PROTEIN CROSS-LINKING AND AMYOTROPHIC LATERAL SCLEROSIS

(An unpublished hypothesis submitted to The Lancet on February 24, 1992, by DL McIlwain, S. Ono, SB Burgess and M Yamauchi, University of North Carolina Schools of Medicine and Dentistry)

Abstract

Recently discovered biochemical abnormalities in the skin of patients with sporadic amyotrophic lateral sclerosis (ALS) indicate that this form of ALS is systemic. Alterations found in specific intermolecular cross-links in skin collagen may help to explain the mechanisms and improve the early diagnosis of this adult motor neurone disease.

Introduction

The pathologic manifestations of the sporadic form of amyotrophic lateral sclerosis (ALS) are not confined to the loss of cerebral and spinal motor neurones. Other neurones, including large primary sensory cells (1-4) and neurones of Clarke’s nucleus (5,6), also degenerate in this disorder. Here we discuss recent biochemical evidence that extends the boundaries of ALS beyond the nervous system by demonstrating that collagen and elastin are altered in the skin in sporadic ALS. We propose that sporadic ALS is a systemic disease, the pathogenesis of which is reflected in an undetermined way by abnormalities in a specific class of intermolecular protein cross-linking recently found in the skin of ALS patients. Possible relationships between the changes in skin and nervous system in sporadic ALS and diagnostic implications of the skin changes are considered.

Skin changes in ALS have been commented upon since the time of Charcot (8), who noted in 1880 the infrequency of decubitus ulcers in ALS patients seen at the Salpetriere hospital. Eighty years later, histological changes in skin collagen were reported by Fullmer et al. (9) in patients suffering from the Guamanian form of ALS. Toyokura and his associates then provided further support for decreased decubitus formation in sporadic ALS (10), as well as light and electron microscopic evidence for loss of collagen and decreased collagen fibril diameter in the dermis of ALS patients in Japan (11). In 1986 Ono et al. (12) introduced new clinical sign for ALS – the delayed return phenomenon – demonstrated a late onset loss of elasticity in ALS skin. In 1990 Ono, Mechanic and Yamauchi (13) presented biochemical evidence for a loss of collagen from skin of ALS patients, but not from the skin of control patients of similar age, sex or duration and severity of illness. Moreover, no evidence of collagen loss was observed in the skin of control patients with compromised nutrition or motor activity, including patients with cachexia from cancer, significant muscle atrophy, or with a history of lengthy confinement to bed. By both morphological and biochemical criteria, the changes in skin collagen in ALS progressed with duration the disease.

Collagen Cross-Linking in ALS

Very recently, Ono and Yamauchi (7) have demonstrated alterations in intermolecular cross-links in type I collagen in ALS skin, but not in control skin from patients with other neuromuscular diseases. One change occurs early in the clinical course of the disease and is characterized by a progressive decrease with duration of disease in the presence of the non-reducible, stable cross-link histidinohydroxylysinonorleucine (HHL) in skin collagen and a concurrent increase in its unstable, reducible precursor, dehydro-hydroxylysinonorleucine (deH-HLNL), an iminium cross-link between Lys ald (5-amino-5-carboxypentanal) and hydroxylysine (Fig.1).

Again, these changes were not seen in the skin of control patients, but were obvious in the earliest biopsies taken 6 months after diagnosis of ALS, when muscle weakness was confined to on or two extremities. These progressive changes in skin collagen are not associated with normal aging. On the contrary, the stable HHL cross-link accumulate with age in normal human skin collagen, increasing more rapidly during the first 50 years of life than thereafter (14). Normally, deH-HLNL predominates in fetal skin collagen, while HHL is present in higher amounts than deH-HLNL in adult type I skin collagen (15,16). In sporadic ALS this ratio is reversed (7). Thus, as ALS progresses, a “rejuvenation” of skin collagen occurs, in opposite manner to normal aging.

HHL is a covalent intermolecular bond that most likely connects three collagen molecules to impart the characteristic tensile strength and visco-elasticity to collagen matrices in skin and corneal tissue. Little, if any HHL is present in bone, ligament, dentin or cartilage (14). The molecular locus of this trifunctional cross-link is alpha 1(I) Lys ald-16c, alpha 1(I)Hyl-87 and alpha 2(I)His-92 of type I collagen (16,17). Figure 1 illustrates three major steps in the formation of an HHL cross-link in type I collagen in normal skin: 1) oxidative deamination by the enzyme lysyl oxidase of the e -amino group of the 16th residue of Lys in the COOH-terminal, non-helical portion of the molecule to form Lys ald; 2) non-enzymatic formation of reducible, labile iminium intermolecular cross-link (deH-HLNL) by condensation between Lysald and alpha 1(I)87 hydroxylysine; and 3) spontaneous condensation of the labile iminium cross-link and the imidazole C-2 carbon atom of alpha 2(I)His-92 to form a stable, non-reducible trifunctional cross-link (18).

Other Iminium-Derived Protein Cross-Links

Two other examples of iminium-derived protein cross-links are.known to exist in skin: the D 6-7-dehydrolysinonorleucine cross-link leading to the synthesis of desmosine and isodesmosine in skin elastin (19) and a second type of reducible iminium cross-link in type I collagen in skin, dehydro-histidinohydroxymerodesmosine (deH-HHMD) (18). The prior oxidative deamination of lysine by lysyl oxidase is also required in the formation of each of these two cross-links. Ono and Yamauchi (7) have shown that the mole fraction of deH-HHMD in skin collagen also increases in patients with sporadic ALS as a function of duration of disease, and that desmosine and isodesmosine are decreased on a dry weight basis in ALS skin (unpublished observations). Again, control skin from patients of comparable age, sex, muscle atrophy and nutritional status did not show these changes, indicating that the changes are not epiphenomena of ALS. Although less conclusive than the findings on HHL with regard to the precise locus of the defect, the changes in deH-HHMD, desmosine and isodesmosine in ALS are also consistent with the possibility of altered iminium-derived cross-linking in both skin collagen and elastin, occurring after the formation of the unstable iminium function. Other forms of iminium-derived cross-links, such as the pyridinoline present in most connective tissues except skin and cornea (20) and its lysyl analogue present in mineralized tissue collagens (21), have not yet been examined in sporadic ALS. However, arterial angiopathy has been reported for patients with sporadic ALS (22).

Possible Mechanisms for Altered Protein Cross-linking in ALS Skin

The biochemical changes identified thus far in ALS skin collagen and to a lesser degree in elastin are consistent with either an increase in the turnover (increased synthesis and degradation) of these proteins or a specific inhibition of mature, non-reducible cross-link formation. In the first case, for example, increased destruction of collagen and synthesis of new collagen could reverse the ratio of deH-HLNL and HHL cross-links. It is well documented that reducible cross-links like deH-HLNL are abundant in newly synthesized collagen of fetal and other tissue with rapid collagen turnover rates (20,24). Consistent with an increased turnover of collagen are reports of increased collagenase activity in ALS skin (25,26) accompanied by increased protein secretion by fibroblasts cultured from ALS skin (26). Mature collagen is normally less susceptible to enzymatic hydrolysis than immature collagen (27), although its susceptibility to destruction might increase in ALS with structural modifications, such as changes in age-dependent aspartic acid racemization (28,29), glycosylation (30,31) or non-enzymatic glycation (32) of collagen. On the other hand, similar structural alterations in immature collagen could sterically hinder HHL formation and result in a reversal of the deH-HLNL/HHL ratio in type I collagen. An in vitro method is available to compare the rate of formation of HHL cross-links in collagen isolated from normal and ALS skin (15). The second possibility, an inhibition of the cross-link pathway prior to the formation of deH-HLNL (Fig. 1, would not explain the increased deH-HLNL/HHL ratio or the lack of change in the total mole fraction of these two cross-links (7) in ALS skin collagen.

Hypothesis

The involvement of skin in this neurological disorder leads us to propose that sporadic ALS is a systemic disease, an early manifestation of which is a change in iminium-derived protein cross-links. We consider two general questions which are relevant to this hypothesis: 1) are defects in iminium-derived protein cross-links also responsible for abnormalities known to exist in nervous tissue in.sporadic ALS? 2) of what clinical value is the new information on skin changes?

Are There Iminium-Derived Cross-Links in Nervous Tissue?

To our knowledge, there are no published reports of the presence or absence of iminium-derived cross-links or lysyl oxidase activity within the CNS. There is, however, indirect evidence from toxicological studies of two aminoproprionitrile compounds suggesting that such cross-links may exist normally within the nervous system. Beta-aminoproprionitrile (BAPN), a component of the sweet pea, Lathyrus odoratus, interferes with the oxidative deamination of lysine in both collagen and elastin (33) by irreversibly inhibiting lysyl oxidase (34), thereby preventing the formation of Lys ald and the subsequent formation of reducible iminium cross-links. The abnormalities which BAPN causes in bone and connective tissue, termed “osteolathyrism” by Selye (35), have long been attributed to the inhibition of iminium-derived collagen cross-links (33), although other actions of BAPN may also be involved (36). BAPN-induced Purkinje cell damage has been observed (37,38) without motor neurone damage (38), although loss spinal motor neurones has been reported after BAPN (39), possibly the result of compression injury secondary to vertebral defects. In vitro effects of BAPN on Schwann cells have also been observed (40,41). IDPN (beta, beta ‘-iminiodiproprionitrile), a synthetic analogue and metabolic precursor (42) of BAPN, is widely recognized in ALS research as an experimental agent that can produce proximal spheroids in motor axons (43,44) similar to those found in the motor axons of some patients with ALS (45-48). Like those in motor neurone disease, IDPN-induced spheroids contain large accumulations of disordered neurofilament.proteins. IDPN also causes the separation of axonal neurofilaments and microtubules and displacement of neurofilaments to subaxolemmal regions (49,50), and inhibition of slow axonal transport (51,52). Sayre et al. (53), Williams and Runge (54) and others have postulated that IDPN or one of its metabolites may disrupt axonal structure and function by interfering with neurofilament-microtubule crosslinks. Axonal neurofilament proteins, like collagen, are long-lived proteins that undergo a stabilization and insolubilization process after their synthesis (55). Rarely, IDPN has been reported to produce signs of osteolathyrism (56). It is possible that IDPN causes spheroids by interfering with the formation of iminium-derived protein cross-links involving neurofilament protein. Nevertheless, the biological effects of BAPN and IDPN differ substantially (35,57), and present toxicological data do not answer the question of whether iminium-dependent cross-links exist in nervous tissue.

It should be emphasized that the biochemical changes in ALS skin described by Ono and Yamauchi do not imply the chronic presence in ALS of aminoproprionitrile lathyrogens or other agents (58) that inhibit lysyl oxidase. On the contrary, neither the normal lysine content in ALS collagen (13) nor the reversed molar ratio of deH-HLNL/HHL with a normal mole fraction of deH-HLNL + HHL in ALS skin collagen (7) would be predicted from the presence of such an agent. It is the similarity of the effects of aminoproprionitriles and ALS on the collagen cross-linking pathway that enhances the possibility that iminium-derived cross-links may exist within the nervous system.

Clinical Uses of Skin Changes in ALS

Whether or not a common mechanism can be found for the changes that are observed in the CNS and skin of sporadic ALS patients, the newly-described skin changes may be of practical value to the clinician. New therapeutic strategies could emerge from knowledge of how the skin changes are initiated. Moreover, the early, opposite changes in skin content of HHL and deH-HLNL (7) may precede the clinical onset of the disease and could be useful in the timely diagnosis of the disease by skin biopsy. As effective therapies for sporadic ALS become available, early diagnosis will become an even more urgent need, to ensure that as many vulnerable neurones as possible are spared.

Supported by NIH grants DEO8522, DEO86ll, DEOO233, ARl9969 and AR30587 to M.Y. and NS12103 to D.L.M.

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