Molecular and immunogenic features of myelin lipids: incitants or modulators of multiple sclerosis?

Myelin lipids have long been thought to play intriguing roles in the pathogenesis of multiple sclerosis (MS). This review summarizes current understanding of the molecular basis of MS with emphasis on the: (i.) physico-chemical properties, organization and accessibility of the lipids and their distribution within the myelin multilayer; (ii.) characterization of myelin lipid structures, and structure-function relationships relevant to MS mechanisms, and; (iii.) immunogenic and other features of lipids in MS including molecular mimicry, lipid enzyme genetic knockouts, glycolipid-reactive NKT cells, and monoclonal antibody-induced remyelination. New findings associate anti-lipid antibodies with pathophysiological biomarkers and suggest clinical utility. The structure of CD1d-lipid complexed with the lipophilic invariant T cell receptor (iTCR) may be crucial to understanding MS pathogenesis, and design of lipid antigen-specific therapeutics. Novel immuno-modulatory tools for treatment of autoimmune diseases including MS in which there is both constraint of inflammation and stimulation of remyelination are now emerging.

Maternal alcohol consumption increases sphingosine levels in the brains of progeny mice.

The effect of 'binge' alcohol upon sphingolipid metabolism in the fetal alcohol syndrome (FAS) was examined in pregnant mice (C57BL/6J) by administering a single dose of alcohol during the third trimester (gestational day 15-16). The control mice were administered a sucrose solution of equal caloric value. Brains from progeny at postnatal days 5, 15, 21 and 30 were dissected into three regions, and sphingolipid concentrations of the brain regions were determined including assay of monoglycosylceramide, ceramide, sphingosine and sphingomyelin. We found that a single dose of ethanol induces an elevation of sphingosine (2-3.5-fold) in the brain of progeny. The level of brain ceramide at a dose of 1.5 g/kg was significantly higher than control. Alcohol consumption during pregnancy induces neuronal loss in progeny brains. Our result suggests that the elevation of sphingosine in progeny brain induced by maternal alcohol consumption may be responsible for observed neuronal loss in FAS.

Relatively low levels of calpain expression in juvenile rat correlate with less neuronal apoptosis after spinal cord injury.

Approximately 5% of spinal cord injuries in the US occur in patients younger than 16 years. These young patients have an increased mortality within the 24 h after trauma but have a greater capacity for functional recovery than adults, suggesting age-related differences in injury tolerance. Unfortunately, the response of the developing cord to secondary injury has not been thoroughly investigated. Calpain, a Ca(2+)-dependent protease, has been implicated in the pathogenesis of spinal cord injury (SCI) in rats. Our current investigation revealed that following SCI, calpain upregulation was qualitatively less in the 21-day-old rats than in adult rats, as shown by immunofluorescent labeling. Decreased levels of TUNEL+ neurons were also noted in juvenile rat spinal cord, indicating that the developing cord may have an increased resistance to injury.

Inhibition of calpain-mediated apoptosis by E-64 d-reduced immediate early gene (IEG) expression and reactive astrogliosis in the lesion and penumbra following spinal cord injury in rats.

Upregulation of calpain, a Ca(2+)-activated cysteine protease, has been implicated in apoptosis and tissue degeneration in spinal cord injury (SCI) that over time spreads from the site of injury to the surrounding regions. We examined calpain content and activity, regulation of immediate early genes (IEGs) such as c-jun and c-fos, reactive astrogliosis as the expression of glial fibrillary acidic protein (GFAP), and apoptosis-related features such as caspase-3 mRNA expression and internucleosomal DNA fragmentation in 1-cm long spinal cord segments (S1, distant rostral; S2, adjacent rostral; S3, lesion or injury; S4, adjacent caudal; and S5, distant caudal) following SCI in rats. Calpain content and production of 150 kD calpain-cleaved alpha-fodrin fragment, expression of IEGs, reactive astrogliosis, and apoptotic features were highly increased in the lesion (S3), moderately in adjacent areas (S2 and S4), and slightly in distant areas (S1 and S5) in SCI rats when compared to sham animals. Administration of the calpain-specific inhibitor E-64-d (1 mg/kg) to SCI rats continuously for 24 h inhibited calpain activity and other factors contributing to apoptosis in the lesion and surrounding areas, indicating that calpain played a key role in the pathophysiology of SCI. The results obtained from this animal model of SCI suggest that calpain inhibitor can provide neuroprotection in patients with SCI.

Cell death in spinal cord injury (SCI) requires de novo protein synthesis. Calpain inhibitor E-64-d provides neuroprotection in SCI lesion and penumbra.

Degradation of cytoskeletal proteins by calpain, a Ca(2+)-dependent cysteine protease, may promote neuronal apoptosis in the lesion and surrounding areas following spinal cord injury (SCI). Clinically relevant moderate (40 g-cm force) SCI in rats was induced at T12 by a standardized weight-drop method. Internucleosomal DNA fragmentation or apoptosis in the lesion was inhibited by 24-h treatment of SCI rats with cycloheximide (1 mg/kg), indicating a requirement for de novo protein synthesis in this process. To prove an involvement of calpain activity in mediation of apoptosis in SCI, we treated SCI rats with a cell-permeable calpain inhibitor E-64-d (1 mg/kg). Following 24-h treatment, a 5-cm-long spinal cord section centered at the lesion was collected, and divided equally into five segments (1 cm each) to determine calpain activity, as shown by degradation of the 68-kD neurofilament protein (NFP), and apoptosis as indicated by internucleosomal DNA fragmentation. Neurodegeneration propagated from the site of injury to neighboring rostral and caudal regions. Both calpain activity and apoptosis were readily detectable in the lesion, and moderately so in neighboring areas of untreated SCI rats, whereas these were almost undetectable in E-64-d-treated SCI rats, and absent in sham animals. Results indicate that apoptosis in the SCI lesion and penumbra is prominently associated with calpain activity and is inhibited by the calpain inhibitor E-64-d providing neuroprotective benefit.

Identification and expression analysis of spastin gene mutations in hereditary spastic paraplegia.

Pure hereditary spastic paraplegia (SPG) type 4 is the most common form of autosomal dominant hereditary SPG, a neurodegenerative disease characterized primarily by hyperreflexia and progressive spasticity of the lower limbs. It is caused by mutations in the gene encoding spastin, a member of the AAA family of ATPases. We have screened the spastin gene for mutations in 15 families consistent with linkage to the spastin gene locus, SPG4, and have identified 11 mutations, 10 of which are novel. Five of the mutations identified are in noninvariant splice-junction sequences. Reverse transcription-PCR analysis of mRNA from patients shows that each of these five mutations results in aberrant splicing. One mutation was found to be "leaky," or partially penetrant; that is, the mutant allele produced both mutant (skipped exon) and wild-type (full-length) transcripts. This phenomenon was reproduced in in vitro splicing experiments, with a minigene splicing-vector construct only in the context of the endogenous splice junctions flanking the splice junctions of the skipped exon. In the absence of endogenous splice junctions, only mutant transcript was detected. The existence of at least one leaky mutation suggests that relatively small differences in the level of wild-type spastin expression can have significant functional consequences. This may account, at least in part, for the wide ranges in age at onset, symptom severity, and rate of symptom progression that have been reported to occur both among and within families with SPG linked to SPG4. In addition, these results suggest caution in the interpretation of data solely obtained with minigene constructs to study the effects of sequence variation on splicing. The lack of full genomic sequence context in these constructs can mask important functional consequences of the mutation.

Chromatographic resolution and quantitative assay of CNS tissue sphingoids and sphingolipids.

Quantitative separation of ceramide, sphingoids (dihydrosphingosine, sphingosine, psychosine), and glycosphingolipids as individual fractions was achieved with silicic acid, Dowex column chromatography, and specific solvent mixtures that have not been previously described. Purified ceramide, resolved as a single band, was assayed by thin-layer chromatography (TLC) followed by gas chromatography (GC) and high performance liquid chromatography (HPLC). Sphingoids, purified by Dowex, were assayed by GC and HPLC without mild alkaline hydrolysis, which reduces the yield by interfering with the free amino group of psychosine and dihydrosphingosine. Several less polar (than cerebroside) alkali-/acid-labile glycosphingolipids that elute with galactosylceramide were also identified. Neutral and acidic glycosphingolipids, quantitatively recovered and purified to homogeneity, were resolved by TLC. We used these techniques to determine sphingolipids and sphingoids of vertebrate central nervous system (CNS) tissue, using as little as 30-50 mg (wet weight) of tissue. In addition, phosphatidylcholine and sphingomyelin, relevant to ceramide metabolism, were quantitatively recovered in pure form and resolved by TLC. This method, used to study CNS sphingolipid content, may well be applicable to determine the sphingolipid composition of other tissues and cell culture, but further experiments are necessary to ascertain this.

Inhibition of proteolysis by a cyclooxygenase inhibitor, indomethacin.

The effect of indomethacin, a non-steroidal anti-inflammatory drug upon purified calpain has been studied. Also, its effects upon Ca2+-mediated degradation of cytoskeletal proteins (neurofilament) in spinal cord homogenate has been investigated. A dose-dependent inhibition of purified calpain activity was observed. A 50% inhibition of 14C-caseinolytic activity was obtained with less than 1.1 mM of indomethacin while the activity was completely inhibited at 3.3 mM concentration. The inhibitory effect of ketorlac, another non-steroidal anti-inflammatory drug, upon calpain was weaker than that of indomethacin. The degradation of myelin basic protein (MBP) by cathepsin B, a lysosomal cysteine protease, was significantly inhibited by indomethacin. It also inhibited the Ca2+-mediated degradation of neurofilament protein (NFP) in spinal cord homogenate. The extent of NFP degradation was analyzed by SDS-PAGE and the inhibition shown by indomethacin was weaker than that observed with leupeptin and the calpain inhibitor E64-d. The inhibitory effect of indomethacin on the activity of multicatalytic proteinase complex was negligible. These results suggest that indomethacin, a non-steroidal anti-inflammatory drug and cyclooxygenase inhibitor also inhibits proteinases, including cathepsin B and calpain.

Increased calpain expression is associated with apoptosis in rat spinal cord injury: calpain inhibitor provides neuroprotection.

Calpain content was investigated in the lesion of rat spinal cord at 1, 4, 24, and 72 h following injury induced by the weight-drop (40 g-cm force) technique. Calpain content was increased in the lesion, and was highest at 24 h following injury. microCalpain mRNA level in the lesion was increased by 58.4% (p = 0.0135) at 24 h following trauma, compared to sham. Alterations in mRNA expression in the lesion increased bax/bcl-2 ratio by 20.8% (p = 0.0395) at this time point, indicating a commitment to apoptosis. Therapeutic effect of the calpain inhibitor E-64-d (1 mg/kg) was studied in SCI rats following administration for 24 h. Internucleosomal DNA fragmentation (apoptosis) was observed in SCI rats, but not in sham or E-64-d treated rats. These results indicate a new information that E-64-d has the therapeutic potential for inhibiting apoptosis in SCI.

Combined TUNEL and double immunofluorescent labeling for detection of apoptotic mononuclear phagocytes in autoimmune demyelinating disease.

Apoptosis is usually associated with genomic DNA fragmentation which can be detected in situ by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. We describe a combined TUNEL and double immunofluorescent labeling technique to determine the fate of inflammatory infiltrates and resident glial cells in the central nervous system following the onset of an autoimmune demyelinating disease such as experimental allergic encephalomyelitis (EAE) in rats. Anti-digoxigenin (anti-DIG) antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) emitting blue fluorescence was used to detect apoptotic cell DNA, which was already labeled by modified TUNEL using alkali-stable DIG-11-dUTP. Anti-mouse IgG secondary antibody conjugated with Texas Red emitting red fluorescence was used to detect anti-rat CD11b primary antibody (clone OX-42) directed to the surface antigen of mononuclear phagocytes including microglia. Using this technique, we detected apoptotic mononuclear phagocytes (co-labeled with blue and red fluorescences) in the spinal cord sections of rats with EAE.

Calpain activity and expression increased in activated glial and inflammatory cells in penumbra of spinal cord injury lesion.

Following traumatic injury of the spinal cord, cells adjacent to the lesion are subject to ischemic cell death as a result of vascular disruption and secondary inflammatory responses. Proteases such as calcium-activated neutral proteinase (calpain) have been implicated in axon and myelin destruction following injury since they degrade structural proteins in the axon-myelin unit. To examine the role of calpain in cell death following spinal cord injury (SCI), calpain activity and translational expression were evaluated using Western blotting techniques. Calpain activity (as measured by specific substrate degradation) was significantly increased in and around the lesion site as early as 4 hr following injury with continued elevation at 48 hr compared to sham controls. Likewise, calpain expression was significantly increased in both the lesion site and penumbra at 4 and 48 hr after injury. Using double immunofluorescent labeling for calpain and cell-specific markers, this increase in calpain expression was found to be due in part to activated glial/inflammatory cells such as astrocytes, microglia, and infiltrating macrophages in these areas. Thus, since calpain degrades many myelin and axonal structural proteins, the increased activity and expression of this enzyme may be responsible for destruction of myelinated axons adjacent to the lesion site following SCI.

E-64-d prevents both calpain upregulation and apoptosis in the lesion and penumbra following spinal cord injury in rats.

Calpain, a Ca(2+)-dependent cysteine protease, has been implicated in cytoskeletal protein degradation and neurodegeneration in the lesion and adjacent areas following spinal cord injury (SCI). To attenuate apoptosis or programmed cell death (PCD) in SCI, we treated injured rats with E-64-d, a cell permeable and selective inhibitor of calpain. SCI was induced on T12 by the weight-drop (40 g-cm force) method. Within 15 min, E-64-d (1 mg/kg) in 1.5% DMSO was administered i.v. to the SCI rats. Following 24 h treatment, a 5-cm long spinal cord section with the lesion in the center was collected. The spinal cord section was divided equally into five 1-cm segments (S1: distant rostral, S2: near rostral, S3: lesion or injury, S4: near caudal and S5: distant caudal) for analysis. Determination of mRNA levels by reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that ratios of bax/bcl-2 and calpain/calpastatin were increased in spinal cord segments from injured rats compared to controls. Degradation of the 68-kD neurofilament protein and internucleosomal DNA fragmentation were also increased. All of these changes were maximally increased in the lesion and gradually decreased in the adjacent areas of SCI rats, while largely undetectable in E-64-d treated rats and absent in sham controls. The results indicate that apoptosis in rat SCI appears to be associated with calpain activity which can be attenuated by the calpain inhibitor E-64-d.

Oxidative stress and Ca2+ influx upregulate calpain and induce apoptosis in PC12 cells.

Calpain, a Ca2+-dependent cysteine protease, has previously been implicated in apoptosis or programmed cell death (PCD) in immune cells. Although oxidative stress and intracellular free Ca2+ are involved in neurodegenerative diseases, the mechanism of neuronal cell death in the central nervous system (CNS) due to these agents has not yet been defined. To explore a possible role for calpain in neuronal PCD under oxidative stress and Ca2+ influx, we examined the effects of H2O2 and A23187 on PC12 cells. Treatments caused PCD (light microscopy and TUNEL assay) with altered mRNA expression (RT-PCR) of bax (pro-apoptotic) and bcl-2 (anti-apoptotic) genes, resulting in a high bax/bcl-2 ratio. Control cells expressed 1.3-fold more microcalpain (requiring microM Ca2+) than mcalpain (requiring mM Ca2+). Expression of mcalpain was significantly increased following exposure to oxidative stress and Ca2+ influx. The mRNA levels of calpastatin (endogenous calpain inhibitor) and beta-actin (house-keeping) genes were not changed. Western analysis indicated degradation of 68 kDa neurofilament protein (NFP), a calpain substrate. Pretreatment of cells with MDL28170 (a cell permeable and selective inhibitor of calpain) prevented increase in bax/bcl-2 ratio, upregulation of calpain, degradation of 68 kDa NFP, and occurrence of PCD. These results suggest a role for calpain in PCD of PC12 cells due to oxidative stress and Ca2+ influx.

Molecular characterization and immunohistochemical localization of IV(4)GalNAcGgOse(4)Cer: a naturally occurring novel neutral glycosphingolipid in bovine brain.

A pair of novel neutral glycosphingolipids (Ngsls) has been identified in bovine brain. Their mobilities on thin layer chromatography were slightly different from a standard pentaglycosylceramide (nLcOse(5)Cer from bovine erythrocytes). The compounds were purified to homogeneity by column chromatography. Their fatty acid and base compositions, their monosaccharide compositions and sugar linkage positions were determined by gas-liquid chromato-graphy/mass spectrometry. Carbohydrate sequence analy-sis by(1)H NMR spectroscopy and stepwise exoglyco-sidase digestion indicated the following pentaglycosyl structure for the oligosaccharide moiety of both Ngsls: GalNAcbeta1-4Galbeta1-3GalNAcbeta1-4Galbeta1-4Gl c. The two Ngsls (abbreviated as IV(4)GalNAcGgOse(4)Cer or GalNAc-GA1), differ in their ceramide compositions, having d18:0 and d18:1 sphingosine as their long chain bases. A monospecific polyclonal anti-GalNAc-GA1 antibody, prepared in rabbit and purified by affinity chromatography, stained the neurons of cerebral cortex and cerebellum including Purkinje cells in adult rat brain, indicating that the novel GalNAc-GA1 is associated with cerebellar and other neurons in vertebrate central nervous system.

Immunohistochemical localization of kininogen in rat spinal cord and brain.

Kininogen localization has been determined by immunocytochemistry in rat spinal cord and brain using a kinin-directed kininogen monoclonal antibody. In the spinal cord, there were immunostained neurons and fibers in laminae I, II, VII, and IX, intensely stained fibers in the superficial layers of the dorsal horn, and immunoreactive glial and endothelial cells. Small neurons, satellite cells, and Schwann cells immunostained distinctly in the dorsal root ganglion. In the brain stem, there were immunoreactive neurons and fibers in the tractus solitarius and nucleus, trigeminal spinal tract and nuclei, periaqueductal gray matter, vestibular nuclei, cochlear nuclei, trapezoid body, medial geniculate nucleus, and red nucleus. Immunostained neurons and fibers were also found in cerebellum (dentate nucleus), cerebral cortex (layers III and V), hippocampus (pyramidal cell layer), and corpus callosum. Glia and endothelial cells stained in all brain regions. The widespread location of kininogen in neurons and their processes, as well as in glial and endothelial cells, indicates more than one functional role, including those proposed as a mediator, a calpain inhibitor, and a kinin precursor, in a variety of neural activities and responses.

Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells.

Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptosis of immune cells. Since central nervous system (CNS) is abundant in calpain, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells exposed to reactive hydroxyl radical (.OH) [formed via the Fenton reaction (Fe2++H2O2+H+-->Fe3++H2O+.OH)], interferon-gamma (IFN-gamma), and calcium ionophore (A23187). Cell death, cell cycle, calpain expression, and calpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) and biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 cells at the G2/M phase of cell cycle. The levels of mRNA expression of six genes were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Diverse stimuli did not alter beta-actin (internal control) expression, but increased calpain expression, and the upregulated bax (pro-apoptotic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in expression of calpastatin (endogenous calpain inhibitor). Western blot analysis showed an increase in calpain content and degradation of myelin-associated glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpression, MAG degradation, and DNA fragmentation. We conclude that calpain overexpression due to.OH stress, IFN-gamma stimulation, or Ca2+ influx is involved in C6 cell death, which is attenuated by a calpain-specific inhibitor.

Calpain activity and translational expression increased in spinal cord injury.

Calpain, a calcium-activated neutral proteinase, has been implicated in myelin and cytoskeletal protein degradation following spinal cord injury. In the present study, we examined the activity and transcriptional expression of calpain in spinal cord injury lesions via Western blotting analysis and RT-PCR, respectively. No increases in transcriptional expression of calpain or calpastatin, the endogenous inhibitor, were observed in the lesion at 1, 4, 24, and 72 h following injury. However, calpain activity (as measured by calpain-specific degradation of the endogenous substrate fodrin) was marginally increased at 4 h and significantly increased by 129.8% at 48 h compared to sham controls after injury. Calpain translational expression was localized in injured spinal cords using double immunofluorescent labeling which revealed increased calpain expression in astrocytes compared to sham controls. These results suggest that calpain produced by astrocytes located in or near spinal cord injury lesions may participate in myelin/axon degeneration following injury.

Calpeptin and methylprednisolone inhibit apoptosis in rat spinal cord injury.

Intracellular free Ca2+ and free radicals are increased following spinal cord injury (SCI). These can activate calpain to degrade cytoskeletal proteins leading to apoptotic and necrotic cell death. Primary injury triggers a cascade of secondary injury, which spreads to rostral and caudal areas. We tested calpain involvement in apoptosis in five 1-cm segments of rat spinal cord with injury (40 g-cm) induced at T12 by weight-drop. Animals were immediately treated with calpeptin (250 micrograms/kg) and methylprednisolone (165 mg/kg) and sacrificed at 48 hr. Untreated SCI rats manifested 68-kD neurofilament protein (NFP) degradation (indicating calpain activity), and internucleosomal DNA fragmentation (indicating apoptosis). Both calpain activity and apoptosis were highest in the lesion, and decreased with increasing distance from the lesion. Treatment decreased 68-kD NFP degradation with reduction in apoptosis in all five areas. Thus, calpeptin and methylprednisolone are found to be neuroprotective in SCI.

Role of calpain in spinal cord injury: effects of calpain and free radical inhibitors.

The demonstration of increased calpain activity, immunostaining, and expression at the gene (mRNA) and protein levels concomitant with ultrastructural degeneration and loss of axon and myelin proteins in lesioned cord have implicated a pivotal role for calpain in tissue destruction in spinal cord injury (SCI). Calpain, stimulated by free radicals, also mediates apoptotic cell death. These findings suggested that the use of calpain and lipid peroxidation drugs as therapeutic agents would protect cells and maintain the axon-myelin structural unit by preventing protein degradation. In order to examine this hypothesis, we treated SCI animals with calpain inhibitors (calpeptin) and/or methlprednisolone (MP), and antiinflammatory and free-radical inhibitor. SCI (40 g/cm) was induced by weight-drop, and 1 mg calpeptin or 165 mg MP/kg were given intravenously (i.v.) for 24 hours. Untreated injured animals receiving vehicle served as controls. Lesion 68-kDa and 200-kDa neurofilament proteins (NFPs) were analyzed by sodium dodecylsulfate polyarcylamide gel electrophoresis (SDS-PAGE) and chemiluminescence, and the extent of protein loss was quantitated. Loss of protein in the lesion of untreated cord amounted to 47% compared to sham control, while that for calpeptin- or MP-treated rats was 25-30%. Combination treatment with calpeptin and MP was slightly more effective in preventing NFP degradation, compared to either when used alone. Apopotic cell death in SCI as characterized by internucleosomal DNA fragmentation was also reduced following treatment with the inhibitors. The inhibition of cytoskeletal protein degradation suggests that calpain and free-radical inhibitors may rescue cells and preserve and maintain membrane structure by preventing protein breakdown, preserving motor function, and being neuroprotective.

Genetic polymorphism and Parkinson's disease in Taiwan: study of debrisoquine 4-hydroxylase (CYP2D6).

Debrisoquine 4-hydroxylase (CYP2D6) is one of the cytochrome P450 enzyme families that catalyze the breakdown of a variety of exogenous and endogenous compounds. Previous reports have suggested that genetic polymorphisms of debrisoquine 4-hydroxylase are associated with susceptibility to Parkinson's disease (PD) in Caucasians. To determine if CYP2D6 also confers susceptibility to PD in Chinese patients, we carried out a study of genetic association using three polymorphic markers of the CYP2D6 gene, 188C/T, 1934G/A (mutant B), and 4268G/C. No differences of allele or genotype frequencies of these three polymorphisms were detected upon comparison of primary PD patients (n=53) with normal controls (n=94). The 1934A allele (mutant B), which accounts for the majority of poor metabolizers in Caucasians, is extremely rare in Chinese. Our data do not support the suggestion that the CYP2D6 gene is related to PD susceptibility in Chinese.