GSK3β phosphorylation is reduced in PD fibroblasts.

<p>(A) Immunoblot and densitometric analyses of (B) total and (C) phosphorylated glycogen synthase kinase 3 beta (GSK3β), p38 MAP Kinase (p38) and p44/42 MAPK (Erk) were performed in whole cell extracts from human fibroblasts deriving from control (CONT, white bars, N = 3), mutated <i>parkin</i> (PARK, dark grey bars, N = 3), mutated <i>LRRK2</i> (LRRK2, light grey bars, N = 3) and idiopathic PD (PD, black bars, N = 3). For the quantitation, values of total protein were normalized on the level of GAPDH of the relative sample, whereas the levels of phosphorylated form were normalized on the values of total protein. All values are expressed as mean ± SEM. *<i>p</i><0.05 and **<i>p</i><0.02 vs control, #<i>p</i><0.05 vs PD according to ANOVA, Tukey HSD <i>post hoc</i> test.</p

Genetic manipulation restores MT stability and rescues fibroblast phenotype.

<p>(A) Representative immunoblot of Triton X-100-soluble (free α-tubulin, S) and -insoluble fraction (α-tubulin incorporated into MTs, I) of fibroblasts collected from patients with parkin mutations (PARK) transfected with control plasmid (VEC) or WT parkin (WT), and of control fibroblasts (CONT) transfected with short hairpin RNA, sh-183 (183) or control shRNA (VEC). (B-D) Morphometric analyses of patients fibroblasts expressing control plasmid (PARK-VEC, N = 4) or WT parkin (PARK-WT, N = 4), and control fibroblasts transfected with control shRNA (CONT-VEC, N = 4) or silenced with sh-183 (CONT-183, N = 4), showing the ratio between maximum and minimum axes (B), the area (C) and the number of overlapping regions between cells (D). ns = not significant, **<i>p</i><0.02 and ***<i>p</i><0.005 according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM. (E) Representative immunoblot of Triton X-100-soluble (free α-tubulin, S) and -insoluble fraction (α-tubulin incorporated into MTs, I) of fibroblasts collected from patients with LRRK2 mutations (LRRK2) transfected with control plasmid (VEC) or WT LRRK2 (WT), and of control fibroblasts (CONT) expressing control plasmid (VEC) or G2019S mutant LRRK2 (MUT). Morphometric analyses of patients fibroblasts expressing control plasmid (LRRK2-VEC, N = 3) or WT LRRK2 (LRRK2-WT, N = 3), or of control fibroblasts transfected with control plasmid (CONT-VEC, N = 3) or G2019S mutant LRRK2 (CONT-MUT, N = 3), showing the ratio between maximum and minimum axes (F), the area (G) and the number of overlapping regions between cells (H). ns = not significant, *<i>p</i><0.05, **<i>p</i><0.02 and ***<i>p</i><0.005 according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM.</p

PD fibroblasts show subtle cytoskeleton differences.

<p>(A) Immunoblot and (B) densitometric analyses of vimentin (Vim), α-tubulin (α-Tub), β-tubulin (β-Tub) and actin (Actin) were performed in whole cell extracts from human fibroblasts deriving from control (CONT, white bars, N = 10), mutated <i>parkin</i> (PARK, dark grey bars, N = 6), mutated <i>LRRK2</i> (LRRK2, light grey bars, N = 6) and idiopathic PD (PD, black bars, N = 3). For the quantitation, values of each protein were normalized on the level of GAPDH of the relative sample. All values are expressed as mean ± SEM. *<i>p</i><0.05 and ***<i>p</i><0.005 vs control, ##<i>p</i><0.02 vs PD, according to ANOVA, Tukey HSD <i>post hoc</i> test. (C) Cultured human fibroblasts were stained with anti-vimentin and anti-α-tubulin primary antibodies or with TRITC-conjugated phalloidin to reveal the organization of intermediate filaments (Vim, top), microtubules (α-Tub, middle) and actin fibers (Actin, bottom), respectively. Concurrent nuclear staining was made by using DAPI (Blue). Scale bar: 20 µm.</p

Morphological alterations characterize PD fibroblasts.

<p>(A) Representative phase contrast micrographs of cultured human fibroblasts of healthy and PD affected people. Scale bar: 25 µm. Morphometric analysis showed reduced ratio between maximum and minimum axes in parkinsonian fibroblast (B) and increased area in the presence of mutated parkin or LRRK2 (C). (D) Histogram showing the increased number of overlapping regions between cells in patient fibroblasts. *<i>p</i><0.05 and ***<i>p</i><0.005 vs control according to ANOVA, Tukey HSD <i>post hoc</i> test. All values are expressed as mean ± SEM. CONT = control (N = 10); PARK = patients with mutations of <i>parkin</i> (N = 6); LRRK2 = patients carrying mutations in <i>LRRK2</i> (N = 6); PD = idiopathic Parkinson’s disease patients (N = 3).</p

Data_Sheet_1_TARDBP mutations in a cohort of Italian patients with Parkinson’s disease and atypical parkinsonisms.docx

BackgroundAggregates of TAR DNA-binding protein of 43 kDa (TDP-43) represent the pathological hallmark of most amyotrophic lateral sclerosis (ALS) and of nearly 50% of frontotemporal dementia (FTD) cases but were also observed to occur as secondary neuropathology in the nervous tissue of patients with different neurodegenerative diseases, including Parkinson’s disease (PD) and atypical parkinsonism. Mutations of TARDBP gene, mainly in exon 6 hotspot, have been reported to be causative of some forms of ALS and FTD, with clinical signs of parkinsonism observed in few mutation carriers.MethodsDirect DNA sequencing of TARDBP exon 6 was performed in a large Italian cohort of 735 patients affected by PD (354 familial and 381 sporadic) and 142 affected by atypical parkinsonism, including 39 corticobasal syndrome (CBS) and 103 progressive sopranuclear palsy (PSP). Sequencing data from 1710 healthy, ethnically matched controls were already available.ResultsFour TARDBP missense variants (p.N267S, p. G294A, p.G295S, p.S393L) were identified in four patients with typical PD and in two individuals with atypical parkinsonism (1 CBS and 1 PSP). None of the detected mutations were found in healthy controls and only the variant p.N267S was previously described in association to idiopathic familial and sporadic PD and to CBS.ConclusionIn this study we provide further insight into the clinical phenotypic heterogeneity associated with TARDBP mutations, which expands beyond the classical ALS and FTD diseases to include also PD and atypical parkinsonism, although with a low mutational frequency, varying considerably in different Caucasian populations. In addition, our study extends the spectrum of TARDBP pathogenetic mutations found in familial and sporadic PD.</p

Image_1.pdf

<p>Mutations in leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson’s disease (PD). LRRK2 is a complex protein that consists of multiple domains, including 13 putative armadillo-type repeats at the N-terminus. In this study, we analyzed the functional and molecular consequences of a novel variant, E193K, identified in an Italian family. E193K substitution does not influence LRRK2 kinase activity. Instead it affects LRRK2 biochemical properties, such as phosphorylation at Ser935 and affinity for 14-3-3ε. Primary fibroblasts obtained from an E193K carrier demonstrated increased cellular toxicity and abnormal mitochondrial fission upon 1-methyl-4-phenylpyridinium treatment. We found that E193K alters LRRK2 binding to DRP1, a crucial mediator of mitochondrial fission. Our data support a role for LRRK2 as a scaffolding protein influencing mitochondrial fission.</p

data_sheet_1.DOC

Background<p>Alpha-synuclein is a constituent of Lewy bodies and mutations of its gene cause familial Parkinson’s disease (PD). A previous study showed that a variant of the alpha-synuclein gene (SNCA), namely the 263 bp allele of Rep1 was associated with faster motor progression in PD. On the contrary, a recent report failed to detect a detrimental effect of Rep1 263 on both motor and cognitive outcomes in PD. Aim of this study was to evaluate the influence of the Rep1 variants on disease progression in PD patients.</p>Methods<p>We recruited and genotyped for SNCA Rep1 426 PD patients with age at onset ≥40 years and disease duration ≥4 years. We then analyzed frequency and time of occurrence of wearing-off, dyskinesia, freezing of gait, visual hallucinations, and dementia using a multivariate Cox’s proportional hazards regression model.</p>Results<p>SNCA Rep1 263 carriers showed significantly increased risk of both dementia (HR = 3.03) and visual hallucinations (HR = 2.69) compared to 263 non-carriers. Risk of motor complications did not differ in the two groups.</p>Conclusion<p>SNCA Rep1 263 allele is associated with a worse cognitive outcome in PD.</p

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Background<p>Alpha-synuclein is a constituent of Lewy bodies and mutations of its gene cause familial Parkinson’s disease (PD). A previous study showed that a variant of the alpha-synuclein gene (SNCA), namely the 263 bp allele of Rep1 was associated with faster motor progression in PD. On the contrary, a recent report failed to detect a detrimental effect of Rep1 263 on both motor and cognitive outcomes in PD. Aim of this study was to evaluate the influence of the Rep1 variants on disease progression in PD patients.</p>Methods<p>We recruited and genotyped for SNCA Rep1 426 PD patients with age at onset ≥40 years and disease duration ≥4 years. We then analyzed frequency and time of occurrence of wearing-off, dyskinesia, freezing of gait, visual hallucinations, and dementia using a multivariate Cox’s proportional hazards regression model.</p>Results<p>SNCA Rep1 263 carriers showed significantly increased risk of both dementia (HR = 3.03) and visual hallucinations (HR = 2.69) compared to 263 non-carriers. Risk of motor complications did not differ in the two groups.</p>Conclusion<p>SNCA Rep1 263 allele is associated with a worse cognitive outcome in PD.</p