Ascorbate availability and neurodegeneration in amyotrophic lateral sclerosis

Medical Hypotheses (1997) 48, 281-296 © Pearson Professional Ltd 1997

Ascorbate availability and neurodegeneration in amyotrophic lateral sclerosis A. B. KOK Science Applications International Corporation, 626 Towne Center Drive, Joppa, Maryland 21085, USA (Te# + 1 410-679-3290; Fax: 410-679-7104; E-mail: [email protected])

Abstract - - Amyotrophic lateral sclerosis is a fatal neurodegenerative disease in which upper and lower motoneurons progressively deteriorate and die. Neuronal damage is most evident in the lower central nervous system, and death generally occurs following central respiratory failure. Proposed and demonstrated mechanisms for amyotrophic lateral sclerosis are diverse, and include altered superoxide dismutase and neurofilament proteins, autoimmune attack, and hyperglutamatergic activity. However, they do not account for the late onset of the disease, its earlier onset in males, and the differential vulnerability of neurons located in the brainstem and spinal cord. It is proposed here that, within the context of a specific defect such as altered superoxide dismutase, age-dependent decline in ascorbate availability triggers the disease. A role for ascorbate, which is found in millimolar levels in neurons, is suggested by a number of consistencies: 1) superoxide radicals being a common substrate for superoxide dismutase and ascorbate; 2) a close association between central nervous system ascorbate levels and injury tolerance; 3) a steady decline in ascorbate plasma levels and cellular availability with age; 4) plasma ascorbate levels being lower in males; 5) an association of ascorbate release with motor activity in central nervous system regions, in vivo; 6) the coupling of brain-cell ascorbate release with glutamate uptake; 7) possible roles for ascorbate modulation of N-methyI-D-aspartate receptor activity; 9) the ability of ascorbate to prevent peroxynitrite anion formation; and 10) evidence supporting the scorbutic guinea pig as a model for amyotrophic lateral sclerosis. Emphasis is placed on the probable competition between superoxide dismutase and ascorbate within the context of a primary defect of metalbinding or metal access in high-concentration proteins such as superoxide dismutase and human heavy neurofilaments. Finally, distinct features of alpha-motoneuronal physiology suggest that cell physiological characteristics such as high metabolic activity and extensive calcium dynamics may render neurons differentially vulnerable in amyotrophic lateral sclerosis.

Introduction

Amyotrophic lateral sclerosis (ALS) is an enigmatic neurodegenerative disease in which patients show a

rapid progression of bulbospinal and upper cortical motoneuronal loss. Over the years, efforts to identify a basis for ALS have resulted in many hypotheses, variously implicating metal-intoxication, trauma,

Date received 17 May 1996 Date accepted 18 June 1996

281

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MEDICAL HYPOTHESES

viral infection, autoimmune response, altered gluta- vulnerable cell types in ALS. While the properties matergic activity, and the ingestion of toxic phyto- of ALS-vulnerable motoneurons vary, those of the compounds. Most recently, genetic studies have ~-motoneuron implicate extensive calcium dynamics implicated mutationally altered Cu/Zn superoxide and high metabolic rate as important contributors dismutase (SOD) or neurofilament proteins as causes to differential neural vulnerability in ALS. Potential of the disease (1,2). Only a small portion of the total therapeutic and investigative leads are discussed, and ALS population - about 2-3% - possess genetically animal models for ALS are proposed. altered SOD, and it is not clear to what extent defects (genetic or post-translationally modified) in SOD Ascorbate and ALS occur in the overall patient population. About 10% of patients possess inherited, or familial, ALS (FALS), A study of the biology of Asc shows a number of and a small portion of spontaneously occurring ALS characteristics consistent with the etiology of ALS. (SALS) patients, possess genetically altered SOD, Combined, they offer a compelling argument for the perhaps through spontaneous mutation or through investigation of Asc decline in triggering the disease. inheritance wherein the defective phenotype is conThe high concentrations of CNS Asc suggest that tingent on other, genetic or environmental factors this compound is fundamental to the preservation of (3). Overall, however, genetic, histopathological and cellular homeostasis under both normal and pathoimmunological differences between SOD-FALS logical conditions. The review will begin by assessing patients, SALS patients, and factions within these the potential relationship of Asc with SOD, since groups (4,5) point to the likelihood that there are altered SOD is an established cause of the disease. multiple etiologies leading to a selective degeneration In this context, a reasonable hypothesis is proposed of motoneurons, with damage being most severe in on the relationship between Asc and altered SOD. the bulbospinal regions of the lower central nervous This discussion is followed by: (1) the extension system (CNS). of the Asc/SOD hypothesis to heavy neurofilamentThis review describes evidence that ascorbate derived ALS; (2) a review of ALS-relevant roles (Asc) availability is the common determinant of for Asc in the CNS; (3) a review of Asc transport ALS onset in individuals rendered vulnerable to the and availability to the CNS under normal and injury disease because of genetic or environmentally derived conditions; and (4) a summary and discussion of changes. The high concentrations of intra- and extrafindings from a scorbutic animal model for ALS. neuronal Asc alone endow considerable importance Before beginning, it should be pointed out that to this reducing compound and free radical scavenger Asc is often inappropriately assumed to possess net in the CNS (6). Resting extracellular levels are on pro-oxidant activity in vivo. This stems in part from the order of 400 ~tM, and are maintained at this its frequent use in in vitro free-radical-generating level at the expense of intracellular stores (7,8). Intrasystems. In vitro systems, in general, are highly artifineuronal levels are 5 to 10-fold higher than outside cial because of poor representation of extracellular the cell, and because whole brain homogenates yield redox conditions and a lack of metal sequestration. Asc levels on the order of 2-3 mM, neuronal levels In such systems, submillimolar Asc, like lower are likely to be even higher; a recent estimate points concentrations of other reducing molecules, including to 7 mM (9). Glial cells, in contrast, appear to possess thiol compounds and probably dopamine, can autolower Asc levels (see 9-11). The biochemical prooxidize and induce cell death (e.g. 12,13), an activity perties of Asc are consistent with critical protective that may be prevented by catalase addition or metal roles in both normal and pathological states, and its sequestration. As the concentrations of the reducing blood and tissue concentrations are consistent with compounds are raised (over 1 mM for Asc), their the demographics of ALS. toxic activities are no longer observed, presumably It should be noted that, with increasing support by their having achieved a net scavenging capability for the notion that ALS is free radical-effected, anti(14-16). Under normal in vivo conditions, howoxidant therapy is likely being pursued by many ALS ever, pro-oxidant activity by Asc in the CNS is very patients. However, there are legitimate concerns over unlikely to occur because free, unbound transition limitations on the ability of this vitamin to reach the metals would be virtually absent, and both sides of neuronal cytoplasm under normal conditions, and even the neural cell membrane are extremely rich in more concern under an injury state. Hence Asc transreducing compounds. port and cellular uptake are reviewed in this article. In addition to reviewing the potential roles of Ascorbate and superoxide dismutase Asc, a tentative model of ALS is offered based on characteristics of the ~-motoneuron, one of the most Given that altered SOD can cause ALS, that both

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ASCORBATE AVAILABILITY AND NEURODEGENERATION IN ALS

Cu/Zn-SOD and Asc are collocated in the cytoplasm (17), and that the relative intraneuronal concentrations of Cu/Zn-SOD and Asc appear to allow competition between these molecules for 02 °- elimination, consideration of Asc in the context of ALS onset and pathogenesis is imperative. In this section, it is argued that the 'gain-of-function' now generally proposed for altered SOD involves the generation of a toxic product, and that superoxide radical (O2°-) is the most likely direct or indirect precursor to that product. While this position is speculative, it represents a reasonable, if not the simplest, hypothesis given the current state of knowledge. What is known regarding the altered SOD of ALS patients? Expression of the altered enzymes in transgenic mice shows no consistent association between normal enzymatic SOD activity levels and the induction of motoneuronal degeneration (1); both deficiency (18) and overexpression (19,20) of SOD activity can be associated with selective motoneuron degradation in the spinal cord anterior horn. Most recently, Dal Canto and Gurney (21) showed in the transgenic mouse model, that the severity of the disease may be determined by the expression level of the altered enzyme. These and other studies have led to the conclusion that the altered SOD gene imparts an additional activity, or 'gain-of-function', leading to the pathological state. Whether this involves the elimination of some essential cell component or the generation of an additional product is unknown. Structural changes in the enzyme offer no clear clues as to what additional activity it may perform, although there is some evidence hinting that additional copper binding sites may occur in at least one mutant (see below). Superoxide radicals, thought to be primarily generated from mitochondrial electron transport activity, can effect several damaging activities. They may react directly with selected cellular components, or generate extremely reactive hydroxyl radicals (HO°) upon interacting with hydrogen peroxide (H202), or generate thiol-reactive peroxynitrite anions after encountering nitric oxide (NO) (see Fig. 1). Both SOD and Asc eliminate O2o-, yielding hydrogen peroxide as a product, as summarized in Figure 2. NO ~ ONOO-

~'~'-Oz...

.o.

~,~Superoxide L Dismutase

MetalReduction and Catalysis

Fig. 1 In additionto its own reactivity,superoxidecan cause the formationof peroxynitriteanions and hydroxylradicals.

A (la) (lb)

AschH-+O2.-+H ÷ A s c . - + O2"-+ 2H ÷

(2)

202"-+ 2H ÷

> A s c . + H202 ) H202+ D H A

> H202+ 02 SOD

B (3) (4) (5)

A s c . - + A s c . - + H÷ > DHA + AscHD H A + GSH ) GSSG + A s c H - + H ÷ D H A + NADH ) NAD* + AscHNADH-Semid e h y d r o a s c o r b a t e reductase (?)

Fig. 2 (A) Superoxideradicaleliminationreactionsby ascorbate

(AscH-) and SOD (DHA:dehydroxyascorbate;Asco-:ascorbyl radical); (B) Ascorbaterestorationreactions(GSH:glutathione; GSSG:glutathionedisulfide).The presenceof NADH-Semidehydroaseorbatereductaseactivityin the CNS is suggested,but not established.

With the possible exception of lymphocytes, neurons appear unique in possessing cytoplasmic Asc levels capable of competing with Cu/Zn-SOD for 02 °elimination. Although neuronal SOD levels are high (in the micromolar range), Asc concentrations in these cells reach the competitive range (9, see 22). That the two scavengers compete at in vivo concentrations has been confirmed in experiments wherein dialysis of ischemic and non-ischemic cortex and caudate slice homogenates results in a dramatic decrease in SOD-type activity (23). These studies by Tagaki et al suggest that neuronal Asc levels may account for half of the superoxide radical elimination activity in these homogenates, and that intraneuronal SOD and Asc, which appear collocated in cytoplasmic fractions, compete for 02 o-. Asc levels corresponding to those seen in vivo can also effect the preservation of cortical and hippocampal brain slices in vitro (24), and indirect evidence is also suggested for Asc protection in vivo under conditions wherein 02 °- is may be generated by xanthine oxidase (25-27). It is reasonable to speculate that, since SOD structure has evolved specificity toward recognizing and interacting with 02% this radical is a likely precursor for any toxic product generated by SOD. This interpretation predicts that intracellular Asc levels provide a competitive shield against the excessive formation of toxic product generated by altered Cu/Zn-SOD; a decline in cytoplasmic Asc levels would increase the share of 02 °- eliminated by SOD, concomitantly allowing more toxic product to form. What product(s) might the altered enzyme form? That SOD serves to eliminate superoxide radicals (02 °-) strengthens the hypothesis that free-radical activity may play an important role in ALS. Evidence consistent with free-radical-mediated oxidative damage has been found in SALS patients (4), and

284 histopathological observations in mice are noted to bear close resemblance to changes in neurons and neuronal processes following ischemia-reperfusion injury (21), a process associated with considerable intracellular free-radical damage. It is interesting that the latter study also revealed mild pathological changes in some motoneurons of mice overexpressing wild-type SOD, although, as in other studies, these animals did not develop ALS. Changes in copper access are an important consideration in ALS in that 02 °- or H202 produced by superoxide elimination at the SOD active site may react with copper to yield HOo or other toxic products (28). While transition metals such as copper are tightly bound by the enzyme, copper access may be altered or, as has been shown by Carri et al (29), ALS-associated altered SOD may exhibit additional copper binding sites. In reducing environments such as those found in vivo, such binding, in the context of the reactants and/or products of SOD, may result in the formation of toxic product(s). It should be noted that the broader SALS population may similarly possess altered SOD through postranslational modification/alteration. The possibility that wild-type enzymes are changed due to some other environmental or genetic change cannot be excluded because normal activity levels of mutant FALS enzymes do not necessarily correlate with the presence of ALS. However, the presence of such enzymes cannot be determined until requisite peptide alterations are better characterized and the 'gain-offunction' of the mutated SOD enzymes is identified. Ascorbate and neurofilament proteins: a common mechanism with superoxide dismutase ?

How, then, might an ALS state result from alterations in human heavy neurofilament proteins (HNFPs) (2,30,31)? One may approach this question by asking what relatively unique features SOD and HNFPs have in common. First, these proteins have very high concentrations. That of SOD is millimolar in the cytoplasm, and HNFPs are not only ubiquitous in the cell, but the altered portion has been localized to a highly repeated region. Second, in the context of the Asc trigger hypothesis, they should be attractive to 02 °- radicals. This description certainly characterizes SOD, and a case can be made for HNFP. HNFP ALS-associated mutations fall within a highly repeated (43 repeats per polypeptide), lysineserine-proline motif in the C-terminal region. Lysine and proline are among the peptides most sensitive to free-radical attack/oxidative stress (see 32). Studies of elastin suggest that such repeated peptide regions may be particularly prone to mutations (33), and

MEDICAL HYPOTHESES

normally exhibit lysine crosslinking (34). Lysine crosslinking of neuronal filaments can be enhanced in the presence of metal ions including Fe, Cu, and Mn (35), and copper-catalyzed, O2.--dependent proline oxidation and hydroxyl radical-effected proline crosslinking has been characterized in collagen (36,37). Free radical-effected proline hydroxylation also has been demonstrated in Alzheimer plaque material (38, 39). Medium neurofilament proteins (MNFPs) also possess these highly repeated, lysine-serine-prolinerich terminal regions. MNFP-metal interaction studies (40) offer a mechanism whereby metal ions, such as A13+, could inappropriately substitute for calcium ions that may normally form intrastrand crosslinks between adjacent HNFPs or MNFPs. Excess metal substitution at phosphorylated serine binding sites of these proteins may not only result in altered filament structure, but could provide increased small-molecule access to bound metal, leading to free-radical formation. In summary, ALS may be the result of inappropriately located or accessible metal-binding sites that can be cytotoxic only in highly common proteins, such as SOD or HNFP/MNFP neurofilaments. This hypothesis points to two features held in common by these proteins: high cytoplasmic concentration, and a potential for increased metal accessibility. Ascorbate then, prevents cytotoxicity through elimination of superoxide, a substrate for generation of the metal-catalyzed product. It should be mentioned that, although altered neurofilament structure alone could affect important cytoskeletal filament-associated activities, such as vesicle transport, or perhaps even glycolysis (41), this hypothesis is less attractive in that it is more difficult to reconcile with other features of the disease. Further roles of ascorbate in the central nervous syktem

Beyond its high brain concentrations and rigorously maintained extracellular levels, important roles for Asc are suggested by its properties in the brain. These properties overlap with proposed and established characteristics of ALS, such as increased glutamate receptor activity, altered intracellular calcium dynamics, and regionally decreased levels of thyrotropin releasing hormone (TRH). Some of these properties are summarized in the table, below. Studies have shown that: Asc levels exhibit considerable extracellular dynamics under normal conditions, Asc is released in close coupling with high affinity glummate uptake and catecholamine release, and Asc may protect against ischemic damage (see 6,42). Several observations point to the importance of

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ASCORBATE AVAILABILITY AND NEURODEGENERATION IN ALS

Table ALS-relevant ascorbate actions in the central nervous system Activity

Potential relationship with ALS

Superoxide radical elimination

1. Competition with SOD; preventing altered SOD access to its substrate 2. Prevention of O2,--derived, toxic molecules such as hydroxyl radicals and peroxynilrite anions Vitamin E, a lipophilic, free-radical scavenger that can prevent lipid peroxidation, has been reported to delay ALS onset in transgenic mice

tx-tocopherol (vitamin E) regeneration NMDA receptor inhibition

1. Regulation of glutamatergic stimulation; 2. Prevention of NO/peroxynitrite formation; 3. Reduction of Ca ~ influx;

Sparing of glutathione

Glutathione loss is closely associated with induction of cell death under stress conditions

Specific maturation factor for TRH

TRH is selectively diminished in injured CNS regions in ALS

Asc to the maintenance of neurons, per se. Whole brain tissue homogenates show an anteroposterior Asc gradient in vertebrates, including man. That Asc levels in the brainstem and spinal cord are considerably lower than those in the forebrain is proposed to reflect the increased proportion of white matter in the former (10). This may similarly be reflected by an interesting complementary relationship found in newborn rats, wherein the CNS contains high Asc, but little glutathione. CNS glutathione levels subsequently rise in association with gliogenesis, overall relative CNS Asc levels fall (11). Extracellular Asc shows activity-dependent variation in several brain regions, including those associated with motor control. Extracellular signals corresponding to Asc release increase in good correlation with normal motor activity in cortical and other regions of freely moving rats (43). Asc biochemistry is best described in the basal ganglia where its release is closely coupled with motor activity, and occurs following both dopamine release and glutamate uptake. Exogenous infusion of Asc into the striatum alters motor activity (see 42). While Asc dynamics have not been measured in vivo in the spinal cord, explants from this region release Asc in vitro (44). In a direct antioxidant role, Asc elimination of the superoxide radical would prevent direct damage induced by that molecule, as well as indirect damage caused by superoxide reactions with H202 to form hydroxyl radicals or with nitric oxide to form glutathione-depleting and damaging peroxynitrite anions. Finally, unlike SOD, Asc can act as a broadrange antioxidant, an important consideration should altered SOD yield some new reactive compound. In an indirect antioxidant role, Asc can restore oxidized vitamin E (~-Tocopherol). The latter has been reported to delay ALS onset in mice transgenic for human ALS-SOD (21). Asc restores vitamin E in spite of their different partitioning within the cell

(in the cytoplasm and in cell membranes, respectively). This activity, which has been demonstrated in vitro, in plasma preparations (46), must take place at the membrane surface, and may be a critical role for Asc, since enzyme regeneration of vitamin E has not been observed (45). Asc can also spare glutathione loss, providing further indirect antioxidant protection. Depletion of glutathione, which can eliminate H 2 0 2 and peroxynitrite anions (47-49), predisposes cells to apoptotic or necrotic cell death (14,15,50,51). Although the nature of the Asc sparing effect is unknown, Asc supplementation to glutathione-deprived animals considerably slows loss of this sulfhydryl antioxidant. In fact, in vivo studies, deprivation of one of these compounds coupled with supplementation by the other considerably slows depletion of the limited compound (47). This sparing relationship has recently been documented, in vitro, in human endothelial cells subjected to exogenously supplied nitric oxide (52). Asc appears to play a protective role under injury conditions. There is considerable evidence supporting Asc-release in injured tissue. This release is generally transient, and follows increases in extracellular glutamate and aspartate. Microdialysis from injured cortical and striatal tissues shows extracellular Asc to reach millimolar levels following unilateral middle cerebral artery occlusion, localized cold injury, or compression-contusion trauma (53-55). This increase to millimolar extracellular Asc is critical, especially under pro-oxidative conditions, such as those associated with an influx of hemoglobin-derived iron from blood breaches into the neuropil, and experimental evidence supports the contention that this Asc is protective (25,26). Extracellular Asc levels are maintained at the expense of intracellular stores, which are over tenfold higher, and inflammatory response may prevent Asc reuptake by cells (see below). Furthermore, Asc concentrations corresponding to normal

286 extracellular levels in vivo show good protection of brain slices (24), and anoxia tolerance over a range of species correlates well with cortical Asc levels, while glutathione concentrations did not vary between species (10). One can also speculate that, given that the total pool of Asc is lower in the brainstem and spinal cord (10), and that maintenance of extracellular Asc levels depends on restoration from intraneuronal stores, it is possible that relative neuronal cell density may contribute to neuronal vulnerability and contribute to regional vulnerability in ALS. Asc is released in close association with highaffinity glutamate uptake (see 6), and both Asc and its oxidized metabolite, dehydroxyascorbate (DHA), can inhibit glutamate binding to NMDA receptors with an IC50 of about 500 +_50 ~tM (56-58). Therefore only a 2 to 3-fold increase in extracellular Asc may be required to effect significant blockade of this receptor, which is well-known for mediating increased intracellular calcium levels (10,56,58,59), and the elevated extracellular Asc levels of ischemia may provide protection against hyperglutamatergic stimulation by blocking NMDA receptors. Asc can also protect cortical cells, in vitro, against NMDA or glutamate exposure (57). A coupling of Asc dynamics with excitatory amino acid (EAA) neurotransmission has important implications for ALS. Whether Asc serves to modulate glutamatergic receptors under normal operating conditions, is unclear, although its release during normal forebrain activity, in vivo, suggests a protective or neuromodulatory role for Asc in excitatory amino-acid transmission. N-methyl-D-aspartate (NMDA) receptors are suggested capable of mediating selective cortical and spinal motoneuron death in studies of the cycad toxin, b-methylamino-t-alanine (BMAA) (60), and can stimulate potentially toxic nitric oxide and peroxynitrite production (61). The latter has recently been implicated in lower motoneuronal paralysis and ALS involvement (62-64). Although rat studies suggest that little nitric oxide synthetase (NOS) expression occurs in the ventral horn of adults under nonpathological conditions (other than in development (see 65)), the NOS gene can be induced in a variety of tissues under injury conditions, and studies of scorbutic animals show a link between NOS products and Asc levels. Asc actions at the NMDA receptor could help to attenuate calcium influx and transmitter release, providing both neuromodulatory and neuroprotective roles for Asc in normal cortical function and brain injury, respectively. While its glutamatergic roles are not well understood (6,66,67), Asc is likely to be very important to glutamatergic activities under both normal and pathological conditions.

MEDICAL HYPOTHESES

Asc also performs important roles, either directly or indirectly, in the biochemistry of other neurotransmitters, regulatory peptides, and inflammatory mediators (42,68-71). Notably, it is specifically required for the post-translational maturation of thyrotropinreleasing hormone (TRH), which is depressed in damaged areas in ALS patients (72). Asc is also released in close association with catecholaminergic activity in the adrenal glands and in the basal ganglia, where its striatal release in response to dopamine elevation is well documented (42). However, basal gangliar damage, specifically in the substantia nigra (73), has only been described for Parkinson's-ALS complex. While intracellular neuronal calcium dynamics may play an important role in ALS-associated cell death, there is no strong evidence supporting Asc regulation of this cation in the cytoplasm, aside from NMDA receptor inhibition. First, the suggestion that Asc regulates the Na÷-dependent Ca ++ exchanger (74), an important excitable cell component (75), seems unlikely, given that intracellular Asc levels far exceed the Km for this activity. Second, kinetic studies of Asc chelation of calcium ions (76) suggest that such complexes are not likely to reach appreciable levels in the cell. Ascorbate availability to the central nervous system and injured central nervous system areas

Having addressed the potentially critical biochemical roles of ACS in the etiology of SOD-effected ALS, this section reviews established and likely limitations on Asc availability to the intracellular milieu of the neuron. Physiological Asc levels correspond well to the demographic characteristics of ALS, and Asc uptake in CNS may be compromised during the course of the disease. Established characteristics of Asc support these contentions with respect to age and gender. First, there is an age-dependent Asc decline in both serum and tissue uptake and levels, consistent with the onset of ALS later in life. Second, men show lower plasma Asc levels, and animal studies have shown greater Asc demand in males under injury conditions. Men, as a group, clearly develop the disease earlier. The biochemistry of Asc transport also suggests that there may be further limitations on Asc uptake following ALS onset. This is suggested by a potential for: reduced Asc uptake into the CNS due to glucose competition in association with elevated blood-glucose levels stemming from hyperglucagonemia and insulin resistance in ALS patients, and, a blockade of cellular Asc uptake into CNS cells in damaged regions as a result of complement activation.

ASCORBATE AVAILABILITYAND NEURODEGENERATION IN ALS

287

Serum levels of Asc decrease with age in humans (see 77), which may reduce available Asc in times of higher demand. Between the ages of 20 and 60, plasma Asc levels, approximately 0.8-1.0 mg/dl at age 20, fall by 50-70% in most individuals, and some, more likely to be elderly and/or male, show levels less than 0.2 or even 0.1 mg/dl (77). The basis for this age-dependent decrease, which is also seen in intracellular leukocyte concentrations (77,78), is unclear. It does not derive from a change in renal threshold (79) and appears to reflect a general change in the homeostatic control of serum/cellular Asc levels. A similar age-dependent decline is also reported in the extracellular fluids of brain and liver in rats (80), a species that synthesizes Asc, suggesting that Asc decline, independent of availability, is a more general mammalian phenomenon. This decrease may be regionally profound in the brain, showing a 60-80% drop in the striatal area in animals between the ages of 3 and 18 months. Furthermore, most studies on human Asc levels show average plasma Asc and leukocyte levels to be lower in men (77,81,82), which is consistent with the clearly increased level of early ALS onset in that group (83), and possibly a more rapid decline (84). Patients with longer survival times after diagnosis have also been reported to be younger and female (84), although a study of clinical indices of musclestrength loss shows little difference between the sexes (85). One explanation for the gender difference may lie in dietary intake, the only source of Asc in man. Studies of rat brain regional Asc levels suggest that, in males, normal Asc levels are higher and more vulnerable to loss following ischemic insult. Glutathione levels fell similarly in both sexes (86). If this observation parallels that for human systems, then dietary intake may be lower or elimination higher in males, although gender differences in human brain Asc have not been examined. Asc intake shows strong dietary dependence, as suggested by seasonal fluctuations of its plasma levels in man, presumably in association with the availability of dietary sources such as fresh vegetables and fruit (87). The route and transfer mechanism of Asc entry into the CNS are unclear but may affect Asc availability to that region. The location of Asc entry was initially proposed to be limited to transfer through the choroid plexus (88,89). Given the anteroposterior, directional flow of cerebrospinal fluid (CSF) (90,91) this scenario would dictate that the brainstem and spinal cord will be the last regions be irrigated and, should overall CNS demand for Asc increase in the face of limiting Asc availability to the choroid plexus from the blood, the brainstem and spinal cord would be the most vulnerable regions to Asc deprivation.

However, a more recent study (92), albeit indirectly, suggests that while the choroid plexus may possess the highest density of Asc transporters at the CNSplasma interface, Asc can also enter the CNS across the general blood-brain barrier surface. This scenario is consistent with the observation that vascular endothelial cells are capable of Asc transport, and would allow a more uniform access, among brain regions, to the serum Asc supply. The mechanism of Asc uptake into the brain is also undescribed, but may have bearing on Asc availability in ALS. Two types of Asc transporters are known: a more selective Asc transporter that depends on certain ions, most notably Na +, and a hexose transporter possessing a similar affinity for both Asc and glucose. Both these transporters may be inhibited under conditions prevalent in the ALS patient. Given current knowledge of Asc uptake from plasma, vascular endothelial uptake in the choroid plexus and blood-brain barrier would most likely be mediated by the hexose transporter, which is characterized by Na÷-independence as well as glucose sensitivity. Studies of Asc uptake in granulocytes, mononuclear leukocytes, erythrocytes, and endothelial cells from both retina and heart show these two characteristics (see 17,93,94). In the only direct studies of Asc transport in choroid plexus tissue (in vitro), Spector and Lorenzo (88,89) used a glucosefree assay medium and did not address Na+-depen dence. Normal physiological levels of serum glucose (about 5.5 mM) can exert a profound inhibitory effect; the K i for granulocyte uptake inhibition by glucose is about 3.7 mM, and the higher plasma glucose levels of diabetes can exert even further inhibition (see 17). Diabetic levels, which may reach 20 mM, would reduce uptake to less than half of that seen under normal glucose levels. Furthermore, patients with diabetes mellitus show low plasma Asc concentrations, increased Asc oxidation (elevated plasma DHA), as well as a lowered rate of Asc uptake (95,96). Based on these observations, uptake in ALS patients may show similar tendencies, although less extreme, due to their hyperglucagonemic and insulinresistant state (97). Extravascular tissue uptake, such as that within the CNS, is most likely mediated through Na+-dependent Asc transport. Adrenal chromaffin cells, retinal epithelium, kidney and intestinal brush border cells, astrocytes, osteoblasts, brain preparations, and retinal cells show Na+-dependent transport (see 17,93, 98-100). Padh and Aleo showed Na+-dependent Asc transport in 3T6 fibroblasts to be exceedingly sensitive to a factor tentatively identified as complement factor C3a (IC50 = 5 nM; 101,102). C3 deposits have been detected in spinal cord and motor cortex

288 of ALS patients (103). Whether this C3 factor derives from glial cell expression (104) or from localized blood-brain barrier permeation is unclear, although its activation, which would yield C3a, is reflected by the detection of both the C3c and C3d (105,106, 107) cleavage products in the affected regions and CSF of ALS patients. The suggestion of Padh and Aleo that this effect may have broader implications for injury and autoimmune disease, may be applied to ALS as well as to Alzheimer's disease, which is similarly associated with the presence of C3 activation products. C3 expression was not seen in all ALS patients and its source is not clear. High levels of C3 are found in blood (1-2 mg/ml) and may offer a source of this factor through localized blood-brain barrier breaches as suggested in Alzheimer's plaques which are closely associated with blood vessels (108). Alternatively, glial cells can be induced to express C3 by IL-lb in vitro (104). Macrophages, which express interleukin (IL)-lb in the periphery, have been found in CNS damage areas in a majority of ALS patients (109,110), and T cells, which are also associated with damaged regions in ALS (111), may promote IL-1 release from macrophages (112) and/or may also be recruited by local IL-lb expression (113). IL-lb expression in the CNS has been well documented in association with both injury and disease. Once released, C3 cleavage may be induced by antibodies, which are found in a majority of ALS patients (>70%) (5), or perhaps by extracellular hydrogen peroxide (114). In this context, it is notable that thyroid releasing hormone (TRH) levels are substantially depressed in tissue regions damaged in ALS (115). TRH maturation specifically requires Asc. Although this loss could be due to factors other than uptake blockade, such as neuronal loss or atrophy, the TRH change is not paralleled by lowered levels of serotonin, which is collocated with TRH in terminals presynaptic to ventral horn motoneurons. Serotonin levels in postmortem tissue are described as unchanged or slightly elevated (116), or reduced (117). In the latter case, given that 5-hydroxyindoleacetic acid (5-HIAA) levels were found to be unchanged, and that both glial-cell numbers and monoamine oxidase levels are increased in ALS spinal tissue, the serotonin reduction was attributed to its increased metabolism (118-120).

The guinea pig revisited: an animal model for amyotrophic lateral sclerosis ? A scorbutic (Asc-deficient) animal model has been described by den Hartog Jager (121) to show selective CNS deterioration in the anterior motor horn and

MEDICAL HYPOTHESES

pyramidal tracts of the guinea pig, a species that, like primates, is unable to synthesize Asc. A subsequent effort to verify the first study did not find this CNS deterioration (122, see also 123). The different diets employed in the two studies led the authors of the second group, Sillevis Smitt et al, to suggest that the high levels of dietary manganese used in the first study may have effected the ALS-like deterioration. Metal intoxication has long been proposed as a causal factor in ALS (e.g. 124,125-127) and is consistent with the addition of aberrant copper-binding sites in the SOD enzyme. Metallothionein production is reported to be elevated in central and peripheral tissues of ALS patients (125), an interesting observation in that this protein not only increases in response to metal binding/sequestration requirements, but has been shown, when expressed in cultured cells, to compensate for Cu/Zn-SOD deficiency by protecting against oxidative stress (128). In an effort to model Guamanian ALS, administration of a high-manganese and -aluminum/low-calcium diet to monkeys is described to effect regional CNS changes corresponding to those affected in ALS (129). The basis for this decrement is unclear. While aluminum does not directly interact with SOD (130), for example, it may react with superoxide to form a strong oxidant (131) or effect iron-displacement where, in the reducing environment of the cell, the latter can form free radicals (see 132). Metals could also act by directly or indirectly altering proteins such as SOD. The den Hartog Jager studies hint that metal intoxication may offer a parallel to defective Cu/Zn-SOD. In either case, a decline in Asc availability could combine with a second, free-radical-stressing factor to effect an ALS-like pathology. It follows that the most logical approach to testing the roles of Asc and metals in ALS would be to test this idea in scorbutic animal models such as the guinea pig or the osteogenic disorder Shionogi (ODS) rat (see 133) which, like man, lacks L-gluconolactone oxidase for Asc synthesis; the combined manipulation of Asc and external factors in these animals may yield an animal model for the study of SALS. It should be noted that, unlike guinea pigs, animals such as the rat synthesize Asc in the liver but, like the guinea pig and man, still must transport it into the CNS. Rats also show the lowest CNS Asc content in the brainstem and spinal cord regions of the CNS (10). Therefore, it is not unexpected to find ALS-like symptoms in Asc-synthesizing animals like mice. It is also remarkable that the decline in extracellular Asc levels in rat brains with age also occurs in the liver, the site of its synthesis (see 80). This suggests

ASCORBATE AVAILABILITYAND NEURODEGENERATION IN ALS

289

that exclusion of this compound from the CNS is not the cause of Asc decline.

tial', remains depolarized (by about 10mV) by a persistent suppression of potassium effiux. This prolonged bursting membrane activity is proposed to function in postural maintenance (140,141). Experiments by Eken et al (141) indicate that this faring can be terminated by sensory feedback inhibition. Motoneuron bistability has recently been proposed to be present in man (143). Serotonin, acting through its numerous receptors on spinal motoneurons (72,144,145), can induce the plateau potential (141,146), although TRH, also present at serotoninergic terminals, possesses characteristics ideal for inducing this state (see 147). TRH also stimulates L-type calcium channels, on which the bistable response is dependent, in cultured pituitary cells. Why TRH levels are considerably decreased in the ventral cords of ALS patients (115) is unclear, perhaps as a secondary damage effect or from locally lowered levels of intracellular Asc, an essential cofactor in TRH post-translational processing. Kiehn (138) notes that NMDA receptor agonists can also effect a persistent, inward current response in motoneurons, including hypoglossal motoneurons, although plateau potentials are not known to occur among the latter. Plateau response activity in motoneurons, like that of other cells showing this activity, depends on a steady influx of calcium ions into the cell (142,148, 149), an activity that may lead to cytotoxic injury or death due to excessive calcium elevation or extreme fluctuations deriving from imbalances in Ca ++ buffering. Recent observations describe cells with low calcium-binding activity to be differentially vulnerable to immunoglobulins from ALS patients (150) and, as noted, low parvalbumin levels have been described to correlate with ALS-vulnerability in motoneurons. While not deleterious per se, intraceUular calcium elevated by a few hundred nanomoles, perhaps two or three times normal peak levels, can trigger apoptotic cell death when superimposed over depleted stores of glutathione or thiol groups (50). In cardiomyocyte apoptosis (50), production of the glutathione reactant H202 follows extramitochondrial calcium elevation. Glutathione depletion can adversely affect antioxidant/free radical status and strongly predisposes cells to apoptotic and/or necrotic cell death (14,15,50-151, 152,153-155). It should be noted that the outcomes of apoptotic vs necrotic cell death are determined by the cellular state (e.g. intracellular calcium levels) or degree of damage, as suggested by Duvall and Wyllie (156), the initiating cause being the same. This much is becoming increasingly clear in recent studies of cell death (157,158). That the motoneuronal bistable response is L-type voltage-gated calcium channel-dependent (VGCC)

The bistable response plateau potential While declining Asc availability may explain regional vulnerability in ALS, it does not account for differential vulnerability among coexisting neuronal types. Recent findings generated in an effort to identify common elements to explain the differential vulnerability of upper and lower motoneurons suggest that cell-specific neuronal vulnerability in ALS is associated with low levels and expression of the calciumbinding protein, parvalbumin (134-137). There is considerable evidence that high intracellular calcium rises can effect death in cells with reduced oxidative defenses, such as those with depleted glutathione. Within the context of an Asc decline, it is reasonable to propose, based on available information, that the most vulnerable cells in the disease may be characterized by a combination of several factors: (1) relatively high metabolic activity, which generates superoxide radicals; (2) considerable calcium dynamics; and (3) a characteristic calcium ion-binding profile such as that with low parvalbumin levels. These characteristics appear to be present in alpha motoneurons of the anterior spinal cord, which are likely to show considerable calcium dynamics during the course of normal function. Here, we briefly review elements of alpha motoneuronal physiology that may be predisposing factors to cell death in ALS. It is important to emphasize that the characteristics of these cells may only serve to model ALS neuronal vulnerability in a general sense, because motoneurons in the brainstem and selected cortical motoneurons, which show distinctly different characteristics, also die. The unusual bistable response capability of alpha motoneurons (see 138), appears to endow these cells with the properties of both extensive calcium dynamics and energy metabolism. Studies in cats, rats, and turtles show these motoneurons to be capable of shifting between two firing modes that differ in both frequency and persistence (139-141). In the first mode, these cells fire in direct correspondence with synaptic input events while, in the second mode, these cells can respond to a transient or brief synaptic input by prolonged, continuous bursting. This bursting is self-sustained and can continue autogenously for many minutes until suppressed by an exogenous (e.g. sensory) input (142). Firing frequency appears higher in this second bursting mode (e.g. 20-25 Hz vs 10-15 Hz (139)), in which the cell membrane baseline potential, or 'plateau poten-

290 (138) is notable because L-channel autoantibodies, which are elevated in about 75% of patients, have been suggested to mediate both upper and lower CNS motoneuronal cell death in the disease (5,111,159). In vitro studies using cerebellar cells suggest that these antibodies depress L-VGCC response while directly enhancing calcium entry through P-type VGCCs (160). It is not known whether motoneurons are particularly rich in L-VGCCs, which could result in elevated autoantibody formation in association with their death (see model, below). Beyond increased calcium dynamics, the second, albeit more speculative predisposing factor pertinent to this activity is that free-radical generation may be favored in these cells if the prolonged cellular firing activity demands high levels of oxidative metabolism. Although other CNS neurons may fire at much higher frequencies than motoneurons, highfrequency firing is usually brief, while alphamotoneuronal firing may persist for many minutes. Neuronal metabolic activity, per se, tends to be higher than that in other body tissues, and the intrinsic metabolic rate of mitochondria supporting this prolonged firing activity may be high. Intracellular free-radical production derives primarily from mitochondrial metabolism, especially under state 4 conditions (abundant substrate and low ADP acceptor levels) (161,162), and recent studies suggest that state 4 superoxide radical production is dependent both on tissue type and on specific metabolic rate (cal/g/day) (163). Although state 4 respiration rates of hippocampal and entorhinal cortex areas are undetermined, comparative studies of these regions suggest an association between high neuronal pathway activity, increased cytochrome oxidase levels, and susceptibility to tissue damage. Chandrasekaran et al (164) show that highly active hippocampal CA1 cells exhibit differential vulnerability to neurodegeneration in Alzheimer's disease. Cytochrome oxidase activity in this disease becomes clearly depressed (165), and such a deficiency within a normally highly active electron transport chain may well enhance free-radical generation (161). If mitochondrial state 4 respiration is the primary source of free radical generation as suggested by Chance et al (161), and is proportional to tissue metabolic rate, as suggested by Sohal and coworkers (163), then one can predict that mitochondrial freeradical production will be proportional to relative cellular activity levels. The presence of (normally) elevated metabolic activity in anterior horn motoneurons can be indirectly tested by analysis of cytochrome oxidase activity levels in healthy tissue which, under normal conditions, correlates with metabolic capability.

MEDICAL HYPOTHESES

A model for amyotrophic lateral sclerosis It is likely that the different pathological ALS profiles share common elements given the unifying characteristic of motoneuronal death. Toward identifying these elements, a model for ALS is proposed here in which neurodegeneration in ALS results from a combination of the following general and specific (cell-type dependent) components: (1) the presence of a causal defect or state, such as inappropriately accessible metal sites in common proteins such as SOD or neurofilaments; (2) the formation of an 02 °-dependent toxic product; (3) an age-dependent decline in Asc availability, triggering disease onset; and (4) local cell-specific characteristics, such as those pertinent to calcium ion binding, that account for differential cell vulnerability. The implication of Asc decline as the 'trigger' of ALS pathogenesis points most strongly to a central role for superoxide radical elimination in ALS etiology; the same pool of cytoplasmic superoxide is subject to shared elimination by CuZn-SOD and Asc. At the molecular level, there is strong evidence, albeit indirect, from brain homogenates, indicating that SOD and Asc compete for 02°- elimination in vivo. CuZn-SOD and Asc are collocalized in the cytoplasm and neuronal Asc and SOD concentrations there fall in a competitive range for 02 °- elimination. At the physiological level, serum (and tissue) Asc levels decline in life, especially in males. As aging progresses and Asc declines, the relative share of 02 °available to SOD increases, which correspondingly increases the generation of some toxic product in the case of SOD-effected ALS. Evidence pointing to an age-dependent tissue Asc decline in rats, a species that synthesizes Asc, indicates that this idea is consistent with findings in neurodegenerative, transgenic mice (which also make Asc) expressing mutant SOD. Thus far, the data are consistent with the idea that Asc prevents the formation of an unknown reactive species derived from O2o-, and generated via an altered or aberrant metal-binding site. In the case of SOD, this could occur in two ways: (1) by altered active site access due to mutationally or chemically induced conformational changes (most mutations are not located directly at the active site); or (2) the creation of additional metal-binding sites, as suggested by evidence for additional copper-binding site in the ALS SOD study of Carri et al (29). Neurofilament proteins may also be subjected to high levels of innapropriately substituted metal species at phosphorylated serine crosslinking sites. While such sites could, conceivably, occur on many proteins, only those with high cellular concentrations and probably

291

ASCORBATEAVAILABILITYAND NEURODEGENERATIONIN ALS

[ASC]

~tt

~MACROPHAGE 1 IL-1 GLIALCELL

FI'IASI21 FHASE ill PI'IASt~ Ill mY/ME

C3A ,~ ~

~C3

Ca++

I

HO" ONOO- "~

-.

NEURON ) _ _

Fig. 3 (A) A general schematic of the ascorbate decline model for ALS initiation; (B) Ascorbate actions and factors that may block ascorbate uptake in regions of injury (*denotes complement activation).

with some attraction to 02*- will yield enough toxic product for cellular degeneration to occur. A model, albeit speculative, can be envisioned in which the disease process can be divided into three phases (see Fig. 3 inset). In phase 1, the afflicted (defect-possessing) individual is asymptomatic because the toxic product(s) formed by altered SOD or another source is either prevented from forming or eliminated by Asc. In phase 2, the age-dependent decline in serum Asc reduces its availability to the brain. For certain ceils, the Asc replacement rate falls beneath the critical O2 e- elimination threshold and cells begin to degenerate. Cell death may be apoptotic or necrotic, depending on the degree of intracellular stress and/or calcium rise. The damage pattern observed by Dal Canto and Gurney (21) showed injury severity to be highest in the spinal cord, followed by the medulla, the pons, and the midbrain (cortical regions were not evaluated). The most vulnerable cells are predicted to show higher metabolism, which generates corresponding levels of superoxide radical, and considerable intracellular calcium dynamics combined with differences in levels of calcium binding proteins such as parvalbumin. Injury initially causes ALS motoneurons to decrease in size (166). Although the functional integrity of motoneurons in ALS is unclear, this decreased size may affect both the response timing of individual motoneurons within their muscle motoneuronal pools (167), and increase excitability following Henneman's

size principle. Both these factors could affect cell vulnerability by increasing calcium dynamics and energy metabolism. In phase 3, cell death spreads to adjacent cells, increasing in rate. Observations by Brooks (168) suggest that motoneuron degeneration in ALS tends to spread to contiguous spinal regions over more distal ones. The mode of cell death, which could determined by the degree of injury or extent of cal-cium rise (158), may influence the spread of damage. Whereas apoptosis would result in an orderly, controlled removal or reabsorption of a dying cell, necrotic cell death with its spillage of cell contents may elicit autoantibody formation or inflammation that could hasten the decline of neighboring cells by further increasing VGCC calcium uptake response through L-VGCC autoantibody action, as proposed by Appel and coworkers (5), and/or preventing local Asc uptake through C3 expression (see Fig. 3B) and activation by antibodies or H202 to form C3a. Increasing CNS demand for Asc, age-associated resistance to serum Asc elevation, and local C3a Asc uptake blockade may render this final and clinical phase of the disease resistant to Asc therapy. The presence of a high level of asymptomatic non-Hodgkin's lymphoma among ALS patients (169) is also notable since, adrenal gland cells aside, leukocytes share with neurons the unique distinction of accumulating high levels of Asc. Age-dependent decline in human leukocyte Asc levels is well supported and appears greater in males (77).

292

Conclusion: implications for research The tissue distribution, age-dependent decline, and biochemistry of Asc would predict that Asc elevation in susceptible individuals may delay or prevent onset of the disease, a prediction that should be tested in animal models. However, Asc supplementation may prove difficult for several reasons: (1) resistance to plasma elevation due to the renal threshold for Asc elimination; (2) increasing resistance to plasma Asc elevation with age; (3) resistance of Asc uptake into the CSF following its elevation in plasma (170); and (4) under normal conditions, a strong resistance of CNS cellular Asc uptake in the face of its increase in the CSF (170). Asc administration will require continuous blood or intracerebroventricular infusion, with careful consideration of its pharmacokinetics. Once the disease process is ongoing, it is unclear whether Asc therapy could be effective. We know little enough regarding the homeostatic regulation of Asc levels in normal individuals, and even less regarding the overall systemic, regional and local Asc demands in the ALS patient. Hyperglycemic glucose elevation may reduce Asc uptake from plasma, and the nanomolar inhibition of Asc uptake by C3a complement protein should be examined, not only in the context of ALS, but with regard to acute neurological injury and Alzheimer disease. The latter shows even greater C3 expression and activation. Increased tissue Asc availability may be achieved through the development of membrane permeant Asc analogs that bypass regulatory controls. One formulation, a calcium ascorbate and threonate mixture identified as 'Ester C', was reported, in studies of scorbutic (ODS) rats (133), to offer higher cell uptake, plasma availability, and lower renal loss, than Asc. Finally, the scorbutic guinea pig model of den Hartog Jager should be more carefully re-examined as a possible model for SALS. The ODS rat may offer an even better model, given that this species is better characterized. Asc therapy has been shown to be efficacious in this model with respect to metal (trimethyl tin) intoxication (171). This research may be critical for the identification of components required to yield an ALS state, perhaps identifying metal species, highly common binding sites, or metal regulatory molecules that could be important in effecting environmentally induced or precipitated SALS. Hopefully, it will lead to the development and/or clarification of ALS models for the identification of sequelae and therapeutic strategies common to different forms of the disease.

Acknowledgements The author wishes to thank SAIC for its support, in part, of this

MEDICAL HYPOTHESES

research and to acknowledgethe generous assistanceof following individuals in providing encouragement and/or reviewing part or all of the document: Margaret Rice, Frank Lebeda, Garry Buettner, Rudy Kuppers and Bruno Papirmeister, and Dan Zirpoli (deceased).

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