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Duplication/triplication mutations of the SNCA locus, encoding alpha-synuclein (ASYN), and loss of function mutations in Nurr1, a nuclear receptor guiding midbrain dopaminergic neuron development, are associated with familial Parkinson's disease (PD). As we age, the expression levels of these two genes in midbrain dopaminergic neurons follow opposite directions and ASYN expression increases while the expression of Nurr1 decreases. We investigated the effect of ASYN and Nurr1 age-related expression alterations in the pathogenesis of PD by coupling Nurr1 hemizygous with ASYN(s) (heterozygote) or ASYN(d) (homozygote) transgenic mice. ASYN(d)/Nurr1+/- (2-hit) mice, contrary to the individual genetic traits, developed phenotypes consistent with dopaminergic dysfunction. Aging '2-hit' mice manifested kyphosis, severe rigid paralysis, L-DOPA responsive movement impairment and cachexia and died prematurely. Pathological abnormalities of phenotypic mice included SN neuron degeneration, extensive neuroinflammation and enhanced ASYN aggregation. Mice with two wt Nurr1 alleles [ASYN(d)/Nurr1+/+] or with reduced ASYN load [ASYN(s)/Nurr1+/-] did not develop the phenotype or pathology. Critically, we found that aging ASYN(d), in contrast to ASYN(s), mice suppress Nurr1-protein levels in a brain region-specific manner, which in addition to Nurr1 hemizygosity is necessary to instigate PD pathogenesis. Our experiments demonstrate that ASYN-dependent PD-related pathophysiology is mediated at least in part by Nurr1 down-regulation. © The Author(s) 2021. Published by Oxford University Press.

Background: New noninvasive and affordable molecular approaches that will complement current practices and increase the accuracy of Parkinson's disease (PD) diagnosis are urgently needed. Circular RNAs (circRNAs) are stable noncoding RNAs that accumulate with aging in neurons and are increasingly shown to regulate all aspects of neuronal development and function. Objectives: Τhe aims of this study were to identify differentially expressed circRNAs in blood mononuclear cells of patients with idiopathic PD and explore the competing endogenous RNA networks affected. Methods: Eighty-seven circRNAs were initially selected based on relatively high gene expression in the human brain. More than half of these were readily detectable in blood mononuclear cells using real-time reverse transcription-polymerase chain reaction. Comparative expression analysis was then performed in blood mononuclear cells from 60 control subjects and 60 idiopathic subjects with PD. Results: Six circRNAs were significantly down-regulated in patients with PD. The classifier that best distinguished PD consisted of four circRNAs with an area under the curve of 0.84. Cross-linking immunoprecipitation-sequencing data revealed that the RNA-binding proteins bound by most of the deregulated circRNAs include the neurodegeneration-associated FUS, TDP43, FMR1, and ATXN2. MicroRNAs predicted to be sequestered by most deregulated circRNAs have the Gene Ontology categories “protein modification” and “transcription factor activity” mostly enriched. Conclusions: This is the first study that identifies specific circRNAs that may serve as diagnostic biomarkers for PD. Because they are highly expressed in the brain and are derived from genes with essential brain functions, they may also hint on the PD pathways affected. © 2021 Biomedical Research Foundation, Academy of Athens. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. © 2021 Biomedical Research Foundation, Academy of Athens. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Background: The SNCA gene encoding α-synuclein (αSyn) is the first gene identified to cause autosomal-dominant Parkinsonʼs disease (PD). Objective: We report the identification of a novel heterozygous A30G mutation of the SNCA gene in familial PD and describe clinical features of affected patients, genetic findings, and functional consequences. Methods: Whole exome sequencing was performed in the discovery family proband. Restriction digestion with Bbvl was used to screen SNCA A30G in two validation cohorts. The Greek cohort included 177 familial PD probands, 109 sporadic PD cases, and 377 neurologically healthy controls. The German cohort included 136 familial PD probands, 380 sporadic PD cases, and 116 neurologically healthy controls. We also conducted haplotype analysis using 13 common single nucleotide variants around A30G to determine the possibility of a founder effect for A30G. We then used biophysical methods to characterize A30G αSyn. Results: We identified a novel SNCA A30G (GRCh37, Chr4:90756730, c.89 C>G) mutation that co-segregated with the disease in five affected individuals of three Greek families and was absent from controls. A founder effect was strongly suggested by haplotype analysis. The A30G mutation had a local effect on the intrinsically disordered structure of αSyn, slightly perturbed membrane binding, and promoted fibril formation. Conclusion: Based on the identification of A30G co-segregating with the disease in three families, the absence of the mutation in controls and population databases, and the observed functional effects, we propose SNCA A30G as a novel causative mutation for familial PD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

BackgroundNew noninvasive and affordable molecular approaches that will complement current practices and increase the accuracy of PD diagnosis are urgently needed. CircRNAs are highly stable non-coding RNAs that accumulate with aging in neurons and are increasingly shown to regulate all aspects of neuronal development and function.ObjectivesThe aims of the present study were to identify differentially expressed circRNAs in PBMCs of idiopathic PD patients and explore the competing endogenous RNA networks affected.MethodsEighty-seven circRNAs were initially selected based on relatively high gene expression in the human brain. Over half of these were readily detectable in PBMCs using RT-qPCR. Comparative expression analysis was then performed in PBMCs from sixty controls and sixty idiopathic subjects with PD.ResultsSix circRNAs derived from MAPK9, HOMER1, SLAIN1, DOP1B, REPS1, and PSEN1 transcripts were significantly downregulated in PD patients. The classifier that best distinguished PD consisted of four circRNAs with an AUC of 0.84. CLIP-Seq data revealed that the RNA binding proteins bound by most of the deregulated circRNAs include the neurodegeneration-associated FUS, TDP43, FMR1 and ATXN2. MicroRNAs predicted to be sequestered by most deregulated circRNAs had the GOslim categories ‘Protein modification’, ‘Transcription factor activity’ and ‘Cytoskeletal protein binding’ mostly enriched.ConclusionsThis is the first study that identifies circRNAs deregulated in the peripheral blood of PD patients. They may serve as diagnostic biomarkers and since they are highly expressed in the brain and are derived from genes with essential brain functions, they may also hint on the PD pathways affected.

Background: A minimally invasive test for early detection and monitoring of Parkinson's disease (PD) is a highly unmet need for drug development and planning of patient care. Blood plasma represents an attractive source of biomarkers. MicroRNAs (miRNAs) are conserved noncoding RNA molecules that serve as posttranscriptional regulators of gene expression. As opposed to ubiquitously expressed miRNAs that control house-keeping processes, brain-enriched miRNAs regulate diverse aspects of neuron development and function. These include neuron-subtype specification, axonal growth, dendritic morphogenesis, and spine density. Backed by a large number of studies, we now know that the differential expression of neuron-enriched miRNAs leads to brain dysfunction. Objectives: The aim was to identify subsets of brain-enriched miRNAs with diagnostic potential for familial and idiopathic PD as well as specify the molecular pathways deregulated in PD. Methods: Initially, brain-enriched miRNAs were selected based on literature review and validation studies in human tissues. Subsequently, real-time reverse transcription polymerase chain reaction was performed in the plasma of 100 healthy controls and 99 idiopathic and 53 genetic (26 alpha-synucleinA53T and 27 glucocerebrosidase) patients. Statistical and bioinformatics analyses were carried out to pinpoint the diagnostic biomarkers and deregulated pathways, respectively. Results: An explicit molecular fingerprint for each of the 3 PD cohorts was generated. Although the idiopathic PD fingerprint was different from that of genetic PD, the molecular pathways deregulated converged between all PD subtypes. Conclusions: The study provides a group of brain-enriched miRNAs that may be used for the detection and differentiation of PD subtypes. It has also identified the molecular pathways deregulated in PD. © 2019 International Parkinson and Movement Disorder Society. © 2019 International Parkinson and Movement Disorder Society

Introduction: The aim of this study is to investigate the association between environmental factors (smoking, coffee, pesticide exposure) and Parkinson's disease (PD) subtypes (early-onset, mid-and-late onset, familial and sporadic) in the Greek population. Methods: The Hellenic Biobank of PD recorded information of PD cases and controls from two centers in Greece during 2006–2017. Patients with the A53T mutation in SNCA or GBA mutations were excluded. Associations of environmental factors with PD overall (and PD subtypes) versus controls were explored with logistic regression models adjusting for age, gender and each environmental factor. Results: 686 patients and 356 controls were included. Smoking was associated with a reduced risk of PD overall (OR 0.48, 95% CI 0.35–0.67), mid-and-late onset (0.46, 0.32–0.66), familial (0.53, 0.34–0.83) and sporadic (0.46, 0.32–0.65), but not early-onset PD. There was an inverse linear association with pack-years of smoking, except for early-onset PD. Early-onset PD was the only PD subtype inversely associated with coffee consumption when dichotomously treated. Compared to never-coffee drinkers, only those at the upper tertile had lower odds for PD overall (0.52, 0.29–0.91), early-onset (0.16, 0.05–0.53) and familial PD (0.36, 0.17–0.75). No associations were found between pesticides and PD. Conclusions: Our study shows that the well-known negative association of smoking with PD occurs across all PD subtypes in the Greek population, apart from early-onset PD. Early-onset PD was also most strongly inversely associated with coffee consumption, highlighting a potential distinct underlying physiopathology in this PD subset that may involve specific gene-environment interactions. © 2019 Elsevier Ltd

Background: Alpha-synuclein aggregation is considered one of the main causes of Parkinson's Disease (PD). Malfunction of autophagy-lysosomal pathways is believed to be an underlying mechanism of α-synuclein aggregation. Although such malfunction has been observed in PD brains, it is unclear whether it may also occur in extraneuronal tissues. Objectives: To assess lysosome-mediated protein degradation in cultured Peripheral Blood Mononuclear Cells (PBMCs) of PD patients and healthy controls. Methods: Total protein degradation in cultured PBMCs was measured by labelling the cells with 3 H-leucine using pulse-chase experiments. Different inhibitors were used to measure a range of autophagic pathways. Results: Protein degradation through the main autophagic pathways is reduced in PD patients (n = 18) compared to age- and sex-matched healthy controls (n = 18), (macroautophagy, p =.018; Chaperone-Mediated autophagy, p =.04; and total lysosomal function, p =.007). Conclusions: Lysosomal dysfunction is present in cultured PBMCs of PD patients, suggesting that it may reflect a systemic feature of the disease. © 2019 Elsevier B.V.

Background: Variations of α-synuclein levels or species have been reported in Parkinson's Disease (PD). There has been little systematic examination of erythrocytes, a rich source of α-synuclein. Methods: Erythrocyte membranes were obtained from PD patients (mutation carriers in the α-synuclein gene (A53T-PD) and glucocerebrosidase gene (GBA-PD) (n = 18 each), and patients without known mutations (GU-PD, n = 56)), and age-/sex-matched controls (n = 56). Levels of monomeric and dimeric α-synuclein were assessed using Western immunoblotting. Results: A statistically significant increase of α-synuclein dimer and dimer to monomer ratio was found in GBA-PD and GU-PD. In contrast, dimer levels of A53T-PD were not different from controls. No difference was found in α-synuclein monomer levels. Conclusions: The increased α-synuclein dimer in GBA-PD and GU-PD is suggestive of an apparent systemic dysfunction causing the dimerization, and potentially oligomerization, of α-synuclein. These results may have implications for PD pathogenesis and biomarker development. © 2017 Elsevier B.V.

Subacute-acute, hyperacute, or even catastrophic and fulminant respiratory events occur in almost all classic connective tissue disorders (CTDs); they may share systemic life-threatening manifestations, may precipitously lead to respiratory failure requiring ventilatory support as well as a combination of specific therapeutic measures, and in most affected patients constitute the devastating end-of-life event. In CTDs, acute respiratory events may be related to any respiratory compartment including the airways, lung parenchyma, alveolar capillaries, lung vessels, pleura, and ventilatory muscles. Acute respiratory events may also precipitate disease-specific extrapulmonary organ involvement such as aspiration pneumonia and lead to digestive tract involvement and heart-related respiratory events. Finally, antirheumatic drug-related acute respiratory toxicity as well as lung infections related to the rheumatic disease and/or to immunosuppression complete the spectrum of acute respiratory events. Overall, in CTDs the lungs significantly contribute to morbidity and mortality, since they constitute a common site of disease involvement; a major site of infections related to the 'mater' disease; a major site of drug-related toxicity, and a common site of treatment-related infectious complications. The extreme spectrum of the abovementioned events, as well as the 'vicious' coexistence of most of the aforementioned manifestations, requires skills, specific diagnostic and therapeutic means, and most of all a multidisciplinary approach of adequately prepared and expert scientists. Avoiding lung disease might represent a major concern for future advancements in the treatment of autoimmune disorders. © 2016 S. Karger AG, Basel.

Lung involvement in connective tissue disorders (CTDs) may present as pleomorphic since any lung compartment may be involved such as airways, exocrine secretory and alveolar epithelia, interstitial lung structure, pulmonary vasculature and pleura as well as, in specific disorders, several tissues of the thoracoabdominal ventilator pump. Any combination of the above anatomic structures may be involved concomitantly although some specific combinations may include a determinant of rheumatic disorders. The diagnosis of a specific CTD requires the fulfilment of clearly defined clinical and laboratory criteria including in most cases positivity for autoantibodies, mostly specific serologic combinations. In this setting, serologic investigation targets mainly, although not exclusively, the detection of antinuclear antibodies. A specific serologic positivity or a combination of autoantibodies constitutes not only a diagnostic criterion for a specific CTD, but may also characterize the pattern of respiratory manifestation in a determinant rheumatic disorder. Therefore, the investigation of lung involvement in CTDs requires adequate skills in the ambit of a multidisciplinary approach and an extended spectrum of diagnostic tools and modalities able to detect both early clinical clues and serologic conversion as well as any pathophysiologic alteration that regards the complexity of respiratory functional status. Although many patients with CTDs suffer from a 'vicious' combination of lung involvement, lung drug toxicity and infections related to the above two as well as to the 'mater' disease, for space reasons this review will focus on the established lung manifestations that regard the 7 major CTDs. © 2015 S. Karger AG, Basel.

Background and purpose Although the first mutation associated with Parkinson’s disease (PD) was identified several years ago in the alpha-synuclein (SNCA) gene in families of Greek and Italian ancestry, a more systematic study of this and other known PD mutations has not been performed in the Greek population. Methods A genetic analysis in 111 familial or sporadic with early-onset (50years, EO) PD patients was performed for the presence of the A53T SNCA mutation. In separate subgroups of these patients, further mutations in the SNCA, LRRK2, Parkin, PINK1 and DJ-1 genes were searched for. Additionally, a subgroup of familial cases was analysed for mutations in the glucocerebrosidase (GBA) gene. Results In total, five patients (4.5% of our whole population) were identified with the A53T SNCA mutation, two with a heterozygote dosage mutation and one with a heterozygote point mutation in the Parkin gene, and seven patients (10.3% of our familial cohort) with GBA gene mutations. Conclusions The A53T mutation in the SNCA gene, although uncommon, does represent a cause of PD in the Greek population, especially of familial EOPD with autosomal dominant inheritance. GBA mutations in the familial cohort tested here were as common as in a cohort of sporadic cases previously examined from the same centres. For the remainder of the genes, genetic defects that could definitively account for the disease were not identified. These results suggest that further Mendelian traits that lead to PD in the Greek population remain to be identified.

Genetic studies have implicated the neuronal ubiquitin C-terminal hydrolase (UCH) protein UCH-L1 in Parkinson's disease (PD) pathogenesis. Moreover, the function of UCH-L1 may be lost in the brains of PD and Alzheimer's disease patients. We have previously reported that the UCH-L1 polymorphic variant S18Y, potentially protective against PD in population studies, demonstrates specific antioxidant functions in cell culture. Albeit genetic, biochemical and neuropathological data support an association between UCH-L1, PD, synaptic degeneration and oxidative stress, the relationship between the dopaminergic system and UCH-L1 status remains obscure. In the current study, we have examined the dopaminergic system of mice lacking endogenous UCH-L1 protein (gracile axonal dystrophy mice). Our findings show that the lack of wild-type (WT) UCH-L1 does not influence to any significant degree the dopaminergic system at baseline or following injections of the neurotoxin methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, using a novel intrastriatal adenoviral injection protocol, we have found that mouse nigral neurons retrogradely transduced with S18Y UCH-L1, but not the WT protein, are significantly protected against MPTP toxicity. Overall, these data provide evidence for an antioxidant and neuroprotective effect of the S18Y variant of UCH-L1, but not of the WT protein, in the dopaminergic system, and may have implications for the pathogenesis of PD or related neurodegenerative conditions, in which oxidative stress might play a role. © The Author 2011. Published by Oxford University Press. All rights reserved.

Objectives: To investigate the resistance mechanisms of meropenem-resistant, ceftazidime-susceptible Pseudomonas aeruginosa isolates, in a clinical setting where VIM-2 or VIM-4 metallo-β-lactamase (MBL)-producing pseudomonads are common. Methods: During May to December 2003, 13 consecutive meropenem-resistant, ceftazidime-susceptible P. aeruginosa isolates were recovered from separate patients at the University Hospital of Larissa, Thessaly, Greece. The isolates were studied by Etest MBL, PCR for blaVIM, blaIMP and blaSPM genes and PFGE. Experiments were performed to detect synergy between meropenem or other antimicrobials and the efflux pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP). The isolates were also tested by PCR and RT-PCR for the expression of the genes mexB and mexY, which encode the efflux pumps MexAB-OprM and MexXY-OprM. Results: Twelve of the isolates, belonging to six distinct PFGE types, gave negative results in the MBL Etest and lacked genes encoding MBLs but exhibited synergy between meropenem and CCCP, indicating that efflux pump activity contributed to the meropenem resistance. All 12 isolates were positive for mexB and 11 were also positive for mexY genes. RT-PCR showed that 10 and five isolates over-expressed mexB and mexY, respectively. One isolate was blaVIM-2-positive and did not show synergy with CCCP, or harbour mexB or mexY. Conclusions: In our hospital, where MBL-producing P. aeruginosa were previously prevalent, meropenem resistance due to the overexpression of efflux pumps has also now emerged. Early recognition of this resistance mechanism should allow the use of alternative β-lactams, such as ceftazidime, which would be inactive even against phenotypically susceptible MBL producers. © The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

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Objectives: The possible contribution of metallo-β-lactamases in the frequent detection of carbapenem-resistant Pseudomonas aeruginosa isolates in a tertiary Greek hospital in Central Greece was investigated. Materials and methods: All carbapenem-resistant (imipenem- and/or meropenem-resistant) P. aeruginosa isolates recovered from separate patients during a 1 year period in the Clinical Microbiology Laboratory at the University Hospital of Thessaly, Larissa, Greece, were studied for metallo-β-lactamases. They were tested by Etest MBL, PCR analysis and nucleotide sequencing. DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE) of Xbaldigested chromosomal DNA. Results: A blaVIM gene was detected in 47 of the 53 (88.7%) carbapenem-resistant P. aeruginosa isolates. PFGE grouped the blaVIM-positive isolates in six unrelated genotypes; one type included two subtypes. Nucleotide sequencing of the PCR amplicons of a randomly selected isolate from each one of the seven subtypes, detected the variant sequences blaVIM-2 in four and blaVIM-4 in three cases, respectively. They were carried as single gene cassettes or along with an aminoglycoside resistance gene (aacA29a) in class 1 integrons. Conclusions: These findings suggest that different strains of P. aeruginosa carrying unrelated metallo-β-lactamase gene variants predominate in our hospital environment.

A Pseudomonas aeruginosa isolate highly resistant to carbapenems was collected from a patient with postsurgical cerebrospinal infection in Greece. The isolate carried a class 1 integron that contained as a sole cassette the gene blaVIM-4, a novel variant of blaVIM-1, with one nucleotide difference resulting in a Ser-to-Arg change at amino acid position 175 of the VIM-1 enzyme. This is the first detection of a VIM-1 variant after its appearance in Italy.