.J Brown et al.

    Nerve conduction slowing precedes demyelination in experimental n-butyl ketone (MBK) neuropathy

  • J.D Burek et al.

    Subchronic toxicity of acrylamide administered to rats in drinking water followed by up to 144 days of recovery

    J. Environ. Pathol. Toxicol.

    (1980)
  • M.J Carden et al.

    2,5-Hexanedione neuropathy is associated with the covalent crosslinking of neurofilament proteins

    Neurochem. Pathol.

    (1986)
  • J.B Cavanagh

    The pattern of recovery of axons in the nervous system of rats following 2,5-hexanediol intoxication: a question of rheology?

    Neuropathol. Appl. Neurobiol.

    (1982)
  • J Chen et al.

    The C-terminal tail domain of neurofilament protein-H NF-H forms the crossbridges and regulates neurofilament bundle formation

    J. Cell Sci.

    (2000)
  • G.Y Ching et al.

    Overexpression of a-internexin causes abnormal neurofilamentous accumulation and motor coordination deficits in transgenic mice

    J. Neurosci.

    (1999)
  • D Couri et al.

    Toxicity and the metabolism of the neurotoxic hexacarbons n-hexane, 2-hexanone and 2,5-hexanedione

    Annu. Rev. Pharmacol. Toxicol.

    (1982)
  • J.G Davenport et al.

    Giant axonal neuropathy caused by industrial chemicals: neurofilamentous axonal masses in man

    Neurology

    (1976)
  • A.P DeCaprio

    Mechanisms of in vitro pyrrole adduct autoxidation in 2,5-hexanedione-treated protein

    Mol. Pharmacol.

    (1986)
  • A.P DeCaprio

    n-Hexane neurotoxicity. A mechanism involving pyrrole adduct formation in axonal cytoskeletal protein

    Neurotoxicology

    (1987)
  • A.P DeCaprio

    n-Hexane, metabolites and derivative

  • A.P DeCaprio et al.

    Mechanism of formation and quantitation of imines, pyrroles, and stable nonpyrrole adducts in 2,5-hexanedione-treated protein

    Mol. Pharmacol.

    (1987)
  • R.L Friede et al.

    Axon caliber related to neurofilaments and microtubules in sciatic nerve fibers of rats and mice

    Anat. Rec.

    (1970)
  • E Fuchs et al.

    A structural scaffolding of intermediate filaments in health and disease

    Science

    (1998)
  • Cited by (30)

    • 2,5-hexanedione-induced deregulation of axon-related microRNA expression in rat nerve tissues

      2020, Toxicology Letters
      Citation Excerpt :

      Axonopathy is the most commonly observed lesion in toxic peripheral neuropathies (Landowski et al., 2016; Valentine, 2019). Based on early morphological studies, the primary neuropathological manifestation of HD-induced neuropathy appeared to be retrograde myelinated axon degeneration in the peripheral central nervous systems (LoPachin and Lehning, 1997; Lehning et al., 2000; LoPachin and DeCaprio, 2004). Axon atrophy involves disruption of the neurophysiological processes responsible for maintaining axon caliber in neurons.

    • The imbalance between dynamic and stable microtubules underlies neurodegeneration induced by 2,5-hexanedione

      2020, Biochimica et Biophysica Acta - Molecular Basis of Disease
      Citation Excerpt :

      Interestingly, 2,5-HD strongly affects neuronal morphology already at 2 mM, whereas mitochondrial defects are detectable only at 20 mM, allowing us to speculate that they could be a possible consequence of the impairment of other frailer systems. In accordance with previous works suggesting that 2,5-HD affects cytoskeletal components [28], we hypothesized that the observed morphological changes probably derived from dysfunction of the overall cytoskeleton. Thus, the characterization of all its components (actin filaments, NFs and MTs) after 24 h of toxin treatment was carried out with both biochemical and immunocytochemical approaches (Fig. 3).

    • Mechanisms of soft and hard electrophile toxicities

      2019, Toxicology
      Citation Excerpt :

      The swellings were presumed to be responsible for the γ-diketone axonopathy associated with subchronic occupational exposure to n-hexane (DeCaprio et al., 1997; Graham et al., 1991). Accordingly, previous studies (reviewed in LoPachin and De Caprio, 2004, 2005) demonstrated the presence of abundant high molecular weight neurofilament derivatives in nervous tissue preparations from 2,5-HD-intoxicated animals. However, more recent research has shown that these abnormal proteins were common to nervous tissue samples from both control and 2,5-HD intoxicated animals.

    • Toxic neuropathies: Mechanistic insights based on a chemical perspective

      2015, Neuroscience Letters
      Citation Excerpt :

      Both parent compounds are used in fabric manufacturing and have been associated with several human outbreaks of neuropathy following subchronic occupational exposure [1]. Early studies of HD and ACR neurotoxicity were based on the premise that distal axon regions were sites of toxicant action and that axonopathy was the pathognomonic outcome of a specific mechanism; e.g., inhibition of axolemmal Na pumps [3,4]. Because axonal swellings and degeneration were assumed to be causally related to the onset of neurotoxicity, substantial effort was devoted to deciphering the respective mechanisms [5,6].

    View all citing articles on Scopus
    View full text