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The activation of the unfolded protein response, particularly via the PERK pathway, has been suggested as a promising therapeutic approach in tauopathies, a group of neurodegenerative disorders characterized by the abnormal phosphorylation and aggregation of tau protein. So far, a shortage of available direct PERK activators has been limiting the progresses in this field. Our study aimed at the development of a cell-free screening assay enabling the detection of novel direct PERK activators. By applying the catalytic domain of recombinant human PERK, we initially determined ideal conditions of the kinase assay reaction, including parameters such as optimal kinase concentration, temperature, and reaction time. Instead of using PERK’s natural substrate proteins, eIF2α and NRF2, we applied SMAD3 as phosphorylation-accepting protein and successfully detected cell-free PERK activation and inhibition by selected modulators (e.g., calcineurin-B, GSK2606414). The developed assay revealed to be sufficiently stable and robust to assess an activating EC50-value. Additionally, our results suggested that PERK activation may take place independent of the active site which can be blocked by a kinase inhibitor. Finally, we confirmed the applicability of the assay by measuring PERK activation by MK-28, a recently described PERK activator. Overall, our data show that a cell-free luciferase-based assay with the recombinant human PERK kinase domain and SMAD3 as substrate protein is capable of detecting PERK activation, which enables to screen large compound libraries for direct PERK activators, in a high-throughput-based approach. These activators will be useful for deepening our understanding of the PERK signaling pathway, and may also lead to the identification of new therapeutic drug candidates for neurodegenerative tauopathies.

Ataxia telangiectasia is a rare autosomal recessive multisystem disorder caused by mutations in the gene of ATM serine/threonine kinase. It is characterized by neurodegeneration, leading to severe ataxia, immunodeficiency, increased cancer susceptibility, and telangiectasia. Here, we discovered a co-segregation of two ATM gene variants with ataxia telangiectasia in an Egyptian family. While one of these variants (NM_000051.4(ATM_i001):p.(Val128*)) has previously been reported as pathogenic, the other one (NM_000051.4(ATM_i001):p.(Val1729Leu)) is regarded as a variant of uncertain significance. Our findings in this family provide additional evidence for causality of the second variant and argue that its status should be changed to pathogenic.

Pathological aggregates of alpha-synuclein are the common hallmarks of synucleinopathies, including Parkinson's disease. There is currently no disease-modifying therapy approved for neurodegenerative synucleinopathies. The induction of macroautophagy by small compounds may be a strategy to reduce the cellular alpha-synuclein burden and to confer neuroprotection. Therefore, in the present study, we investigated a broad spectrum of druggable molecular signaling pathways reported to induce macroautophagy in human cells and compared their protective efficacy against alpha-synuclein-induced toxicity in cultured human postmitotic dopaminergic neurons. Several compounds affecting different pathways were able to activate macroautophagy. All compounds that activated autophagy also protected against alpha-synuclein-induced toxicity. The compounds with the lowest effective concentrations were PI-103, L-690,330, and NF 449, making them particularly interesting for further investigations, including in vivo models. Our findings demonstrate that activation of macroautophagy, as a neuroprotective approach in synucleinopathies, is accessible to pharmacotherapy. Moreover, pharmacological activation of macroautophagy via diverse signaling pathways is effective to protect human dopaminergic neurons against alpha-synuclein-induced toxicity.

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare hereditary neurodegenerative disease characterized by an accumulation of iron within the brain. In the present report, we describe a family with 4 affected siblings presenting with variable clinical manifestations, e.g., parkinsonian features, dystonia and slow disease progression over 5 years. Exome sequencing revealed a causative variant in the pantothenate kinase 2 gene (PANK2). Variant NM_024960.6:c.710C > T was homozygous in all affected subjects. Our report describes the first genetically confirmed cases of PKAN in the Egyptian population. Studying genetics of neurodegenerative diseases in different ethnicities is very important for determining clinical phenotypes and understanding pathomechanisms of these diseases., Pantothenate kinase-associated neurodegeneration; PANK2; Neurodegeneration iron accumulation; Genetics.

Growing evidence suggests that epigenetic mechanisms like microRNA-mediated transcriptional regulation contribute to the pathogenesis of parkinsonism. In order to study the influence of microRNAs (miRNAs), we analyzed the miRNome 2 days prior to major cell death in α-synuclein-overexpressing Lund human mesencephalic neurons, a well-established cell model of Parkinson’s disease (PD), by next-generation sequencing. The expression levels of 23 miRNAs were significantly altered in α-synuclein-overexpressing cells, 11 were down- and 12 upregulated (P < 0.01; non-adjusted). The in silico analysis of known target genes of these miRNAs was complemented by the inclusion of a transcriptome dataset (BeadChip) of the same cellular system, revealing the G0/G1 cell cycle transition to be markedly enriched. Out of 124 KEGG-annotated cell cycle genes, 15 were present in the miRNA target gene dataset and six G0/G1 cell cycle genes were found to be significantly altered upon α-synuclein overexpression, with five genes up- (CCND1, CCND2, and CDK4 at P < 0.01; E2F3, MYC at P < 0.05) and one gene downregulated (CDKN1C at P < 0.001). Additionally, several of these altered genes are targeted by miRNAs hsa-miR-34a-5p and hsa-miR-34c-5p, which also modulate α-synuclein expression levels. Functional intervention by siRNA-mediated knockdown of the cell cycle gene cyclin D1 (CCND1) confirmed that silencing of cell cycle initiation is able to substantially reduce α-synuclein-mediated cytotoxicity. The present findings suggest that α-synuclein accumulation induces microRNA-mediated aberrant cell cycle activation in post-mitotic dopaminergic neurons. Thus, the mitotic cell cycle pathway at the level of miRNAs might offer interesting novel therapeutic targets for PD.

Aggregation of alpha-synuclein (αSyn) is a crucial event underlying the pathophysiology of synucleinopathies. The existence of various intracellular and extracellular αSyn species, including cleaved αSyn, complicates the quest for an appropriate therapeutic target. Hence, to develop efficient disease-modifying strategies, it is fundamental to achieve a deeper understanding of the relevant spreading and toxic αSyn species. Here, we describe comparative and proof-of-principle approaches to determine the involvement of αSyn fragments in intercellular spreading. We demonstrate that two different αSyn fragments (1–95 and 61–140) fulfill the criteria of spreading species. They efficiently instigate formation of proteinase-K-resistant aggregates from cell-endogenous full-length αSyn, and drive it into different aggregation pathways. The resulting aggregates induce cellular toxicity. Strikingly, these aggregates are only detectable by specific antibodies. Our results suggest that αSyn fragments might be relevant not only for spreading, but also for aggregation-fate determination and differential strain formation.

Pesticide exposure is associated with increased risk of Parkinson’s disease (PD). We investigated in Egypt whether common variants in genes involved in pesticide detoxification or transport might modify the risk of PD evoked by pesticide exposure. We recruited 416 PD patients and 445 controls. Information on environmental factors was collected by questionnaire-based structured interviews. Candidate single-nucleotide polymorphisms (SNPs) in 15 pesticide-related genes were genotyped. We analyzed the influence of environmental factors and SNPs as well as the interaction of pesticide exposure and SNPs on the risk of PD. The risk of PD was reduced by coffee consumption [OR = 0.63, 95% CI: 0.43–0.90, P = 0.013] and increased by pesticide exposure [OR = 7.09, 95% CI: 1.12–44.01, P = 0.036]. The SNP rs1126680 in the butyrylcholinesterase gene BCHE reduced the risk of PD irrespective of pesticide exposure [OR = 0.38, 95% CI: 0.20–0.70, P = 0.002]. The SNP rs1803274, defining K-variant BCHE, interacted significantly with pesticide exposure (P = 0.007) and increased the risk of PD only in pesticide-exposed individuals [OR = 2.49, 95% CI: 1.50–4.19, P = 0.0005]. The K-variant BCHE reduces serum activity of butyrylcholinesterase, a known bioscavenger for pesticides. Individuals with K-variant BCHE appear to have an increased risk for PD when exposed to pesticides.

BACKGROUND The microtubule-associated tau protein is the defining denominator of a group of neurodegenerative diseases termed tauopathies. OBJECTIVE Provide a timely state of the art review on recent scientific advances in the field of tauopathies. MATERIAL AND METHODS Systematic review of the literature from the past 10 years. RESULTS Tau proteins are increasingly being recognized as a highly variable protein, underlying and defining a spectrum of molecularly defined diseases, with a clinical spectrum ranging from dementia to hypokinetic movement disorders. Genetic variation at the tau locus can trigger disease or modify disease risk. Tau protein alterations can damage nerve cells and propagate pathologies through the brain. Thus, tau proteins may serve both as a serological and imaging biomarker. Tau proteins also provide a broad spectrum of rational therapeutic interventions to prevent disease progression. This knowledge has led to modern clinical trials. CONCLUSION The field of tauopathies is in a state of dynamic and rapid progress, requiring close interdisciplinary collaboration.

The RNA‐like endoplasmic reticulum kinase (PERK) is genetically associated with the tauopathy progressive supranuclear palsy (PSP). To elucidate the functional mechanisms underlying this association, we explored PERK activity in brains of PSP patients and its function in three tauopathy models (cultured human neurons overexpressing 4‐repeat wild‐type tau or treated with the environmental neurotoxin annonacin, and P301S tau transgenic mice). In vitro, treatment with a pharmacological PERK activator CCT020312 or PERK overexpression reduced tau phosphorylation, tau conformational change and 4‐repeat tau isoforms, and increased cell viability. In vivo, the PERK activator significantly improved memory and locomotor function, reduced tau pathology, and prevented dendritic spine and motoneuron loss in P301S tau mice. Importantly, the PERK substrate EIF2A, mediating some detrimental effects of PERK signaling, was downregulated in PSP brains and tauopathy models, suggesting that the alternative PERK–NRF2 pathway accounts for these beneficial effects in the context of tauopathies. In summary, PERK activation may be a novel strategy to treat PSP and eventually other tauopathies.

ObjectiveTo verify the previously reported association between long-term use of β2-adrenoreceptor (β2AR) agonist and antagonist with reduced and increased risk of Parkinson disease (PD), respectively.MethodsWe obtained odds ratios (ORs) associating time of β2AR agonist and antagonist use with PD risk in nationwide Danish health registries.ResultsWe included 2,790 patients with PD and 11,160 controls. Long-term β2AR agonist use was associated with reduced PD risk (OR 0.57, 95% confidence interval [CI] 0.40–0.82) in this cohort. Unexpectedly, short-term β2AR agonist use was equally associated (OR 0.64, 95% CI 0.42–0.98). Because β2AR agonists are prescribed mostly for chronic obstructive pulmonary disease (COPD), often caused by long-term nicotine abuse, we analyzed other markers of smoking. Diagnosis of COPD (OR 0.51, 95% CI 0.37–0.69) and use of inhaled corticosteroids (OR 0.78, 95% CI 0.59–1.02) or inhaled anticholinergics (OR 0.41, 95% CI 0.25–0.67) were also inversely associated with PD. Increased PD risk was not found for all β2AR antagonists but only for propranolol and metoprolol. Associations were markedly stronger for short-term than long-term use.ConclusionWe confirmed β2AR agonist use to be associated with reduced PD risk and β2AR antagonist use with increased PD risk. However, our data indicate the association of β2AR agonists to be indirectly mediated by smoking, which is repeatedly associated with reduced risk of PD. The association of β2AR antagonists indicates reverse causation, with PD symptoms triggering their prescription rather than β2AR antagonists causing PD. Thus, current epidemiologic data do not support a causal link between β2AR agonists and antagonists and PD risk.

Androgen receptor (AR) antagonists are used for hormone therapy of prostate cancer (PCa). However resistance to the treatment occurs eventually. One possible reason is the occurrence of AR mutations that prevent inhibition of AR-mediated transactivation by antagonists. To offer in future more options to inhibit AR signaling, novel chemical lead structures for new AR antagonists would be beneficial. Here we analyzed structure-activity relationships of a battery of 36 non-steroidal structural variants of methyl anthranilate including 23 synthesized compounds. We identified structural requirements that lead to more potent AR antagonists. Specific compounds inhibit the transactivation of wild-type AR as well as AR mutants that render treatment resistance to hydroxyflutamide, bicalutamide and the second-generation AR antagonist enzalutamide. This suggests a distinct mode of inhibiting the AR compared to the clinically used compounds. Competition assays suggest binding of these compounds to the AR ligand binding domain and inhibit PCa cell proliferation. Moreover, active compounds induce cellular senescence despite inhibition of AR-mediated transactivation indicating a transactivation-independent AR-pathway. In line with this, fluorescence resonance after photobleaching (FRAP) - assays reveal higher mobility of the AR in the cell nuclei. Mechanistically, fluorescence resonance energy transfer (FRET) - assays indicate that the amino-carboxy (N/C)-interaction of the AR is not affected, which is in contrast to known AR-antagonists. This suggests a mechanistically novel mode of AR-antagonism. Together, these findings indicate the identification of a novel chemical platform as a new lead structure that extends the diversity of known AR antagonists and possesses a distinct mode of antagonizing AR-function.

We appreciate the comments from Drs. Scherzer, Riise, and Locascio on our article1 on the effects of β2-adrenoceptor (β2AR) antagonists and agonists and risk of Parkinson disease (PD). Since the initial publication,2 this hypothesis has been scrutinized by multiple groups, yielding conflicting results.2–5

Tauopathies are neurodegenerative brain diseases that are characterized by the formation of intraneuronal inclusions containing the microtubule-associated protein tau. This major hallmark defines tau pathology which is predominant in primary tauopathies, while in secondary forms additional driving forces are involved. In the course of the disease, different brain areas degenerate and lead to severe defects of language, behavior and movement. Although neuropathologically heterogeneous, primary tauopathies share a common feature, which is the generation of abnormal tau species that aggregate and progress into filamentous deposits in neurons. Mechanisms that are involved in this disease-related process offer a broad range of targets for disease-modifying therapeutics. The present review provides an up-to-date overview of currently known targets in primary tauopathies and their possible therapeutic modulation. It is structured into four major targets, the post-translational modifications of tau and tau aggregation, protein homeostasis, disease propagation, and tau genetics. Chances, as well as obstacles in the development of effective therapies are highlighted. Some therapeutic strategies, e.g., passive or active immunization, have already reached clinical development, raising hopes for affected patients. Other concepts, e.g., distinct modulators of proteostasis, are at the ready to be developed into promising future therapies. This article is part of the special issue entitled 'The Quest for Disease-Modifying Therapies for Neurodegenerative Disorders'.

Accumulation of pathological α-synuclein aggregates plays a major role in Parkinson’s disease. Macroautophagy is a mechanism to degrade intracellular protein aggregates by wrapping them into autophagosomes, followed by fusion with lysosomes. We had previously shown that pharmacological activation of macroautophagy protects against α-synuclein-induced toxicity in human neurons. Here, we hypothesized that inhibition of macroautophagy would aggravate α-synuclein-induced cell death.Unexpectedly, inhibition of autophagosome formation by silencing of ATG5 protected from α-synuclein-induced toxicity. Therefore, we studied alternative cellular mechanisms to compensate for the loss of macroautophagy. ATG5 silencing did not affect the ubiquitin–proteasome system, chaperone systems, chaperone-mediated autophagy, or the unfolded protein response. However, ATG5 silencing increased the secretion of α-synuclein via exosomes. Blocking exosomal secretion exacerbated α-synuclein-induced cell death.We conclude that exosomal secretion of α-synuclein is increased after impaired formation of autophagosomes to reduce the intracellular α-synuclein burden. This compensatory mechanism prevents α-synuclein-induced neuronal cell death.

Dietary supplements containing plant materials of Annonaceae species ( Annona muricata L., A. squamosa L., A. mucosa JACQ., A. squamosa × cherimola Mabb.) were extracted by hot, pressurized ethyl acetate and analyzed for their effect in vitro on Lund human mesencephalic neurons. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell death was determined by lactate dehydrogenase levels. Three supplements strongly decreased the cell viability at extract concentrations of 1 µg/mL, of which 1 decreased cell viability at 0.1 µg/µL. Also, strong neuronal toxicities of these supplements were found. Cell death was observed at concentrations of 10 µg/mL. The degree of toxicity was comparable to the ones found in Annonaceous fruit extracts. Two fruit pulps of Annonaceae ( A. muricata and A. squamosa) showed a reduction in cell viability at lower concentrations. The fruit pulp extract of A. muricata revealed the strongest neurotoxic effect, with 67% cell death at a concentration of 1 µg/mL. A high reduction in cell viability coupled with pronounced cell death was found at 0.1 µg/mL for an Annonaceous seed extract. These results demonstrate that the intake of dietary supplements containing plant material from Annonaceae may be hazardous to health in terms of neurotoxicity.

Previously, we demonstrated that systemically injected extracellular domain of neuregulin-1β1 (Nrg1β1), a nerve growth and differentiation factor, passes the blood-brain barrier and rescues dopaminergic neurons of substantia nigra in the 6-hydroxydopamine-mouse model of Parkinson's disease (PD). Here, we studied the effects of peripherally administered Nrg1β1 in another toxin-based mouse model of PD. For this purpose, (i) nigrostriatal pathway injury was induced by treatment of adult wild-type mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in acute and subchronic paradigms; and (ii) Nrg1β1 or saline (control) were administered 1 h before each MPTP injection. We found that Nrg1β1 significantly reduced the loss of nigral dopaminergic neurons in both intoxication paradigms (7 days post-injection). However, Nrg1β1 did not reverse MPTP-induced decrease in dopamine levels and dopaminergic fibers in the striatum. We also show that MPTP conversion to its toxic metabolite 1-methyl-4-phenylpyridinium as well as levels of dopamine transporter, mediating intracellular uptake of 1-methyl-4-phenylpyridinium, are unaffected by Nrg1β1. Finally, neuroprotective properties of Nrg1β1 on nigral dopaminergic neurons are specifically mediated by ErbB4 as revealed through the study of ErbB4 knockout mice. In conclusion, systemically administered Nrg1β1 protects midbrain dopaminergic neurons against this PD-related toxic insult. Thus, Nrg1β1 may have a benefit in the treatment of PD patients. Previously, we demonstrated that systemically administered neuregulin-1β1 (Nrg1β1) passes the blood-brain barrier, phosphorylates ErbB4 receptors and elevates dopamine (DA) levels in the nigrostriatal system of healthy mice. Nrg1β1 protects nigral DAergic neurons in the 6-hydroxydopamine (6-OHDA) mouse model of Parkinson's disease (PD). Here, we show that Nrg1β1 rescues nigral DAergic neurons also against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced cell death. ErbB4 expression is essential for the neuroprotective effect of Nrg1β1 on midbrain DAergic neurons. Nrg1β1 might be beneficial in PD treatment.

Aim P301S MAPT transgenic mice (P301S mice) are a widely used model of frontotemporal dementia and parkinsonism linked to chromosome 17 with tau pathology (FTDP-17-tau). However, a systematic correlation between cognitive deficits and cellular tau pathology at different ages is still missing. Therefore, our study investigated memory deficits of P301S mice in relation to pathological tau species and dendritic spine pathology throughout adulthood. Methods We analysed P301S mice behaviourally with the novel open field, rotarod, and Morris water maze tests to measure deficits in locomotion, balance and cognition, respectively; immunohistochemically with different tau antibodies for specific tau species; and with Golgi staining for dendritic spine pathology. Results We confirmed the occurrence of locomotor deficits at an age of 5 months and newly report memory deficits from 2.5 months of age onwards. At this early age, MC1 and CP13, but not AT180 immunoreactivity, was prominent in the hippocampus of P301S mice. Neuronal cell loss in the hippocampus of P301S mice was not observed to occur till 6 months of age. However, there was a significant reduction in the density of dendritic spines from young adulthood onwards in hippocampal pyramidal neurones. Conclusion In P301S mice, memory deficits precede the onset of locomotor dysfunction and coincide with the appearance of conformationally changed, S202-phosphorylated tau and reduced spine density in the absence of neuronal cell loss in the hippocampus. Our finding provides insights into the toxic effects of different tau species in vivo and may facilitate the development of new therapies against neurodegenerative tauopathies.

Tau is a microtubule-associated protein with versatile functions in the dynamic assembly of the neuronal cytoskeleton. Four-repeat (4R-) tauopathies are a group of neurodegenerative diseases defined by cytoplasmic inclusions predominantly composed of tau protein isoforms with four microtubule-binding domains. Progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease or glial globular tauopathy belong to the group of 4R-tauopathies. The present review provides an introduction in the current concept of 4R-tauopathies, including an overview of the neuropathological and clinical spectrum of these diseases. It describes the genetic and environmental etiological factors, as well as the contemporary knowledge about the pathophysiological mechanisms, including post-translational modifications, aggregation and fragmentation of tau, as well as the role of protein degradation mechanisms. Furthermore, current theories about disease propagation are discussed, involving different extracellular tau species and their cellular release and uptake mechanisms. Finally, molecular diagnostic tools for 4R-tauopathies, including tau-PET and fluid biomarkers, and investigational therapeutic strategies are presented. In summary, we report on 4R-tauopathies as overarching disease concept based on a shared pathophysiological concept, and highlight the challenges and opportunities on the way towards a causal therapy.

International audience; In the pathogenesis of tauopathies, genetic and environmental factors have been identified. While familial clustering led to the identification of mutations in MAPT encoding the microtubule‐associated protein tau, the high incidence of a sporadic tauopathy endemic in Guadeloupe was linked to the plant‐derived mitochondrial complex I inhibitor annonacin. The interaction of both factors was studied in the present work in a realistic paradigm over a period of 12 months. Mice over‐expressing either human wild‐type tau or R406W mutant tau as well as non‐transgenic mice received either regular drinking water or commercially available tropical fruit juice made of soursop (Annona muricata L.) as dietary source of neurotoxins. HPLC‐MS analysis of this juice identified several Annonaceous acetogenins, mainly annonacin (16.2 mg/L), and 41 isoquinoline alkaloids (18.0 mg/L, mainly asimilobine and reticuline). After 12 month of juice consumption, several brain regions showed an increased number of neurons with phosphorylated tau in the somatodendritic compartment of R406W mice and, to a much lesser extent, of non‐transgenic mice and mice over‐expressing human wild‐type tau. Moreover, juice drinking was associated with a reduction in synaptophysin immunoreactivity, as well as an increase in 3‐nitrotyrosine (3NT) reactivity in all three genotypes. The increase in 3NT suggests that Annona muricata juice promotes the generation of reactive nitrogen species. This study provides first experimental evidence that long‐lasting oral ingestion of a widely consumed environmental factor can induce somatodendritic accumulation of hyperphosphorylated tau in mice expressing rodent or human wild‐type tau, and can accelerate tau pathology in R406W‐MAPT transgenic mice.

Neuregulin-1 (NRG1) belongs to a large family of growth and differentiation factors with a key role in the development and maintenance of the brain. Genetic association of NRG1 within brain disorders such as Alzheimer's disease, schizophrenia and neuroprotective properties of certain NRG1 isoforms have led to a variety of studies in corresponding disease models. In the present work, we investigated NRG1 with regard to its peripheral and central biodistribution after systemic application. We first-time radiolabeled the entire biologically active extracellular domain of NRG1 isotype-β1 (NRG1-β1 ECD; aa 2-246) with iodine-125 and administered it peripherally to healthy adult C57Bl6 mice. Blood kinetics and relative organ distribution of (125)I-labeled NRG1-β1 ECD were determined. The blood level of NRG1-β1 ECD peaked within the first hour after intraperitoneal (i.p.) application. The brain-blood ratios of (125)I-labeled NRG1-β1 ECD were time-dependently 150-370% higher compared to the brain impermeable control, (131)I-labeled bovine serum albumin. Autoradiographs of brain slices demonstrated that (125)I-labeled NRG1-β1 ECD accumulated in several regions of the brain e.g. frontal cortex, striatum and ventral midbrain containing the substantia nigra. In addition we found histochemical and biochemical evidence that phosphorylation of the NRG1 prototype receptor ErbB4 was increased in these regions after systemic application of NRG1-β1 ECD. Our data suggest that NRG1-β1 ECD passes the blood-brain barrier and activates cerebral ErbB4 receptors.

Neuregulin-1 (Nrg1) is genetically linked to schizophrenia, a disease caused by neurodevelopmental imbalance in dopaminergic function. The Nrg1 receptor ErbB4 is abundantly expressed on midbrain dopaminergic neurons. Nrg1 has been shown to penetrate blood-brain barrier, and peripherally administered Nrg1 activates ErbB4 and leads to a persistent hyperdopaminergic state in neonatal mice. These data prompted us to study the effect of peripheral administration of Nrg1 in the context of Parkinson's disease, a neurodegenerative disorder affecting the dopaminergic system in the adult brain. We observed that systemic injections of the extracellular domain of Nrg1s(1) (Nrg1s(1) -ECD) increased dopamine levels in the substantia nigra and striatum of adult mice. Nrg1s(1) -ECD injections also significantly protected the mouse nigrostriatal dopaminergic system morphologically and functionally against 6-hydroxydopamine-induced toxicity in vivo. Moreover, Nrg1s(1) -ECD also protected human dopaminergic neurons in vitro against 6-hydroxydopamine. In conclusion, we have identified Nrg1s(1) -ECD as a neurotrophic factor for adult mouse and human midbrain dopaminergic neurons with peripheral administratability, warranting further investigation as therapeutic option for PD patients. (Less)

A plant extract from the fruits of saw palmetto, which is currently used to treat the androgen-dependent benign prostatic hyperplasia and PCa, served as source for new structure variants. We investigated the antiandrogenic potential of an ethanolic total extract and one of its main aromatic components anthranilic acid. An androgen receptor-antagonistic (antiandrogenic) effect of the extract was evident, and although anthranilic acid itself revealed no remarkable effect, its methyl ester, methyl anthranilate, exhibited antiandrogenic potential. Based on this chemical structure, we synthesized and investigated the antiandrogenic activity of four AnA ester derivatives, which were either novel or only little described in literature. These AnA esters inhibit the androgen-dependent transactivation of both the wild-type (wt) androgen receptor and the androgen receptor point mutant T877A, which often occurs in refractory PCa. Moreover, they inhibit the androgen receptor-induced expression of the endogenous prostate-specific antigen. Importantly, AnA esters repress the growth of human PCa cells. Deletion analyses of androgen receptor propose that the antiandrogenic effect of anthranilic acid esters is mediated by the ligand-binding domain, most likely through direct binding, without affecting androgen receptor protein levels. Taken together, the data suggest antiandrogenic potential of anthranilic acid ester derivatives, which can serve as lead structures for novel antiandrogens.

Zonisamide is widely used as an antiepileptic drug. Two studies published recently in Experimental Neurology focus on the drug's neuroprotective effect. In the present commentary, we discuss the significance of their findings and aspects of zonisamide in neuroprotection.

The P301S mutation in exon 10 of the tau gene causes a hereditary tauopathy. While mitochondrial complex I inhibition has been linked to sporadic tauopathies. Fenazaquin, 4-[[4(1,1-dimethylethyl) phenyl] ethoxy] quinazoline, is a low dose emerging acaricide used to control mite attack in different vegetables. With the widespread use, long term persistence in the soil and the mitochondrial complex I inhibitory effects, fenazaquin has a possible link to some neurodegenerative disorders. The aim of this study was to determine whether there is a pathogenic interaction of the environmental toxin fenazaquin and the P301S mutation. Transgenic mice expressing human tau with the P301Smutation (P301S+/+) and wild-type mice at 12 weeks of age were treated orally with vehicle (N = 10 P301S+/+, N = 10 wild-type) or fenazaquin (N = 10 P301S+/+, N = 10 wild-type mice) at a dose of 0.5 mg/kg/d for a period of 54 days. Tau pathology was measured by stereological counts of cells immunoreactive with antibodies against phosphorylated tau (AD2, AT8, AT180, and AT100) and corresponding Western blot analysis. Fenazaquin significantly increased the number of phospho-tau immunoreactive cells in the cerebral cortex in P301S+/+ mice, but only to a variable and mild extent in wild-type mice. Furthermore, fenazaquin led to increased levels of pathologically phosphorylated tau only in P301S+/+ mice. While we observed no apparent cell loss in the frontal cortex, the synaptic density was reduced by fenazaquin treatment in P301S+/+ mice. This study shows that exposure to fenazaquin aggravates the course of genetically determined tau pathology, providing experimental support for the concept of gene environment interaction in the etiology of tauopathies. This research was supported by the German Egyptian Research Fund (GERF: GEF 10-54) funded mutually by the German Ministry of Education and Research (BMBF), Science and Technology Development Fund (STDF).

Objective:To elucidate the genetic cause of an Egyptian family with dopa-responsive dystonia (DRD), a childhood-onset dystonia, responding therapeutically to levodopa, which is caused by mutations in various genes.Methods:Rare variants in all coding exons ofGCH1were excluded by Sanger sequencing. Exome sequencing was applied for 1 unaffected and 2 affected family members. To investigate the functional consequences of detected genetic variants, urinary sepiapterin concentrations were determined by high-performance liquid chromatography.Results:A heterozygous rare nonsynonymous variant in exon 1 of sepiapterin reductase (SPR, c.207C>G, p.Asp69Glu) was found in all affected family members. Urinary concentrations of sepiapterin were above the standard of normal controls in mostSPRmutation carriers, suggesting functional biochemical consequences of the mutation. Variant filtering of all genes involved in the tetrahydrobiopterin pathway, required for levodopa synthesis, revealed an additional common variant in dihydrofolate reductase (DHFR, rs70991108). The presence of both variants was significantly stronger associated with the biochemical abnormality and the clinical disease state as opposed to 1 variant only.Conclusions:The rareSPRmutation can cause autosomal dominant DRD with incomplete penetrance. The commonDHFRvariant might have synergistic effects on production of tetrahydrobiopterin and levodopa, thereby increasing penetrance.

Environment Gene Interaction in Parkinson's disease (EGI-PD) Mohamed M. Salama1, Thomas W. Rosler2, Ali Shalash3, Abdelhaleem Tantawy4, , Gunter U. Hoglinger2,5 1Toxicology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt 2Department for Translational Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE), Munich, Germany 3. Department of Neurology, Faculty of Medicine, Ain Shams University, Cairo, Egypt 4. Department of Neurology, Faculty of Medicine, Mansoura University, Mansoura, Egypt 3 Department of Neurology, Technical University, Munich, Germany Introduction: The prevalence of PD is increased in Egypt compared to reports in other countries. Both genetic and environmental causes might be responsible. E.g. a high rate of LRRK2 mutations has been reported in other North African countries, but has not been systematically studied in Egypt. Also, several epidemiologic studies have supported the hypothesis that broadly defined pesticide exposure may increase the risk of PD. A high degree of pesticide use occurs in Egypt due to the limited availability of agricultural areas in the country. Moreover, lack of precautionary measures on applying pesticides leads to increased pesticide exposures. We aimed to study the link between genes encoding detoxifying enzymes and PD-risk genes on one hand and exposure to pesticides on the other hand in the etiology of PD in Egypt. Methods: This work is undertaken in collaboration between the Technische Universtitat Munchen, Germany and a network of 16 Egyptian Universities leaded by Mansoura University (Egyptian Network of Neurodegenerative Diseases [ENND]). PD cases from all over Egypt are recruited. Half of the cases were chosen in areas of low pesticide exposure, the other half with high pesticides exposure (farmers 50 years of age or older). Similar numbers of non-PD controls are chosen from two sources; first, patients arriving to outpatient clinic who are not suffering from any other neurodegenerative disorder; secondly, persons accompanying participating PD patients. Controls are further stratified into low pesticides and high pesticides controls, as described for the PD cases. Patients are assessed with a standardized questionnaire to evaluate past exposure to pesticides or other Parkinson-related environmental factors, a standardized clinical neurological examination to verify presence of PD (UK brain bank criteria) and to quantify disease severity (Hoehn & Yahr stage, UPDRS motor part) and genotyping for presence or absence of risk-carrying alleles in detoxification and Parkinson's associated genes. Results: We were able to establish ENND which is the first collaborative network of this type in Egypt. Moreover, we established a functional Standard Operation Procedure to collect and ship clinical data and blood for analysis to the German cooperation partner at TUM. So far, we collected blood and epidemiological data from 70 cases and 80 controls. Preliminary data on the analysis of gene-environment interaction analysis will be presented. Conclusion: This work will provide further insights into the mechanisms of gene environment interplay in the etiology of PD and hopefully insights which allow the establishment of primary preventive measures. Funding: DAAD-funded Al-Tawasul project EGI-PD

P301S MAPT transgenic mice (P301S mice) are a widely used model of frontotemporal dementia and parkinsonism linked to chromosome 17 with tau pathology (FTDP-17-tau). However, a systematic correlation between cognitive deficits and cellular tau pathology at different ages is still missing. Therefore, our study investigated memory deficits of P301S mice in relation to pathological tau species and dendritic spine pathology throughout adulthood.We analysed P301S mice behaviourally with the novel open field, rotarod, and Morris water maze tests to measure deficits in locomotion, balance and cognition, respectively; immunohistochemically with different tau antibodies for specific tau species; and with Golgi staining for dendritic spine pathology.We confirmed the occurrence of locomotor deficits at an age of 5 months and newly report memory deficits from 2.5 months of age onwards. At this early age, MC1 and CP13, but not AT180 immunoreactivity, was prominent in the hippocampus of P301S mice. Neuronal cell loss in the hippocampus of P301S mice was not observed to occur till 6 months of age. However, there was a significant reduction in the density of dendritic spines from young adulthood onwards in hippocampal pyramidal neurones.In P301S mice, memory deficits precede the onset of locomotor dysfunction and coincide with the appearance of conformationally changed, S202-phosphorylated tau and reduced spine density in the absence of neuronal cell loss in the hippocampus. Our finding provides insights into the toxic effects of different tau species in vivo and may facilitate the development of new therapies against neurodegenerative tauopathies.

With the aim to develop beneficial tracers for cerebral tumors, we tested two novel 5-iodo-2'-deoxyuridine (IUdR) derivatives, diesterified at the deoxyribose residue. The substances were designed to enhance the uptake into brain tumor tissue and to prolong the availability in the organism. We synthesized carrier added 5-[125I]iodo-3',5'-di-O-acetyl-2'-deoxyuridine (Ac2[125I]IUdR), 5-[125I]iodo-3',5'-di-O-pivaloyl-2'-deoxyuridine (Piv2[125I]IUdR) and their respective precursor molecules for the first time. HPLC was used for purification and to determine the specific activities. The iodonucleoside tracer were tested for their stability against human thymidine phosphorylase. DNA integration of each tracer was determined in 2 glioma cell lines (Gl261, CRL2397) and in PC12 cells in vitro. In mice, we measured the relative biodistribution and the tracer uptake in grafted brain tumors. Ac2[125I]IUdR, Piv2[125I]IUdR and [125I]IUdR (control) were prepared with labeling yields of 31-47% and radiochemical purities of >99% (HPLC). Both diesterified iodonucleoside tracers showed a nearly 100% resistance against degradation by thymidine phosphorylase. Ac2[125I]IUdR and Piv2[125I]IUdR were specifically integrated into the DNA of all tested tumor cell lines but to a less extend than the control [125I]IUdR. In mice, 24 h after i.p. injection, brain radioactivity uptakes were in the following order Piv2[125I]IUdR>Ac2[125I]IUdR>[125I]IUdR. For Ac2[125I]IUdR we detected lower amounts of radioactivities in the thyroid and stomach, suggesting a higher stability toward deiodination. In mice bearing unilateral graft-induced brain tumors, the uptake ratios of tumor-bearing to healthy hemisphere were 51, 68 and 6 for [125I]IUdR, Ac2[125I]IUdR and Piv2[125I]IUdR, respectively. Esterifications of both deoxyribosyl hydroxyl groups of the tumor tracer IUdR lead to advantageous properties regarding uptake into brain tumor tissue and metabolic stability.

Suppression of tau protein expression has been shown to improve behavioral deficits in mouse models of tauopathies, offering an attractive therapeutic approach. Experimentally this had been achieved by switching off the promoters controlling the transgenic human tau gene (MAPT), which is not possible in human patients. The aim of the present study was therefore to evaluate the effectiveness of small interfering RNAs (siRNAs) and their cerebral delivery to suppress human tau expression in vivo, which might be a therapeutic option for human tauopathies. We used primary cortical neurons of transgenic mice expressing P301S-mutated human tau and Lund human mesencephalic (LUHMES) cells to validate the suppressive effect of siRNA in vitro. For measuring the effect in vivo, we stereotactically injected siRNA into the brains of P301S mice to reveal the suppression of tau by immunochemistry (AT180, MC1, and CP13 antibodies). We found that the Accell™ SMART pool siRNA against MAPT can effectively suppress tau expression in vitro and in vivo without a specific delivery agent. The siRNA showed a moderate distribution in the hippocampus of mice after single injection. NeuN, GFAP, Iba-1, MHC II immunoreactivities and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed neither signs of neurotoxicity or neuroinflammation nor apoptosis when MAPT siRNA is present in the hippocampus. Our data suggest that siRNA against MAPT can serve as a potential tool for gene therapy in tauopathies.

The chemical composition of the hydrodistillate won by steam distillation from the fruits of Serenoa repens (W. Bartram), a well known phytomedicine against benign prostatic hyperplasia (BPH), was analyzed by GC-MS. It resulted in the identification of 144 steam-volatile components including about 100 structures which have not been described as constituents of the fruits from S. repens so far. The main component detected was lauric acid (40.4 %).

Neuregulin-1 (Nrg1) is genetically linked to schizophrenia, a disease caused by neurodevelopmental imbalance in dopaminergic function. The Nrg1 receptor ErbB4 is abundantly expressed on midbrain dopaminergic neurons. Nrg1 has been shown to penetrate blood-brain barrier, and peripherally administered Nrg1 activates ErbB4 and leads to a persistent hyperdopaminergic state in neonatal mice. These data prompted us to study the effect of peripheral administration of Nrg1 in the context of Parkinson's disease, a neurodegenerative disorder affecting the dopaminergic system in the adult brain. We observed that systemic injections of the extracellular domain of Nrg1β(1) (Nrg1β(1)-ECD) increased dopamine levels in the substantia nigra and striatum of adult mice. Nrg1β(1)-ECD injections also significantly protected the mouse nigrostriatal dopaminergic system morphologically and functionally against 6-hydroxydopamine-induced toxicity in vivo. Moreover, Nrg1β(1)-ECD also protected human dopaminergic neurons in vitro against 6-hydroxydopamine. In conclusion, we have identified Nrg1β(1)-ECD as a neurotrophic factor for adult mouse and human midbrain dopaminergic neurons with peripheral administratability, warranting further investigation as therapeutic option for Parkinson's disease patients.

A plant extract from the fruits of saw palmetto, which is currently used to treat the androgen-dependent benign prostatic hyperplasia and PCa, served as source for new structure variants. We investigated the antiandrogenic potential of an ethanolic total extract and one of its main aromatic components anthranilic acid. An androgen receptor-antagonistic (antiandrogenic) effect of the extract was evident, and although anthranilic acid itself revealed no remarkable effect, its methyl ester, methyl anthranilate, exhibited antiandrogenic potential. Based on this chemical structure, we synthesized and investigated the antiandrogenic activity of four AnA ester derivatives, which were either novel or only little described in literature. These AnA esters inhibit the androgen-dependent transactivation of both the wild-type (wt) androgen receptor and the androgen receptor point mutant T877A, which often occurs in refractory PCa. Moreover, they inhibit the androgen receptor-induced expression of the endogenous prostate-specific antigen. Importantly, AnA esters repress the growth of human PCa cells. Deletion analyses of androgen receptor propose that the antiandrogenic effect of anthranilic acid esters is mediated by the ligand-binding domain, most likely through direct binding, without affecting androgen receptor protein levels. Taken together, the data suggest antiandrogenic potential of anthranilic acid ester derivatives, which can serve as lead structures for novel antiandrogens.

The average interparticle voidage or porosity (epsilon(inter)) in cylindrical capillaries is studied in dependence on the column diameter (d(c)) to particle diameter (d(p)) ratio for 5d(c)/d(p)50. Using optimized slurry and packing solvents, high pressure and ultrasonication, 5 mum-sized porous C18-silica particles were slurry-packed into fused-silica capillaries having ids from 30 to 250 mum. Packing densities are assessed by a polystyrene standard which is size-excluded from the intraparticle pore space of the packings. For d(c)/d(p)35 densely packed beds are realized (epsilon(inter) = 0.36-0.37), while for decreasing aspect ratios an exponential increase in epsilon(inter )is observed reaching epsilon(inter ) approximately 0.47 at d(c)/d(p) = 5. This behaviour is ascribed to a combination of the geometrical wall effect operating in the direct vicinity of the column wall, caused by the inability of the particles to form a dense packing against the hard surface of the column wall, and particle characteristics like the size distribution, shape and surface roughness. Results are compared with the literature data to address also the importance of absolute particle size in studying structure-transport relations in packed beds in dependence on the aspect ratio d(c)/d(p).