Oliver Kaut

Different methylation of the TNF-alpha promoter in cortex and substantia nigra: Implications for selective neuronal vulnerability

Increasing evidence has linked inflammatory processes to neurodegenerative disorders, including Alzheimers and Parkinsons disease (PD). Tumor necrosis factor alpha (TNF-alpha) is a key inflammatory cytokine and several studies linked increased TNF-alpha to dopaminergic cell death in PD. The TNF-alpha promoter sequence contains several CpG dinucleotides located within or next to transcription factor binding sites. To test the hypothesis whether the methylation state of the TNF-alpha promoter contributes to increased expression of TNF-alpha in PD we compared DNA from different brain regions (substantia nigra pars compacta (SNpc) and cortex) of PD patients and neurologically healthy, age and sex matched controls by bisulfite sequencing of the TNF-alpha promoter region. The TNF-alpha promoter DNA from SNpc was significantly less methylated in comparison to DNA from cortex; however both in PD patients and controls. Although there was a tendency for hypomethylation in PD, our analysis of the 10 CpGs in the TNF-alpha core promoter region (-258 to -35 relative to the TSS) revealed no particular pattern in PD patients compared to control and identified no particular hypomethylated position in cortex or SNpc DNA. Electrophoretic mobility shift and luciferase reporter assays showed that methylation of specific solitary CpG in the TNF-alpha promoter resulted in reduced binding of the transcription factors AP-2 and Sp1, respectively, and suppressed TNF-alpha promoter activity. The brain region specific methylation state of solitary CpG in the TNF-alpha promoter thus determines transcription factor binding efficacy and TNF-alpha expression. A lesser degree of methylation of the TNF-alpha promoter in SNpc cells could underlie the increased susceptibility of dopaminergic neurons to TNF-alpha mediated inflammatory reactions.

Comparative study of the neurotrophic effects elicited by VEGF-B and GDNF in preclinical in vivo models of Parkinson's disease

Vascular endothelial growth factor B (VEGF-B) has recently been shown to be a promising novel neuroprotective agent for several neurodegenerative conditions. In the current study we extended previous work on neuroprotective potential for Parkinsons disease (PD) by testing an expanded dose range of VEGF-B (1 and 10 μg) and directly comparing both neuroprotective and neurorestorative effects of VEGF-B in progressive unilateral 6-hydroxydopamine (6-OHDA) PD models to a single dose of glial cell line-derived neurotrophic factor (GDNF, 10 μg), that has been established by several groups as a standard in both preclinical PD models. In the amphetamine-induced rotational tests the treatment with 1 and 10 μg VEGF-B resulted in significantly improved motor function of 6-OHDA-lesioned rats compared to vehicle-treated 6-OHDA-lesioned rats in the neuroprotection paradigm. Both doses of VEGF-B caused an increase in tyrosine hydroxylase (TH)-positive cell and fiber count in the substantia nigra (SN) and striatum in the neuroprotective experiment. The effect size was comparable to the effects seen with GDNF. In the neurorestoration paradigm, VEGF-B injection had no significant effect in either the behavioral or the immunohistochemical analyses, whereas GDNF injection significantly improved the amphetamine-induced rotational behavior and reduced TH-positive neuronal cell loss in the SN. We also present a strong positive correlation (p=1.9e-50) of the expression of VEGF-B with nuclear-encoded mitochondrial genes involved in fatty acid metabolism in rat midbrain, pointing to the mitochondria as a site of action of VEGF-B. GDNF showed a positive correlation with nuclear-encoded mitochondrial genes that was not nearly as strong (p=0.018). VEGF-B counteracted rotenone-induced reduction of (a) fatty acid transport protein 1 and 4 levels and (b) both Akt protein and phosphorylation levels in SH-SY5Y cells. We further verified VEGF-B expression in the human SN pars compacta of healthy controls and PD patients, in neuronal cells that show co-expression with neuromelanin. These results have demonstrated that VEGF-B has potential as a neuroprotective agent for PD therapy and should be further investigated.

L‐dopa increases α‐synuclein DNA methylation in Parkinson's disease patients in vivo and in vitro

Increasing gene dosages of α‐synuclein induce familial Parkinsons disease (PD); thus, the hypothesis has been put forward that regulation of gene expression, in particular altered α‐synuclein gene methylation, might be associated with sporadic PD and could be used as a biological marker.

Aberrant NMDA receptor DNA methylation detected by epigenome-wide analysis of hippocampus and prefrontal cortex in major depression

Abstract Current perspectives on the molecular underpinnings of major depressive disorder (MDD) posit a mechanistic role of epigenetic DNA modifications in mediating the interaction between environmental risk factors and a genetic predisposition. However, conclusive evidence for differential methylation signatures in the brain’s epigenome of MDD patients as compared to controls is still lacking. To address this issue, we conducted a pilot study including an epigenome-wide methylation analysis in six individuals diagnosed with recurrent MDD and six control subjects matched for age and gender, with a priori focus on the hippocampus and prefrontal cortex as pathophysiologically relevant candidate regions. Our analysis revealed differential methylation profiles of 11 genes in hippocampus and 20 genes in prefrontal cortex, five of which were selected for replication of the methylation status using pyrosequencing. Among these replicated targets, GRIN2A was found to be hypermethylated in both prefrontal cortex and hippocampus. This finding may be of particular functional relevance as GRIN2A encodes the glutamatergic N-methyl-d-aspartate receptor subunit epsilon-1 (NR2A) and is known to be involved in a plethora of synaptic plasticity-related regulatory processes probably disturbed in MDD.

DNA methylation levels of α-synuclein intron 1 in the aging brain

DNA methylation patterns change with age, and aging itself is a major confounding risk factor for Parkinsons disease (PD). Duplication and triplication, that is, increased expression of the α-synuclein (SNCA) gene, cause familial PD, and demethylation of SNCA intron 1 has been shown to result in increased expression of SNCA. We thus hypothesized that age-related alterations of SNCA methylation might underly the increased susceptibility toward PD in later life. The present study sought to determine (1) whether alterations of SNCA intron 1 methylation occurred during aging, (2) whether the methylation pattern differed between men and women, and (3) whether purified neurons compared with non-neuronal cells exhibited different methylation patterns. The analysis of DNA from brain tissue and fluorescence activated cell sorting-sorted purified neurons of 41 individuals revealed only a minor increase of SNCA intron 1 DNA methylation levels in presumably healthy individuals during aging but no significant difference between men and women. Interestingly enough, methylation of SNCA intron 1 was higher in neurons compared with non-neuronal cells, although non-neuronal cells express lower levels of SNCA. Therefore, the normal pattern of SNCA methylation during aging should not result in increased expression of α-synuclein protein. It is thus likely that additional, yet not identified, mechanisms contribute to the tissue specificity of SNCA expression and the presumed dysregulation in PD.

Stochastic resonance therapy in Parkinson's disease.

OBJECTIVE To test the effects of stochastic whole body vibration (WBV) we performed a double-blind randomized controlled study. METHODS Patients were allocated either to the experimental or sham group. The experimental group received 5 cycles of stochastic WBV on three days, each cycle consisting of 5 stimulus trains of 60 seconds duration (frequency 6.5 Hz) and 60 seconds resting time between stimuli. Patients allocated to the control group received a sham treatment with 1 Hz. Unified Parkinsons Disease Rating Scale, part III (UPDRSIII) was performed after treatment at baseline, after the first series on day 1 and on day 5. RESULTS The reduction of subscores included in UPDRS III relative to baseline served as primary outcome measure. After the five-day course bradykinesia was improved in 14 of 18 patients (77.8%) and postural stability in 8 of 18 (44.4%). Speech and facial expression remained unchanged in both groups. Tremor (p=0.027) and postural stability (p=0.048) showed a reduction also, but did not reached level of significance (p < 0.01); UPDRSIII sum score was improved by 26.7%. CONCLUSION Stochastic whole body vibration may offer a supplementation to canonical physical treatments of PD motor symptoms.

51-year-old female with steroid-responsive optic neuropathy: a new case of chronic relapsing inflammatory optic neuropathy (CRION)

JO N 2919 the left eye. Vision was reduced to finger counting on the left and 100 % of the right eye. Ophthalmological diseases were ruled out by an ophthalmologist. Visual-evoked responses were mildly delayed on the left side. MRI of the optic nerves was normal. MRI of the brain showed small unspecific signals in the white matter in FLAIR and T2-weighted sequences. Intravenous prednisolone (1 g for 3 days) improved vision rapidly to almost normal vision and relieved pain. Three days later, vision deteriorated, but rapidly improved after a second trial of intravenous prednisolone. Prednisolone was then continued orally for 2 weeks. After cessation of prednisolone, vision deteriorated for the third time, and again improved with steroids. A couple of days later, vision started to worsen again. With the left eye, only finger counting was possible; on the right side vision was 100 %. At this time, she was transferred to our department, but refused to undergo a fourth treatment with steroids. Nevertheless, she accepted diagnostic tests. Swinging flash light test was abnormal on the left side; in detail the direct pupillary light reflex was weaker on the left side. Visual-evoked responses were not evoked on the left side. CSF showed normal cell count, protein content and negative oligoclonal bands. Serological tests for Borrelia burgdorferi-IgG and -IgM were negative. CRP, ESR (erythrocyte sedimentation rate), glucose, FBC (full blood cell count), hepatitis A-, B-, C-screening, markers of vasculitis (ANA, ANCA, dsDNA-antibody), HIV-1/2 antibody and marker of Devic syndrome (aquaporin-4) were normal. Uveitis and ocular manifestation of lymphoma were again ruled out by ophthalmological examination. Screening for systemic sarcoidosis with chest X-ray and serum angiotensin converting Oliver Kaut Thomas Klockgether

DJ-1 is a redox sensitive adapter protein for high molecular weight complexes involved in regulation of catecholamine homeostasis

&NA; DJ‐1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinsons disease (PD), but growing evidence suggests involvement of DJ‐1 in idiopathic PD. The key clinical features of PD, rigidity and bradykinesia, result from neurotransmitter imbalance, particularly the catecholamines dopamine (DA) and noradrenaline. We report in human brain and human SH‐SY5Y neuroblastoma cell lines that DJ‐1 predominantly forms high molecular weight (HMW) complexes that included RNA metabolism proteins hnRNPA1 and PABP1 and the glycolysis enzyme GAPDH. In cell culture models the oxidation status of DJ‐1 determined the specific complex composition. RNA sequencing indicated that oxidative changes to DJ‐1 were concomitant with changes in mRNA transcripts mainly involved in catecholamine metabolism. Importantly, loss of DJ‐1 function upon knock down (KD) or expression of the PD associated form L166P resulted in the absence of HMW DJ‐1 complexes. In the KD model, the absence of DJ‐1 complexes was accompanied by impairment in catecholamine homeostasis, with significant increases in intracellular DA and noraderenaline levels. These changes in catecholamines could be rescued by re‐expression of DJ‐1. This catecholamine imbalance may contribute to the particular vulnerability of dopaminergic and noradrenergic neurons to neurodegeneration in PARK7‐related PD. Notably, oxidised DJ‐1 was significantly decreased in idiopathic PD brain, suggesting altered complex function may also play a role in the more common sporadic form of the disease.

Spontaneous unwelcome orgasms due to pramipexole and ropinirole

Dopamine agonists like pramipexole and rotigotine are associated with various side effects including somnolence, hallucinations, and leg edema. Reported hypersexual behavior has been categorized as an impulse control disorder (ICD). Here, we expand the spectrum of sexual side effects associated with dopamine agonists by events outside the spectrum of ICD. A patient with dopa-responsive dystonia experienced spontaneous unwelcome orgasms from pramipexole and ropinirole without hypersexual behavior or increased libido. A 52-year-old woman presented with multifocal dystonia manifesting as cervical dystonia, writer’s cramp, and dystonic posturing of the left foot. Family history and brain MRI were normal. Genetic testing was unremarkable for dopa-responsive dystonia (GCH1, TH, and SPR mutations), DYT1, and Wilson’s disease. At 600 mg of levodopa, dystonia severity decreased dramatically. However, levodopa had to be stopped because of severe diarrhea, weight loss, and orthostatic hypotension. Dystonia returned promptly after discontinuation of levodopa. Three months later, treatment with pramipexole was initiated. On the third day (pramipexole dose of 0.088 mg twice a day), she experienced 4 spontaneous orgasms. Three occurred during the day while doing housework, each approximately 1 hour after intake of pramipexole, and one at night, waking her up. Orgasms occurred without any premonition, perceivable stimulation, or increased sexual desire. They were experienced as undesired and highly embarrassing. Pramipexole was discontinued, and no further undesired orgasms occurred. Ten weeks later, treatment with ropinirole was initiated. At 0.5 mg per day, spontaneous orgasms reappeared, stopping again at 0.25 mg daily. Unwelcome orgasms are rare symptoms of neurological disease. They have also been observed during antidepressant treatment, for example with paroxetine. So far, spontaneous orgasms from pramipexole or ropinirole have not been reported, although treatment with dopamine agonists may be complicated by hypersexual behavior. Sexual arousal has been related to increased dopaminergic neurotransmission. The reproducible induction of recurrent orgasms (and, in our case, further dopamine-related effects such as diarrhea and orthostatic hypotension) may indicate our patient has intrinsic sensitivity to dopaminergic stimulation. Interestingly, there was no evidence of ICD. Our patient experienced neither sexual arousal nor increase in libido. In a 28-year-old woman, painful clitoral tumescence from bromocriptine was reported. Presumably, both cases are based on similar mechanisms influencing the physiology of female orgasms outside the spectrum of ICD. Although spontaneous orgasms were unwelcome in our case, sexual health and ability to experience orgasm are important predictors of quality of life. They are often compromised in neuropsychiatric disorders. In depression, selective serotonin reuptake inhibitors are known to negatively affect all steps of the sexual response cycle and to cause anorgasmia. Our observation may confirm the idea of DRD3 stimulation via pramipexole in the management of these potentially unpleasant side effects.

Tremor in Parkinson's disease is not associated with the DRD3 Ser9Gly polymorphism.

A common subset of genetic risk factors for Parkinsons disease (PD) and essential tremor (ET) has been postulated. Recently, an association between the dopamine D(3) receptor (DRD3) Ser9Gly polymorphism and ET has been reported. We studied whether PD tremor is influenced by Ser9Gly in a genetic association study based on the gene bank of the German Competence Network on Parkinsons disease. The study included analyses of motor predominance (mixed, hypokinetic, and tremor), and tremor type (resting, postural, and action). We did not identify any effect of DRD3 Ser9Gly on tremor in PD, even when regarding various symptom combinations to avoid missing a weak effect on the phenotype. Additional studies incorporating symptoms at disease onset, and grading of tremor response to dopaminergic therapy, are warranted.