The Role of Protein Adduction in Toxic Neuropathies of Exogenous and Endogenous Origin

The peripheral (axonal) neuropathy associated with repeated exposure to aliphatic and aromatic solvents that form protein-reactive γ-diketones shares some clinical and neuropathological features with certain metabolic neuropathies, including type-II diabetic neuropathy and uremic neuropathy, and with the largely sub-clinical nerve damage associated with old age. These conditions may be linked by metabolites that adduct and cross-link neuroproteins required for the maintenance of axonal transport and nerve fiber integrity in the peripheral and central nervous system.


Introduction
This paper discusses the presence of neurotoxic γ-diketones in normal subjects and in peripheral neuropathies resulting from overexposure to certain hydrocarbon solvents. Also considered is the possibility that γ-diketones of endogenous origin contribute to the etiology and pathogenesis of certain metabolic neuropathies, including type-II diabetic neuropathy and uremic neuropathy, and to the largely subclinical peripheral nerve changes that accumulate with age.

Experimental Animal Neuropathy
The human disease is readily and accurately reproduced in animals (rats, cats) treated systemically with aliphatic C6 compounds that form γ-diketone metabolites [23][24][25]. In rats treated subcutaneously with 2,5-hexanedione, the onset of clinical signs is insidious and occurs symmetrically in the hindlimbs. Nerve fiber degeneration is prominent in the distal tibial nerve and its branches, with few changes in more proximal regions. Lumbosacral motor neurons and dorsal root ganglion cells appear histologically normal. Myelinated tibial nerve fibers show focal distal axonal swellings, often on the proximal sides of nodes of Ranvier, secondary demyelination and remyelination, and distal atrophy. Axonal swellings of myelinated and unmyelinated axons contain excessive numbers of 10 nm neurofilaments. Distal regions with advanced nerve fiber degeneration are associated with endoneurial and sub-perineurial edema [26][27][28]. In the spinal cord/medulla oblongata, swollen axons are most prominent in the gracile nucleus, although in animals with early neuropathy, similar pathological changes are evident in the cuneate nucleus and in a superficial ventrolateral zone corresponding to dorsal and ventral spinocerebellar tracts. These findings demonstrate that γ-diketone neuropathy is a central-peripheral distal axonopathy [24,29].

γ-Diketone Protein Adduction and Cross-Linking
Whereas 1,3-diacetylbenzene was non-chromogenic and non-neurotoxic, the widespread bluish discoloration of tissues of rats treated systemically with 1,2-diacetylbenzene indicated that the γ-diketone reacted with amino groups of proteins globally [46]. Measurement of chromogenic product in vitro showed that, of 22 tested amino acids, 1,2-diacetylbenzene was most reactive with L-lysine, glycine, γ-aminobutyric acid, and ornithine, a non-protein amino acid and higher homolog of L-lysine. L-Cysteine was among the least reactive with 1,2-diacetylbenzene, and 1,3-diacetylbenzene failed to react with any of the amino acids or with ornithine [46].
Proteomic studies showed that 1,2-diacetylbenzene, but not 1,3-diacetylbenzene, also reacted with mouse brain stathmin (15.5% lysine), which binds to tubulin and inhibits microtubule assembly [33,51]. Stathmin and proteins involved in stathmin regulation showed increased abundance in C57 mouse brain one week following single intraperitoneal treatment with 50 mg/kg 1,2-diacetylbenzene. At this time, marked 1,2-diacetylbenzene-specific changes were noted in the relative abundance of brain proteins involved in microtubule interaction (notably lysine-rich actin α−2) and energy metabolism, especially malate dehydrogenase [33], which also participates in gluconeogenesis. Noteworthy is that aged stathmin knock-out mice developed axonal degeneration and secondary demyelination [52] comparable with the neuropathology induced by blue-chromogenic acetyl-ethyltetramethyl tetralin [53]. Additionally, lysine-acetylated actin interacts with cyclase-associated protein to form an inhibitor of inverted formin 2, dominant missense mutations of which are linked with Charcot-Marie-Tooth disease, an inherited human axonal neuropathy associated specifically with glycolysis-and gluconeogenesis-related processes [54,55].
The vulnerability of proteins to γ-diketone attack is focused on their differential content of L-lysine, the epsilon-amino acid group of which is believed to be its primary vulnerability. 2,5-Hexanedione and 1,2-diacetylbenzene undergo selective Paal-Knorr reactions with lysine in axonal cytoskeleton proteins to form 2,5-dimethylpyrrole and 1,2diacetylisoindole adducts, respectively [33,56] (Figures 2 and 3). These adducts undergo progressive oxidative protein crosslinking, which can be inter-or intramolecular, depending on the adduct site [57,58]. Adduct oxidation is minimized in the absence of air or the presence of antioxidants [59,60], and hyperbaric oxygen accelerates the neurotoxicity of 2,5-hexanedione [61]. Rats treated orally with 2,5-hexanedione develop pyrrole adducts in hair, urine and serum in a dose-response relationship [63], and serum and urine pyrrole levels are correlated with time to onset of neuropathy induced by n-hexane administered to rats repeatedly by gavage [64]. The highest level of 2,5-hexanedione is found in urine and the lowest in the sciatic nerve, while the highest level of pyrrole adducts occurs in the liver and the lowest in serum. Pyrrole adducts in serum show the most significant correlation with free 2,5-hexanedione or pyrrole adducts in tissues [65].

Tissue Susceptibility to γ-Diketones
Systemic treatment with γ-diketones triggers pathological changes in the testes, as well as the nervous system. Extensive experimental studies of Sertoli cells in rats treated orally with 2,5-hexanedione point to alterations in microtubule-mediated functions, including microtubule assembly (which is associated with the status of stathmin phosphorylation), as well as kinesin and dynein motor-dependent transport [62,66]. The transport functions of axonal microtubules are also implicated in the genesis of nerve fiber degeneration. Injection of 2,5-hexanedione into the rat sciatic nerve results in rapid (<30 s) local reorganization of axoplasm, specifically the central accumulation of microtubules and peripheral relocation and disorientation of neurofilaments [67]. Microtubule cross-linking side arms are possible targets of lysine-reactive γ-diketones. Noteworthy is that spindle microtubule crosslinks contain PRC1 (protein regulator of cytokinesis 1), which binds microtubules via a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain, and PRC1 shares the same binding site on the tubulin dimer as the motor proteins kinesin and dynein [68,69]. Clustered microtubules, sometimes associated with mitochondria, are often observed in the axons of peripheral nerve fibers of animals treated systemically with n-hexane, 2-hexanone, 2,5-hexanedione, or 1,2-diacetylbenzene [70,71], as well in a number of other experimental toxic neuropathies, human giant axonal neuropathy, and in hereditary distal motor and sensory neuropathy or Charcot-Marie-Tooth disease [72]. Since local 2,5-hexanedione nerve injection shows that the resulting local cytoskeletal reorganization precedes the formation of neurofilament-filled focal axonal swellings, microtubule displacement may mark sites at which anterograde transport of axonal neurofilaments is disrupted. Thus, intrathecal 2,5hexanedione interfered with the slow axonal transport of neurofilaments [73], and systemic treatment with 3,4-dimethyl-2,5-hexanedione reduced the rate of transport of neurofilament proteins 75-90%, while tubulin and other proteins were only modestly retarded [74]. Similarly, anterograde neurofilament transport was slowed, but the velocities and amounts of labeled proteins in the fast anterograde component of the sciatic nerve were normal 2-5 h after eight once-weekly systemic treatments with 2,5-hexanedione [75]. Additionally, a single high intraperitoneal dose of 2,5-hexanedione produced a time-dependent decrease of fast retrograde transport velocity in mouse sciatic nerves (~65% inhibition between 2.0-2.5 h) with a reversal to normal rate 3-5 h after toxin administration, while retrograde transport velocity was halved in animals with hindlimb paralysis after repeated treatment with 3,4-dimethyl-2,5-hexanedione [76]. Motor nerves showed earlier and greater changes than sensory nerves in the accumulation of retrogradely transported materials [75,77].
Changes in anterograde neurofilament transport are consistent with initial nerve fiber changes in rats administered n-hexane, 2-hexanol, or 2,5-hexanedione. Teased fiber studies revealed that neurofilament-rich giant axonal swellings developed first on the proximal sides of multiple paranodes in distal non-terminal regions of large-diameter myelinated fibers. Paranodal myelin sheaths were often retracted at sites of axonal swellings, while adjacent distal internodes were attenuated. Axonal swellings later developed at internodal sites. Demyelinated paranodes apparently underwent local shrinkage and remyelination prior to the onset of distal nerve fiber breakdown. Smaller myelinated and unmyelinated fibers also contained multifocal, giant axonal swellings with abnormally large numbers of 10 nm neurofilaments. [70].
While neurofilaments are prominent in the pathological process of γ-diketone neuropathy, distal axonal degeneration also occurs in transgenic mice that lack axonal neurofilaments [78,79]. Taken in concert, these findings suggest that γ-diketones primarily disrupt microtubule-associated proteins (MAP) and MAP-microtubule binding [80], perhaps with preferential effect at nodes of Ranvier, and secondarily cause changes in bidirectional axonal transport associated with changes in neurofilament distribution that may result locally in axonal swelling or atrophy.

Summary
γ-Diketones react preferentially with lysine residues to form chromogenic oxidized pyrrole or isoindole adducts that can cross-link and disrupt the function of proteins, notably microtubule-associated proteins responsible for protein transport within cells. Neurons are vulnerable because they are required to transport proteins bidirectionally along elongate axons in the central and peripheral nervous system. γ-Diketone potency determines whether blockade of slow anterograde axonal transport of neurofilaments occurs proximally (3,4-dimethyl-2,5-hexanedione, 1,2-diacetylbenzene) or distally (2,5-hexanedione), with resulting focal neurofilament-filled axonal swellings that displace myelin sheaths resulting in localized demyelination and remyelination, nerve fiber and axonal attenuation and, eventually, distal axonal degeneration resulting in denervation of sensory end-organs and muscle fibers. Comparable changes occur later in smaller (and shorter) myelinated and unmyelinated nerve fibers. Contemporaneously, elongate ascending and descending axons in the spinal cord undergo comparable distal changes. Whereas, in humans, distal nerve fiber degeneration induced by aliphatic hexacarbons (n-hexane, 2-hexanone) manifests as a slowly ascending distal symmetrical peripheral neuropathy that may continue to evolve for some time after exposure ceases, sensation and muscle strength are restored in the opposite direction in association with axonal regeneration and end-organ reinnervation. However, failure of spinal motor pathways to regenerate may result in persistent spasticity in persons who have otherwise recovered from severe neuropathy [28,29]. The distal ends of other elongate nerve fibers in the central nervous system, including the optic nerve and mammillary bodies, may also develop pathological changes (neurofilament-filled axons) in γ-diketone intoxication [81][82][83][84].
These findings point to the endogenous production of reactive γ-diketones in healthy people, perhaps from lipid peroxidation [87,90] or other sources, including the endogenous production of n-hexane (and other straight-chain alkanes) from probable lipid peroxidation, as reported in humans and laboratory rats [104][105][106].
Urinary excretion of free 2,5-hexanedione did not seem to be influenced by gender (52M, 47F), age (39.6 ± 12.8 years, range: 17-67 years), smoking habit, or area of residence (urban vs. rural) [88]. Levels of acidified urinary 2,5-hexanedione were found to be similar in a small study of male and female Germans [90] but modestly higher in Chinese males than females [99]. Levels were somewhat higher in tobacco smokers but were apparently unaffected by diet or alcohol consumption [97,99]. Fluctuations typical of a circadian rhythm were not observed for 2,5-hexanedione in blood or urine. Excretion of 2,5hexanedione was inversely related to age among Chinese subjects [99], while a comparison of two independent studies suggested that levels of urinary pyrroles were markedly higher in older persons [101,102].

Central-Peripheral Distal Axonopathies
This term was introduced 45 years ago to describe the pathological substrate of distal symmetrical sensorimotor neuropathies (often with an autonomic component) that occur in a number of toxic, metabolic, nutritional, and inherited states [24,29]. The distal axonopathies comprise a group of diseases of varying etiology, of slow or rapid development, in which there is largely symmetrical axonal degeneration, commencing distally, spreading proximally, and rarely involving neuronal cell bodies. In the peripheral nervous system, secondary demyelination and remyelination are found proximal (sometimes including the spinal roots) to the region of active nerve fiber degeneration. On the central side, degenerative changes are symmetrical, tract-oriented, and involve, to a varying degree, the distal regions of the dorsal columns, especially the gracile tracts, the corticospinal tracts, and the spinocerebellar tracts. The central changes are usually clinically silent until post-exposure recovery from peripheral nerve damage is complete when, in severe cases, unrepaired and thus persistent damage to the central motor pathway may manifest in the form of lower-extremity hyperreflexia or even spasticity. Clinically silent features often include reduced or loss of vibratory perception that is greater in the lower than upper extremities, a result both of degenerative changes in nerve terminals innervating Pacinian corpuscles and in the terminal regions of their corresponding centrally projecting axons terminating in the gracile nucleus.
While the molecular etiology of peripheral neuropathies associated with centralperipheral distal axonopathy is often obscure, the exception being toxic γ-diketone neuropathy, certain peripheral neuropathies of metabolic origin are also associated with the endogenous production of molecules that form adducts with proteins and neuroproteins. Prominent among these metabolic disorders are type-II diabetes mellitus and, to a lesser extent, uremia, both of which are associated with distal symmetrical peripheral neuropathies that evolve as a consequence of metabolic imbalance. Similar molecular mechanisms may also have relevance to the etiology of age-related changes that resemble those found in mild central-peripheral distal axonopathy.

Clinical and Neuropathological Features
Distal symmetrical sensorimotor axonal neuropathy is the most common form of diabetic neuropathy [107,108], the incidence of which increases with age and duration of the diabetic state [109,110]. The prevalence of T2DN was 61.8% among Chinese patients (>30 years old, n = 435) seen in downtown Shanghai [111]. Most relevant here is the painless T2DN that manifests with impaired light touch sensation, position sense, vibratory perception, and diminished or absent ankle reflexes [112]. Sensorimotor polyneuropathy first affects the distal parts of the lower extremities and then spreads proximally [113]. Early loss of vibration perception is prominent in T2DN [114][115][116] and has a pivotal role in the early detection of nerve fiber degeneration [117]. The vibration perception threshold has high sensitivity and specificity relative to nerve conduction studies in the early detection of T2DN [118]. The neuropathology of T2BN conforms to a central-peripheral distal axonopathy [24,119].
Risk factors for diabetic neuropathy include age, male gender, height, duration of diabetes, uncontrolled glycemia, insulin resistance, and metabolic syndrome components, such as hypertriglyceridemia, hypertension, abdominal obesity, and low high-density lipoprotein level [109,120]. The pathogenesis of T2DN is not understood. Pathophysiological concepts include hypoxia, immune complexes, oxidative stress, reduced neurotrophic factors, mitochondrial dysfunction, inflammation, and inositol depletion leading to the accumulation of sorbitol and fructose [112,120,121].
In recent years, we and our colleagues have begun to examine more critically whether endogenous γ-diketones are involved in the genesis of T2DN. We assayed the urinary concentration of γ-diketone pyrroles in age-and gender-matched elderly (60-84 years) Chinese persons with and without (n = 267/group) indicators of diabetes mellitus. Subjects were drawn from a community of 9411 adults aged ≥ 60 years in the city of Shenzhen, Guangdong, China [102]. Subjects with diabetes mellitus relative to healthy controls had significantly higher levels of fasting blood glucose, glycated hemoglobin A1c, urinary ketone bodies, and urinary γ-diketone pyrroles. The median concentration of urinary γ-diketone pyrrole adducts ( Figure 5) was significantly higher (p < 0.0001) in individuals with diabetes (7.5 µM or 0.99 mg/g urine creatinine) than in healthy controls (5.9 µM or 0.70 mg/g urine creatinine). Significant linear associations and non-linear associations were observed between urinary pyrrole adduct concentrations and diabetic indices (DM status, fasting blood glucose levels, or HbA1c levels). Studies are now underway to assess whether there is a correlation between urinary γ-diketone pyrroles and clinically measurable neuropathy. In summary, from the time of onset of diabetes mellitus, glucose forms covalent adducts with plasma proteins through a non-enzymatic process known as glycation [141]. Protein glycation and formation of AGEs are considered to play an important role in the pathogenesis of diabetic complications, including neuropathy and retinopathy [142,143]. However, rats treated with intraperitoneal injection of AGE for 12 weeks failed to induce structural changes in tibial nerves other than interstitial edema [144]. Neuroprotein modifications triggered by endogenous levels of a pyrrole-forming γ-diketone might also participate in the genesis of T2DN.

Uremic Neuropathy
End-stage renal disease is commonly associated (16-60% of patients) with a slowly progressive symmetrical sensorimotor disorder of the axons of elongate large-diameter myelinated fibers [145]. Nerve conduction studies demonstrate findings consistent with a generalized neuropathy of the axonal type. Early clinical signs include distal loss of position and vibration sense plus a decreased Achilles reflex. Sensory deficits ascend to the knees, whereupon the hands may become involved. Advanced disease is associated with additional muscle atrophy and eventual paralysis. Patients may also develop autonomic features, with postural hypotension, impaired sweating, diarrhea, constipation, or impotence [145]. In sum, like T2DN, uremic neuropathy has features characteristic of a metabolic central-peripheral distal axonopathy.
The etiology of uremic neuropathy is unknown but has been related in part to elevated levels of blood urea [146]. Carbonyl stress may contribute to the long-term complications of chronic renal failure. AGEs (carboxymethyllysine and pentosidine) are markedly increased in plasma proteins and skin collagen of uremic patients, whether or not diabetes or elevated blood glucose is present [146][147][148]. Breath analysis of patients with end-stage renal disease undergoing dialysis included 4-heptanal, 4-heptanone, and 2-heptanone [149,150]. Another study of exhaled breath and blood identified 60 volatile organic compounds including n-alkanes (C4-C7), 2-ethyl-hexanol, 3-methyl-2-hexanone, 2-hexanone, and 3-heptanone, the latter two of which, plus the C6 alkane n-hexane, have the potential to form a neurotoxic γ-diketone.

Age-Associated Distal Axonopathy
By the seventh decade of life, most persons display evidence of peripheral nervous system dysfunction in the form of decreased vibration sensation, two-point discrimination, ankle reflexes, muscle bulk, and nerve conduction [151][152][153]. While the possible causes of these changes are manifold, including repeated trauma, focal nerve entrapment, and ischemia, the normal process of biological aging is thought to be an important contributing factor. The most suggestive evidence of central-peripheral distal axonopathy is the progressive increase in vibratory threshold with age, more in the lower limb (great toe) than in the upper limb (finger) [154][155][156][157][158][159][160]. Vibration perception also decreases with body height, which is consistent with a length-dependent axonopathy [160]. Other signs, such as distal impairment of tendon reflexes follow the same pattern in aging as in many distal axonopathies. Studies of very old rats reveal an age-associated axonopathy featured by axonal swelling and distal retrograde degeneration of the gracile tract, similar but less affected cuneate nuclei, spinocerebellar tracts, and corticospinal tracts [161]. While the etiology of these neuropathological changes is unknown, it is noteworthy that age-related changes are found in levels of 2,5-hexanedione (decrease) and associated pyrroles (increase) in acidified human urine [99,102]. This might indicate age-dependent increased binding of 2,5-hexanedione to target proteins, including neuroproteins responsible for maintenance of axonal integrity. AGEs are also formed in high amounts during aging [143,[162][163][164][165].

Summary
The molecular mechanisms of peripheral neuropathy resulting from overexposure to certain organic solvents are largely understood. Several studies have shown that 2,5hexanedione, the common γ-diketone metabolite of n-hexane and 2-hexanone, reacts with amino groups of proteins, including neuroproteins, to form pyrrolated products that undergo oxidation and cross-link proteins. Lysine-rich neuroproteins are particularly vulnerable, including microtubule-associated proteins required for axonal transport. Disruption of axonal transport results in sequential degenerative changes, notably focal neurofilament accumulation resulting in localized demyelination and remyelination, with initial changes in the largest and longest axons in peripheral nerves and the spinal cord, with similar changes in shorter nerve fibers at a later stage. This results in distal symmetrical sensorimotor neuropathy underpinned by central-peripheral distal axonopathy. Potent neurotoxic methylated aliphatic and aromatic γ-diketones disrupt axonal transport more proximally, which results in neurofilament blockade in spinal roots (leading to localized demyelination and remyelination) with distal nerve fiber atrophy.
Low levels of the γ-diketone 2,5-hexanedione appear to be a component of the normal metabolome. Unlike low tissue levels of other potentially neurotoxic substances with established physiological functions, such as carbon monoxide, cyanide, formaldehyde, and nitric oxide, endogenous 2,5-hexanedione has no known function. Urinary levels of 2,5-hexanedione seem to decline with the advance of age while its protein reaction product increases, which raises the possibility of an etiological relationship with the usually subclinical distal axonopathy associated with advancing age. Preliminary findings also show elevated urinary γ-diketone pyrroles in T2DM, a metabolic state that commonly results in a progressive distal symmetrical axonopathy. A similar length-dependent neuropathy develops in chronic renal disease, but tissue γ-diketone levels in this condition are unknown and uninvestigated. Other endogenous metabolites, notably methylglyoxal (a dicarbonyl with both keto and aldehyde groups), that bind to lysine and other protein residues have been demonstrated with the advance of age, in uremia, and especially in T2DM. However, pyralline was not detected (<1.2 pmol) in one study of serum albumin isolated from the serum samples of diabetic and non-diabetic subjects [166]. Further research is therefore needed to determine whether distal axonal degeneration in these conditions is triggered by the formation of advanced glycation products, 2,5-hexanedione-pyrrolated proteins, or both.
Note Added in Proof: Recently published data show that prolonged treatment of rats with large doses (400 mg/day, 5 days/week, for 5 weeks) of 2,5-hexanedione induces evidence of increased neuronal apoptosis in spinal anterior horn cells in association with severe limb weakness [167] indicative of marked central-peripheral distal axonopathy. Apoptotic changes in neuronal somata, which probably occur secondary to the onset of distal axonal degeneration/regeneration, involve changes in nerve growth factor and associated cell signal pathways [167][168][169].
Author Contributions: X.C. led the work on diabetic neuropathy, revised the manuscript drafted by P.S.S., and drew the chemical formulae. All authors have read and agreed to the published version of the manuscript.