Ullrich Wüllner

Elevated cerebrospinal fluid and blood concentrations of oxytocin following its intranasal administration in humans

There has been an unprecedented interest in the modulatory effects of intranasal oxytocin on human social cognition and behaviour, however as yet no study has actually demonstrated that this modality of administration increases concentrations of the peptide in the brain as well as blood in humans. Here using combined blood and cerebrospinal fluid (CSF) sampling in subjects receiving either 24 IU of oxytocin (n = 11) or placebo (n = 4) we have shown that oxytocin levels significantly increased in both plasma and CSF. However, whereas oxytocin plasma concentrations peaked at 15 min after intranasal administration and decreased after 75 min, CSF concentrations took up to 75 min to reach a significant level. Moreover, there was no correlation (r = <0.10) between oxytocin plasma and CSF concentrations. Together, these data provide crucial insights into the plasma and CSF kinetics of intranasally administered oxytocin.

Sleep attacks, daytime sleepiness, and dopamine agonists in Parkinson's disease

To study the putative association of dopamine agonists with sleep attacks in patients with Parkinsons disease (PD) and their relation to daytime sleepiness, we performed a survey of 2,952 PD patients in two German counties. In 177 patients, sudden, unexpected, and irresistible sleep episodes while engaged in some activity were identified in a structured telephone interview. Ninety‐one of these patients denied the occurrence of appropriate warning signs. A total of 133 patients (75%) had an Epworth Sleepiness Scale (ESS) score >10; 65 (37%) >15. Thirty‐one patients (18%) had an ESS score ≤10 and yet experienced sleep attacks without warning signs. Thus, although a significant proportion of patients at risk for sleep attacks might be identified using the ESS, roughly 1% of the PD patient population seems to be at risk for sleep attacks without appropriate warning signs and without accompanying daytime sleepiness. Sleep attacks occurred with all dopamine agonists marketed in Germany (α‐dihydroergocryptine, bromocriptine, cabergoline, lisuride, pergolide, pramipexole, ropinirole), and no significant difference between ergot and nonergot drugs was evident. Levodopa (L‐dopa) monotherapy carried the lowest risk for sleep attacks (2.9%; 95% confidence interval [CI], 1.7–4.0%) followed by dopamine agonist monotherapy (5.3%; 95% CI, 1.5–9.2%) and combination of L‐dopa and a dopamine agonist (7.3%; 95% CI, 6.1–8.5%). Neither selegeline nor amantadine or entacapone appeared to influence the occurrence of sleep attacks. A high ESS score, intake of dopamine agonists, and duration of PD were the main influencing factors for the occurrence of sleep attacks. The odds ratio for dopamine agonist therapy was 2.9 compared to 1.9 with L‐dopa therapy and 1.05 for a 1‐year‐longer disease duration.

Multiple regions of alpha-synuclein are associated with Parkinson's disease.

α‐Synuclein is considered to play an important role in the pathogenesis of both the rare familial and the common sporadic forms of Parkinsons disease. Previous reports primarily have tested the association of α‐synuclein promoter polymorphisms with idiopathic Parkinsons disease, but results are controversial. We first characterized the linkage disequilibrium structure of the α‐synuclein gene region with a dense set of 56 genetic markers and subsequently performed two independent case–control association analyses using tagging markers. We could distinguish two large linkage disequilibrium blocks spanning the α‐synuclein gene. Several markers within the 3′‐block around exons 5 and 6 showed strong association with Parkinsons disease (p = 0.00009). Effects of the associated variants might be mediated by regulatory elements in this highly conserved region or by a frequency shift in a previously described splice variant lacking exon 5. A direct association with promoter polymorphisms could not be replicated in our sample set. A second set of markers in the 5′‐block of the gene were also significantly associated with Parkinsons disease, when young patients and female subjects were analyzed separately. These findings indicate locus heterogeneity for the pathogenesis of Parkinsons disease in different genetic or physiological environments, related to sex and age. Ann Neurol 2005;57:535–541

SNCA Variants Are Associated with Increased Risk for Multiple System Atrophy

To test whether the synucleinopathies Parkinsons disease and multiple system atrophy (MSA) share a common genetic etiology, we performed a candidate single nucleotide polymorphism (SNP) association study of the 384 most associated SNPs in a genome‐wide association study of Parkinsons disease in 413 MSA cases and 3,974 control subjects. The 10 most significant SNPs were then replicated in additional 108 MSA cases and 537 controls. SNPs at the SNCA locus were significantly associated with risk for increased risk for the development of MSA (combined p = 5.5 × 1012; odds ratio 6.2). Ann Neurol 2009;65:610–614

Excitation-induced ataxin-3 aggregation in neurons from patients with Machado–Joseph disease

Machado–Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that l-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca2+-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na+ and K+ channels as well as ionotropic and voltage-gated Ca2+ channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.

Induction of Nitric Oxide Synthase and Nitric Oxide-Mediated Apoptosis in Neuronal PC12 Cells After Stimulation with Tumor Necrosis FActor-α/Lipopolysaccharide

Abstract: Exposure of neuronal PC12 cells, differentiated by nerve growth factor, to tumor necrosis factor‐α (TNF‐α) and bacterial lipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differntiated PC12 cells was associated with cell death characterized by features of apoptosis, The NOS inhibitors N‐monomethylarginine, aminoguanidine, and 2‐amino‐5,6‐dihydro‐6‐methyl‐4H‐1,3‐thiazine HCl prevented TNF‐α/LPS‐induced cell death and DNA fragmentation, suggesting that the TNF‐α/LPS‐induced cell death is mediated by iNOS‐derived NO. This hypothesis is supported by the finding that addition of l‐arginine, which serves as a precursor and limiting factor of enzyme‐derived NO production, potentiated TNF‐α/LPS‐induced loss of viability.

Glutathione depletion and neuronal cell death: the role of reactive oxygen intermediates and mitochondrial function.

Glutathione (GSH) levels are supposed to determine the vulnerability of many cells towards a wide array of insults. We investigated the effects of chronic inhibition of GSH synthesis and acute depletion of GSH on cerebellar granule neurons in vitro and determined cytoplasmic and mitochondrial GSH with relation to mitochondrial function and generation of reactive oxygen intermediates (ROI). l-buthionine sulfoximine (BSO), which irreversibly blocks gamma-glutamyl-cysteine synthase, led to a time- and concentration-dependent loss of cytoplasmic GSH, while mitochondrial GSH was relatively preserved. No increased generation of ROI was detected over 48 h and the mitochondrial membrane potential was largely maintained. Neuronal degeneration occurred when mitochondrial GSH levels had fallen below 50% of control after 24-36 h. In contrast, direct conjugation of mitochondrial and cytoplasmic GSH with etacrynic acid (EA), resulted in immediate loss of mitochondrial GSH, a large increase of ROI within 2 h, subsequent collapse of the mitochondrial membrane potential and complete cell death within 4-8 h. Electron microscopy studies revealed an as yet unknown change of the chromatin structure to a homogeneous granular pattern after BSO, while EA resulted in typical necrotic changes. No typical features of apoptosis, i.e., no chromatin condensation or DNA fragmentation were detected after GSH depletion after BSO or EA treatment.

Peroxisome proliferator-activated receptor gamma agonists protect cerebellar granule cells from cytokine-induced apoptotic cell death by inhibition of inducible nitric oxide synthase

Cerebellar granule cells (CGCs) can express the inducible isoform of nitric oxide synthase (iNOS) in response to inflammatory stimuli. We demonstrate that induction of iNOS in CGCs by bacterial lipopolysaccharide and pro-inflammatory cytokines results in cell death that was potentiated by excess L-arginine and inhibited by the selective iNOS inhibitor, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine. The NO-mediated cell death was accompanied by increased caspase-3-like activity, DNA fragmentation and positive terminal transferase dUTP nick end labeling (TUNEL), suggesting that apoptosis mediates CGC cell death. Incubation of CGCs with the non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen or indomethacin, or with 15-deoxy-delta12,14 prostaglandin J2 (PGJ2) downregulates iNOS expression and reduces subsequent cell death. Since in other cell types, both NSAIDs and PGJ2 can activate the peroxisome proliferator-activated receptor-gamma (PPARgamma) and downregulate cytokine levels and iNOS expression, and since CGCs express PPARgamma in vivo and in vitro, our data suggest that activation of CGC PPARgamma mediates iNOS suppression and reduced cell death. Because PPARgamma is expressed in brains of Alzheimers Disease (AD) patients, in which neuronal iNOS expression and apoptotic cell death have been described, these results may help explain the basis for the beneficial effects of NSAIDs in AD.

Glutathione depletion potentiates MPTP and MPP+ toxicity in nigral dopaminergic neurones

Glutathione levels are decreased in the substantia nigra of patients with Parkinsons disease. We studied whether glutathione depletion contributes to dopaminergic cell death using a specific inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (BSO). We found no significant reduction of tyrosine hydroxylase-positive cells in the substantia nigra pars compacta (SNpc) when BSO was administered systemically to preweanling mice or locally to the SNpc of adult rats. However, the combination of BSO with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in preweanling mice and the combination of nigral injections of BSO with intrastriatal injections of MPP+ (1-methyl-4-phenylpyridinium), the active metabolite of MPTP in adult rats, potentiated the toxic effects of MPTP and MPP+ on nigral neurones. Our data show that glutathione depletion can result in cell death if the nigrostriatal system is metabolically compromised.

An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis

Arginine/lysine‐rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin‐3 (Atx‐3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx‐3 in a concentration‐dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx‐3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine‐rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx‐3. Together, these results define the VCP–Atx‐3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.