Takao Takeshima

Plasma homocysteine and MTHFR C677T genotype in levodopa-treated patients with PD

To the Editor: The paper by Yasui et al.1 showing elevated plasma homocysteine levels in levodopa-treated patients with PD is interesting and confirms other reports2 and our unpublished findings (table). It also shows that patients with the TT-genotype of the C677T polymorphism of methylenetetrahydrofolate reductase (MTHFR) have especially high homocysteine levels. These findings have to be related to the metabolism of levodopa and methyl groups and may have importance in the future treatment of PD with levodopa. Homocysteine is the demethylated derivative of methionine and is only formed in numerous transmethylation reactions (e.g., catecholamine O-methyltransferase [COMT]) that use S-adenosylmethionine (SAM) as the methyl donor.3 Most administered levodopa is COMT-methylated to 3O-methyldopa and the methylation of 1000 mg of levodopa consumes 5 mMol of SAM methyls and produces 5 mMol of S-adenosylhomocysteine (SAH), which then is deadenosylated to homocysteine. In the study of Yasui et al., the average levodopa dose was 1500 mg. Therefore, it is most likely that levodopa treatment contributed to hyperhomocysteinemia in their patients with PD. In this context it is important to note that humans normally consume about 15 mMol/day of SAM methyls for methylation of endogenous compounds such as phospholipids, DNA, myeline, and proteins. About 10 mMol of these methyls are supplied from food, mainly as methionine and choline, and about 5 mMol are de novo synthesized in one-carbon (folate) metabolism and are transferred by a vitamin B12-dependent reaction to homocysteine, again forming methionine and SAM.4 As we can assume that levodopa-treated PD patients do not increase their intake of labile methyls, they must de novo synthesize all their extra methyls consumed for levodopa methylation. In the above 1000 mg example, the patient must increase his or her de novo synthesis of methyls by about 100%. If methyl neogenesis is hampered by poor folate intake, especially if the patient is a TT-homozygote for C677T/MTHFR polymorphism (about 12% of the Caucasian population), or has a low vitamin B12 level, production of SAM methyls may not be sufficient for both methylation of endogenous compounds and levodopa. This may lead to hypomethylation of essential neural components, and might explain the neuropsychiatric symptoms in folate and vitamin B12 deficiency.5 This mechanism may contribute to side effects of levodopa. If this is the case, early combination of levodopa with COMT inhibitors (tolcapone, entacapone) would prove to be beneficial. Moreover, studies on the relationship between complications to levodopa therapy in PD and homocysteine, folate, vitamin B12, and C677T/MTHFR genotypes are warranted.Plasma homocysteine and cysteine levels were measured in 90 patients with PD with the MTHFR C677T (T/T) genotype. The authors found that the levels of homocysteine-a possible risk factor for vascular disease-were elevated by 60% in levodopa-treated patients with PD, with the most marked elevation occurring in patients with the T/T genotype. Cysteine levels in subjects with PD did not differ from levels in control subjects. In the T/T genotype patients, homocysteine and folate levels were inversely correlated. Increased homocysteine might be related to levodopa, MTHFR genotype, and folate in PD.

Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of glial cell line-derived neurotrophic factor

Glial cell line‐derived neurotrophic factor (GDNF) has been shown to protect cranial and spinal motoneurons, that suggests potential uses of GDNF in the treatment of spinal cord injury and motor neuron diseases. We examined neuroprotective effect of human GDNF encoded by an adenovirus vector (AxCAhGDNF) on the death of lesioned adult rat spinal motoneurons. The seventh cervical segment (C7) ventral and dorsal roots and dorsal root ganglia of adult Fisher 344 rats were avulsed, and AxCAhGDNF, AxCALacZ (adenovirus encoding β‐galactosidase gene) or PBS was inoculated in C7 vertebral foramen. One week after the avulsion and treatment with AxCALacZ, the animals showed expression of β‐galactosidase activity in lesioned spinal motoneurons. Animals avulsed and treated with AxCAhGDNF showed intense immunolabeling for GDNF in lesioned spinal motoneurons and expression of virus‐induced human GDNF mRNA transcripts in the lesioned spinal cord tissue. Nissl‐stained cell counts revealed that the treatment with AxCAhGDNF significantly prevented the loss of lesioned ventral horn motoneurons 2 to 8 weeks after avulsion, as compared to AxCALacZ or PBS treatment. Furthermore, the AxCAhGDNF treatment ameliorated choline acetyltransferase immunoreactivity in the lesioned motoneurons after avulsion. These results indicate that the adenovirus‐mediated gene transfer of GDNF may prevent the degeneration of motoneurons in adult humans with spinal cord injury and motor neuron diseases. J. Neurosci. Res. 60:511–519, 2000

Hypertrophy of IMC of carotid artery in Parkinson's disease is associated with L-DOPA, homocysteine, and MTHFR genotype.

In recent years, an intense interest has developed in the association between Parkinsons disease (PD) and hyperhomocysteinemia. Homocysteine (Hcy) is a neuronal excitotoxic amino acid, and is well known as a risk factor for vascular diseases. Some reports suggest that the administration of L-DOPA may promote hyperhomocysteinemia and idiopathic atherosclerosis. In this study, we report that a mild hypertrophy of the intima-media complex (IMC) of the carotid artery, which has been established as a marker for systemic atherosclerosis, is observed in PD patients compared with normal subjects. PD patients that were treated with L-DOPA for long durations showed a hypertrophic IMC, while the patients that were not treated with L-DOPA did not show any hypertrophic changes in the IMC. These hypertrophic changes were observed primarily in patients with a Hoehn-Yahr stage of 3-5. PD patients with hypertrophic IMC of the carotid artery also exhibited elevated plasma levels of Hcy associated with the C677T genotype of 5,10-methylenetetrahydrofolate reductase (MTHFR). Moreover, a prolonged duration of treatment with L-DOPA in patients with MTHFR T/T genotype enhanced the hypertrophy of IMC, compared with patients with the C/C or C/T genotype. These results suggest that hyperhomocysteinemia promoted by the C677T genotype of MTHFR and prolonged treatment with L-DOPA enhances atherosclerosis in PD patients and affects their general condition.

Diagnostic significance of tau protein in cerebrospinal fluid from patients with corticobasal degeneration or progressive supranuclear palsy

Distinguishing corticobasal degeneration (CBD) from progressive supranuclear palsy (PSP) is clinically and pathologically difficult, and a useful biological marker to discriminative these two diseases has been a subject of clinical interest. In the present study, we assessed tau protein levels in cerebrospinal fluids by sandwich ELISA to distinguish CBD from PSP. The subjects consisted of 27 cases of CBD, 30 cases of PSP, and 36 healthy controls (CTL). The tau values in CBD were significantly higher than those in PSP (P<0.001) and those in CTL (P<0.001). The assay of CSF tau provided diagnostic sensitivity of 81.5% and specificity of 80.0% between CBD and PSP according to receiver-operating characteristic (ROC) curve analysis. When values were compared separately with respect to stage of the disease, differences in the values for moderate CBD vs. moderate PSP had the greatest significance (P<0.001 sensitivity 92.3%, specificity 100.0%), followed by cases of mild CBD and PSP (P<0.005, sensitivity 100.0%, specificity 87.5%). The values in severe CBD and PSP were not significantly different (P=0.07, sensitivity 100%, specificity 75.0%). Using data obtained from a larger number of disease cases, we confirmed our previous findings that tau protein levels in cerebrospinal fluids in patients with CBD are significantly higher than those in patients with PSP. Because tau protein levels in cerebrospinal fluids are significantly higher in early CBD cases than in early PSP cases, measurement of tau protein levels in cerberospinal fluids may be useful for the differential diagnosis of early CBD from early PSP.

Standardized methods to bioassay neurotrophic factors for dopaminergic neurons

The search for specific neurotrophic factors that will eventually be used to reduce or arrest the rate of degeneration of dopaminergic neurons in Parkinsons disease is being pursued by first testing the ability of putative compounds to increase the survival of dopaminergic neurons in primary cultures of the fetal, ventral mesencephalon. This research has intensified in recent years. The experimental procedures used by different laboratories in these studies differ widely, and meaningful comparisons of the results obtained are accordingly difficult to make. Some important experimental variables include the age of the fetal tissue used; the dissection technique used to isolate the ventral mesencephalon; the percentage of dopaminergic neurons present in the culture initially; handling of the tissue during dissection; the technique used to disperse the cells; the use of serum; the technique of plating the cells; the attachment factors used; detachment and loss of cells during the staining procedure; the age of the cultures at the time of analysis; the uneven distribution of cells at the time of analysis and the use of imaging techniques in the analysis. We show that when the E14 rat embryo is used, it is possible to consistently obtain a culture with 20% of tyrosine hydroxylase-positive neurons. Neither the plating density in the range of 7.8 x 10(3) to 1.25 x 10(5) cells/cm2, nor the percentage of serum in the growth medium affected the percentage of cells that expressed TH initially, at 4 or 12 h after plating. When the cells were plated as 25 microliters droplets, called microislands (area approximately 12.5 mm2), and allowed to attach before additional growth medium was added, cell density remained uniform at the center of the microisland for the duration of the culture. Restriction of the analysis of cell survival to the center of the microisland therefore helped to decrease the variability in counting that could occur when cells are dispersed over a larger area. In contrast, in an 8-well chamber slide or 35 mm petri dish, in which the whole area is plated, cell density was consistently higher at the edge (edge effect), versus the centre, by a factor of about three. The use of microisland cultures also has the additional benefit of increasing by a factor of about five the number of individual cultures that can be set up per liter, and a proportionate reduction in the number of animals used per experiment. When the percentage of serum in the growth medium was 0% always, or 10% for the first 12 h, and 0% thereafter, or 10% always, the number of TH-pos neurons per field (using a x 20 objective, column factor 1.25; area 320 microns2) after 5 days in culture (DIV5) was < 1,3-8 and 14-22, respectively. Under the same experimental conditions, the number of neurons (MAP2-positive) per field was 5-8, 18-30 and 45-65 (N = 10 in all cases), respectively. Serum deprivation therefore has a highly deleterious effect on neuronal survival in culture. We suggest that cultures that were exposed to serum at any stage of the experiment, should not be referred to as serum-free, since even a brief exposure to serum exerts a protective effect on neurons, and especially on dopaminergic neurons. Instead, the percentage and kind of serum used, the exact usage, and the duration of exposure of the cells to serum should be stated. Finally, it is suggested that where possible, an imaging system with manual count and journaling capabilities be used in the analysis. The methods described are illustrated by dose-response curves of the neurotrophic effects of BDNF, NGF-beta and IL-6 versus percentage survival on dopaminergic neurons, when grown in serum-free medium throughout.

Static stabilometry in patients with migraine and tension-type headache during a headache-free period

Abstract The vestibulospinal system was evaluated using a stabilometric method in patients with migraine and episodic tension‐type headache during headache‐free periods. Migraine patients often complain of dizziness or vertigo during headache attacks and some exhibit these symptoms between attacks. Computerized static stabilometry is a reliable and non‐invasive technique to evaluate the equilibrium function in various diseases. The subjects consisted of 21 patients with migraine, 12 patients with episodic tension‐type headache and, age‐ and sex‐matched controls. We performed two sets of static stabilometric measurements with eyes open (EO) and eyes closed (EC) for 30 s. The averages of two sessions of the following six stabilometric parameters were used for the analysis: locus length (LNG), environmental area (ENV‐AREA), rectangle area (REC‐AREA), locus length per second, locus length per environ area (L/E), and root mean square area. Romberg quotients (EC/EO) of these six parameters were also analyzed. The mean values of LNG, ENV‐AREA and REC‐AREA in the EC session in the migraine group were significantly greater than those in the controls (P < 0.05, Mann–Whitney rank sum test). Romberg quotients of all stabilometric parameters except the L/E in the migraine group were significantly greater than in the controls. Patients with episodic tension‐type headache did not show any differences in the stabilometric study from the controls. The present findings suggest that patients with migraine show a significant increase of the body sway during the EC session, which indicates an underlying dysfunction in the vestibulospinal system.

GAPDH knockdown rescues mesencephalic dopaminergic neurons from MPP+ -induced apoptosis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) has a number of diverse functions apart from glycolytic function. We explored the possible involvement of GAPDH in 1-methyl-4-phenylpyridinium (MPP+)-induced death of mesencephalic dopaminergic neurons (MDNs) in culture. MPP+ (10 and 20 μM, 24 h) exposure selectively decreased the survival of tyrosine hydroxylase positive (TH+) MDNs, which manifested apoptotic features including shrinkage of the cell body, chromatin condensation and nuclear fragmentation. Two types of GAPDH antisense oligonucleotides almost completely rescued MDNs from MPP+ toxicity. GAPDH was strongly expressed in apoptotic TH+ neurons, and MPP+ exposure significantly increased the percentage of TH+ neurons in which GAPDH is over-expressed. Confocal microscopic analysis demonstrated the nuclear accumulation of GAPDH in neurons undergoing MPP+-induced apoptosis. These results suggest that MPP+ causes apoptosis of MDNs, concomitant with the over-expression and nuclear accumulation of GAPDH.

Cosinor Analysis of Heart Rate Variability in Ambulatory Migraineurs

Objective.—To clarify whether the circadian rhythm of heart rate variability parameters can be identified in patients with migraine during a headache‐free period and to identify any specific pattern of the circadian rhythm of heart rate variability, using time‐domain and spectral analysis and cosinor rhythm analysis of heart rate variability during normal daily activity.

Genetic Analysis of Vascular Factors in Alzheimer's Disease

Abstract: Genetic risk factors for Alzheimers disease (AD) have been extensively examined. Several risk factors for AD are shared with vascular dementia (VaD). We performed genetic case‐control studies on polymorphisms of the apolipoprotein E (ApoE) gene, the methylene tetrahydrofolate reductase (MTHFR) gene, and the angiotensin‐converting enzyme (ACE) gene. The most acceptable genetic risk factor for the development of AD is the ApoE epsilon‐4 (ApoE ε4) allele. ApoE promoter polymorphisms have also been reported to be associated with AD. As expected, the ApoE ε4 allele had strong association with AD in our samples. The ApoE ε4 allele was also estimated as a risk factor for VaD. An ApoE promoter polymorphism (−291T/G) did not show positive association with AD or any other diseases. Common MTHFR phenotypes are thought to genetically regulate blood homocysteine level, which has been associated with AD. We failed to show independent associations between AD and the common MTHFR polymorphisms (C677T and A1298C). A deletion polymorphism at intron 16 of the ACE gene has also been associated with AD. In our study, we found a significant ethnic difference of the genotype distribution, but failed to replicate the positive association between the I allele and AD.

Double-blind crossover study of branched-chain amino acid therapy in patients with spinocerebellar degeneration

To determine whether treatment with branched-chain amino acids (BCAA) can improve the condition of patients with ataxia, a double-blind crossover study of BCAA therapy was performed in 16 patients with spinocerebellar degeneration (SCD). The patients were treated with BCAA in oral doses of 1.5, 3.0, or 6.0 g or with placebo daily for 4 weeks in each study phase. The order of treatment phases (placebo or BCAA) was assigned randomly. An International Cooperative Ataxia Rating Scale (ICARS) was used to quantify the severity of symptoms of SCD. The mean ICARS score improved significantly with BCAA treatment compared with the mean pretreatment score (p<0.01). In addition, the improvement in the mean global ICARS score was significant in the middle-dose group compared with that in the placebo group (p<0.02). The estimated improvement in kinetic functions compared with pretreatment (p<0.01) was significant after treatment with BCAA, 1.5 and 3.0 g. All of the responders manifested predominantly cerebellar symptoms, especially those with spinocerebellar ataxia type 6 (SCA6). Thus, treatment with BCAA may be effective in patients with the cerebellar form of SCD.