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Accumulating data from experimental studies indicate that oxidative stress has a major role in the pathogenesis of multiple sclerosis (MS). It has been suggested that local production of reactive oxygen species, probably by macrophages, mediates axonal damage in both MS patients and the mouse model experimental autoimmune encephalomyelitis (EAE). We have shown previously that our novel brain-penetrating antioxidant, N-acetylcysteine amide (AD4), reduces the clinical and pathological symptoms, including inflammation and axonal damage in myelin oligodendrocyte glycoprotein (MOG)-induced chronic EAE in mice. The aim of this study was to examine the molecular mechanism by which AD4 exerts protection in MOG-induced EAE mice. Therefore, we analyzed gene-expression profile in the spinal cords of MOG-induced chronic EAE mice and compared them with MOG-induced mice treated with AD4, using a cDNA microarray. We found that MOG treatment up-regulated genes encoding growth factors, cytokines, death receptors, proteases, and myelin structure proteins, whereas MOG- and AD4-treated mice demonstrated gene expression profiles similar to that seen in naive healthy mice. In conclusion, our study shows that chronic AD4 administration suppresses the induction of various pathological pathways that play a role in EAE and probably in MS.

The purpose of our study was to prospectively evaluate the striatal uptake of 123I-labeled N-(3-fluoropropyl)-2beta-carbomethoxy-3beta-(4-iodophenyl)nortropane (FP-CIT) and the response to l-dopa therapy in patients with cerebrovascular disease (CVD) who develop clinical symptoms of vascular parkinsonism (VP).Twenty consecutive patients who developed VP in the course of CVD were prospectively enrolled in the study. All patients had CT evidence of CVD (17 patients had lacunar infarcts, 3 patients had territorial strokes). The clinical stage of the patients was assessed using the Hoehn and Yahr scale, and the severity of the symptoms was measured using the Unified Parkinson's Disease Rating Scale score. Ten age-matched subjects were used as controls. SPECT was performed 180 min after injection of 185 MBq 123I-FP-CIT using a dual-head gamma-camera. The ratio of the mean specific-to-nonspecific striatal binding for the entire striatum, caudate, and putamen was calculated in all patients and compared with that of controls. Putamen-to-caudate binding ratios were compared as well. The response to therapy was compared between patients with normal and abnormal 123I-FP-CIT binding.No correlation was found between any of the clinical variables and response to therapy in patients with VP. Nine patients had normal striatal 123I-FP-CIT binding with no significant differences in striatal or subregional binding ratios compared with those of the controls. In contrast, 11 patients had significantly diminished striatal binding compared with that of controls (P0.001). Subanalyses showed significantly decreased binding in the caudate (P0.04 and P0.01 for the right and left caudate, respectively), diminished binding in the putamen (P0.04 and P0.01 for the right and left putamen, respectively), and a decreased putamen-to-caudate ratio on the right side (P0.001). The latter ratio was not significant on the left. Two of the 3 patients with territorial strokes had significantly diminished striatal 123I-FP-CIT binding in the hemisphere contralateral to the CT lesion. All 9 patients with normal scan findings had a poor response to L-dopa. Six of 11 patients with abnormal studies had no response to L-dopa, whereas 5 patients had a good response (P0.03).The diagnosis of VP cannot be accurately confirmed on the basis of clinical features alone because CVD may alter the typical presentation of PD. Functional imaging with 123I-FP-CIT is highly recommended in patients with CVD who develop symptoms of VP to confirm or exclude the existence of nigrostriatal dopaminergic degeneration. Identifying a subset of patients with reduced 123I-FP-CIT binding in the striatum is important for better treatment selection.

We describe a unique gait phenomenon of bent knees in 9 patients with idiopathic Parkinson disease (mean age, 73.1 +/- 11.1 years), 3 of whom also manifested tiptoeing. The bent-knee posture appeared only during ambulation; in the recumbent position, full or nearly full extension was possible in all patients. The abnormality emerged after long-standing disease (6-23 years from onset) and failed to respond to dopaminergic treatment. Most of the patients also had bent spine (camptocormia). The pathogenesis of these phenomena are unknown, but they might represent a rare type of dystonia.

Levodopa is the most effective symptomatic agent in the treatment of Parkinson's disease (PD) and the "gold standard" against which new agents must be compared. However, there remain two areas of controversy: (1) whether levodopa is toxic, and (2) whether levodopa directly causes motor complications. Levodopa is toxic to cultured dopamine neurons, and this may be a problem in PD where there is evidence of oxidative stress in the nigra. However, there is little firm evidence to suggest that levodopa is toxic in vivo or in PD. Clinical trials have not clarified this situation. Levodopa is also associated with motor complications. Increasing evidence suggests that they are related, at least in part, to the short half-life of the drug (and its potential to induce pulsatile stimulation of dopamine receptors) rather than to specific properties of the molecule. Treatment strategies that provide more continuous stimulation of dopamine receptors provide reduced motor complications in MPTP monkeys and PD patients. These studies raise the possibility that more continuous and physiological delivery of levodopa might reduce the risk of motor complications. Clinical trials to test this hypothesis are underway. We review current evidence relating to these areas of controversy.

Pain is common in patients with Parkinson disease (PD) and can precede the diagnosis of the disease. Experimental studies and clinical evidence indicate involvement of basal ganglia and dopaminergic pathways in central pain processing.To quantitatively assess and compare pain perception in patients with unilateral PD with and without pain and in patients with response fluctuations.Thirty-six patients with PD (mean age, 61.8 +/- 11.2 years) with predominantly unilateral disease, 15 patients with response fluctuations (mean age, 65.3 +/- 10.4 years), and 28 age-matched healthy control subjects participated in the study. Subjective pain was assessed using the visual analog scale with von Frey filaments for tactile thresholds and contact thermode for warm sensation (WS) and heat pain thresholds (HPTs).Tactile and WS thresholds did not differ between patients in both patient groups and control subjects nor between sides. HPT was lower in patients with PD who experienced pain (n = 21) compared with those who did not (42.6 +/- 3.0 degrees C vs 45.6 +/- 2.8 degrees C; p0.01) and those who experienced pain in the more affected side (41.4 +/- 2.6 degrees C vs 43.7 +/- 3.3 degrees C; p0.0001). In patients with fluctuations there were no side differences in WS and HPT or between "on" and "off" periods.Endogenous pain in patients with Parkinson disease is accompanied by increased sensitivity to some painful stimuli, suggesting that basal ganglia abnormality also involves pain encoding.

Increasing evidence suggests that enhanced production of reactive oxygen species (ROS) activates the MAP kinases, c-Jun N-terminal protein kinase (JNK) and mitogen-activated protein kinase MAPK (p38). These phosphorylated intermediates at the stress-activated pathway induce expression of matrix metalloproteinases (MMPs), leading to inflammatory responses and pathological damages involved in the etiology of multiple sclerosis (MS). Here we report that N-acetylcysteine amide (AD4) crosses the blood-brain barrier (BBB), chelates Cu(2+), which catalyzes free radical formation, and prevents ROS-induced activation of JNK, p38 and MMP-9. In the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, oral administration of AD4 drastically reduced the clinical signs, inflammation, MMP-9 activity, and protected axons from demylination damages. In agreement with the in vitro studies, we propose that ROS scavenging by AD4 in MOG-treated animals prevented MMP's induction and subsequent damages through inhibition of MAPK pathway. The low toxicity of AD4 coupled with BBB penetration makes this compound an excellent potential candidate for the therapy of MS and other neurodegenerative disorders.

Mutations in familial Parkinson's disease (PD) have been associated with the failure of protein degradation through the ubiquitin-proteasome system (UPS). Impairment of proteasome function has also been suggested to play a role in the pathogenesis of sporadic PD. We examined the proteasome activity in PC12 cells treated with 6-hydroxydopamine (6-OHDA), the dopamine synthetic derivate used in models of PD. We found that 6-OHDA treatment increased protein oxidation, as indicated by carbonyl group accumulation, and increased caspase-3 activity. In addition, there was an increase in trypsin-, chymotrypsin-, and postacidic-like proteasome activities in cells treated with 10-100 microM 6-OHDA, whereas higher doses caused a marked decline. 6-OHDA exposure also increased mRNA expression of the 19S regulatory subunit in a dose-dependent manner, whereas the expression of 20S- and 11S-subunit mRNAs did not change. Administration of the antioxidant N-acetylcysteine to 6-OHDA-treated cells prevented the alteration in proteasome functions. Moreover, reduction in cell viability owing to administration of proteasome inhibitor MG132 or lactacystin was partially prevented by the endogenous antioxidant-reduced glutathione. In conclusion, our data indicate that mild oxidative stress elevates proteasome activity in response to increase in protein damage. Severe oxidative insult might cause UPS failure, which leads to protein aggregation and cell death. Moreover, in the case of UPS inhibition or failure, the blockade of physiological reactive oxygen species production during normal aerobic metabolism is enough to ameliorate cell viability. Control of protein clearance by potent, brain-penetrating antioxidants might act to slow down the progression of PD.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by demyelination and axonal damage. Although the exact pathophysiology is unknown, apoptosis plays a crucial role. Here, we studied the role of the pro-apoptotic gene Bax in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), the animal model for MS. We demonstrate that the clinical signs were markedly reduced in the EAE Bax-deficient mice as compared to wild type (2.3 +/- 0.5 vs. 1.02 +/- 0.32, respectively, P < 0.05). Bax-deficient mice demonstrated less inflammatory infiltration and axonal damage, although they showed similar T-cell immune potency. In conclusion, ablation of the bax gene attenuates the severity of MOG-induced EAE and emphasizes the importance of apoptosis in the pathogenesis of EAE and MS.

"Dose failures" and "delayed on" phenomena following an intake of levodopa dose in patients with Parkinson's disease (PD) with motor fluctuations may be caused by stagnation of poorly soluble levodopa in the atonic stomach. Etilevodopa is a unique, highly soluble prodrug of levodopa. When ingested, etilevodopa is more readily dissolved in the stomach than levodopa. It passes unchanged through the stomach to the duodenum, where it is rapidly hydrolyzed by local esterases and rapidly absorbed as levodopa. To compare the pharmacokinetics of three different modes of etilevodopa/carbidopa administration with standard levodopa/carbidopa tablets in fluctuating PD patients, 29 patients with PD and response fluctuations were enrolled in an open-label, randomized, four-way crossover study of single doses of 4 treatments: swallowed etilevodopa/carbidopa tablets, etilevodopa/carbidopa tablets dissolved in water, etilevodopa oral solution with carbidopa tablets, and standard levodopa/carbidopa tablets. To measure the maximal concentration (Cmax), time to Cmax (tmax), and area under the curve (AUC) of plasma levodopa, etilevodopa, and carbidopa, blood samples were drawn before drug administration and at intervals up to 240 minutes thereafter. Plasma levodopa tmax was significantly shorter with all three modes of administration of etilevodopa (mean of about 30 minutes) than with levodopa treatment (mean of 54 minutes). During the first 45 minutes after drug ingestion, plasma levodopa AUC was significantly greater after etilevodopa administration than after levodopa administration. Levodopa AUC for 0 to 1 hour and 0 to 2 hours were also significantly greater following administration of etilevodopa/carbidopa swallowed tablets than following administration of levodopa/carbidopa tablets. Mean levodopa Cmax was in the range 2.3 to 2.7 microg/mL for all treatments. Levodopa Cmax was significantly greater following treatment with etilevodopa swallowed tablets than with levodopa tablets. Etilevodopa/carbidopa was well tolerated, with a safety profile comparable to that of levodopa/carbidopa. The shorter levodopa tmax observed with etilevodopa potentially translates to a shorter time to "on". Clinical trials with etilevodopa/carbidopa tablets should be carried out in PD patients with response fluctuations such as "delayed on" and "dose failures".

The causes for the highly selective loss of dopaminergic neurons in the substantia nigra pars compacta in Parkinson's disease (PD) are still unknown. However, a major advance has been recently made with the introduction of the concept of apoptosis as the route leading this specific neuronal population to degeneration. Apoptosis, or programmed cell death (PCD), is an active, controlled program inherent in every living cell. Upon receiving certain signals, cells that are destined to die undergo a highly characteristic process of "suicide." This process consists of massive biochemical and morphological alterations, including cell shrinkage, loss of cell-to-cell contacts, blebbing of cell membranes, cytoskeletal rearrangements, and DNA condensation and fragmentation. It culminates in cell conversion to membrane-bound particles (apoptotic bodies) that are ready to be digested by neighboring macrophages (1-3).

Recent studies suggest that glutamate neurotoxicity is involved in the pathogenesis of multiple sclerosis (MS), and that treatment with glutamate receptor (AMPA/kainate) antagonists inhibits experimental autoimmune encephalomyelitis (EAE), the conventional model of MS. Therefore, we examined whether riluzole, an inhibitor of glutamate transmission, affects the pathogenesis and clinical features of MS-like disease in myelin oligodendrocyte glycoprotein (MOG)-induced EAE in mice. Here we report that riluzole (10 mg/kgx2/day, i.p.), administered before and even after the appearance of clinical symptoms, dramatically reduced the clinical severity of MOG-induced EAE, while all the MOG-immunized control mice developed significant clinical manifestations. Moreover, the riluzole-treated mice demonstrated only mild focal inflammation, and less demyelination, compared to MOG-treated mice, using histological methods. Furthermore, riluzole markedly reduced axonal disruption, as assessed by Bielshowesky's silver staining and by antibodies against non-phosphorylated neurofilaments (SMI-32). No difference was detected in the immune system potency, as T-cell proliferative responses to MOG were similar in both groups. In conclusion, our study demonstrates, for the first time, that riluzole can reduce inflammation, demyelination and axonal damage in the CNS and attenuate the clinical severity of MOG-induced EAE. These results suggest that riluzole, a drug used in amyotrophic lateral sclerosis (ALS), might be beneficial for the treatment of MS.

Accumulating data from experimental and human studies indicate that oxidative stress (OS) plays a major role in the pathogenesis of Alzheimer's disease (AD). The production of reactive oxygen species (ROS), which leads to OS, can occur very early, even before the appearance of symptoms and molecular events (beta-amyloid plaques and neurofibrillary tangles), leading to tissue damage via several different cellular molecular pathways. ROS can cause damage to cardinal cellular components such as lipids, proteins, and nucleic acids (e.g., RNA, DNA), causing cell death by modes of necrosis or apoptosis. The damage can become more widespread because of the weakened cellular antioxidant defense systems. Therefore, treatment with antioxidants might theoretically act to prevent propagation of tissue damage and improve both survival and neurological outcome. Indeed, several studies preformed to date examined whether dietary intake of several antioxidants, mainly vitamins, might prevent or reduce the progression of AD. Although a few of the antioxidants showed some efficacy in these trials, no answer is yet available as to whether antioxidants are truly protective against AD. Reasons for these results might include, in part, blood-brain barrier (BBB) permeability, inappropriate timing of administration, or suboptimal drug levels at the target site in the central nervous system. Thus, antioxidant cocktails or antioxidants combined with other drugs may have more successful synergistic effects. Further, well-designed intervention, as well as observational investigations based on large cohorts studied over a long period of time with several methods for assessing antioxidant exposure, including relation to BBB penetration, are needed to test this hypothesis.

Progressive neurodegenerative disorders share common mechanisms of cell death, and in all likelihood multiple factors are involved in every disease. Therefore, several neuroprotective agents are being investigated with the purpose of slowing or preventing further deterioration of cell loss. These include experimental animal and clinical studies on the neuroprotective effects of caspase inhibitors, antioxidants, glutamate antagonists, anti-inflammatory agents and trophic factors in several neurodegenerative diseases. At present there is limited clinical evidence for direct neuroprotective effects against these diseases, but much effort is being invested in research on novel technologies and compounds.

The authors compared the two portions of the OFF period in patients with Parkinson disease and response fluctuations: time to ON (the latency from levodopa intake to turning ON) and wearing off (time from termination of the beneficial dose effect until the time when the next dose was taken). Time to ON was more than twice the duration of wearing off. Although underrecognized, time to ON is the major component of total daily OFF.

The exact pathogenesis of neuronal death following bleeding in brain parenchyma is still unknown. Hemoglobin (Hb) toxicity has been postulated to be one of the underlying mechanisms. The purpose of this study was to examine the possible contribution to neurotoxicity of each of the Hb compounds and to characterize the death pathway. Pheochromocytoma (PC12) and neuroblastoma (SH- SY5Y) cell lines were exposed to Hb, globin, hemin, protoporphyrin IX and iron for 1.5- 24 h. We found that Hb and hemin are highly toxic (LD(50) of 8 and 20mgr; mol/l, respectively) as compared to globin that was not toxic. In addition, protoporphyrin IX and iron, compounds of hemin, were less toxic than hemin itself (LD(50) of 962 and 2070mgr; mol/l respectively). We also demonstrated that non-specific protein digestion with proteinase-K, markedly increased Hb toxicity. Hemin-treated cells caused a typical apoptotic cell death pattern as indicated by DNA fragmentation, caspase activation and reduction in the mitochondrial membrane potential. Treatment with the antioxidant N-acetyl-L-cysteine or iron chelator, deferoxamine, diminished hemin-induced cell death, indicating a role of oxidative stress in this deleterious process. Thus, therapeutic strategies, based on antioxidant, iron chelator and anti-apoptotic agents may be effective in counteracting Hb neurotoxicity.

Parkinson's disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Although PD has been heavily researched, the precise etiology of nigral cell loss is still unknown and, consequently, treatment is largely symptomatic rather than preventive. There are conflicting data regarding the mode of dopaminergic cell death in PD and, hence, this remains controversial. Several mutations in specific genes have recently been linked with hereditary forms of PD. Although none of these mutations are seen in idiopathic disease cases, the elucidation of these genetic defects sheds light on the nature of idiopathic PD. It is possible that dopaminergic neurogenesis also contributes to the etiology of idiopathic PD. In addition, intracellular as well as extracellular substances found in the SNc are believed to function as damaging pathogenetic factors. These factors, and the interactions among them, might hold the secret to the underlying causes of the selective death of dopaminergic neurons in PD.

Mesenchymal stem cells in the adult bone marrow are differentiated to connective tissue, muscle, bone, cartilage, and fat cells. Recent studies in cultures, animal models, and humans demonstrated the plasticity of these cells and their capacity to express neuronal markers. However, questions were raised as to whether the neuronal phenotypes reflect transient changes or even fusion with neurons. In this study, we induced the differentiation of mouse stromal cells to neuron-like cells and observed the activation of the tissue-specific promoter of neuron-specific enolase (NSE). We used transgenic (Tg) mice that carry the antiapoptotic human bcl-2 gene, expressed only in neurons under the NSE promoter. Some previous studies have indicated that the transgene induces neuroprotection in various animal models of neurodegenerative diseases. We found that following induction, the mouse stromal cells demonstrate neuronal phenotype and express the neuronal marker, NeuN (neural nuclei protein). However, most of the stromal cells derived from the Tg mice, but not the wild type, also expressed human Bcl-2, as indicated by immunocytochemistry. Furthermore, these induced neuron-like cells were more resistant to cell death induced by dopamine. In conclusion, our experimental models showed that stromal cells might be induced to neuronal phenotypes and activate neuronal-specific promoters. Moreover, neurons targeted over expression of the human bcl-2 gene and provided high resistance against such apoptotic insults. This novel strategy reveals a new horizon in the improvement of gene therapy, based on stem cell transplantation in neurodegenerative diseases.

Damage to the optic nerve in mammals induces retrograde degeneration and apoptosis of the retinal ganglion cell (RGC) bodies. The mechanisms that mediate the response of the neuronal cells to the axonal injury are still unknown. We have previously shown that semaphorins, axon guidance molecules with repulsive cues, are capable of mediating apoptosis in cultured neuronal cells (Shirvan, A., Ziv, I., Fleminger, G., Shina, R., He, Z., Brudo, I., Melamed, E., and Brazilai, A. (1999) J. Neurochem. 73, 961-971). In this study, we examined the involvement of semaphorins in an in vivo experimental animal model of complete axotomy of the rat optic nerve. We demonstrate that a marked induction of type III semaphorin proteins takes place in ipsilateral retinas at early stages following axotomy, well before any morphological signs of RGC apoptosis can be detected. Time course analysis revealed that a peak of expression occurred after 2-3 days and then declined. A small conserved peptide derived from semaphorin 3A that was previously shown to induce neuronal death in culture was capable of inducing RGC loss upon its intravitreous injection into the rat eye. Moreover, we demonstrate a marked inhibition of RGC loss when axotomized eyes were co-treated by intravitreous injection of function-blocking antibodies against the semaphorin 3A-derived peptide. Marked neuronal protection from degeneration was also observed when the antibodies were applied 24 h post-injury. We therefore suggest that semaphorins are key proteins that modulate the cell fate of axotomized RGC. Neutralization of the semaphorin repulsive function may serve as a promising new approach for treatment of traumatic injury in the adult mammalian central nervous system or of ophthalmologic diseases such as glaucoma and ischemic optic neuropathy that induce apoptotic RGC death.

Down's syndrome (trisomy 21) is the most common chromosomal aberration and is one of the major reasons for mental retardation. Down's syndrome has manifestations in many systems. The syndrome has many neurological complications, which include structural changes, mental retardation, young-onset Alzheimer's disease, strokes and basal ganglia damage. Epileptic seizures are more prevalent in Down's syndrome patients than in the general population. Cervical spinal cord instability, which is common in Down's syndrome, can cause neurological complications resulting from cervical spinal cord compression. The aim of this article is to review the neurological complications of Down's syndrome in order to increase awareness and assist doctors in diagnosis and counseling the patients' families.

Alpha-synuclein, a presynaptic protein, was found to be the major component in the Lewy bodies (LB) in both inherited and sporadic Parkinson's disease (PD). Furthermore, rare mutations of alpha-synuclein cause autosomal-dominant PD. However, it is unknown how alpha-synuclein is involved in the pathogenesis of nigral degeneration in PD. In this study, we examine the protein-protein interactions of wild-type and mutant (A53T) a-synuclein with adult human brain cDNA expression library using the yeast two-hybrid technique. We found that both normal and mutant alpha-synuclein specifically interact with the mitochondrial complex IV enzyme, cytochrome C oxidase (COX). Wild-type and mutant alpha-synuclein genes were further fused with c-Myc tag and translated in rabbit reticulocyte lysate. Using anti-c-Myc antibody, we demonstrated that both wild-type and mutant alpha-synuclein, coimmunoprecipitated with COX. We also showed that potassium cyanide, a selective COX inhibitor, synergistically enhanced the sensitivity of SH-SY5Y neuroblastoma cells to dopamine-induced cell death. In conclusion, we found specific protein-protein interactions of alpha-synuclein, a major LB protein, to COX, a key enzyme of the mithochondrial respiratory system. This interaction suggests that alpha-synuclein aggregation may contribute to enhance the mitochondrial dysfunction, which might be a key factor in the pathogenesis of PD.

Levodopa ethylester (LDEE), a highly soluble prodrug of levodopa, may overcome the impaired absorption of regular levodopa, due mainly to a combination of levodopa's poor solubility and delayed gastric emptying. We conducted a double-blind, levodopa-controlled, multicenter study of oral LDEE solution compared with standard levodopa-carbidopa (LD-CD) tablets. Sixty-two patients with Parkinson's disease who had "delayed on" and "no-on" subtypes of response fluctuations were randomly assigned for treatment with LDEE-CD or LD-CD 250/25 mg for 4 weeks (phase A). Only the first morning and first post-lunch dose of LD were replaced. This was followed by a 2-week extension with a supplementation of carbidopa (25 mg) to each replaced dose (phase B). Patients filled home diaries 2 weeks before and during the trial period in which times of turning on and off for the two doses were reported. In phase A, mean latency to turning on was reduced by 21% (morning dose) and 17% (post-lunch dose) in the LDEE-CD group. Percentage of no-on episodes after the post-lunch dose was decreased by 21% in the LDEE-CD group but increased by 36% in the LD-CD group (P < 0.01). In phase B, LDEE-CD decreased latencies to on after the morning and post-lunch doses and no-on episodes after the post-lunch dose. The beneficial effects of LDEE were supported by the pharmacokinetic data. Results indicate that LDEE solution is beneficial in ameliorating delayed on and no-on response fluctuations. This effect of LDEE is due to more rapid levodopa absorption.

Some patients with Parkinson's disease (PD) suffer from autonomic dysfunction, even in the early stage of the disease. We examined the skin wrinkling response following immersion of the hands in warm water in 18 patients with hemiparkinsonism. This test evaluates the function of the sympathetic autonomic system. Mean age of the patients was 61 +/- 10 and mean disease duration 5.5 +/- 3.5 years. Both hands of each patient were immersed in warm water for 30 minutes and the number of skin ridges of the fingertip of each finger was counted. The results of each hand were compared to those of nine healthy controls. The mean number of the ridges of the less affected hand was significantly decreased as compared to the affected hand and controls (6.1 +/- 6.8 vs 13.1 +/- 6.8 and 15.3 +/- 8.5, respectively; P0.01). These results suggest that autonomic dysfunction is prevalent in the less affected side of patients with PD and can be simply tested by the skin response test.

Rasagiline mesylate (TVP-1012) is a potent, selective, non-reversible MAO-B inhibitor, without the tyramine-potentiating effect and with neuroprotective activities. The benefit of rasagiline as monotherapy in patients with early Parkinson's disease (PD) has already been reported. To evaluate the safety, tolerability, and clinical effect of rasagiline as adjunctive therapy to levodopa, a multicenter, double-blind, randomized, placebo-controlled, parallel-group study (0.5, 1, and 2 mg/d) was conducted for 12 weeks in 70 patients with PD (mean age, 57.4 y; mean disease duration, 5.7 y; 32 patients had motor fluctuations). A beneficial clinical effect was observed in fluctuating patients treated with rasagiline (all doses), expressed as a decrease in total Unified Parkinson's Disease Rating Scale (UPDRS) score (23.0% vs 8.5% in the placebo group). The treatment effect was still evident 6 weeks after drug discontinuation (in all doses). The safety and tolerability of rasagiline were good. Adverse events were no different than those of patients taking placebo. Almost complete platelet MAO-B inhibition was obtained at all rasagiline doses. This study has demonstrated that rasagiline (up to 2 mg/day) has a good safety profile and a beneficial clinical effect in fluctuating patients with PD when given as an add-on to chronic levodopa therapy.

Available evidence on the practice of acute pharmacological challenge tests in parkinsonian patients was reviewed by a committee of experts, which achieved a general consensus. The published data deal mainly with the acute administration of levodopa and apomorphine in Parkinson's disease. Such challenge may serve different purposes, e.g., research, diagnosis, or tailoring of treatment. Unique protocols describing the clinical setting and practice parameters are not available. The present paper describes the scientific background and supplies practical guidelines, whenever possible, to perform and evaluate acute challenge tests in parkinsonian syndromes. With the appropriate indication and setting, acute challenge tests are useful in diagnosis and therapy of Parkinson's disease and related disorders.

Glutaredoxin 2 (Grx2) from Escherichia coli protects cerebellar neurons from dopamine-induced apoptosis via nuclear factor kappa B (NF-kappaB) activation, which is mediated by the expression of redox factor-1 (Ref-1). An analysis of the mechanisms underlying Grx2 protective activity revealed dual activation of signal transduction pathways. Grx2 significantly activated the Ras/phosphoinositide 3-kinase/Akt/NF-kappaB cascade in parallel to the Jun N-terminal kinase (JNK)/AP1 cascade. Dopamine, in comparison, down-regulated both pathways. Treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate Akt and AP-1 but had no effect on the phosphorylation of JNK1/2, suggesting that Akt/NF-kappaB and AP-1 are regulated by Ref-1. Exposure of the neurons to JNK1/2 antisense oligonucleotide in the presence of Grx2 significantly reduced AP-1 and NF-kappaB DNA binding activities and abolished Grx2 protection. These results demonstrate that dual activation of Ras/phosphoinositide 3-kinase and AP-1 cascades, which are mediated by Ref-1, is an essential component of the Grx2 mechanism of action.

Endothelins are a recently discovered group of most powerful vasoconstrictor peptides. Endothelin-1 is produced by endothelial cells, and endothelin-3 is derived from neuronal tissue. Theoretically, endothelin-mediated vasoconstriction may enhance ischemic neuronal damage. This study aimed to measure plasma levels of both endothelins in patients with acute nonhemorrhagic cerebral infarction. Plasma levels of endothelin-1 and endothelin-3 were measured by radioimmunoassay in 16 consecutive patients within the first 72 hours after the onset of nonhemorrhagic cerebral infarct, as diagnosed clinically and by computed tomography. There was a marked (fourfold) elevation in plasma endothelin-1 levels in the patients (median, 11.7 pg/ml; 25th and 75th centiles, 5.4 and 13.2 pg/ml) compared with those in a control group of 13 age-matched subjects (median, 2.56 pg/ml; 25th and 75th centiles, 2.4 and 3.0 pg/ml; p less than 0.0001). The first 24 hours after stroke onset were associated with higher endothelin-1 levels, and there was a trend to elevated levels with more severe neurological deficits. In all patients and controls endothelin-3 levels were below 0.5 pg/ml. Ischemic stroke is associated with acute and marked increases in plasma levels of endothelin-1. This may reflect enhanced production by damaged endothelial cells within the infarcted tissue. Local leakage of endothelin-1 may induce severe and prolonged constriction of collateral vessels and may therefore have a deleterious role in the pathogenesis and final outcome of cerebral infarction.

The neurotransmitter dopamine (DA) induces apoptosis via its oxidative metabolites. This study shows that glutaredoxin 2 (Grx2) from Escherichia coli and human glutaredoxin could protect cerebellar granule neurons from DA-induced apoptosis. E. coli Grx2, which catalyzes glutathione-disulfide oxidoreduction via its -Cys-Pro-Tyr-Cys- active site, penetrates into cerebellar granule neurons and exerts its activity via NF-kappaB activation. Analysis of single and double cysteine to serine substitutions in the active site of Grx2 showed that both cysteine residues were essential for activity. Although DA significantly reduced NF-kappaB binding activity, Grx2 could stimulate the binding of NF-kappaB to DNA by: (i) translocating NF-kappaB from the cytoplasm to the nucleus after promoting the phosphorylation and degradation of I-kappaBalpha, and (ii) activating the binding of pre existing nuclear NF-kappaB. The DNA binding activity of NF-kappaB itself was essential for neuronal survival. Overexpression of I-kappaB dominant negative gene (I-kappaB-DeltaN) in granule neurons significantly reduced their viability, irrespective of the presence of Grx2. Ref-1 expression was down-regulated by DA but up-regulated by Grx2, while treatment of neurons with Ref-1 antisense oligonucleotide reduced the ability of Grx2 to activate NF-kappaB binding activity. These results show that Grx2 exerts its anti apoptotic activity through the activation of Ref-1, which then activates NF-kappaB.

Auto-oxidation of levodopa generates toxic metabolites, such as free radicals, semiquinones and quinones. In vitro, levodopa is a powerful toxin that is lethal to cultures of neurones. This raises the concern that levodopa may also be toxic in vivo, and that chronic treatment with levodopa could induce further damage to nigrostriatal neurones in patients with Parkinson's disease, accelerating the natural predetermined rate of disease progression. Although a few animal studies have shown that chronic levodopa may be toxic in vivo, most others report that it is not. The few available clinical studies also indicate that the course of Parkinson's disease is not accelerated by chronic systemic treatment with levodopa.

The neurotransmitter dopamine (DA) is capable of inducing apoptosis in post-mitotic sympathetic neurons via its oxidative metabolites. The differential display method was applied to cultured sympathetic neurons in an effort to detect genes whose expression is transcriptionally regulated during the early stages of DA-triggered apoptosis. One of the up-regulated genes was identified as the chick homologue to T-complex polypeptide-1delta (TCP-1delta), a member of the molecular chaperone family of proteins. Each chaperone protein is a complex of seven to nine different subunits. A full-length clone of 1.9 kilobases was isolated containing an open reading frame of 536 amino acids with a predicted molecular weight of 57,736. Comparison with the mouse TCP-1delta revealed 78 and 91% homology on the DNA and protein levels, respectively. Northern blot analysis disclosed a steady and significant increase in mRNA levels of TCP-1delta after DA administration, reaching a peak between 4 and 9 h and declining thereafter. Induction of the TCP-1delta protein levels was also observed as a function of DA treatment. Overexpression of TCP-1delta in sympathetic neurons accelerated DA-induced apoptosis; inhibition of TCP-1delta expression in these neurons using antisense technology significantly reduced DA-induced neuronal death. These findings suggest a functional role for TCP-1delta as a positive mediator of DA-induced neuronal apoptosis.

Neurodegenerative processes are generally characterized by the long-lasting course of neuronal death and the selectivity of the neuronal population or brain structure involved in the lesion. Two main common forms of cell death that have been described in neurons as in other vertebrate tissues i.e., necrosis and apoptosis. Necrosis is the result of cellular “accidents”, such as those occurring in tissues subjected to chemical trauma. The necrotizing cells swell, rupture and provoke an inflammatory response. Apoptosis, on the other hand, is dependent on the cell’s “decision” to commit suicide and die, and therefore is referred to as “programmed cell death” (PCD). The course of apoptotic death is characterized by a massive morphological change, including cell shrinkage, nuclear (chromosome) condensation and DNA degradation. Activation of PCD in an individual cell is based on its own internal metabolism, environment, developmental background and its genetic information. Such a situation occurs in most of the neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases and amyotrophic lateral sclerosis (ALS). In these pathological situations, specific neurons undergo apoptotic cell death characterized by DNA fragmentation, increased levels of pro-apoptotic genes and “apoptotic proteins” both, in human brain and in experimental models. It is of utmost importance to conclusively determine the mode of cell death in neurodegenerative diseases, because new “antiapoptotic” compounds may offer a means of protecting neurons from cell death and of slowing the rate of cell degeneration and illness progression.

Shrinkage and collapse of the neuritic network are often observed during the process of neuronal apoptosis. However, the molecular and biochemical basis for the axonal damage associated with neuronal cell death is still unclear. We present evidence for the involvement of axon guidance molecules with repulsive cues in neuronal cell death. Using the differential display approach, an up-regulation of collapsin response mediator protein was detected in sympathetic neurons undergoing dopamine-induced apoptosis. A synchronized induction of mRNA of the secreted collapsin-1 and the intracellular collapsin response mediator protein that preceded commitment of neurons to apoptosis was detected. Antibodies directed against a conserved collapsin-derived peptide provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. Moreover, neuronal apoptosis was inhibited by antibodies against neuropilin-1, a putative component of the semaphorin III/collapsin-1 receptor. Induction of neuronal apoptosis was also caused by exposure of neurons to semaphorin III-alkaline phosphatase secreted from 293EBNA cells. Anti-collapsin-1 antibodies were effective in blocking the semaphorin III-induced death process. We therefore suggest that, before their death, apoptosis-destined neurons may produce and secrete destructive axon guidance molecules that can affect their neighboring cells and thus transfer a “death signal” across specific and susceptible neuronal populations.

Camptocormia is characterized by severe forward flexion of the thoracolumbar spine which increases while walking and disappears in the recumbent position. We describe for the first time eight patients with presumed idiopathic Parkinson's disease (mean age 66+/-5 yrs; mean symptom duration 13.1+/-5.1 yrs) who developed camptocormia. This impressive abnormal posture emerged 4-14 years from disease onset, and in some patients stooped posture was the prominent symptom at diagnosis. There was no clear correlation between camptocormia and levodopa treatment. In some patients the camptocormic posture improved, and in others it was unchanged or even aggravated following levodopa administration. Three patients reported worsening of this symptom during "off" periods and also with fatigue. The pathogenesis of this phenomenon is unknown but might represent either a rare type of dystonia or an extreme form of rigidity.

Motor fluctuations after long-term administration of levodopa may be due to central pharmacodynamic mechanisms such as reduced striatal synthesis and storage of dopamine from exogenous levodopa and subsensitization of postsynaptic dopaminergic receptors. Peripheral pharmacokinetic mechanisms may be equally important, particularly in motor fluctuations of the “delayed on” (increased time latencies from dose intake to start-up of clinical benefit) and “no-on” (complete failure of a levodopa dose to exert an “on” response) types. Levodopa itself has a very poor solubility. In addition, there is delayed gastric emptying in many advanced patients. Therefore, an oral dose of levodopa may remain in the stomach for long periods of time before it passes into the duodenum where there is immediate absorption. Consequently, in order to overcome response fluctuations caused by impaired pharmacokinetic mechanisms and to improve its absorption, we recommend that levodopa be taken in multiple small doses, on an empty stomach, preferably crushed and mixed with a lot of liquid. Protein intake should be minimized. Prokinetic drugs such as prepulsid (Cisaprid) could be used to facilitate gastric motility and levodopa transit time. Administration of crushed levodopa through nasoduodenal or gastrojejunostomy tubes may be helpful in certain circumstances. Bypassing the stomach with subcutaneous injections of apomorphine may provide dramatic rescue from difficult “off” situations. Oral and s.c. administration of novel, extremely soluble prodrugs of levodopa, e.g., levodopa ethylester, may offer a new approach to overcome difficulties in levodopa absorption. Addition of dopamine agonists, MAO-B inhibitors, COMT inhibitors and controlled release levodopa preparations may be helpful in prolonging the duration of efficacy of each single levodopa dose.

After cessation of long-term treatment with haloperidol, a 77-year-old man developed severe dyskinetic-dystonic movements involving mainly the tongue and associated also with oromandibular dystonia and blepharospasm that were manifested exclusively during the process of eating and that interfered with feeding. There were no spontaneous involuntary movements nor were they apparent during any other simple or complex activations of the orofacial musculature.

Objective: To measure fatigue quantitatively during continuous motor performance in patients with PD. Background: Enhanced fatigue on performance of motor tasks is a very frequent and disabling complaint of PD patients, and is poorly characterized and understood. Recent evidence suggests a role for mitochondrial dysfunction in the pathogenesis of PD. Reduced exercise capacity is one of the hallmarks of systemic mitochondrial impairment. Methods: The authors used an automated system to measure muscle fatigue during a continuous (30-second), maximal, isometric forearm flexion in 17 PD patients and 10 age-matched control subjects. A fatigue index (FI) was then calculated. Peak force (PF) was measured as an internal standard of the examination. Measurements were performed before and 2 hours after an oral dose levodopa/carbidopa (125 mg/12.5 mg). Results: In PD patients there was a 50% increase in FI. The increased FI was often asymmetric and more pronounced on the side more affected by the disease. FI was significantly responsive to, and improved after, an oral dose of levodopa. The rate of improvement in FI induced by levodopa correlated with disease severity, as measured by the Unified Parkinson9s Disease Rating Scale. No significant alterations in PF were observed. Conclusions: Enhanced muscle fatigue should be recognized as an integral part of the spectrum of motor impairment of PD. However, our data argue for its association with a central dopamine deficiency rather than with a muscle mitochondrial abnormality.

Objective: To examine the presence of anti-L-type calcium channel antibodies in the serum of ALS patients.Background: Autoimmunity has been hypothesized as one of the mechanisms underlying the pathogenesis of sporadic ALS. Previous studies reported that sera from patients with sporadic ALS contain antibodies against voltagegated calcium channels (L-type and P-type), but others do not support these findings.Methods: Regulated secretion of tritiated dopamine ([3H]DA) in PC12 cells in mediated exclusively by calcium entry through L-type calcium channels. To examine whether purified ALS immunoglobulin G (IgG) inhibits [3H]DA release by interfering with calcium entry through L-type calcium channels, evoked release in PC12 cells was determined in the presence of ALS IgG. This functional assay provides a sensitive way to examine L-type calcium channel interaction with IgG from ALS patients.Results: A significant inhibition of depolarization-evoked [3H]DA release (32 ± 4%) was observed by purified IgG from ALS patients compared with control subjects (11 ± 2%; p < 0.01). Significant inhibition by IgG occurred in 79% (15/19) of the ALS patients compared with only 29% (5/17) in the control group (p < 0.01). The level of calcium channel inhibition by ALS IgG correlated positively with disease duration (r = 0.68; p < 0.01) and correlated negatively with age (r = -0.48; p < 0.05).Conclusions: These results confirm the presence of antibodies against the L-type calcium channel in the majority of sera from ALS patients, supporting their role in the pathogenesis of ALS.

More than 50% of patients with Parkinson's disease (PD) develop response fluctuations following prolonged treatment with levodopa. Some are due to central pharmacodynamic mechanisms such as reduced striatal synthesis and storage of dopamine from exogenous levodopa and subsensitization of post synaptic dopaminergic receptors. Other fluctuations, especially the "delayed on" (increased time latencies from dose intake to turning "on") and "no on" (complete failure of levodopa dose to induce an "on" response) are caused by peripheral pharmacokinetic mechanisms. Patients with PD, especially those with response fluctuations, have gastric atony. The reduced motility of the stomach, combined with the poor solubility of levodopa, is the cause for the delayed and incomplete absorption of levodopa. The best strategy to overcome central pharmacodynamic mechanisms and to increase daily "on" hours can be achieved by using dopamine agonists, controlled release preparations, MAO-B and COMT inhibitors. Therapeutic strategies that improve levodopa absorption are needed to overcome response fluctuations that are caused by peripheral mechanisms. This can be achieved by crushing levodopa and drinking it as a suspension. Administration of crushed levodopa or levodopa/carbidopa/ascorbic acid solutions orally or through gastroduodenal or gastrojejunostomy tubes may also be helpful. Prokinetic drugs, such as prepulsid, improve absorption of levodopa by enhancing gastric motility. Bypassing the stomach by subcutaneous dopamine agonists (apomorphine and lisuride pumps) or by the novel prodrug of levodopa, i.e., levodopa ethylester, may produce dramatic rescue from incapacitating "off" states.

The protooncogene bcl-2 inhibits neuronal apoptosis during normal brain development as well as that induced by cytotoxic drugs or growth factor deprivation. We have previously demonstrated that neurons of mice deficient in Bcl-2 are more susceptible to neurotoxins and that the dopamine (DA) level in the striatum after systemic 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration was significantly lower than in wild-type mice. In the present study we have used transgenic mice overexpressing human Bcl-2 under the control of neuron-specific enolase promoter (NSE- hbcl -2) to test the effects of the neurotoxins 6-hydroxydopamine (6-OHDA) and MPTP on neuronal survival in these mice. Primary cultures of neocortical neurons from normal and transgenic mice were exposed to these dopaminergic neurotoxins. Addition of 6-OHDA resulted in cell death of essentially all neurons from normal mice. In contrast, in cultures generated from heterozygous NSE- hbcl-2 transgenic mice, only 69% of the cells died while those generated from homozygous transgenic mice were highly resistant and exhibited only 34% cell death. A similar effect was observed with neurons treated with MPP + . Moreover, while the striatal dopamine level after MPTP injections was reduced by 32% in the wild type, the concentration remained unchanged in the NSE- hbcl-2 heterozygous mice. In contrast levels of glutathione-related enzymes were unchanged. In conclusion, overexpression of Bcl-2 in the neurons provided protection, in a dose-dependent manner, against neurotoxins known to selectively damage dopaminergic neurons. This study provides ideas for inhibition of neuronal cell death in neurodegenerative diseases and for the development of efficient neuroprotective gene therapy.

The neurotransmitter dopamine is capable of inducing apoptosis in postmitotic sympathetic neurons via its oxidative metabolites. To detect genes whose expression is transcriptionally regulated during the early stages of dopamine-triggered apoptosis, we applied the differential display method to cultured sympathetic neurons. One of the up-regulated genes was identified as cyclin B2, which exhibited two waves of induction and destruction, both at the mRNA and protein levels, resembling the sequential oscillations typical of two successive mitotic events in proliferating cells. The time window between the two waves was characterized by a change in expression of other cell-cycle stage-specific genes, and oscillations in proliferating cell nuclear antigen and alterations in cyclin A were observed. Cyclin D1 and cyclin-dependent kinases were undetected and no sign of active DNA synthesis could be observed, indicating that activation of cell-cycle components is incomplete. In comparison with a normal cell cycle, temporal expression profile of these mediators was unsynchronized. Whereas the first wave of cell-cycle changes occurred prior to the commitment of the cells to the death process and could be tolerated by the cells, the second wave of changes coincided with the death commitment point. Our findings indicate that inappropriate and incomplete activation of some cell cycle-related genes in postmitotic neurons occurs during dopamine-triggered neuronal apoptosis.

The function of neuromelanin (NM), the oxidized dopamine (DA) polymer, within the DA-producing cells in the human and primate substantia nigra (SN), is still an enigma. Some studies show that the vulnerability of nigral neurons in Parkinson's disease is correlated to their toxic NM content, while others suggest that it contributes to cellular protection. We showed recently that DA, the endogenous nigral neurotransmitter, triggers apoptosis, an active program of cellular self-destruction, in neuronal cultures. In the present study, we exposed cells to synthetic dopamine-melanin (DA-M) and analysed the cellular and genetic changes. We found that exposure of PC12 cells to DA-M (0.5 mg/ml for 24 h) caused 50% cell death, as indicated by trypan blue exclusion assay and 3H-thymidine incorporation. Gel electrophoresis DNA analysis of PC12 cells treated with DA-M showed the typical apoptotic DNA ladder, indicating inter-nucleosomal DNA degradation. The DNA fragmentation also was visualized histochemically in situ by DNA end-labeling staining (the TUNEL method). The FeCl2 (0.05 mM) significantly increased DA-M toxicity, while desferrioxamine, an iron chelator, totally abolished the additive toxicity of iron. The contribution of oxidative stress in this model of DA-M-induced cell death was examined using various antioxidants. In contrast to DA, inhibition of DA-M toxicity antioxidants by reduced glutathione (GSH), N-acetyl cysteine, catalase and Zn/Cu superoxide dismutase (SOD) was very limited. In conclusion, we found that DA-M may induce typical apoptotic death in PC12 cells. Our findings support a possible role of NM in the vulnerability of the dopaminergic neural degeneration in Parkinson's disease. The differential protective effect by antioxidants against toxicity of DA and DA-M may have implications for future neuroprotective therapeutic approaches for this common neurological disorder.

The cause for the rather selective degeneration of the nigrostriatal dopaminergic (DA) neurons in Parkinson's disease (PD) is still enigmatic. The major current hypothesis suggests that nigral neuronal death in PD is due to excessive oxidant stress generated by auto- and enzymatic oxidation of DA, formation of neuromelanin and presence of high concentrations of iron. Such cell death is generally regarded as a passive, necrotic process, mainly resulting from membrane lipid peroxidation, leading to its dysfunction and rupture and then to neuronal disintegration. We suggest a novel approach, that views neuronal degeneration in PD as an active process that occurs mainly the nuclear level. Our concept is based on the following observations: (1) Nigral histopathology in PD is characterized by a slow, protracted degeneration of individual neurons. We propose that it may be due to apoptosis [programmed cell-death (PCD), an active, genetically-controlled, intrinsic program of cell "suicide"] rather than to necrotic cell death. (2) DA exerts antitumor effect on melanoma and neuroblastoma cells. (3) Many anticancer drugs, trigger PCD by causing DNA damage. (4) DA has been shown to be genotoxic. (5) We recently first showed that DA, the endogenous neurotransmitter in the nigra, can trigger apoptosis in cultured, postmitotic sympathetic neurons. (6) We have also shown that PC-12 cells, transfected with the bcl-2 gene (a proto-oncogene that inhibits PCD) are relatively resistant to DA-apoptotic effect. Degeneration of nigrostriatal neurons in PD may therefore be linked to dysregulation of the control mechanisms that normally restrain the PCD-triggering-potential of their own neurotransmitter.

Apoptosis is an active, intrinsic cell suicide program. We recently suggested that it may have a role in the death of nigrostriatal dopaminergic neurons in Parkinson's disease (PD). We now report that levodopa, the current major therapy for PD, is a potent inducer of apoptosis in cultured postmitotic chick sympathetic neurons. Levodopa, in a concentration range of 0.01-0.3 mM, caused the characteristic apoptotic cascade of cell shrinkage, massive membrane blebbing, and nuclear fragmentation, as evident by nuclear flow cytometry and fluorescence microscopy. Levodopa-induced apoptosis was inhibited by antioxidants, indicating that it may be mediated by autooxidation-reactive species. Levodopa treatment for PD may therefore constitute an additional challenge for the defective apoptosis-inhibiting systems in the nigrostriatal neurons. Despite reassuring data from some, but not all, previous studies, these findings suggest that the possible in vivo toxic effects of levodopa on the survival of the remaining nigral neurons should be further explored.