Low-Dose Levodopa Protects Nerve Cells from Oxidative Stress and Up-Regulates Expression of pCREB and CD39

Objective

This study aimed to investigate the influence of low-dose levodopa (L-DOPA) on neuronal cell death under oxidative stress.

Methods

PC12 cells were treated with L-DOPA at different concentrations. We detected the L-DOPA induced reactive oxygen species (ROS). Meanwhile, MTT and LDH assay were performed to determine the proliferation and growth of PC12 cells with or without ROS scavenger. In addition, after pretreatment with L-DOPA at different concentrations alone or in combination with CD39 inhibitor, PC12 cells were incubated with hydrogen peroxide (H₂O₂) and the cell viability was evaluated by MTT and LDH assay. In addition, the expression of pCREB and CD39 was detected by immunofluorescence staining and Western blot assay in both cells and rat’s brain after L-DOPA treatment.

Results

After treatment with L-DOPA for 3 days, the cell proliferation and growth were promoted when the L-DOPA concentration was <30 µM, while cell proliferation was comparable to that in control group when the L-DOPA concentration was >30 µM. Low dose L-DOPA could protect the PC12 cells from H₂O₂ induced oxidative stress, which was compromised by CD39 inhibitor. In addition, the expression of CD39 and pCREB increased in both PC12 cells and rats’ brain after L-DOPA treatment.

Conclusions

L-DOPA at different concentrations has distinct influence on proliferation and growth of PC12 cells, and low dose (<30 µM) L-DOPA protects PC12 cells against oxidative stress which might be related to the up-regulation of CD39 and pCREB expression.     

Dopamine Reduction in the Substantia Nigra of Parkinson's Disease Patients Confirmed by In Vivo Magnetic Resonance Spectroscopic Imaging

Objectives

Metabolic changes in the substantia nigra of patients with Parkinson''s disease were previously investigated in different molecular-pathological examinations. The aim of our study was the in vivo measurement of these alterations using three-dimensional magnetic resonance spectroscopic imaging.

Methods

21 patients with Parkinson''s disease and 24 controls were examined using magnetic resonance spectroscopic imaging at 3 Tesla. The spectra of rostral and caudal substantia nigra regions were analyzed using LCModel. For spectral fitting, an adjusted basis data set with pathology-specific metabolites and macromolecules was used to better reproduce the in vivo spectra. To assess differences between both groups more accurately, especially in metabolites at lower concentrations, group-averaged spectra were evaluated in addition to the analysis of individual data.

Results

We found significantly decreased N-acetylaspartate, choline, creatine, myo-inositol, glutathione and dopamine concentrations in patients with Parkinson''s disease compared to controls, whereas glutamine+glutamate, γ-aminobutyric acid, and homovanillic acid were slightly increased. According to anatomical features, clear differences in the biochemical profiles were found between rostral and caudal substantia nigra voxels in both groups.

Conclusions

Reduced N-acetylaspartate and dopamine concentrations result from progressive degeneration of dopamine-producing neurons within the substantia nigra pars compacta. Decreased creatine levels can be interpreted as impaired energy metabolism due to mitochondrial dysfunction. Lower glutathione concentrations might be a cause or consequence of oxidative stress. Furthermore, slightly increased glutamine+glutamate and γ-aminobutyric acid levels are expected based on post mortem data in Parkinson''s disease. To the best of our knowledge, this is the first non-invasive confirmation of these metabolic changes.     

Dopaminergic Neuronal Imaging in Genetic Parkinson's Disease: Insights into Pathogenesis

Objectives

To compare the dopaminergic neuronal imaging features of different subtypes of genetic Parkinson''s Disease.

Methods

A retrospective study of genetic Parkinson''s diseases cases in which DaTSCAN (123I-FP-CIT) had been performed. Specific non-displaceable binding was calculated for bilateral caudate and putamen for each case. The right:left asymmetry index and striatal asymmetry index was calculated.

Results

Scans were available from 37 cases of monogenetic Parkinson''s disease (7 glucocerebrosidase (GBA) mutations, 8 alpha-synuclein, 3 LRRK2, 7 PINK1, 12 Parkin). The asymmetry of radioligand uptake for Parkinson''s disease with GBA or LRRK2 mutations was greater than that for Parkinson''s disease with alpha synuclein, PINK1 or Parkin mutations.

Conclusions

The asymmetry of radioligand uptake in Parkinsons disease associated with GBA or LRRK2 mutations suggests that interactions with additional genetic or environmental factors may be associated with dopaminergic neuronal loss.     

Distinct Changes in cAMP and Extracellular Signal-Regulated Protein Kinase Signalling in L-DOPA-Induced Dyskinesia

Background

In rodents, the development of dyskinesia produced by L-DOPA in the dopamine-depleted striatum occurs in response to increased dopamine D1 receptor-mediated activation of the cAMP - protein kinase A and of the Ras-extracellular signal-regulated kinase (ERK) signalling pathways. However, very little is known, in non-human primates, about the regulation of these signalling cascades and their association with the induction, manifestation and/or maintenance of dyskinesia.

Methodology/Results

We here studied, in the gold-standard non-human primate model of Parkinson''s disease, the changes in PKA-dependent phosphorylation of DARPP-32 and GluR1 AMPA receptor, as well as in ERK and ribosomal protein S6 (S6) phosphorylation, associated to acute and chronic administration of L-DOPA. Increased phosphorylation of DARPP-32 and GluR1 was observed in both L-DOPA first-ever exposed and chronically-treated dyskinetic parkinsonian monkeys. In contrast, phosphorylation of ERK and S6 was enhanced preferentially after acute L-DOPA administration and decreased during the course of chronic treatment.

Conclusion

Dysregulation of cAMP signalling is maintained during the course of chronic L-DOPA administration, while abnormal ERK signalling peaks during the initial phase of L-DOPA treatment and decreases following prolonged exposure. While cAMP signalling enhancement is associated with dyskinesia, abnormal ERK signalling is associated with priming.     

CpG demethylation enhances alpha-synuclein expression and affects the pathogenesis of Parkinson's disease

Background

Alpha-synuclein (SNCA) gene expression is an important factor in the pathogenesis of Parkinson''s disease (PD). Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD. CpG methylation in the promoter region may also influence SNCA expression.

Methodology/Principal Findings

By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression. Postmortem brain analysis revealed regional non-specific methylation differences in this CpG region in the anterior cingulate and putamen among controls and PD; however, in the substantia nigra of PD, methylation was significantly decreased.

Conclusions/Significance

This CpG region may function as an intronic regulatory element for SNCA gene. Our findings suggest that a novel epigenetic regulatory mechanism controlling SNCA expression influences PD pathogenesis.     

Dopamine transporter binding is unaffected by L-DOPA administration in normal and MPTP-treated monkeys

Background

Radiotracer imaging of the presynaptic nigrostriatal dopaminergic system is used to assess disease progression in Parkinson''s disease (PD) and may provide a useful adjunct to clinical assessment during therapeutic trials of potential neuroprotective agents. Several clinical trials comparing dopamine agonists to L-DOPA or early vs. late L-DOPA have revealed differences between clinical assessment and imaging of the presynaptic dopaminergic system, hence questioning the comparability of these measures as neuroprotection outcome variables. Thus, results of these studies may have been affected by factors other than the primary biological process investigated.

Methodology/Principal Findings

We tested the possibility that L-DOPA might interfere with DAT binding. Post-mortem DAT binding was conducted in normal and MPTP-treated macaque monkeys that were administered L-DOPA, acutely or chronically. In parallel, DAT SPECT was conducted in MPTP-treated animals that were administered chronic L-DOPA. [99mTc]TRODAT-1 SPECT binding was similarly reduced in all MPTP monkeys regardless of L-DOPA treatment. L-DOPA had no significant effect on post-mortem DAT binding either in saline or in MPTP-lesioned animals.

Conclusions/Significance

These data indicate that L-DOPA does not induce modifications of DAT expression detectable by SPECT of by DAT binding autoradiography, suggesting that differences between clinical assessment and radiotracer imaging in clinical trials may not be specifically related to L-DOPA treatment.     

L-DOPA Neurotoxicity Is Mediated by Up-Regulation of DMT1?IRE Expression

Background

The mechanisms underlying neurotoxicity caused by L-DOPA are not yet completely known. Based on recent findings, we speculated that the increased expression of divalent metal transporter 1 without iron-response element (DMT1−IRE) induced by L-DOPA might play a critical role in the development of L-DOPA neurotoxicity. To test this hypothesis, we investigated the effects of astrocyte-conditioned medium (ACM) and siRNA DMT-IRE on L-DOPA neurotoxicity in cortical neurons.

Methods and Findings

We demonstrated that neurons treated with L-DOPA have a significant dose-dependent decrease in neuronal viability (MTT Assay) and increase in iron content (using a graphite furnace atomic absorption spectrophotometer), DMT1−IRE expression (Western blot analysis) and ferrous iron (55Fe(II)) uptake. Neurons incubated in ACM with or without L-DOPA had no significant differences in their morphology, Hoechst-33342 staining or viability. Also, ACM significantly inhibited the effects of L-DOPA on neuronal iron content as well as DMT1−IRE expression. In addition, we demonstrated that infection of neurons with siRNA DMT-IRE led to a significant decrease in DMT1−IRE expression as well as L-DOPA neurotoxicity.

Conclusion

The up-regulation of DMT1−IRE and the increase in DMT1−IRE-mediated iron influx play a key role in L-DOPA neurotoxicity in cortical neurons.     

The Monoamine Re-Uptake Inhibitor UWA-101 Improves Motor Fluctuations in the MPTP-Lesioned Common Marmoset

Background

The wearing-OFF phenomenon is a common motor complication of chronic L-3,4-dihydroxyphenylalanine (L-DOPA) therapy for Parkinson’s disease. We recently described the discovery of UWA-101, a dual serotonin (SERT) and dopamine (DAT) transporter inhibitor, which increases the duration of “good quality” ON-time provided by L-DOPA in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned primate. Here, we further characterise the effects of UWA-101 on this extension of ON-time in terms of L-DOPA-induced side-effects in the MPTP-lesioned common marmoset.

Methods

Marmosets were rendered parkinsonian by MPTP injection and “primed” by repeated L-DOPA administration, to exhibit dyskinesia and psychosis-like behaviours. Animals were then administered acute challenges of L-DOPA in combination with UWA-101 (1, 3, 6 and 10 mg/kg) or vehicle.

Results

In combination with L-DOPA, UWA-101 (3, 6 and 10 mg/kg) significantly increased duration of ON-time (by 28%, 28%, and 33%, respectively; all P<0.05). UWA-101 (10 mg/kg) significantly extended duration of ON-time without disabling dyskinesia (by 62%, P<0.01). UWA-101 did not exacerbate the severity of dyskinesia (P>0.05). However, at the highest doses (6 and 10 mg/kg), UWA-101 increased the severity of psychosis-like behaviours (P<0.05).

Conclusions

Our results demonstrate that dual SERT/ DAT inhibitors can effectively enhance L-DOPA anti-parkinsonian action, without exacerbating dyskinesia and, as such, represent a promising new therapeutic class for wearing-OFF. However, at higher doses, dual SERT/ DAT inhibitors may exacerbate dopaminergic psychosis.     

Enteric Neuropathy Can Be Induced by High Fat Diet In Vivo and Palmitic Acid Exposure In Vitro

Objective

Obese and/or diabetic patients have elevated levels of free fatty acids and increased susceptibility to gastrointestinal symptoms. Since the enteric nervous system is pivotal in regulating gastrointestinal functions alterations or neuropathy in the enteric neurons are suspected to occur in these conditions. Lipid induced intestinal changes, in particular on enteric neurons, were investigated in vitro and in vivo using primary cell culture and a high fat diet (HFD) mouse model.

Design

Mice were fed normal or HFD for 6 months. Intestines were analyzed for neuronal numbers, remodeling and lipid accumulation. Co-cultures of myenteric neurons, glia and muscle cells from rat small intestine, were treated with palmitic acid (PA) (0 – 10⁻³ M) and / or oleic acid (OA) (0 – 10⁻³ M), with or without modulators of intracellular lipid metabolism. Analyses were by immunocyto- and histochemistry.

Results

HFD caused substantial loss of myenteric neurons, leaving submucous neurons unaffected, and intramuscular lipid accumulation in ileum and colon. PA exposure in vitro resulted in neuronal shrinkage, chromatin condensation and a significant and concentration-dependent decrease in neuronal survival; OA exposure was neuroprotective. Carnitine palmitoyltransferase 1 inhibition, L-carnitine- or alpha lipoic acid supplementation all counteracted PA-induced neuronal loss. PA or OA alone both caused a significant and concentration-dependent loss of muscle cells in vitro. Simultaneous exposure of PA and OA promoted survival of muscle cells and increased intramuscular lipid droplet accumulation. PA exposure transformed glia from a stellate to a rounded phenotype but had no effect on their survival.

Conclusions

HFD and PA exposure are detrimental to myenteric neurons. Present results indicate excessive palmitoylcarnitine formation and exhausted L-carnitine stores leading to energy depletion, attenuated acetylcholine synthesis and oxidative stress to be main mechanisms behind PA-induced neuronal loss.High PA exposure is suggested to be a factor in causing diabetic neuropathy and gastrointestinal dysregulation.     

AFM Probing the Mechanism of Synergistic Effects of the Green Tea Polyphenol (?)-Epigallocatechin-3-Gallate (EGCG) with Cefotaxime against Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli

Background

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae poses serious challenges to clinicians because of its resistance to many classes of antibiotics.

Methods and Findings

The mechanism of synergistic activity of a combination of (−)-epigallocatechin-3-gallate (EGCG) and β-lactam antibiotics cefotaxime was studied on Extended-spectrum β-lactamase producing Escherichia coli (ESBL-EC), by visualizing the morphological alteration on the cell wall induced by the combination using atomic force microscopy (AFM). Cells at sub-MICs (sub-minimum inhibitory concentrations) of cefotaxime were initially filamentated but recovered to the normal shape later, whereas cells at sub-MICs of EGCG experienced temporal disturbance on the cell wall such as leakage and release of cellular debris and groove formation, but later recovered to the normal shape. In contrast, the combination of cefotaxime and EGCG at their respective sub-MICs induced permanent cellular damages as well as continuous elongation in cells and eventually killed them. Flow cytometry showed that intracellular oxidative stress levels in the cell treated with a combination of EGCG and cefotaxime at sub-MICs were higher than those in the cells treated with either cefotaxime or EGCG at sub-MICs.

Conclusions

These results suggest that the synergistic effect of EGCG between EGCG and cefotaxime against ESBL-EC is related to cooperative activity of exogenous and endogenous reactive oxygen species (ROS) generated by EGCG and cefotaxime, respectively.     

The Effect of (-)-Epigallocatechin 3-O - Gallate In Vitro and In Vivo in Leishmania braziliensis: Involvement of Reactive Oxygen Species as a Mechanism of Action

Background

Leishmaniasis is a parasitic disease associated with extensive mortality and morbidity. The treatment for leishmaniasis is currently based on pentavalent antimonials and amphotericin B; however, these drugs result in numerous adverse side effects. Natural compounds have been used as novel treatments for parasitic diseases. In this paper, we evaluated the effect of (-)-epigallocatechin 3-O-gallate (EGCG) on Leishmania braziliensis in vitro and in vivo and described the mechanism of EGCG action against L. braziliensis promastigotes and intracellular amastigotes.

Methodology/Principal Finding

In vitro activity and reactive oxygen species (ROS) measurements were determined during the promastigote and intracellular amastigote life stages. The effect of EGCG on mitochondrial membrane potential (ΔΨ_m) was assayed using JC-1, and intracellular ATP concentrations were measured using a luciferin-luciferase system. The in vivo experiments were performed in infected BALB/c mice orally treated with EGCG. EGCG reduced promastigote viability and the infection index in a time- and dose-dependent manner, with IC₅₀ values of 278.8 µM and 3.4 µM, respectively, at 72 h and a selectivity index of 149.5. In addition, EGCG induced ROS production in the promastigote and intracellular amastigote, and the effects were reversed by polyethylene glycol (PEG)-catalase. Additionally, EGCG reduced ΔΨ_m, thereby decreasing intracellular ATP concentrations in promastigotes. Furthermore, EGCG treatment was also effective in vivo, demonstrating oral bioavailability and reduced parasitic loads without altering serological toxicity markers.

Conclusions/Significance

In conclusion, our study demonstrates the leishmanicidal effects of EGCG against the two forms of L. braziliensis, the promastigote and amastigote. In addition, EGCG promotes ROS production as a part of its mechanism of action, resulting in decreased ΔΨ_m and reduced intracellular ATP concentrations. These actions ultimately culminate in parasite death. Furthermore, our data suggest that EGCG is orally effective in the treatment of L. braziliensis-infected BALB/c mice without altering serological toxicity markers.     

Levodopa-induced dyskinesia is associated with increased thyrotropin releasing hormone in the dorsal striatum of hemi-parkinsonian rats

Background

Dyskinesias associated with involuntary movements and painful muscle contractions are a common and severe complication of standard levodopa (L-DOPA, L-3,4-dihydroxyphenylalanine) therapy for Parkinson''s disease. Pathologic neuroplasticity leading to hyper-responsive dopamine receptor signaling in the sensorimotor striatum is thought to underlie this currently untreatable condition.

Methodology/Principal Findings

Quantitative real-time polymerase chain reaction (PCR) was employed to evaluate the molecular changes associated with L-DOPA-induced dyskinesias in Parkinson''s disease. With this technique, we determined that thyrotropin releasing hormone (TRH) was greatly increased in the dopamine-depleted striatum of hemi-parkinsonian rats that developed abnormal movements in response to L-DOPA therapy, relative to the levels measured in the contralateral non-dopamine-depleted striatum, and in the striatum of non-dyskinetic control rats. ProTRH immunostaining suggested that TRH peptide levels were almost absent in the dopamine-depleted striatum of control rats that did not develop dyskinesias, but in the dyskinetic rats, proTRH immunostaining was dramatically up-regulated in the striatum, particularly in the sensorimotor striatum. This up-regulation of TRH peptide affected striatal medium spiny neurons of both the direct and indirect pathways, as well as neurons in striosomes.

Conclusions/Significance

TRH is not known to be a key striatal neuromodulator, but intrastriatal injection of TRH in experimental animals can induce abnormal movements, apparently through increasing dopamine release. Our finding of a dramatic and selective up-regulation of TRH expression in the sensorimotor striatum of dyskinetic rat models suggests a TRH-mediated regulatory mechanism that may underlie the pathologic neuroplasticity driving dopamine hyper-responsivity in Parkinson''s disease.     

Radiating Amyloid Fibril Formation on the Surface of Lipid Membranes through Unit-Assembly of Oligomeric Species of α-Synuclein

Background

Lewy body in the substantia nigra is a cardinal pathological feature of Parkinson''s disease. Despite enormous efforts, the cause-and-effect relationship between Lewy body formation and the disorder is yet to be explicitly unveiled.

Methodology/Principal Findings

Here, we showed that radiating amyloid fibrils (RAFs) were instantly developed on the surface of synthetic lipid membranes from the β-sheet free oligomeric species of α-synuclein through a unit-assembly process. The burgeoning RAFs were successfully matured by feeding them with additional oligomers, which led to concomitant dramatic shrinkage and disintegration of the membranes by pulling off lipid molecules to the extending fibrils. Mitochondria and lysosomes were demonstrated to be disrupted by the oligomeric α-synuclein via membrane-dependent fibril formation.

Conclusion

The physical structure formation of amyloid fibrils, therefore, could be considered as detrimental to the cells by affecting membrane integrity of the intracellular organelles, which might be a molecular cause for the neuronal degeneration observed in Parkinson''s disease.     

Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells

Background

Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson''s disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.

Methodology/Principal Findings

In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/− cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

Conclusion/Significance

Parkinson''s disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.     

Lipid pathway alterations in Parkinson's disease primary visual cortex

Background

We present a lipidomics analysis of human Parkinson''s disease tissues. We have focused on the primary visual cortex, a region that is devoid of pathological changes and Lewy bodies; and two additional regions, the amygdala and anterior cingulate cortex which contain Lewy bodies at different disease stages but do not have as severe degeneration as the substantia nigra.

Methodology/Principal Findings

Using liquid chromatography mass spectrometry lipidomics techniques for an initial screen of 200 lipid species, significant changes in 79 sphingolipid, glycerophospholipid and cholesterol species were detected in the visual cortex of Parkinson''s disease patients (n = 10) compared to controls (n = 10) as assessed by two-sided unpaired t-test (p-value <0.05). False discovery rate analysis confirmed that 73 of these 79 lipid species were significantly changed in the visual cortex (q-value <0.05). By contrast, changes in 17 and 12 lipid species were identified in the Parkinson''s disease amygdala and anterior cingulate cortex, respectively, compared to controls; none of which remained significant after false discovery rate analysis. Using gas chromatography mass spectrometry techniques, 6 out of 7 oxysterols analysed from both non-enzymatic and enzymatic pathways were also selectively increased in the Parkinson''s disease visual cortex. Many of these changes in visual cortex lipids were correlated with relevant changes in the expression of genes involved in lipid metabolism and an oxidative stress response as determined by quantitative polymerase chain reaction techniques.

Conclusions/Significance

The data indicate that changes in lipid metabolism occur in the Parkinson''s disease visual cortex in the absence of obvious pathology. This suggests that normalization of lipid metabolism and/or oxidative stress status in the visual cortex may represent a novel route for treatment of non-motor symptoms, such as visual hallucinations, that are experienced by a majority of Parkinson''s disease patients.     

Epistasis Analysis for Estrogen Metabolic and Signaling Pathway Genes on Young Ischemic Stroke Patients

Background

Endogenous estrogens play an important role in the overall cardiocirculatory system. However, there are no studies exploring the hormone metabolism and signaling pathway genes together on ischemic stroke, including sulfotransferase family 1E (SULT1E1), catechol-O-methyl-transferase (COMT), and estrogen receptor α (ESR1).

Methods

A case-control study was conducted on 305 young ischemic stroke subjects aged ≦ 50 years and 309 age-matched healthy controls. SULT1E1 -64G/A, COMT Val158Met, ESR1 c.454−397 T/C and c.454−351 A/G genes were genotyped and compared between cases and controls to identify single nucleotide polymorphisms associated with ischemic stroke susceptibility. Gene-gene interaction effects were analyzed using entropy-based multifactor dimensionality reduction (MDR), classification and regression tree (CART), and traditional multiple regression models.

Results

COMT Val158Met polymorphism showed a significant association with susceptibility of young ischemic stroke among females. There was a two-way interaction between SULT1E1 -64G/A and COMT Val158Met in both MDR and CART analysis. The logistic regression model also showed there was a significant interaction effect between SULT1E1 -64G/A and COMT Val158Met on ischemic stroke of the young (P for interaction = 0.0171). We further found that lower estradiol level could increase the risk of young ischemic stroke for those who carry either SULT1E1 or COMT risk genotypes, showing a significant interaction effect (P for interaction = 0.0174).

Conclusions

Our findings support that a significant epistasis effect exists among estrogen metabolic and signaling pathway genes and gene-environment interactions on young ischemic stroke subjects.     

Standardized Handwriting to Assess Bradykinesia,Micrographia and Tremor in Parkinson's Disease

Objective

To assess whether standardized handwriting can provide quantitative measures to distinguish patients diagnosed with Parkinson''s disease from age- and gender-matched healthy control participants.

Design

Exploratory study. Pen tip trajectories were recorded during circle, spiral and line drawing and repeated character ‘elelelel’ and sentence writing, performed by Parkinson patients and healthy control participants. Parkinson patients were tested after overnight withdrawal of anti-Parkinsonian medication.

Setting

University Medical Center Groningen, tertiary care, the Netherlands.

Participants

Patients with Parkinson''s disease (n = 10; mean age 69.0 years; 6 male) and healthy controls (n = 10; mean age 68.1 years; 6 male).

Interventions

Not applicable.

Main Outcome Measures

Movement time and velocity to detect bradykinesia and the size of writing to detect micrographia. A rest recording to investigate the presence of a rest-tremor, by frequency analysis.

Results

Mean disease duration in the Parkinson group was 4.4 years and the patients were in modified Hoehn-Yahr stages 1–2.5. In general, Parkinson patients were slower than healthy control participants. Median time per repetition, median velocity and median acceleration of the sentence task and median velocity of the elel task differed significantly between Parkinson patients and healthy control participants (all p<0.0014). Parkinson patients also wrote smaller than healthy control participants and the width of the ‘e’ in the elel task was significantly smaller in Parkinson patients compared to healthy control participants (p<0.0014). A rest-tremor was detected in the three patients who were clinically assessed as having rest-tremor.

Conclusions

This study shows that standardized handwriting can provide objective measures for bradykinesia, tremor and micrographia to distinguish Parkinson patients from healthy control participants.     

Effect of Ca2EDTA on Zinc Mediated Inflammation and Neuronal Apoptosis in Hippocampus of an In Vivo Mouse Model of Hypobaric Hypoxia

Background

Calcium overload has been implicated as a critical event in glutamate excitotoxicity associated neurodegeneration. Recently, zinc accumulation and its neurotoxic role similar to calcium has been proposed. Earlier, we reported that free chelatable zinc released during hypobaric hypoxia mediates neuronal damage and memory impairment. The molecular mechanism behind hypobaric hypoxia mediated neuronal damage is obscure. The role of free zinc in such neuropathological condition has not been elucidated. In the present study, we investigated the underlying role of free chelatable zinc in hypobaric hypoxia-induced neuronal inflammation and apoptosis resulting in hippocampal damage.

Methods

Adult male Balb/c mice were exposed to hypobaric hypoxia and treated with saline or Ca₂EDTA (1.25 mM/kg i.p) daily for four days. The effects of Ca₂EDTA on apoptosis (caspases activity and DNA fragmentation), pro-inflammatory markers (iNOS, TNF-α and COX-2), NADPH oxidase activity, poly(ADP ribose) polymerase (PARP) activity and expressions of Bax, Bcl-2, HIF-1α, metallothionein-3, ZnT-1 and ZIP-6 were examined in the hippocampal region of brain.

Results

Hypobaric hypoxia resulted in increased expression of metallothionein-3 and zinc transporters (ZnT-1 and ZIP-6). Hypobaric hypoxia elicited an oxidative stress and inflammatory response characterized by elevated NADPH oxidase activity and up-regulation of iNOS, COX-2 and TNF-α. Furthermore, hypobaric hypoxia induced HIF-1α protein expression, PARP activation and apoptosis in the hippocampus. Administration of Ca₂EDTA significantly attenuated the hypobaric hypoxia induced oxidative stress, inflammation and apoptosis in the hippocampus.

Conclusion

We propose that hypobaric hypoxia/reperfusion instigates free chelatable zinc imbalance in brain associated with neuroinflammation and neuronal apoptosis. Therefore, zinc chelating strategies which block zinc mediated neuronal damage linked with cerebral hypoxia and other neurodegenerative conditions can be designed in future.     

Glutamatergic Dysbalance and Oxidative Stress in In Vivo and In Vitro Models of Psychosis Based on Chronic NMDA Receptor Antagonism

Background

The psychotomimetic effects of N-methyl-D-aspartate (NMDA) receptor antagonists in healthy humans and their tendency to aggravate psychotic symptoms in schizophrenic patients have promoted the notion of altered glutamatergic neurotransmission in the pathogenesis of schizophrenia.

Methods

The NMDA-receptor antagonist MK-801 was chronically administered to rats (0.02 mg/kg intraperitoneally for 14 days). In one subgroup the antipsychotic haloperidol (1 mg/kg) was employed as a rescue therapy. Glutamate distribution and 3-NT (3-nitrotyrosine) as a marker of oxidative stress were assessed by immunohistochemistry in tissue sections. In parallel, the effects of MK-801 and haloperidol were investigated in primary embryonal hippocampal cell cultures from rats.

Results

Chronic NMDA-R antagonism led to a marked increase of intracellular glutamate in the hippocampus (126.1 +/− 10.4% S.E.M of control; p = 0.037), while 3-NT staining intensity remained unaltered. No differences were observed in extrahippocampal brain regions. Essentially these findings could be reproduced in vitro.

Conclusion

The combined in vivo and in vitro strategy allowed us to assess the implications of disturbed glutamate metabolism for the occurrence of oxidative stress and to investigate the effects of antipsychotics. Our data suggest that oxidative stress plays a minor role in this model than previously suggested. The same applies to apoptosis. Moreover, the effect of haloperidol seems to be mediated through yet unidentified mechanisms, unrelated to D2-antagonism. These convergent lines of evidence indicate that further research should be focused on the glutamatergic system and that our animal model may provide a tool to explore the biology of schizophrenia.