Levels of striatal dopamine, DOPAC and HVA are unaffected by α-synuclein siRNA infusion.

<p>α-Synuclein siRNA was unilaterally infused into the left substantia nigra. Values are expressed as ng/mg protein. Data are shown as mean ± SEM. LC, lateral caudate; MC, medial caudate; DP, dorsal putamen; VP, ventral putamen.</p

Effect of α-synuclein siRNA on α-synuclein protein in the monkey substantia nigra.

<p>α-Synuclein or luciferase siRNA was unilaterally infused into the left substantia nigra. Midbrain sections were immunostained with an antibody against α-synuclein. Representative images from an animal receiving α-synuclein siRNA show more robust α-synuclein immunoreactivity within the neuropil of the right (untreated, <b>A</b>) <i>vs.</i> left (siRNA-infused, <b>B</b>) substantia nigra. Scale bar = 5 µm. (<b>C</b>) Optical density measurements of nigral α-synuclein immunoreactivity. Data are expressed as percent of the control value in the right (untreated) substantia nigra and represent mean ± SEM. A significant decrease is caused by α-synuclein but not luciferase siRNA in the left (siRNA-infused) hemisphere. *p<0.03.</p

Reduction of α-synuclein mRNA in the substantia nigra infused with α-synuclein siRNA.

<p>Squirrel monkeys received a unilateral nigral infusion of siRNA targeting α-synuclein (<b>A</b>) or luciferase (<b>B</b>). Midbrain sections at the level of the exit of the 3^rd nerve were used for α-synuclein <i>in situ</i> hybridization using digoxigenin-labeled antisense riboprobes. Representative images compare α-synuclein mRNA in the right (untreated) <i>vs.</i> left (siRNA-infused) substantia nigra. Scale bar = 100 µm.</p

The number of nigral dopaminergic neurons is not affected by siRNA-induced α-synuclein suppression.

<p>Squirrel monkeys received a unilateral nigral infusion of siRNA targeting α-synuclein. Both the number of TH-immunoreactive cells and the total number of dopaminergic neurons were counted stereologically in the substantia nigra. Values (mean ± SEM) were not different between the right (untreated) and left (siRNA-infused) hemisphere.</p

Lack of microglial activation following siRNA infusion.

<p>α-Synuclein siRNA was unilaterally infused through a cannula positioned approximately 1 mm dorsal to the substantia nigra. Representative midbrain sections were immunostained for microglial cells using an antibody against ionizing calcium-binding adaptor molecule 1 (Iba-1, brown) and counterstained with cresyl violet (purple). Images are from the right (untreated, <b>A</b> and <b>C</b>) and left (siRNA-infused, <b>B</b> and <b>D</b>) substantia nigra. At higher magnification (<b>C</b> and <b>D</b>), Iba-1-positive cells with morphological features of resting microglia are shown close to dopaminergic neurons containing neuromelanin (black granules). The arrows indicate one of these neurons in each panel. Scale bars = 20 µm (A and B) and 10 µm (C and D). (<b>E</b>) The number of Iba-1-immunoreactive cells was counted in the right (R) and left (L) substantia nigra. Data are shown as mean ± SEM. (<b>F</b>) A representative section from the left midbrain shows Iba-1 immunoreactivity close to the tip of the infusion cannula (arrow) but not within the nearby parenchyma. This robust immunoreactivity was observed within cells with morphological characteristics of activated microglia (inset). Scale bars = 250 µm (panel F) and 10 µm (inset).</p

Treatment of squirrel monkeys with siRNA.

<p>Animals were unilaterally implanted with a cannula connected to an Alzet minipump delivering siRNA into the left substantia nigra. (<b>A</b>) Sequence of the α-synuclein siRNA. “A,C,G,U” indicate ribonucleotides, “T” designates deoxythymidine, “c” and “u” specify 2′-O-Me-modified pyrimidines and “s” denotes a phosphorothioate linkage. (<b>B</b>) Midbrain sections were immunostained for tyrosine hydroxylase (brown) and counterstained with cresyl violet (purple). A representative section shows placement of the cannula approximately 1 mm dorsal to the substantia nigra (SN). The location of the cannula is indicated by the square box, and the asterisk denotes the exit of the third nerve. Scale bar = 800 µm.</p

Age- and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease

<p>Although exact causes of Parkinson disease (PD) remain enigmatic, mitochondrial dysfunction is increasingly appreciated as a key determinant of dopaminergic neuron susceptibility in both familial and sporadic PD. Two genes associated with recessive, early-onset PD encode the ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase PRKN/PARK2/Parkin, which together orchestrate a protective mitochondrial quality control (mitoQC) pathway. Upon stress, both enzymes cooperatively identify and decorate damaged mitochondria with phosphorylated poly-Ub (p-S65-Ub) chains. This specific label is subsequently recognized by autophagy receptors that further facilitate mitochondrial degradation in lysosomes (mitophagy). Here, we analyzed human post-mortem brain specimens and identified distinct pools of p-S65-Ub-positive structures that partially colocalized with markers of mitochondria, autophagy, lysosomes and/or granulovacuolar degeneration bodies. We further quantified levels and distribution of the ‘mitophagy tag’ in 2 large cohorts of brain samples from normal aging and Lewy body disease (LBD) cases using unbiased digital pathology. Somatic p-S65-Ub structures independently increased with age and disease in distinct brain regions and enhanced levels in LBD brain were age- and Braak tangle stage-dependent. Additionally, we observed significant correlations of p-S65-Ub with LBs and neurofibrillary tangle levels in disease. The degree of co-existing p-S65-Ub signals and pathological PD hallmarks increased in the pre-mature stage, but decreased in the late stage of LB or tangle aggregation. Altogether, our study provides further evidence for a potential pathogenic overlap among different forms of PD and suggests that p-S65-Ub can serve as a biomarker for mitochondrial damage in aging and disease.</p> <p><b>Abbreviations:</b> BLBD: brainstem predominant Lewy body disease; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DLB: dementia with Lewy bodies; DLBD: diffuse neocortical Lewy body disease; EOPD: early-onset Parkinson disease; GVB: granulovacuolar degeneration body; LB: Lewy body; LBD: Lewy body disease; mitoQC: mitochondrial quality control; nbM: nucleus basalis of Meynert; PD: Parkinson disease; PDD: Parkinson disease with dementia; p-S65-Ub: PINK1-phosphorylated serine 65 ubiquitin; SN: substantia nigra; TLBD: transitional Lewy body disease; Ub: ubiquitin </p

Forest plot of the meta-analysis of rs7077361 in <i>ITGA8</i>.

<p>Study-specific allelic odds ratios (ORs, black squares) and 95% confidence intervals (CIs, lines) were calculated for each included dataset. The summary OR and CI was calculated using the DerSimonian Laird random-effects model (grey diamond) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002548#pgen.1002548-DerSimonian1" target="_blank">[31]</a>. C = Caucasian ancestry.</p

Manhattan plot of all meta-analysis results performed in PDGene.

<p>This summary combines association results from 7,123,986 random-effects meta-analyses based on the March 31^st 2011 datafreeze of the PDGene database. Results are plotted as −log₁₀<i>P</i>-values (y-axis) against physical chromosomal location (x-axis). Black and grey dots indicate results originating exclusively from the three fully publicly available GWAS datasets <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002548#pgen.1002548-Maraganore2" target="_blank">[10]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002548#pgen.1002548-Pankratz1" target="_blank">[12]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002548#pgen.1002548-SimnSnchez1" target="_blank">[13]</a> (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002548#s4" target="_blank">Methods</a>), while green dots are based on a combination of smaller scale studies, supplemented by GWAS datasets (where applicable). Gene annotations are provided for genes highlighted in the main text.</p

Overview of genome-wide association studies (GWAS) published in PD until March 31, 2011.

<p>The overview is based on content on the PDGene website (<a href="http://www.pdgene.org" target="_blank">http://www.pdgene.org</a>; current on March 31^st, 2011). Studies are listed in order of publication date. ‘# PD GWAS’ and ‘# CTRL GWAS’ refers to sample sizes used in the initial GWAS datasets, whereas ‘Follow-up’ refers to the total number of replication samples where applicable. ‘Featured genes’ are those genes/loci that were declared as ‘associated’ in the original publication; note that criteria for declaring association varies across studies. Genetic loci in bold font denote genes showing genome-wide significant results (<i>P</i><5×10⁻⁸) in the PDGene meta-analyses.</p