- Email: [email protected]
A radical hypothesis for neurodegeneration
p erspectives
o n di s e a s e
A radicalhypothesisfor neurode eration C. W. Olanow
Point mutationsin the cytosolicCu/Zn superoxidedismutase(SOD-1) &ene have been detected in associabon with familial amyotrophic lateral sclerosis(FALS). SOD clearssuperoxide radical and is one of the body'sprincipal defensemechanismsagainstoxygen toxicity. The finding of SOD variantsin FALSis consistentwith the hypothesisthat free radicalscontribute to the pathosenesisof FAL5, and possibly to the pathogenesis of other neurodegenerative disorders such as Parkinson'sdisease,in which there is substan~alevidenceof oxidant stress. The implication of free radicals in the pathogenesis of neurodegenerative disorders raises the possibility that antioxidants might provide neuroprotectNe therapy.
hydroxyl radical is a particularly reactive oxidizing agent and is thought to be the prime mediator of oxygen toxicity. 02
+e+e+e+e' ) O2- ~ ' ~ H202 :)OH" + OH)2H20
+2H+
+2H+
(1)
A series of naturally occurring antioxidant defense mechanisms normally prevent or limit freeradical production and tissue damage. (1) Oxidative phosphorylation primarily takes place within mitochondria where reactive oxidant species are tightly Rosen et aL have recently reported 1 eleven different bound and can be safely reduced to water. point mutations involving single amino acids in (2) Superoxide radical is dismutated by superoxide the cytosolic copper/zinc superoxide dismutase dismutase (SOD) to H202, which can then be (50D-1) gene on chromosome 21 in 13 families cleared by either catalase or glutathione peroxidase with familial amyotrophic lateral sclerosis (FALS). (GPO). (3) Chain-breaking antioxidants or freeThese changes were not detected in more than 100 radical scavengers such as alpha-tocopherol (vitcontrol individuals and were not considered to amin E) and ascorbate can react directly with free represent normal allelic variants. The authors con- radicals and thereby spare more critical molecules. In cluded that mutations in the 50D-1 gene cause addition, the configuration of the oxygen molecule discourages spontaneous auto-oxidation. Molecular FALS. Amyotrophic lateral sclerosis (ALS) is a progress- oxygen is a di-radical which, in its unexcited ground ive degenerative disorder of motor neurons in the state, contains two unpaired electrons, each housed spinal cord, brain stem and motor cortex character- in a separate orbital and having an identical spin. ized by varying degrees of weakness, atrophy, This creates a 'spin restriction' and limits the fasciculations and spasticity. Like Parkinson's capacity of molecular oxygen to react directly with disease (PD), ALS is a disorder of a subset of most molecules whose paired electrons rotate in neurons that appear to be selectively vulnerable to a opposite directions and do not conform to the degenerative process. Approximately 10% of ALS vacancies in the orbitals of the oxygen molecule. cases are familial (FALS). The detection of 11 Molecular oxygen is more likely to react by acceptdifferent defects in the SOD-1 gene in FALS implies ing a single electron from a transition metal, such as that an alteration in this enzyme is critical to the iron, that can exist in more than one valence state. development of FALS and not simply a co-expressed These metals have a loosely bound electron and can marker. These findings also support the notion that accept or donate a single electron to promote redox free radicals contribute to the pathogenesis of other reactions and free-radical formation. Oxidation neurodegenerative disorders such as PD in which reactions can be influenced by the regional concenthere is substantial evidence of oxidant stress2. tration of a transition metal. Increased concentrations of iron are associated with an increased Free radicals and oxidant stress probability that a redox reaction involving molecular Oxidation and reduction (Redox) reactions in- oxygen will occur. Recycling of iron from its oxidized volve the transfer of electrons and can, at times, state to its reduced state by tissue ascorbate, generate by-products known as free radicals. Free glutathione, or dopamine can drive oxidation reradicals are atoms or molecules which contain an actions and the formation of a cascade of free orbital with an unpaired electron 3. Some free radi- radicals. Iron is more likely to participate in redox cals are highly reactive and capable of extracting an reactions when it is in a 'reactive' or low molecular electron from neighboring molecules in order to fill weight state, usually complexed to ATP or citrate. the vacancy in their orbital. These reactions can Proteins such as transferrin or ferritin which bind damage a variety of critical biological molecules, iron and maintain it in a relatively non-reactive state including DNA, essential cellular proteins and thus serve as another important anti-oxidant membrane lipids 4. In addition, they can initiate defense mechanism. Normally, there is an equichain reactions, such as lipid peroxidation, that can librium between factors that promote free-radical alter the structural integrity of cell membranes and formation and the anti-oxidant defense mechanultimately cause cell death. The reduction of mol- isms. An imbalance in this equilibrium favoring free ecular oxygen to water during the course of radical formation is defined as a state of oxidant oxidative-phosphorylation involves the formation stress. The finding of mutations in the SOD-1 gene of superoxicle radical (02-), hydrogen peroxide in FALS suggests that free-radical defenses may be (H202) and hydroxyl radical (OH'), known collec- compromised in this disorder and that oxidant stress tively as reactive oxidant species (Equation 1). The may contribute to its pathogenesis.
TINS, VoL 16, No. 11, 1993
© 1993,ElsevierSciencePublic;hers Ltd.(UK)
C, W. Olanow,
Deptsof Neurology, Psychiatry, Pharmaco/ogyand Experimenta/ Therapeutics, Universityof South Florida,4 Columbia Drive#410, Tampa, FL33606,USA.
439
Superoxide dismutase and ALS Superoxide dismutase catalyzes the dismutation of 02- to H202 (Equation 2) and represents the first line of defense against oxygen toxicity. Three forms of SOD protein are known to exist in man; a cytosolic Cu/Zn SOD-1 which has been implicated in FALS, a mitochondrial manganese (Mn)-dependent SOD-2 and an extracellular Cu/Zn SOD-3. Cu/Zn SOD is produced constituently even in anaerobic conditions and presumably represents a ready defense against oxygen toxicity. In contrast, Mn-SOD is inducible and rises in response to an oxygen challenge. 02- + 02- + 2H + SOD H202 4- 02
(2)
SOD was first detected by McCord and Fridovich and provided the initial support for the concept that an enzymatic system had evolved to defend aerobic cells from free radicals5. Indeed, it has been proposed that SOD is essential for normal aerobic life. Escherichia coli which have been genetically manipulated to lack SOD are highly sensitive to oxidizing agents such as paraquat or H202 (Ref. 6). Their aerobic growth is inhibited in a minimal-glucose medium but can be restored by removing oxygen or by introducing an SOD-overproducing plasmid. Growth can also be enhanced by supplementing the media with essential amino acids, thus indicating that these molecules are targets of oxidative damage. Drosophila which are deficient in CulZn SOD can survive in an aerobic environment but they are infertile, have a short life span and are highly sensitive to paraquat7. The mechanism responsible for tissue damage associated with reduced SOD activity remains to be defined. Direct toxicity due to 02- is probably minor in comparison to OH" because it is much less reactive. More likely, excess 02- leads to tissue damage by promoting OH" formation. One potential mechanism is the iron-catalyzed Haber-Weiss reaction (Equation 3). However, the rate constant for the reduction of Fe3÷ by 02-is slow in comparison to other cellular reducing agents such as ascorbate or dopamine and it is likely that, in vivo, OH" generated by the Haber-Weiss reaction is limited. Iron-mediated Haber-Weiss reaction 02- + l e - + 2H ÷ --> H202
02- + Fe3+ --> 02 + Fe2+
(3)
H202 + Fe2+ --> OH" + OH- + Fe3+ Alternatively, 02- might lead to OH" formation through an interaction with endogenously formed nitric oxide (NO') (Ref. 8). The interaction of NO" with 02- leads to the formation of peroxynitrite (ONOO-) which can be pronated to form the nitrosyl radical (ONOOH) which decomposes to form OH" (Equation 4). Peroxynitrite itself is a highly reactive oxidizing agent capable of causing tissue damage. Peroxynitrite can oxidize methionine residues in proteins and peptides as well as thiols and thioethers 9'~°. It can also react with conserved SOD to form a nitronium-like intermediate which nitrates tyrosine residues ~ . The latter may be of significance 440
in ALS as receptors for a number of trophic molecules, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), which act on motoneurons, are receptor tyrosine kinases (trk) 12. Nitrated tyrosine might interfere with receptor function and diminish trophic support of motoneurons. 0:2- + NO" -.~ ONOOONOO- + H + --* ONOOH
(4)
ONOOH --* OH" + NO2"
Ca2+generated
Both 02- and NO" can be in response to a rise in cytosolic free . Nitric oxide is formed from arginine by a Ca2÷-activated nitric oxide synthase (NOS) (Equation 5). Increased intracellular Ca2+ can also induce the irreversible conversion of xanthine dehydrogenase to xanthine oxidase by a Ca2+-activated protease. Xanthine oxidase in turn catalyzes the oxidation of xanthine to provide a source of 02- capable of reacting with NO" (Equation 6). Ca2+-activated
Arginine
NOS ~- Citrulline + NO"
(5)
Xanthine dehydrogenase
~ Ca2+.activated
protease Xanthine oxidase Xanthine + 02 ~. 0 2 - 4- H202 4- urea
(6)
A reaction between 02- and NO" has been proposed to mediate toxicity associated with excitatory amino acids (EAA) and with ischemia and reperfusion, both of which promote a rise in intracellular Ca2÷ (Refs 13, 14). Tissue damage associated with models of excitotoxicity or ischemia and reperfusion can be blocked, not only by antagonists of EAA receptors, but also by agents that inhibit either 02- or NO" formation. Such approaches include agents that inhibit NOS or xanthine oxidase as well as agents that clear or scavenge 02- or NO" (see Fig. 1). Exogenously administered human recombinant Cu/Zn SOD ameliorates the development of neuronal damage in models of ischemia and reperfusion is. Similarly, ischemic brain damage is significantly diminished in transgenic mice that overexpress the human 50D-1 gene 16. These observations are of par~icular interest in view of the finding of SOD-mutations in FALS. It is noteworthy that in models of excitotoxicity as well as in a number of neurodegenerative disorders, including Huntington's disease, Alzheimer's disease and PD, NADPH-diaphorase-staining cells are selectively spared, possibly because they are enriched in Mn-SOD which clears 02- and prevents its interaction with NO" (Refs 13, 17, 18). These observations suggest that O2- may be critical to the toxicity associated with EAA and ischemia and reperfusion and that a deficiency in SOD might promote tissue damage. This mechanism may be relevant to FALS as motor neurons are normally exposed to EAA and may thus be particularly vulnerable to a deficiency in SOD activity ~9. TINS, VoL 16, No. 11, 1993
~i~ ~ i~~
"
~. . . . . .
'~ . . . . . . . . . .
~
~i ~ ~¸.i. . . .~. ~ ~ ~ ~ ~i~
Increased SOD activity could also theoretically induce tissue damage by excessively converting 02to H202 thereby promoting the formation of OH" by means of the Fenton reaction (see below). Overexpression of SOD-1 in transgenic mice results in a specific defect in distal motor neuron terminals of the tongue and hindlimb muscles2°. These animals show increased lipid peroxidation but greater resistance to paraquat, an 02- generator2~. It is not yet known whether the genetic mutations observed in FALS are associated with underexpression or over-expression of SOD enzymatic activity. Under-expression of a gene is typically seen with recessive disorders or in a dominantly transmitted disorder involving highly proliferative cells. For these reasons, over-expression of SOD is more likely in FALS, although this does not necessarily imply increased SOD activity. Mutant SOD may be functionally defective or have a dominant negative effect. This latter explanation is appealing as Rosen et al. detected only mis-sense mutations in FALS~. The authors point out that a dimeric coupling of normal and abnormal SOD protein could result in a loss of functional SOD activity that is greater than would occur with complete loss of a single gene product. While mutations in SOD-1 might exist only in a small percentage of all FALS cases, this important finding suggests that an alteration in antioxidant defenses can contribute to neuronal death in this disorder and that free radicals must be seriously considered as the primary cause of neurodegeneration. This finding also has important implications for other neurodegenerative disorders such as PD, where there is evidence of oxidant stress and free radicals are suspected of contributing to cell degeneration. Oxidant stress and PD Brain cells appear to be at particular risk from free radical damage. Polyunsaturated fatty acids are a major constituent of cell membranes and a substrate for lipid peroxidation. In addition, iron which promotes cytotoxic radical formation, accumulates in specific brain regions, such as the globus pallidus and substantia nigra, in concentrations which exceed those found in liver22, Defense mechanisms, on the other hand, are relatively deficient. The brain contains almost no catalase and, in comparison to liver, has reduced concentrations of glutathione (GSH), glutathione peroxidase (GPO) and vitamin E (Refs 2, 3). In blood plasma, there is normally sufficient transferrin to completely bind iron and to limit the likelihood of iron-mediated OH" formation. This might not be the case in the CNS, where ironbinding capacity appears to be limited and reactive iron is normally detected in the cerebrospinal fluid 2~. Neurons of the substantia nigra pars compacta (SNc) may be particularly vulnerable to oxidant stress because the oxidative metabolism of dopamine has the potential to generate cytotoxic free radicals. Dopamine can be oxidized by either monoamine oxidase (MAO) or auto-oxidation to generate H202 (Equation 7). Hydrogen peroxide is normally detoxified by GSH in a reaction catalyzed by GPO TINS, Vol. 16, No. 11, 1993
i~ ~
~
~~ ~
"'~ ' ~ ~!!~ ~
~~¸i ~ ~ 1~~ ¸ ~ ~ i i ~ iii ~ ! ~ ~ ~ i~~~ ! ! ~
i~ ~
i~!~i~i~
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i ¸¸
~iiiii!i! ~ !i: i
aminlacids Excitatory
1"Cytosolic Ca2+ ,~ \ / " Arginine \ ~v Ca2+-activated NOS Ca2+-activated calpain
Xanthine dehydrogenase
Xanthine oxidase Xanthine
NO" + Citrulline
Urea + H202 + O 2-
OH"
Fig. 1. Schematic diagram illustrating how a rise in cytosofic calcium due to EAA could lead to OH" formation. That 02- and NO" formation each play a critical role is illustrated by studies demonstrating that tissue damage in models of excitotoxicity can be blocked by: (1) NOS inhibitors which block NO" formation; (2) reduced hemoglobin which clears NO'; (3) xanthine oxidase inhibitors which block 02- formation; and (4) SOD which clears 02-. This mechanism might be pertinent to FALS, where a deficiency in SOD might fail to clear 02-and permit its interaction with NO" to form peroxynitrite (ONO0-) and the highly reactive hydroxyl radical (Equation 8; SQ, semiquinone), but an increase in the rate of dopamine turnover or a deficiency in the pool of reduced GSH could result in an increase in the steady state concentration of H202 and, in the presence of reactive iron, lead to OH" formation according to the Fenton reaction (Equation 9). Enzymatic oxidation of dopamine
(7.1)
DA + 02 + H20 MAO 3,4 dihydroxyphenylacetaldehyde + NH3 + H202 Auto-oxidation of dopamine
(7.2)
D A + O 2 - - * S Q ' + O 2 - + H+ DA + 02- + 2H + --* SQ" + H202 Clearance of peroxide
(8)
2 GSH + H202 GPO GSSG + 2H20 Fenton reaction
(9)
H202 + Fe2+ --> OH" + OH- + Fe3+ Free radicals have been implicated in PD because: (1) dopamine turnover is likely to be increased in surviving neurons with a resultant increase in the formation of H202; (2) levels of reduced and total glutathione are decreased in the SNc suggesting decreased protection against OH" formation; (3) iron concentration is increased in the SNc thereby increasing the likelihood of an interaction with H202 and consequent OH" formation; and (4) lipid peroxidation is increased in the SNc (see reviews in Refs 2, 24). These findings indicate that in PD the SNc is in a state of oxidant stress. Further, a decrease in reduced GSH has been found in patients who have incidental Lewy bodies at autopsy and are 441
disease
on $ GSH
Mitochondrial damage
$ Free radical
defenses (9 ALS)
.=
1" H202
Free radicals
//
'1
Fe2+
DA
=
1" Free radical
~
producti PD)on(?
Mitochondrial Celldeath~ Ca2+-activated damage degradativeenzymes
\
Excitotoxicity
/
1"Cytosolic Ca2*
Fig. 2. Theoretical model demonstrating how free radicals generated by either reduced defenses (? ALS) or increased formation (? PD) might induce a cycle of neuronal degeneration and continued free-radical production. This also illustrates how free radicals consequent to different etiologic mechanisms could represent a common pathogenic mechanism for neurodegeneration. Abbreviations: DA, dopamine; GSH, glutathione.
site so that it can then participate in redox reactions. The finding of increased iron and aluminium within neuromelanin granules of nigral neurons lends credence to the possibility that iron-induced oxidant stress contributes to cell death in PD. Further, direct infusion of iron into the SNc of rodents induces a parkinsonian syndrome with neuronal degeneration and a dose-related decline in striatal dopamine 3°'31. These effects are associated with lipid peroxidation and are attenuated by coadministration of the iron binding protein transferrin, suggesting that damage occurs consequent to oxidant stress32. More recently, we have shown that a single infusion of a low concentration of iron into the SNc can induce progressive histological, biochemical and behavioral changes33. These findings suggest that iron-induced oxidant stress might more closely model PD than do 6-hydroxydopamine or 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP). A decrease in complex I of the mitochondrial respiratory chain has also been identified in the SNc of patients with PD34'35. A similar defect has been observed in association with MPTP-induced parkinsonism 36. Complex I comprises 26 peptides, seven of which are encoded by mitochondrial DNA, and is highly vulnerable to free-radical damage. A freeradical-induced bioenergetic deficiency could promote a rise in cytosolic free Ca2+ by diminishing the voltage-dependent Mg 2+ blockade of EAA receptors and inactivating the ATP-dependent capacity of the cell to extrude or sequester Ca2+. Subsequent Ca2÷ activation of destructive protease, lipase and endonuclease enzymes could lead to neurodegeneration 37. In addition, a defect in mitochondrial complex I could inhibit ATP synthesis and result in a decrease in GSH formation, a decrease in the clearance of H202 and an increase in free-radical formation (see Fig. 2). Studies of SOD in PD are of interest in view of the findings in FALS. One report noted an increase in mitochondrial SOD activity in the SNc38. A second study described an increase in cytosolic SOD activity 39, although this may have been due to SOD leaking from mitochondria. It is most likely that, in PD, changes in SOD represent a compensatory rise secondary to oxidant stress rather than a fundamental change in constituent levels. However, Ceballos etal., using in situ hybridization, found that SOD-1 is preferentially expressed in the neuromelanincontaining SNc neurons that are prone to degenerate in PD4°. This suggests that biochemical pathways within these cells actively generate 02-. A recent report also indicates that transgenic mice which overexpress the human SOD-1 gene are resistant to the effects of MPTP41. Thus, the possibility must be considered that 02- may also play a role in the development of oxidant stress in PD.
presumed to have preclinical PD24. This suggests that oxidant stress may be present at the earliest stages of the disorder and that a deficiency in GSH could be crucial to neurodegeneration. Interest has also focused on the possibility that iron may be the primary cause of oxidant stress in PD because of its known propensity to promote cytotoxicity 25. Whether the increased iron found in PD is in a reactive state and poses a threat to dopamine neurons is not currently known. Dexter et al. observed that ferritin levels are reduced in PD26. Similarly, Connor et aL found that in PD, levels of both L- and H-chain ferritin in the SNc were not increased despite a rise in iron concentration 27. As ferritin binds iron and maintains it in a relatively unreactive form, these findings suggest that in PD iron is present in a low molecular weight form capable of promoting the formation of free radicals. Youdim et aL have proposed that neuromelanin might account for the site-specific accumulation of iron and its reduction into the Fe2+ form which promotes oxidant stress2s. Using the laser microprobe mass analyzer (LAMMA) we have recently demonstrated that iron accumulation in PD is indeed primarily localized to neuromelanin granules within SNc neurons 2s. These studies also demonstrated that in PD there is a dramatic accumulation of aluminium within neuromelanin granules. The concentration of aluminium in the brain is normally less than one to two parts per million and it does not serve any known physiological role. Aluminium also exists in only a single valence state and does not act as a transition metal. However, the addition of low concentrations of an aluminium salt can enhance Concludingremarks iron-induced lipid peroxidation by almost an order A scenario can be envisioned in which oxidant of magnitude 29. The mechanism responsible for this stress due to diminished antioxidant defenses or effect is not known, although it has been speculated increased free-radical production could initiate a that aluminium may displace iron from its binding cycle of events leading to cell death and continued 442
TINS, Vol. 16, No. 11, 1993
free-radical formation (see Fig. 2). However, it remains to be established whether free radicals represent the driving force leading to the neurodegenerative process or whether free-radical formation occurs as a consequence of cell degeneration due to a different etiologic process. The fact that mutations in the S O D - 1 gene have been found in FALS suggests that in at least some cases of neurodegeneration free radicals are at the top of the pathogenetic cascade and must be seriously considered as the primary cause. Oxidant stress in a given patient might result from different etiologic factors that preferentially damage specific neuronal populations. This might account for how free radicals could constitute a common pathogenetic mechanism in disorders as seemingly diverse as ALS and PD. Motor neurons might be specifically vulnerable to alterations in SOD because they interact with EAA and rely upon SOD to clear 02-. Failure to clear 02- could result in tissue damage due to peroxynitrite or OH" formed in a reaction between 0 2 - and NO'. On the other hand, nigral neurons could be specifically vulnerable to increased iron or decreased GSH because they preferentially bind iron and are rich in peroxide derived from dopamine metabolism. Failure to clear H202 or an increase in reactive iron could result in tissue damage due to OH" generated by the Fenton reaction. Thus, neurodegeneration in FALS and PD could be due to a similar pathogenetic mechanism resulting from different etiologies. In this regard it is noteworthy that overlap syndromes with clinical and pathological features of both ALS and PD have been described 42. Based on the genetic findings in FALS it is reasonable to examine patients with non-familial ALS and PD for mutations in the S O D - 1 gene, although there is no reason to believe that alterations in other genetic or environmental factors that promote free-radical formation might not be equally likely to cause neurodegeneration in an individual patient. From the clinical perspective, the relevance of the free-radical hypothesis is that it provides a basis for considering antioxidant strategies that might be neuroprotective and interfere with the natural progression of these diseases. Such strategies include: (1) approaches designed to inhibit or diminish free radical formation; (2) free-radical scavengers that react directly with free radicals to spare more important biological molecules and inhibit radical-perpetuated chain reactions; and (3) iron chelators that maintain iron in a nonreactive form. Clinical trials of antioxidants as possible neuroprotective therapy for neurodegenerative disorders have been initiated. The recently completed DATATOP study evaluated the free-radical scavenger oc-tocopherol and the selective MAO-B inhibitor deprenyl in PD43. Alpha-tocopherol had no effect on disease progression, although it is not clear that sufficient levels in the brain were attained. Deprenyl was selected based on its potential to inhibit peroxide formation derived from the oxidative metabolism of dopamine and thereby to limit OH" formation. The study demonstrated clearly and TINS, Vol. 16, No. 11, 1993
unequivocally that deprenyl significantly delays the development of disability in untreated patients with early PD. However, symptomatic effects were also observed that confound our ability to document a neuroprotective basis for these observations 44. Still, deprenyl-treated patients experienced a delay in developing disability regardless of whether or not symptomatic effects were observed and had less deterioration from baseline following drug withdrawal than did placebo patients 43. These benefits are not readily explained by a symptomatic effect and the full mechanism of action of deprenyl remains to be elucidated. As deprenyl does not inhibit peroxide derived from M A O - A or autooxidation of dopamine, it is possible that more striking neuroprotection could be accomplished with non-specific inhibition of dopamine metabolism, It is likely that these and other antioxidant strategies will soon be tested in ALS and PD as well as in other neurodegenerative disorders. The finding of a genetic defect in one of the principal oxidant defense mechanisms in FALS coupled with evidence of oxidative stress in PD provide a basis for optimism that these approaches may prove successful. Note added in proof. Since the preparation of this manuscript, a study has been published that indicates that alterations in mutant SOD genes in patients with FALS affect regions critical to the 13-barrel fold and dimer contact45, suggesting that these mutations induce a structurally defective SOD dimer. This interpretation is supported by the finding that mean SOD activity in red blood cells of affected individuals is less than half (41%) that of normal controls. This study supports the notion that structurally defective SOD in FALS results in reduced clearance of 02- and consequent free-radical-induced neurodegeneration.
Selected references 1 Rosen,D. R. eta/. (1993) Nature 362, 59-62 20lanow, C. W. (1992) Ann. Neurol. 32, 2-9 3 Halliwell, B. and Gutteridge, J. M. C. (1985) Trends Neurosci. 8, 22-29 4 Halliwell, B. and Gutteridge, J. M. C, (1984)J. Biochem. 219, 1-14 5 McCord, J. M. and Fridovich, I. (1969) J. Biol. Chem. 244, 6049--6055 6 Carlioz, A. and Touati, D. (1986) EMBO J. 5, 623-630 7 Phillips,J. P., Campbell,S. D., Michaud, D,, Charbonneau,M. and Hilliker, A. J. (1989) Proc. Natl Acad. 5ci. USA 86, 2761-2765
8 Beckman,J. S., Beckman,T. W., Chen,J., Marshall, P. A. and Freeman, B. A. (1990) Proc. Natl Acad. ScL USA 87, 1620-1624 9 Moreno, J. J. and Pryor, W. A. (1992) Chem. Res. Toxicol. 5, 425-431 10 Koppenol, W. H. et al. (1992) Chem. Res. Toxicol. 5, 834-842 11 Beckman,J. S. etal. (1992) Biochem. Biophys. 298, 438-445 12 Glass,D. J. and Yancopoulos,G. (1993) Trends Cell BioL 3, 262-268 13 Dawson,T. M., Dawson,V. L. and Snyder,S. H. (1992)Ann. NeuroL 32, 297-311 14 McCord, J. M. (1985) N. Engl. J. Med. 312, 159-163 15 Uyama, O., Matsuyama, T., Michishita, H., Nakamura, H. and Sugita, M. (1992) Stroke 23, 75-81 16 Kinouchi, H. et aL (1991) Proc. Natl Acad. 5ci. USA 88, 11158-11162 17 Hyman, B. T. etaL (1992)Ann. NeuroL 32,818-820 443
18 Brandabur, M. M. and Mufson, E. J. (1993) Neurology 43, A408 19 Appel, S. H. (1993) Trends Neurosci. 16, 3-5 20 Avaraham, K. B. etaL (1991)J. Neurocytol. 20, 208-215 21 Elroy-Stein, O., Bernsetin, Y. and Groner, Y. (1986) EMBOJ. 5, 615-622 22 Hallgren, B. and Sourander, P. (1958) J. Neurochem. 3, 41-51 23 Gutteridge, J. M. C. (1992) Clin. Sci. 82, 315-320 24 Jenner, P., Schapira, A. H. V. and Marsden, C. D. (1992) Neurology 42, 2241-2250 25 Youdim, M. B. H., Ben-Shachar, D. and Riederer, P. (1993) Movement Disorders 8, 112 26 Dexter, D. T. etaL (1990)J. Neurochem. 55, 16-20 27 Connor, J. R., Menzies, S. L., Snyder, B. S., Loeffler, D. A. and LeWitt, P. A. J. Neurosci. (in press) 28 Good, P. F., Olanow, C. W. and Perl, D. P. (1992) Brain Res. 593, 343-346 29 Gutteridge, J. M., Quinlan, G. J., Clark, I. and Halliwell, B. (1985) Biochim. Biophys. Acta 835, 441-447 30 Sengstock, G. J., Olanow, C. W., Dunn, A. J. and Arendash, G. W. (1992) Brain Res. Bull. 28, 645-649
31 Ben-Shachar, D. and Youdim, M. B. H. (1991)J. Neurochem. 57, 2133-2135 32 Sengstock, G. J. et aL (1991) Movement Disorders 6, 272 33 Sengstock, G., Olanow, C. W. and Arendash, G. (1993) Neurology 43, A389 34 Mizuno, Y. et al. (1989) Biochem. Biophys. Res. Commun. 163, 1450-1455 35 Schapira, A. H. V. et al. (1989) Lancet i, 1269 36 Nicklas, W. J., Vyas, I. and Heikkila, R. E. (1985) Life Sci. 36, 2503-2508 37 Orrenius, S., Burkitt, M. J., Kass, G. E., Dypbukt, J. M. and Nicotera, P. (1993)Ann. Neurol. 32, $33-$42 38 Saggu, H. et al. (1989)J. Neurochem. 53, 692-697 39 Marttila, R. J., Lorentaa, H. and Rinne, U. K. (1988) Neurol. Sci. 86, 321-331 40 Ceballos, I. et al. (1990) Lancet 335, 1035-1036 41 Przedborski, S. et al. (1992)J. Neurosci. 12, 1658-1667 42 Hudson, A. J. (1981) Brain 104, 217-247 43 Parkinson's Study Group (1993) N. Engl. J. /vled. 328, 176-183 44 Olanow, C. W. and Calne, D. B. (1991) Neurology42, 13-26 45 Deng, H. X. etal. (1993) Science 261, 1047-1051
The following debate covers a long-standing controversy in neuroscience, namely, whether the Editor cerebellum, which has traditionally been considered to participate only in motor functions, is involved in so-called higher functions, such as cognition and language. Henrietta Leiner, Alan Leiner and Robert Dow consider the evidence in favour of the latter view, by examining the evolutionarily enlarged dentate nucleus in humans.
The first debate in this new section of TINS covered another topic of higher cognitive processes, olfactory learning, and resulted in several letters from readers, many of which could not be published as a result of lack of space. Points of interest and comments from readers are always welcome, as are any suggestions for topics of interesting controversy for possible debate in future issues. Please contact the editor.
debate Gavin J. Swanson
Cognitiveandlanguagefundionsof thehumancerebellum Henrietta C. Leiner, Alan L. Leiner and Robert S. Dow Alan and Henrietta teinerare at ChanningHouse,850 WebsterStreet(635), PaloAlto, CA94301, USA.RobertS.Dow is at the R.5. Dow NeurologicalSciences Institute, Good SamaritanHospital andMedicalCenter, 1040NW 22nd Avenue(L450), Portland,OR97210, USA.
444
Traditional/v, the human cerebellum has been regarded as a motor mechanism, but this view of its function is being challenged by a growing body of data on the non-motor functions of the cerebellum. Some of these data are presented in this article, which reviews neuroanatomical, neuroimaging and behavioral reports of cerebellar involvementin cognitive and language functions. Thearticle proposes that this functional expansion is a consequence of specific cerebel/ar structural changes that evolved during hominid evolution and that could have been a prerequisite for the evolution of human language.
certain cognitive and language functions 4-6. How this functional expansion could occur as a consequence of the structural enlargement is discussed in this article.
The mystery of the human dentate nucleus In the evolution of the human cerebellum, the most lateral part enlarged both its cortical and subcortical structures 7. The lateral part of the cerebellar cortex sends its output to a lateral In research on the neural basis of cognitive and nucleus, called the dentate nucleus in humans, language skills, one of the large structures in the which is embedded in the white matter beneath the human brain has often been overlooked. This cortex (Fig. 1). During the evolution of this nucleus, structure is located in the lateral part of the human a significant differentiation occurred, which concerebellum, which enlarged enormously in the trasts with the evolution of the cerebellar cortex. course of hominid evolution 1. Why this part of the While the newly-evolved part of the cortex is similar cerebellum grew to enormous size in humans has histologically to the older parts of the cortex, the been a long-standing mystery. At first it was newly-evolved part of the dentate nucleus is difassumed that this part conferred a motor benefit on ferent from the older part of the nucleus. This humans; but the motor assumption was thrown into differentiation between the newly-evolved part doubt when functional imaging techniques made it (ventrolateral) and the older part (dorsomedial) is possible to visualize cerebellar activity. These images based on morphological, histological, embryological, showed that, even in the complete absence of any histochemical, and pathological evidence 7. motor activity, the cerebellum was activated when Because this neodentate part of the cerebellum humans performed certain cognitive and language grew to enormous size in humans, an obvious tasks2'3. This cerebellar participation in mental tasks question has arisen about its function in the human offers an explanation for the cerebellar enlarge- brain. Neurosurgeons have provided a provocative ment: it may have provided the neural basis for a piece of evidence by reporting that stereotaxic functional expansion of the cerebro-cerebellar sys- lesions, when placed in the neodentate, fail to tem, in which the lateral cerebellum can improve produce some classical motor signs of cerebellar ~) 1993,ElsevierSciencePubWishersLtd,(UK)
TINS, VOI. 16, NO. 11, 1993
o n di s e a s e
A radicalhypothesisfor neurode eration C. W. Olanow
Point mutationsin the cytosolicCu/Zn superoxidedismutase(SOD-1) &ene have been detected in associabon with familial amyotrophic lateral sclerosis(FALS). SOD clearssuperoxide radical and is one of the body'sprincipal defensemechanismsagainstoxygen toxicity. The finding of SOD variantsin FALSis consistentwith the hypothesisthat free radicalscontribute to the pathosenesisof FAL5, and possibly to the pathogenesis of other neurodegenerative disorders such as Parkinson'sdisease,in which there is substan~alevidenceof oxidant stress. The implication of free radicals in the pathogenesis of neurodegenerative disorders raises the possibility that antioxidants might provide neuroprotectNe therapy.
hydroxyl radical is a particularly reactive oxidizing agent and is thought to be the prime mediator of oxygen toxicity. 02
+e+e+e+e' ) O2- ~ ' ~ H202 :)OH" + OH)2H20
+2H+
+2H+
(1)
A series of naturally occurring antioxidant defense mechanisms normally prevent or limit freeradical production and tissue damage. (1) Oxidative phosphorylation primarily takes place within mitochondria where reactive oxidant species are tightly Rosen et aL have recently reported 1 eleven different bound and can be safely reduced to water. point mutations involving single amino acids in (2) Superoxide radical is dismutated by superoxide the cytosolic copper/zinc superoxide dismutase dismutase (SOD) to H202, which can then be (50D-1) gene on chromosome 21 in 13 families cleared by either catalase or glutathione peroxidase with familial amyotrophic lateral sclerosis (FALS). (GPO). (3) Chain-breaking antioxidants or freeThese changes were not detected in more than 100 radical scavengers such as alpha-tocopherol (vitcontrol individuals and were not considered to amin E) and ascorbate can react directly with free represent normal allelic variants. The authors con- radicals and thereby spare more critical molecules. In cluded that mutations in the 50D-1 gene cause addition, the configuration of the oxygen molecule discourages spontaneous auto-oxidation. Molecular FALS. Amyotrophic lateral sclerosis (ALS) is a progress- oxygen is a di-radical which, in its unexcited ground ive degenerative disorder of motor neurons in the state, contains two unpaired electrons, each housed spinal cord, brain stem and motor cortex character- in a separate orbital and having an identical spin. ized by varying degrees of weakness, atrophy, This creates a 'spin restriction' and limits the fasciculations and spasticity. Like Parkinson's capacity of molecular oxygen to react directly with disease (PD), ALS is a disorder of a subset of most molecules whose paired electrons rotate in neurons that appear to be selectively vulnerable to a opposite directions and do not conform to the degenerative process. Approximately 10% of ALS vacancies in the orbitals of the oxygen molecule. cases are familial (FALS). The detection of 11 Molecular oxygen is more likely to react by acceptdifferent defects in the SOD-1 gene in FALS implies ing a single electron from a transition metal, such as that an alteration in this enzyme is critical to the iron, that can exist in more than one valence state. development of FALS and not simply a co-expressed These metals have a loosely bound electron and can marker. These findings also support the notion that accept or donate a single electron to promote redox free radicals contribute to the pathogenesis of other reactions and free-radical formation. Oxidation neurodegenerative disorders such as PD in which reactions can be influenced by the regional concenthere is substantial evidence of oxidant stress2. tration of a transition metal. Increased concentrations of iron are associated with an increased Free radicals and oxidant stress probability that a redox reaction involving molecular Oxidation and reduction (Redox) reactions in- oxygen will occur. Recycling of iron from its oxidized volve the transfer of electrons and can, at times, state to its reduced state by tissue ascorbate, generate by-products known as free radicals. Free glutathione, or dopamine can drive oxidation reradicals are atoms or molecules which contain an actions and the formation of a cascade of free orbital with an unpaired electron 3. Some free radi- radicals. Iron is more likely to participate in redox cals are highly reactive and capable of extracting an reactions when it is in a 'reactive' or low molecular electron from neighboring molecules in order to fill weight state, usually complexed to ATP or citrate. the vacancy in their orbital. These reactions can Proteins such as transferrin or ferritin which bind damage a variety of critical biological molecules, iron and maintain it in a relatively non-reactive state including DNA, essential cellular proteins and thus serve as another important anti-oxidant membrane lipids 4. In addition, they can initiate defense mechanism. Normally, there is an equichain reactions, such as lipid peroxidation, that can librium between factors that promote free-radical alter the structural integrity of cell membranes and formation and the anti-oxidant defense mechanultimately cause cell death. The reduction of mol- isms. An imbalance in this equilibrium favoring free ecular oxygen to water during the course of radical formation is defined as a state of oxidant oxidative-phosphorylation involves the formation stress. The finding of mutations in the SOD-1 gene of superoxicle radical (02-), hydrogen peroxide in FALS suggests that free-radical defenses may be (H202) and hydroxyl radical (OH'), known collec- compromised in this disorder and that oxidant stress tively as reactive oxidant species (Equation 1). The may contribute to its pathogenesis.
TINS, VoL 16, No. 11, 1993
© 1993,ElsevierSciencePublic;hers Ltd.(UK)
C, W. Olanow,
Deptsof Neurology, Psychiatry, Pharmaco/ogyand Experimenta/ Therapeutics, Universityof South Florida,4 Columbia Drive#410, Tampa, FL33606,USA.
439
Superoxide dismutase and ALS Superoxide dismutase catalyzes the dismutation of 02- to H202 (Equation 2) and represents the first line of defense against oxygen toxicity. Three forms of SOD protein are known to exist in man; a cytosolic Cu/Zn SOD-1 which has been implicated in FALS, a mitochondrial manganese (Mn)-dependent SOD-2 and an extracellular Cu/Zn SOD-3. Cu/Zn SOD is produced constituently even in anaerobic conditions and presumably represents a ready defense against oxygen toxicity. In contrast, Mn-SOD is inducible and rises in response to an oxygen challenge. 02- + 02- + 2H + SOD H202 4- 02
(2)
SOD was first detected by McCord and Fridovich and provided the initial support for the concept that an enzymatic system had evolved to defend aerobic cells from free radicals5. Indeed, it has been proposed that SOD is essential for normal aerobic life. Escherichia coli which have been genetically manipulated to lack SOD are highly sensitive to oxidizing agents such as paraquat or H202 (Ref. 6). Their aerobic growth is inhibited in a minimal-glucose medium but can be restored by removing oxygen or by introducing an SOD-overproducing plasmid. Growth can also be enhanced by supplementing the media with essential amino acids, thus indicating that these molecules are targets of oxidative damage. Drosophila which are deficient in CulZn SOD can survive in an aerobic environment but they are infertile, have a short life span and are highly sensitive to paraquat7. The mechanism responsible for tissue damage associated with reduced SOD activity remains to be defined. Direct toxicity due to 02- is probably minor in comparison to OH" because it is much less reactive. More likely, excess 02- leads to tissue damage by promoting OH" formation. One potential mechanism is the iron-catalyzed Haber-Weiss reaction (Equation 3). However, the rate constant for the reduction of Fe3÷ by 02-is slow in comparison to other cellular reducing agents such as ascorbate or dopamine and it is likely that, in vivo, OH" generated by the Haber-Weiss reaction is limited. Iron-mediated Haber-Weiss reaction 02- + l e - + 2H ÷ --> H202
02- + Fe3+ --> 02 + Fe2+
(3)
H202 + Fe2+ --> OH" + OH- + Fe3+ Alternatively, 02- might lead to OH" formation through an interaction with endogenously formed nitric oxide (NO') (Ref. 8). The interaction of NO" with 02- leads to the formation of peroxynitrite (ONOO-) which can be pronated to form the nitrosyl radical (ONOOH) which decomposes to form OH" (Equation 4). Peroxynitrite itself is a highly reactive oxidizing agent capable of causing tissue damage. Peroxynitrite can oxidize methionine residues in proteins and peptides as well as thiols and thioethers 9'~°. It can also react with conserved SOD to form a nitronium-like intermediate which nitrates tyrosine residues ~ . The latter may be of significance 440
in ALS as receptors for a number of trophic molecules, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), which act on motoneurons, are receptor tyrosine kinases (trk) 12. Nitrated tyrosine might interfere with receptor function and diminish trophic support of motoneurons. 0:2- + NO" -.~ ONOOONOO- + H + --* ONOOH
(4)
ONOOH --* OH" + NO2"
Ca2+generated
Both 02- and NO" can be in response to a rise in cytosolic free . Nitric oxide is formed from arginine by a Ca2÷-activated nitric oxide synthase (NOS) (Equation 5). Increased intracellular Ca2+ can also induce the irreversible conversion of xanthine dehydrogenase to xanthine oxidase by a Ca2+-activated protease. Xanthine oxidase in turn catalyzes the oxidation of xanthine to provide a source of 02- capable of reacting with NO" (Equation 6). Ca2+-activated
Arginine
NOS ~- Citrulline + NO"
(5)
Xanthine dehydrogenase
~ Ca2+.activated
protease Xanthine oxidase Xanthine + 02 ~. 0 2 - 4- H202 4- urea
(6)
A reaction between 02- and NO" has been proposed to mediate toxicity associated with excitatory amino acids (EAA) and with ischemia and reperfusion, both of which promote a rise in intracellular Ca2÷ (Refs 13, 14). Tissue damage associated with models of excitotoxicity or ischemia and reperfusion can be blocked, not only by antagonists of EAA receptors, but also by agents that inhibit either 02- or NO" formation. Such approaches include agents that inhibit NOS or xanthine oxidase as well as agents that clear or scavenge 02- or NO" (see Fig. 1). Exogenously administered human recombinant Cu/Zn SOD ameliorates the development of neuronal damage in models of ischemia and reperfusion is. Similarly, ischemic brain damage is significantly diminished in transgenic mice that overexpress the human 50D-1 gene 16. These observations are of par~icular interest in view of the finding of SOD-mutations in FALS. It is noteworthy that in models of excitotoxicity as well as in a number of neurodegenerative disorders, including Huntington's disease, Alzheimer's disease and PD, NADPH-diaphorase-staining cells are selectively spared, possibly because they are enriched in Mn-SOD which clears 02- and prevents its interaction with NO" (Refs 13, 17, 18). These observations suggest that O2- may be critical to the toxicity associated with EAA and ischemia and reperfusion and that a deficiency in SOD might promote tissue damage. This mechanism may be relevant to FALS as motor neurons are normally exposed to EAA and may thus be particularly vulnerable to a deficiency in SOD activity ~9. TINS, VoL 16, No. 11, 1993
~i~ ~ i~~
"
~. . . . . .
'~ . . . . . . . . . .
~
~i ~ ~¸.i. . . .~. ~ ~ ~ ~ ~i~
Increased SOD activity could also theoretically induce tissue damage by excessively converting 02to H202 thereby promoting the formation of OH" by means of the Fenton reaction (see below). Overexpression of SOD-1 in transgenic mice results in a specific defect in distal motor neuron terminals of the tongue and hindlimb muscles2°. These animals show increased lipid peroxidation but greater resistance to paraquat, an 02- generator2~. It is not yet known whether the genetic mutations observed in FALS are associated with underexpression or over-expression of SOD enzymatic activity. Under-expression of a gene is typically seen with recessive disorders or in a dominantly transmitted disorder involving highly proliferative cells. For these reasons, over-expression of SOD is more likely in FALS, although this does not necessarily imply increased SOD activity. Mutant SOD may be functionally defective or have a dominant negative effect. This latter explanation is appealing as Rosen et al. detected only mis-sense mutations in FALS~. The authors point out that a dimeric coupling of normal and abnormal SOD protein could result in a loss of functional SOD activity that is greater than would occur with complete loss of a single gene product. While mutations in SOD-1 might exist only in a small percentage of all FALS cases, this important finding suggests that an alteration in antioxidant defenses can contribute to neuronal death in this disorder and that free radicals must be seriously considered as the primary cause of neurodegeneration. This finding also has important implications for other neurodegenerative disorders such as PD, where there is evidence of oxidant stress and free radicals are suspected of contributing to cell degeneration. Oxidant stress and PD Brain cells appear to be at particular risk from free radical damage. Polyunsaturated fatty acids are a major constituent of cell membranes and a substrate for lipid peroxidation. In addition, iron which promotes cytotoxic radical formation, accumulates in specific brain regions, such as the globus pallidus and substantia nigra, in concentrations which exceed those found in liver22, Defense mechanisms, on the other hand, are relatively deficient. The brain contains almost no catalase and, in comparison to liver, has reduced concentrations of glutathione (GSH), glutathione peroxidase (GPO) and vitamin E (Refs 2, 3). In blood plasma, there is normally sufficient transferrin to completely bind iron and to limit the likelihood of iron-mediated OH" formation. This might not be the case in the CNS, where ironbinding capacity appears to be limited and reactive iron is normally detected in the cerebrospinal fluid 2~. Neurons of the substantia nigra pars compacta (SNc) may be particularly vulnerable to oxidant stress because the oxidative metabolism of dopamine has the potential to generate cytotoxic free radicals. Dopamine can be oxidized by either monoamine oxidase (MAO) or auto-oxidation to generate H202 (Equation 7). Hydrogen peroxide is normally detoxified by GSH in a reaction catalyzed by GPO TINS, Vol. 16, No. 11, 1993
i~ ~
~
~~ ~
"'~ ' ~ ~!!~ ~
~~¸i ~ ~ 1~~ ¸ ~ ~ i i ~ iii ~ ! ~ ~ ~ i~~~ ! ! ~
i~ ~
i~!~i~i~
~!~ ~iiii ~ii ii~il !~i¸¸ ~i~ i!~ii ! ! i ~i~¸¸!¸ •
i ¸¸
~iiiii!i! ~ !i: i
aminlacids Excitatory
1"Cytosolic Ca2+ ,~ \ / " Arginine \ ~v Ca2+-activated NOS Ca2+-activated calpain
Xanthine dehydrogenase
Xanthine oxidase Xanthine
NO" + Citrulline
Urea + H202 + O 2-
OH"
Fig. 1. Schematic diagram illustrating how a rise in cytosofic calcium due to EAA could lead to OH" formation. That 02- and NO" formation each play a critical role is illustrated by studies demonstrating that tissue damage in models of excitotoxicity can be blocked by: (1) NOS inhibitors which block NO" formation; (2) reduced hemoglobin which clears NO'; (3) xanthine oxidase inhibitors which block 02- formation; and (4) SOD which clears 02-. This mechanism might be pertinent to FALS, where a deficiency in SOD might fail to clear 02-and permit its interaction with NO" to form peroxynitrite (ONO0-) and the highly reactive hydroxyl radical (Equation 8; SQ, semiquinone), but an increase in the rate of dopamine turnover or a deficiency in the pool of reduced GSH could result in an increase in the steady state concentration of H202 and, in the presence of reactive iron, lead to OH" formation according to the Fenton reaction (Equation 9). Enzymatic oxidation of dopamine
(7.1)
DA + 02 + H20 MAO 3,4 dihydroxyphenylacetaldehyde + NH3 + H202 Auto-oxidation of dopamine
(7.2)
D A + O 2 - - * S Q ' + O 2 - + H+ DA + 02- + 2H + --* SQ" + H202 Clearance of peroxide
(8)
2 GSH + H202 GPO GSSG + 2H20 Fenton reaction
(9)
H202 + Fe2+ --> OH" + OH- + Fe3+ Free radicals have been implicated in PD because: (1) dopamine turnover is likely to be increased in surviving neurons with a resultant increase in the formation of H202; (2) levels of reduced and total glutathione are decreased in the SNc suggesting decreased protection against OH" formation; (3) iron concentration is increased in the SNc thereby increasing the likelihood of an interaction with H202 and consequent OH" formation; and (4) lipid peroxidation is increased in the SNc (see reviews in Refs 2, 24). These findings indicate that in PD the SNc is in a state of oxidant stress. Further, a decrease in reduced GSH has been found in patients who have incidental Lewy bodies at autopsy and are 441
disease
on $ GSH
Mitochondrial damage
$ Free radical
defenses (9 ALS)
.=
1" H202
Free radicals
//
'1
Fe2+
DA
=
1" Free radical
~
producti PD)on(?
Mitochondrial Celldeath~ Ca2+-activated damage degradativeenzymes
\
Excitotoxicity
/
1"Cytosolic Ca2*
Fig. 2. Theoretical model demonstrating how free radicals generated by either reduced defenses (? ALS) or increased formation (? PD) might induce a cycle of neuronal degeneration and continued free-radical production. This also illustrates how free radicals consequent to different etiologic mechanisms could represent a common pathogenic mechanism for neurodegeneration. Abbreviations: DA, dopamine; GSH, glutathione.
site so that it can then participate in redox reactions. The finding of increased iron and aluminium within neuromelanin granules of nigral neurons lends credence to the possibility that iron-induced oxidant stress contributes to cell death in PD. Further, direct infusion of iron into the SNc of rodents induces a parkinsonian syndrome with neuronal degeneration and a dose-related decline in striatal dopamine 3°'31. These effects are associated with lipid peroxidation and are attenuated by coadministration of the iron binding protein transferrin, suggesting that damage occurs consequent to oxidant stress32. More recently, we have shown that a single infusion of a low concentration of iron into the SNc can induce progressive histological, biochemical and behavioral changes33. These findings suggest that iron-induced oxidant stress might more closely model PD than do 6-hydroxydopamine or 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP). A decrease in complex I of the mitochondrial respiratory chain has also been identified in the SNc of patients with PD34'35. A similar defect has been observed in association with MPTP-induced parkinsonism 36. Complex I comprises 26 peptides, seven of which are encoded by mitochondrial DNA, and is highly vulnerable to free-radical damage. A freeradical-induced bioenergetic deficiency could promote a rise in cytosolic free Ca2+ by diminishing the voltage-dependent Mg 2+ blockade of EAA receptors and inactivating the ATP-dependent capacity of the cell to extrude or sequester Ca2+. Subsequent Ca2÷ activation of destructive protease, lipase and endonuclease enzymes could lead to neurodegeneration 37. In addition, a defect in mitochondrial complex I could inhibit ATP synthesis and result in a decrease in GSH formation, a decrease in the clearance of H202 and an increase in free-radical formation (see Fig. 2). Studies of SOD in PD are of interest in view of the findings in FALS. One report noted an increase in mitochondrial SOD activity in the SNc38. A second study described an increase in cytosolic SOD activity 39, although this may have been due to SOD leaking from mitochondria. It is most likely that, in PD, changes in SOD represent a compensatory rise secondary to oxidant stress rather than a fundamental change in constituent levels. However, Ceballos etal., using in situ hybridization, found that SOD-1 is preferentially expressed in the neuromelanincontaining SNc neurons that are prone to degenerate in PD4°. This suggests that biochemical pathways within these cells actively generate 02-. A recent report also indicates that transgenic mice which overexpress the human SOD-1 gene are resistant to the effects of MPTP41. Thus, the possibility must be considered that 02- may also play a role in the development of oxidant stress in PD.
presumed to have preclinical PD24. This suggests that oxidant stress may be present at the earliest stages of the disorder and that a deficiency in GSH could be crucial to neurodegeneration. Interest has also focused on the possibility that iron may be the primary cause of oxidant stress in PD because of its known propensity to promote cytotoxicity 25. Whether the increased iron found in PD is in a reactive state and poses a threat to dopamine neurons is not currently known. Dexter et al. observed that ferritin levels are reduced in PD26. Similarly, Connor et aL found that in PD, levels of both L- and H-chain ferritin in the SNc were not increased despite a rise in iron concentration 27. As ferritin binds iron and maintains it in a relatively unreactive form, these findings suggest that in PD iron is present in a low molecular weight form capable of promoting the formation of free radicals. Youdim et aL have proposed that neuromelanin might account for the site-specific accumulation of iron and its reduction into the Fe2+ form which promotes oxidant stress2s. Using the laser microprobe mass analyzer (LAMMA) we have recently demonstrated that iron accumulation in PD is indeed primarily localized to neuromelanin granules within SNc neurons 2s. These studies also demonstrated that in PD there is a dramatic accumulation of aluminium within neuromelanin granules. The concentration of aluminium in the brain is normally less than one to two parts per million and it does not serve any known physiological role. Aluminium also exists in only a single valence state and does not act as a transition metal. However, the addition of low concentrations of an aluminium salt can enhance Concludingremarks iron-induced lipid peroxidation by almost an order A scenario can be envisioned in which oxidant of magnitude 29. The mechanism responsible for this stress due to diminished antioxidant defenses or effect is not known, although it has been speculated increased free-radical production could initiate a that aluminium may displace iron from its binding cycle of events leading to cell death and continued 442
TINS, Vol. 16, No. 11, 1993
free-radical formation (see Fig. 2). However, it remains to be established whether free radicals represent the driving force leading to the neurodegenerative process or whether free-radical formation occurs as a consequence of cell degeneration due to a different etiologic process. The fact that mutations in the S O D - 1 gene have been found in FALS suggests that in at least some cases of neurodegeneration free radicals are at the top of the pathogenetic cascade and must be seriously considered as the primary cause. Oxidant stress in a given patient might result from different etiologic factors that preferentially damage specific neuronal populations. This might account for how free radicals could constitute a common pathogenetic mechanism in disorders as seemingly diverse as ALS and PD. Motor neurons might be specifically vulnerable to alterations in SOD because they interact with EAA and rely upon SOD to clear 02-. Failure to clear 02- could result in tissue damage due to peroxynitrite or OH" formed in a reaction between 0 2 - and NO'. On the other hand, nigral neurons could be specifically vulnerable to increased iron or decreased GSH because they preferentially bind iron and are rich in peroxide derived from dopamine metabolism. Failure to clear H202 or an increase in reactive iron could result in tissue damage due to OH" generated by the Fenton reaction. Thus, neurodegeneration in FALS and PD could be due to a similar pathogenetic mechanism resulting from different etiologies. In this regard it is noteworthy that overlap syndromes with clinical and pathological features of both ALS and PD have been described 42. Based on the genetic findings in FALS it is reasonable to examine patients with non-familial ALS and PD for mutations in the S O D - 1 gene, although there is no reason to believe that alterations in other genetic or environmental factors that promote free-radical formation might not be equally likely to cause neurodegeneration in an individual patient. From the clinical perspective, the relevance of the free-radical hypothesis is that it provides a basis for considering antioxidant strategies that might be neuroprotective and interfere with the natural progression of these diseases. Such strategies include: (1) approaches designed to inhibit or diminish free radical formation; (2) free-radical scavengers that react directly with free radicals to spare more important biological molecules and inhibit radical-perpetuated chain reactions; and (3) iron chelators that maintain iron in a nonreactive form. Clinical trials of antioxidants as possible neuroprotective therapy for neurodegenerative disorders have been initiated. The recently completed DATATOP study evaluated the free-radical scavenger oc-tocopherol and the selective MAO-B inhibitor deprenyl in PD43. Alpha-tocopherol had no effect on disease progression, although it is not clear that sufficient levels in the brain were attained. Deprenyl was selected based on its potential to inhibit peroxide formation derived from the oxidative metabolism of dopamine and thereby to limit OH" formation. The study demonstrated clearly and TINS, Vol. 16, No. 11, 1993
unequivocally that deprenyl significantly delays the development of disability in untreated patients with early PD. However, symptomatic effects were also observed that confound our ability to document a neuroprotective basis for these observations 44. Still, deprenyl-treated patients experienced a delay in developing disability regardless of whether or not symptomatic effects were observed and had less deterioration from baseline following drug withdrawal than did placebo patients 43. These benefits are not readily explained by a symptomatic effect and the full mechanism of action of deprenyl remains to be elucidated. As deprenyl does not inhibit peroxide derived from M A O - A or autooxidation of dopamine, it is possible that more striking neuroprotection could be accomplished with non-specific inhibition of dopamine metabolism, It is likely that these and other antioxidant strategies will soon be tested in ALS and PD as well as in other neurodegenerative disorders. The finding of a genetic defect in one of the principal oxidant defense mechanisms in FALS coupled with evidence of oxidative stress in PD provide a basis for optimism that these approaches may prove successful. Note added in proof. Since the preparation of this manuscript, a study has been published that indicates that alterations in mutant SOD genes in patients with FALS affect regions critical to the 13-barrel fold and dimer contact45, suggesting that these mutations induce a structurally defective SOD dimer. This interpretation is supported by the finding that mean SOD activity in red blood cells of affected individuals is less than half (41%) that of normal controls. This study supports the notion that structurally defective SOD in FALS results in reduced clearance of 02- and consequent free-radical-induced neurodegeneration.
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The following debate covers a long-standing controversy in neuroscience, namely, whether the Editor cerebellum, which has traditionally been considered to participate only in motor functions, is involved in so-called higher functions, such as cognition and language. Henrietta Leiner, Alan Leiner and Robert Dow consider the evidence in favour of the latter view, by examining the evolutionarily enlarged dentate nucleus in humans.
The first debate in this new section of TINS covered another topic of higher cognitive processes, olfactory learning, and resulted in several letters from readers, many of which could not be published as a result of lack of space. Points of interest and comments from readers are always welcome, as are any suggestions for topics of interesting controversy for possible debate in future issues. Please contact the editor.
debate Gavin J. Swanson
Cognitiveandlanguagefundionsof thehumancerebellum Henrietta C. Leiner, Alan L. Leiner and Robert S. Dow Alan and Henrietta teinerare at ChanningHouse,850 WebsterStreet(635), PaloAlto, CA94301, USA.RobertS.Dow is at the R.5. Dow NeurologicalSciences Institute, Good SamaritanHospital andMedicalCenter, 1040NW 22nd Avenue(L450), Portland,OR97210, USA.
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Traditional/v, the human cerebellum has been regarded as a motor mechanism, but this view of its function is being challenged by a growing body of data on the non-motor functions of the cerebellum. Some of these data are presented in this article, which reviews neuroanatomical, neuroimaging and behavioral reports of cerebellar involvementin cognitive and language functions. Thearticle proposes that this functional expansion is a consequence of specific cerebel/ar structural changes that evolved during hominid evolution and that could have been a prerequisite for the evolution of human language.
certain cognitive and language functions 4-6. How this functional expansion could occur as a consequence of the structural enlargement is discussed in this article.
The mystery of the human dentate nucleus In the evolution of the human cerebellum, the most lateral part enlarged both its cortical and subcortical structures 7. The lateral part of the cerebellar cortex sends its output to a lateral In research on the neural basis of cognitive and nucleus, called the dentate nucleus in humans, language skills, one of the large structures in the which is embedded in the white matter beneath the human brain has often been overlooked. This cortex (Fig. 1). During the evolution of this nucleus, structure is located in the lateral part of the human a significant differentiation occurred, which concerebellum, which enlarged enormously in the trasts with the evolution of the cerebellar cortex. course of hominid evolution 1. Why this part of the While the newly-evolved part of the cortex is similar cerebellum grew to enormous size in humans has histologically to the older parts of the cortex, the been a long-standing mystery. At first it was newly-evolved part of the dentate nucleus is difassumed that this part conferred a motor benefit on ferent from the older part of the nucleus. This humans; but the motor assumption was thrown into differentiation between the newly-evolved part doubt when functional imaging techniques made it (ventrolateral) and the older part (dorsomedial) is possible to visualize cerebellar activity. These images based on morphological, histological, embryological, showed that, even in the complete absence of any histochemical, and pathological evidence 7. motor activity, the cerebellum was activated when Because this neodentate part of the cerebellum humans performed certain cognitive and language grew to enormous size in humans, an obvious tasks2'3. This cerebellar participation in mental tasks question has arisen about its function in the human offers an explanation for the cerebellar enlarge- brain. Neurosurgeons have provided a provocative ment: it may have provided the neural basis for a piece of evidence by reporting that stereotaxic functional expansion of the cerebro-cerebellar sys- lesions, when placed in the neodentate, fail to tem, in which the lateral cerebellum can improve produce some classical motor signs of cerebellar ~) 1993,ElsevierSciencePubWishersLtd,(UK)
TINS, VOI. 16, NO. 11, 1993