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1.
PLoS One ; 18(5): e0283943, 2023.
Article in English | MEDLINE | ID: mdl-37200357

ABSTRACT

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.


Subject(s)
Endoplasmic Reticulum Stress , Tauopathies , Humans , Unfolded Protein Response , Signal Transduction , Protein Kinase Inhibitors , Phosphorylation , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolism , Eukaryotic Initiation Factor-2/metabolism
2.
Acta Neuropathol Commun ; 11(1): 40, 2023 03 11.
Article in English | MEDLINE | ID: mdl-36906636

ABSTRACT

The MAPT gene, encoding the microtubule-associated protein tau on chromosome 17q21.31, is result of an inversion polymorphism, leading to two allelic variants (H1 and H2). Homozygosity for the more common haplotype H1 is associated with an increased risk for several tauopathies, but also for the synucleinopathy Parkinson's disease (PD). In the present study, we aimed to clarify whether the MAPT haplotype influences expression of MAPT and SNCA, encoding the protein α-synuclein (α-syn), on mRNA and protein levels in postmortem brains of PD patients and controls. We also investigated mRNA expression of several other MAPT haplotype-encoded genes. Postmortem tissues from cortex of fusiform gyrus (ctx-fg) and of the cerebellar hemisphere (ctx-cbl) of neuropathologically confirmed PD patients (n = 95) and age- and sex-matched controls (n = 81) were MAPT haplotype genotyped to identify cases homozygous for either H1 or H2. Relative expression of genes was quantified using real-time qPCR; soluble and insoluble protein levels of tau and α-syn were determined by Western blotting. Homozygosity for H1 versus H2 was associated with increased total MAPT mRNA expression in ctx-fg regardless of disease state. Inversely, H2 homozygosity was associated with markedly increased expression of the corresponding antisense MAPT-AS1 in ctx-cbl. PD patients had higher levels of insoluble 0N3R and 1N4R tau isoforms regardless of the MAPT genotype. The increased presence of insoluble α-syn in PD patients in ctx-fg validated the selected postmortem brain tissue. Our findings in this small, but well controlled cohort of PD and controls support a putative biological relevance of tau in PD. However, we did not identify any link between the disease-predisposing H1/H1 associated overexpression of MAPT with PD status. Further studies are required to gain a deeper understanding of the potential regulatory role of MAPT-AS1 and its association to the disease-protective H2/H2 condition in the context of PD.


Subject(s)
Genetic Predisposition to Disease , Parkinson Disease , tau Proteins , Humans , Brain/metabolism , Genotype , Haplotypes , Parkinson Disease/metabolism , Polymorphism, Single Nucleotide , RNA, Messenger/genetics , tau Proteins/genetics
3.
Heliyon ; 7(7): e07469, 2021 Jul.
Article in English | MEDLINE | ID: mdl-34286134

ABSTRACT

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.

4.
Neurogenetics ; 22(2): 143-147, 2021 05.
Article in English | MEDLINE | ID: mdl-33779842

ABSTRACT

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.


Subject(s)
Ataxia Telangiectasia/genetics , Mutation, Missense , Point Mutation , Causality , Egypt , Female , Genotype , Humans , Male , Pedigree , Phenotype , Exome Sequencing , alpha-Fetoproteins/genetics
5.
Front Cell Dev Biol ; 9: 561086, 2021.
Article in English | MEDLINE | ID: mdl-33748099

ABSTRACT

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.

7.
Cell Death Dis ; 11(2): 84, 2020 02 03.
Article in English | MEDLINE | ID: mdl-32015326

ABSTRACT

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.


Subject(s)
Peptide Fragments/metabolism , Protein Aggregation, Pathological/metabolism , alpha-Synuclein/metabolism , Cell Line , Extracellular Space/metabolism , Gene Knockout Techniques , Humans , Neurons/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/toxicity , Protein Aggregates , Protein Domains , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/toxicity , alpha-Synuclein/chemistry , alpha-Synuclein/genetics , alpha-Synuclein/toxicity
8.
Neuropharmacology ; 167: 107842, 2020 05 01.
Article in English | MEDLINE | ID: mdl-31704274

ABSTRACT

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'.


Subject(s)
Genetic Therapy/trends , Immunotherapy/trends , Tauopathies/genetics , Tauopathies/metabolism , tau Proteins/genetics , tau Proteins/metabolism , Animals , Brain/drug effects , Brain/metabolism , Genetic Therapy/methods , Humans , Immunotherapy/methods , Protein Processing, Post-Translational , Tauopathies/therapy , tau Proteins/antagonists & inhibitors
9.
Sci Rep ; 9(1): 13359, 2019 09 16.
Article in English | MEDLINE | ID: mdl-31527656

ABSTRACT

Neuroblasts born in the subventricular zone of adult mammals migrate via the rostral migratory stream into the granular cell layer or periglomerular layer of the olfactory bulb to differentiate into interneurons. To analyze if new neurons in the granular cell layer or periglomerular layer have different origins, we inserted a physical barrier into the rostral migratory stream, depleted cell proliferation with cytarabine infusions, labeled newborn cells with bromodeoxyuridine, and sacrificed mice after short-term (0, 2, or 14 days) or long-term (55 or 105 days) intervals. After short-term survival, the subventricular zone and rostral migratory stream rapidly repopulated with bromodeoxyuridine+ cells after cytarabine-induced depletion. Nestin, glial fibrillary acidic protein and the PAX6 were expressed in bromodeoxyuridine+ cells within the rostral migratory stream downstream of the physical barrier. After long-term survival after physical barrier implantation, bromodeoxyuridine+ neurons were significantly reduced in the granular cell layer, but bromodeoxyuridine+ and dopaminergic neurons in the periglomerular layer remained unaffected by the physical barrier. Thus, newborn neurons for the granular cell layer are mainly recruited from neural stem cells located in the subventricular zone, but new neurons for the periglomerular layer with dopaminergic predisposition can rise as well from neuronal stem or precursor cells in the rostral migratory stream.


Subject(s)
Cell Movement/physiology , Neural Stem Cells/metabolism , Olfactory Bulb/metabolism , Animals , Bromodeoxyuridine/metabolism , Cell Differentiation/physiology , Cell Proliferation/physiology , Dopamine/metabolism , Dopaminergic Neurons/metabolism , Interneurons/metabolism , Lateral Ventricles/metabolism , Male , Mice , Mice, Inbred C57BL , Neural Stem Cells/physiology
10.
Prog Neurobiol ; 180: 101644, 2019 09.
Article in English | MEDLINE | ID: mdl-31238088

ABSTRACT

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.


Subject(s)
Alzheimer Disease/metabolism , Brain/metabolism , Neurons/metabolism , Tauopathies/metabolism , tau Proteins/metabolism , Humans , Neuropathology/methods
12.
Neurology ; 93(2): e135-e142, 2019 07 09.
Article in English | MEDLINE | ID: mdl-31127070

ABSTRACT

OBJECTIVE: To verify the previously reported association between long-term use of ß2-adrenoreceptor (ß2AR) agonist and antagonist with reduced and increased risk of Parkinson disease (PD), respectively. METHODS: We obtained odds ratios (ORs) associating time of ß2AR agonist and antagonist use with PD risk in nationwide Danish health registries. RESULTS: We included 2,790 patients with PD and 11,160 controls. Long-term ß2AR 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 ß2AR agonist use was equally associated (OR 0.64, 95% CI 0.42-0.98). Because ß2AR 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 ß2AR antagonists but only for propranolol and metoprolol. Associations were markedly stronger for short-term than long-term use. CONCLUSION: We confirmed ß2AR agonist use to be associated with reduced PD risk and ß2AR antagonist use with increased PD risk. However, our data indicate the association of ß2AR agonists to be indirectly mediated by smoking, which is repeatedly associated with reduced risk of PD. The association of ß2AR antagonists indicates reverse causation, with PD symptoms triggering their prescription rather than ß2AR antagonists causing PD. Thus, current epidemiologic data do not support a causal link between ß2AR agonists and antagonists and PD risk.


Subject(s)
Adrenergic beta-Agonists/therapeutic use , Adrenergic beta-Antagonists/therapeutic use , Parkinson Disease/epidemiology , Administration, Inhalation , Adrenal Cortex Hormones/therapeutic use , Aged , Aged, 80 and over , Cholinergic Antagonists/therapeutic use , Denmark/epidemiology , Duration of Therapy , Female , Humans , Male , Metoprolol/therapeutic use , Middle Aged , Propranolol/therapeutic use , Protective Factors , Pulmonary Disease, Chronic Obstructive/epidemiology , Registries , Risk Factors , Smoking/epidemiology
13.
Neuropharmacology ; 149: 13-26, 2019 05 01.
Article in English | MEDLINE | ID: mdl-30731136

ABSTRACT

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.


Subject(s)
Autophagy/drug effects , Dopaminergic Neurons/drug effects , alpha-Synuclein/drug effects , alpha-Synuclein/metabolism , Benzenesulfonates/pharmacology , Caspase 3/metabolism , Caspase 7/metabolism , Cell Line , Cell Survival/drug effects , Diphosphonates/pharmacology , Furans/pharmacology , Humans , Microtubule-Associated Proteins/metabolism , Neuroprotection , Parkinson Disease/drug therapy , Parkinson Disease/metabolism , Pyridines/pharmacology , Pyrimidines/pharmacology , Signal Transduction
14.
J Steroid Biochem Mol Biol ; 188: 59-70, 2019 04.
Article in English | MEDLINE | ID: mdl-30615932

ABSTRACT

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.


Subject(s)
Androgen Receptor Antagonists/chemistry , Androgen Receptor Antagonists/pharmacology , ortho-Aminobenzoates/chemistry , ortho-Aminobenzoates/pharmacology , Animals , COS Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Chlorocebus aethiops , Halogenation , Humans , Male , Mutation , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolism
15.
Sci Rep ; 8(1): 16525, 2018 11 08.
Article in English | MEDLINE | ID: mdl-30410011

ABSTRACT

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.


Subject(s)
Butyrylcholinesterase/genetics , Environmental Exposure/adverse effects , Parkinson Disease/genetics , Pesticides/adverse effects , Polymorphism, Single Nucleotide , Adult , Aged , Butyrylcholinesterase/blood , Case-Control Studies , Egypt , Female , Gene-Environment Interaction , Genetic Predisposition to Disease , Humans , Male , Middle Aged , Parkinson Disease/etiology , Surveys and Questionnaires
16.
Cell Death Dis ; 9(7): 757, 2018 07 09.
Article in English | MEDLINE | ID: mdl-29988147

ABSTRACT

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.


Subject(s)
Autophagy/physiology , Exosomes/metabolism , alpha-Synuclein/metabolism , Autophagosomes/metabolism , Blotting, Western , Cell Line , Humans , RNA, Small Interfering/metabolism , Real-Time Polymerase Chain Reaction
17.
Neurol Genet ; 3(6): e197, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29147684

ABSTRACT

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 of GCH1 were 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 most SPR mutation 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 rare SPR mutation can cause autosomal dominant DRD with incomplete penetrance. The common DHFR variant might have synergistic effects on production of tetrahydrobiopterin and levodopa, thereby increasing penetrance.

18.
Sci Rep ; 7(1): 11469, 2017 09 13.
Article in English | MEDLINE | ID: mdl-28904388

ABSTRACT

α-synuclein-induced neurotoxicity is a core pathogenic event in neurodegenerative synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy. There is currently no disease-modifying therapy available for these diseases. We screened 1,600 FDA-approved drugs for their efficacy to protect LUHMES cells from degeneration induced by wild-type α-synuclein and identified dipyridamole, a non-selective phosphodiesterase inhibitor, as top hit. Systematic analysis of other phosphodiesterase inhibitors identified a specific phosphodiesterase 1 inhibitor as most potent to rescue from α-synuclein toxicity. Protection was mediated by an increase of cGMP and associated with the reduction of a specific α-synuclein oligomeric species. RNA interference experiments confirmed PDE1A and to a smaller extent PDE1C as molecular targets accounting for the protective efficacy. PDE1 inhibition also rescued dopaminergic neurons from wild-type α-synuclein induced degeneration in the substantia nigra of mice. In conclusion, this work identifies inhibition of PDE1A in particular as promising target for neuroprotective treatment of synucleinopathies.


Subject(s)
Drug Discovery , Enzyme Inhibitors/pharmacology , Phosphodiesterase I/antagonists & inhibitors , Protein Aggregation, Pathological/metabolism , alpha-Synuclein/metabolism , Animals , Cell Line , Dipyridamole/pharmacology , Drug Evaluation, Preclinical , High-Throughput Screening Assays , Humans , Mice , Neurons/drug effects , Neurons/metabolism , Neuroprotective Agents/pharmacology , Protein Aggregation, Pathological/drug therapy , Vinca Alkaloids/pharmacology , alpha-Synuclein/antagonists & inhibitors
19.
EMBO Mol Med ; 9(3): 371-384, 2017 03.
Article in English | MEDLINE | ID: mdl-28148553

ABSTRACT

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.


Subject(s)
Tauopathies/physiopathology , eIF-2 Kinase/metabolism , tau Proteins/metabolism , Animals , Brain/pathology , Cell Survival , Cells, Cultured , Humans , Locomotion , Memory , Mice, Transgenic , Phosphorylation , Protein Conformation , Protein Processing, Post-Translational , Tauopathies/pathology , tau Proteins/chemistry
20.
J Neurochem ; 139(4): 624-639, 2016 11.
Article in English | MEDLINE | ID: mdl-27569447

ABSTRACT

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.


Subject(s)
Annona , Brain/metabolism , Fruit and Vegetable Juices , Plant Extracts/administration & dosage , tau Proteins/biosynthesis , Animals , Annona/adverse effects , Brain/drug effects , Cell Line , Fruit and Vegetable Juices/adverse effects , Humans , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation/drug effects , Phosphorylation/physiology , Plant Extracts/adverse effects , Random Allocation , tau Proteins/genetics
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