Impairment of Motor Function Correlates with Neurometabolite and Brain Iron Alterations in Parkinson's Disease.

We took advantage of magnetic resonance imaging (MRI) and spectroscopy (MRS) as non-invasive methods to quantify brain iron and neurometabolites, which were analyzed along with other predictors of motor dysfunction in Parkinson's disease (PD). Tapping hits, tremor amplitude, and the scores derived from part III of the Movement Disorder Society-Sponsored Revision of the Unified Parkinson Disease Rating Scale (MDS-UPDRS3 scores) were determined in 35 male PD patients and 35 controls. The iron-sensitive MRI relaxation rate R2* was measured in the globus pallidus and substantia nigra. γ-aminobutyric acid (GABA)-edited and short echo-time MRS was used for the quantification of neurometabolites in the striatum and thalamus. Associations of R2*, neurometabolites, and other factors with motor function were estimated with Spearman correlations and mixed regression models to account for repeated measurements (hands, hemispheres). In PD patients, R2* and striatal GABA correlated with MDS-UPDRS3 scores if not adjusted for age. Patients with akinetic-rigid PD subtype (N = 19) presented with lower creatine and striatal glutamate and glutamine (Glx) but elevated thalamic GABA compared to controls or mixed PD subtype. In PD patients, Glx correlated with an impaired dexterity when adjusted for covariates. Elevated myo-inositol was associated with more tapping hits and lower MDS-UPDRS3 scores. Our neuroimaging study provides evidence that motor dysfunction in PD correlates with alterations in brain iron and neurometabolites.

Association of exposure to manganese and iron with striatal and thalamic GABA and other neurometabolites - Neuroimaging results from the WELDOX II study.

OBJECTIVE: Magnetic resonance spectroscopy (MRS) is a non-invasive method to quantify neurometabolite concentrations in the brain. Within the framework of the WELDOX II study, we investigated the association of exposure to manganese (Mn) and iron (Fe) with γ-aminobutyric acid (GABA) and other neurometabolites in the striatum and thalamus of 154 men. MATERIAL AND METHODS: GABA-edited and short echo-time MRS at 3T was used to assess brain levels of GABA, glutamate, total creatine (tCr) and other neurometabolites. Volumes of interest (VOIs) were placed into the striatum and thalamus of both hemispheres of 47 active welders, 20 former welders, 36 men with Parkinson's disease (PD), 12 men with hemochromatosis (HC), and 39 male controls. Linear mixed models were used to estimate the influence of Mn and Fe exposure on neurometabolites while simultaneously adjusting for cerebrospinal fluid (CSF) content, age and other factors. Exposure to Mn and Fe was assessed by study group, blood concentrations, relaxation rates R1 and R2* in the globus pallidus (GP), and airborne exposure (active welders only). RESULTS: The median shift exposure to respirable Mn and Fe in active welders was 23µg/m3 and 110µg/m3, respectively. Airborne exposure was not associated with any other neurometabolite concentration. Mn in blood and serum ferritin were highest in active and former welders. GABA concentrations were not associated with any measure of exposure to Mn or Fe. In comparison to controls, tCr in these VOIs was lower in welders and patients with PD or HC. Serum concentrations of ferritin and Fe were associated with N-acetylaspartate, but in opposed directions. Higher R1 values in the GP correlated with lower neurometabolite concentrations, in particular tCr (exp(ß)=0.87, p<0.01) and choline (exp(ß)=0.84, p=0.04). R2* was positively associated with glutamate-glutamine and negatively with myo-inositol. CONCLUSIONS: Our results do not provide evidence that striatal and thalamic GABA differ between Mn-exposed workers, PD or HC patients, and controls. This may be due to the low exposure levels of the Mn-exposed workers and the challenges to detect small changes in GABA. Whereas Mn in blood was not associated with any neurometabolite content in these VOIs, a higher metal accumulation in the GP assessed with R1 correlated with generally lower neurometabolite concentrations.

Association of exposure to manganese and iron with relaxation rates R1 and R2*- magnetic resonance imaging results from the WELDOX II study.

OBJECTIVE: Magnetic resonance imaging is a non-invasive method that allows the indirect quantification of manganese (Mn) and iron (Fe) accumulation in the brain due to their paramagnetic features. The WELDOX II study aimed to explore the influence of airborne and systemic exposure to Mn and Fe on the brain deposition using the relaxation rates R1 and R2* as biomarkers of metal accumulation in regions of interest in 161 men, including active and former welders. MATERIAL AND METHODS: We obtained data on the relaxation rates R1 and R2* in regions that included structures within the globus pallidus (GP), substantia nigra (SN), and white matter of the frontal lobe (FL) of both hemispheres, as well as Mn in whole blood (MnB), and serum ferritin (SF). The study subjects, all male, included 48 active and 20 former welders, 41 patients with Parkinson's disease (PD), 13 patients with hemochromatosis (HC), and 39 controls. Respirable Mn and Fe were measured during a working shift for welders. Mixed regression models were applied to estimate the effects of MnB and SF on R1 and R2*. Furthermore, we estimated the influence of airborne Mn and Fe on the relaxation rates in active welders. RESULTS: MnB and SF were significant predictors of R1 but not of R2* in the GP, and were marginally associated with R1 in the SN (SF) and FL (MnB). Being a welder or suffering from PD or HC elicited no additional group effect on R1 or R2* beyond the effects of MnB and SF. In active welders, shift concentrations of respirable Mn>100µg/m3 were associated with stronger R1 signals in the GP. In addition to the effects of MnB and SF, the welding technique had no further influence on R1. CONCLUSIONS: MnB and SF were significant predictors of R1 but not of R2*, indicative of metal accumulation, especially in the GP. Also, high airborne Mn concentration was associated with higher R1 signals in this brain region. The negative results obtained for being a welder or for the techniques with higher exposure to ultrafine particles when the blood-borne concentration was included into the models indicate that airborne exposure to Mn may act mainly through MnB.

Epitope Mapping Using Peptide Microarray in Autoantibody Profiling.

The use of peptide microarrays for epitope mapping of autoantibodies greatly facilitates the early diagnosis of allergic, cytotoxin-associated diseases and especially inflammatory diseases. A common approach to create the microarrays utilizes nitrocellulose-coated glass slides for peptide probe binding, which is based on surface adsorption. Advantages of this method include excellent peptide binding capacity and long-term stability. To ensure equal accessibility to all antibodies on the peptide microarray during epitope mapping, all probes are immobilized in a random manner, thus avoiding concentration-dependent effects on signal intensity.In this chapter, we provide a step-by-step protocol on how to construct the peptide microarrays and perform epitope mapping of autoantibodies using them. Finally we present a comparative approach for the evaluation of the data.

Effects of fatigue on cognitive control in neurosarcoidosis.

Fatigue is a usual reaction to prolonged performance but also a major symptom in various neuroimmunological diseases. In neurosarcoidosis fatigue is a core symptom, but little is known about the relevance of fatigue on cognitive functions in this disease. Previous results in healthy subjects suggest that fatigue strongly affects cognitive control processes. However, fatigue is not a uni-dimensional construct but consists of different facets. It is unknown which of these facets are most important for mechanisms of cognitive control. In the current study we investigate conflict monitoring and response selection processes in neurosarcoidosis patients as a 'model disease' of fatigue and healthy controls in relation to the impact of 'cognitive' and 'motor fatigue' on these processes using event-related potentials (ERPs). We focus on ERPs reflecting attentional selection (P1, N1) and conflict monitoring/response selection processes (N2). ERPs reflecting attentional selection processes were unchanged. The N2 on incompatible trials was reduced in neurosarcoidosis suggesting that response selection and conflict monitoring functions are dysfunctional. Of note, fatigue strongly modulates responses selection processes in conflicting situations (N2) in controls and neurosarcoidosis, but the effect of fatigue on these processes was stronger in neurosarcoidosis. Neuroimmunological parameters like TNF-α and soluble interleukin-2 receptor serum concentrations do not seem to modulate the pattern of results. Concerning fatigue it seems to be the 'cognitive' dimension and not the 'motor' dimension that is of relevance for the modulation of response selection in conflicting situations.

Detection of patient subgroups with differential expression in omics data: a comprehensive comparison of univariate measures.

Detection of yet unknown subgroups showing differential gene or protein expression is a frequent goal in the analysis of modern molecular data. Applications range from cancer biology over developmental biology to toxicology. Often a control and an experimental group are compared, and subgroups can be characterized by differential expression for only a subgroup-specific set of genes or proteins. Finding such genes and corresponding patient subgroups can help in understanding pathological pathways, diagnosis and defining drug targets. The size of the subgroup and the type of differential expression determine the optimal strategy for subgroup identification. To date, commonly used software packages hardly provide statistical tests and methods for the detection of such subgroups. Different univariate methods for subgroup detection are characterized and compared, both on simulated and on real data. We present an advanced design for simulation studies: Data is simulated under different distributional assumptions for the expression of the subgroup, and performance results are compared against theoretical upper bounds. For each distribution, different degrees of deviation from the majority of observations are considered for the subgroup. We evaluate classical approaches as well as various new suggestions in the context of omics data, including outlier sum, PADGE, and kurtosis. We also propose the new FisherSum score. ROC curve analysis and AUC values are used to quantify the ability of the methods to distinguish between genes or proteins with and without certain subgroup patterns. In general, FisherSum for small subgroups and t-test for large subgroups achieve best results. We apply each method to a case-control study on Parkinson's disease and underline the biological benefit of the new method.

Soluble CSF interleukin 2 receptor as indicator of neurosarcoidosis.

Neurosarcoidosis (NS) represents an important differential diagnosis of multiple sclerosis (MS). However, thus far no reliable laboratory marker of neurosarcoidosis exists. The objective of this study was to evaluate whether cerebrospinal fluid (CSF) levels of soluble interleukin 2 receptor (sIL2-R) distinguish NS and other inflammatory disorders of the central nervous system. For this purpose, 139 paired CSF and serum samples from 11 patients with NS, 21 with MS, 10 with CNS vasculitis, 22 with bacterial meningitis, 17 with viral meningitis/encephalitis, seven with neurotuberculosis, and 18 healthy donors were assessed for sIL2-R using an enzyme-linked immunosorbent assay. We found that sIL2-R CSF levels above 150 pg/ml identified untreated NS patients with an overall accuracy of 93% against a group of non-infectious CNS-diseases. Furthermore, an increase in sIL2-R in the CSF was associated with and preceded the outbreak of new neurological symptoms. In conclusion, these findings suggest that sIL2-R measurement in the CSF may be a valuable tool in the diagnosis and follow-up of patients with suspected and proven neurosarcoidosis.

Reduced basal autophagy and impaired mitochondrial dynamics due to loss of Parkinson's disease-associated protein DJ-1.

BACKGROUND: Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2. CONCLUSIONS/SIGNIFICANCE: We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson's disease.

A comprehensive genetic study of the proteasomal subunit S6 ATPase in German Parkinson's disease patients.

Dysfunction of proteasomal protein degradation is involved in neurodegeneration in Parkinson's disease (PD). Recently we identified the regulatory proteasomal subunit S6 ATPase as a novel interactor of synphilin-1, which is a substrate of the ubiquitin-ligase Parkin (PARK2) and an interacting protein of alpha-synuclein (PARK1). To further investigate a potential role in the pathogenesis of PD, we performed a detailed mutation analysis of the S6 ATPase gene in a large sample of 486 German sporadic and familial PD patients. Direct sequencing revealed two novel intronic variants. An insertion/deletion variant in intron 5 of the S6 ATPase gene was more frequent in patients compared to controls. Moreover, this variant was significantly more frequent in early-onset compared to late-onset PD patients. The identification of a genetic link between a regulatory proteasomal subunit and PD further underscores the relevance of disturbed protein degradation in PD.

Endurance exercise modulates levodopa induced growth hormone release in patients with Parkinson's disease.

Acute levodopa (LD) application and exercise release human growth hormone (GH). An earlier trial showed, that combined stimulus of exercise and LD administration is the best provocative test for GH response in healthy participants. Objective was to show this combined effect of LD application and exercise on GH response and to investigate the impact on LD metabolism in 20 previously treated patients with Parkinson's disease (PD). We measured GH- and LD plasma concentrations following soluble 200 mg LD/50 mg benserazide administration during endurance exercise and rest on two separate consecutive days. GH concentrations significantly increased on both days, but GH release was significantly delayed during rest. LD metabolism was not altered due to exercise in a clinical relevant manner. Exercise induced a significant faster LD stimulated GH release in comparison with the rest condition. We did not find the supposed increase of LD induced GH release by endurance exercise. We assume, that only a limited amount of GH is available for GH release in the anterior pituitary following an acute 200 mg LD administration. GH disposal also depends on growth hormone releasing hormone (GHRH), which is secreted into hypothalamic portal capillaries. During the exercise condition, the resulting higher blood pressure supports blood flow and thus GHRH transport towards the GH producing cells in the pituitary. This might additionally have caused the significant faster GH release during exercise.

Metabolism of the A1 adenosine receptor PET ligand [18F]CPFPX by CYP1A2: implications for bolus/infusion PET studies.

The A1 adenosine receptor positron emission tomography (PET) ligand 8-cyclopentyl-3-(3-[18F]fluoropropyl)-1-propylxanthine ([18F]CPFPX, ) undergoes a fast hepatic metabolism. An optimal design of PET quantitation approaches (e.g., bolus/infusion studies) necessitates the knowledge of factors that influence this metabolism. Metabolites of were separated by radio thin-layer chromatography. Metabolism in vivo, in pooled human liver microsomes and in recombinant human cytochrome isoenzyme preparations was studied. Dynamic PET studies using were performed on three controls and two patients, one treated with the antidepressant and inhibitor of cytochrome CYP1A2 fluvoxamine, the other suffering from liver cirrhosis. CPFPX is metabolized by cytochrome CYP1A2 with high selectivity [KM=1.1 microM (95% confidence interval, or CI, 0.6-2.0 microM) and Vmax=243 pmol min(-1) mg(-1) (95% CI, 112-373 pmol min(-1) mg(-1)) corresponding to 2.4 pmol min(-1) pmol(-1) cytochrome P-450]. This metabolism can competitively be inhibited by fluvoxamine with KI=68 nM (95% CI, 34-138 nM). At least eight compounds found in human plasma and in the CYP1A2 in vitro preparations have an identical migration pattern and account together for >90% and >80% of the respective metabolite yield. Metabolism was considerably delayed in the two patients. In conclusion, is metabolized by cytochrome CYP1A2. Its metabolism is therefore subdued to disease-related or xenobiotic-induced changes of CYP1A2 activity. The identification of the metabolic pathway of 1 allows to optimize image quantification in A1 adenosine receptor PET studies.

Novel homozygous p.E64D mutation in DJ1 in early onset Parkinson disease (PARK7).

Mutations in the parkin gene have been identified as a common cause of autosomal recessive inherited Parkinson disease (PD) associated with early disease manifestation. However, based on linkage data, mutations in other genes contribute to the genetic heterogeneity of early-onset PD (EOPD). Recently, two mutations in the DJ1 gene were described as a second cause of autosomal recessive EOPD (PARK7). Analyzing the PARK7/DJ1 gene in 104 EOPD patients, we identified a third mutation, c.192G>C (p.E64D), associated with EOPD in a patient of Turkish ancestry and characterized the functional significance of this amino acid substitution. In the patient, a substantial reduction of dopamine uptake transporter (DAT) binding was found in the striatum using [(18)F]FP-CIT and PET, indicating a serious loss of presynaptic dopaminergic afferents. His sister, homozygous for E64D, was clinically unaffected but showed reduced dopamine uptake when compared with a clinically unaffected brother, who is heterozygous for E64D. We demonstrate by crystallography that the E64D mutation does not alter the structure of the DJ1 protein, however we observe a tendency towards decreased levels of the mutant protein when overexpressed in HEK293 or COS7 cells. Using immunocytochemistry in contrast to the homogenous nuclear and cytoplasmic staining in HEK293 cells overexpressing wild-type DJ1, about 5% of the cells expressing E64D and up to 80% of the cells expressing the recently described L166P mutation displayed a predominant nuclear localization of the mutant DJ1 protein.