Tracking peripheral biomarkers identified from the causal mapping of PD brain.

<p>(<b>a</b>) High confidence biomarkers consistently identified for PD cortex, striatum, and substantia nigra (S. nigra) using microarray analysis. Upregulated biomarkers are shown on the left together with fold changes in the three brain regions, downregulated biomarkers on the right. (<b>b</b>) Functional biomarker panel that combines DEGs, high confidence biomarkers shown in (a) and genes from the causal map. (<b>c</b>) Assessment of biomarkers in brain (top panels)(substantia nigra, SN) and blood (bottom panels) from age-matched control and PD patients using QuantiGene technology from Panomics. * <i>p</i>≤0.05 and ** <i>p</i>≤0.01 as determined using a two-tailed unpaired t-test with Welch's correction.</p

Overlap between pathways significantly enriched with differentially expressed genes identified by microarray and RNA-sequencing.

<p>(<b>a</b>) Overlap of pathways enriched with upregulated genes together with the five most significant overlapping pathways. (<b>b</b>) Overlap of pathways enriched with downregulated genes together with the five most significant overlapping pathways.</p

Identification of pathways dysregulated in PD-brain regions, and the overlap of differentially expressed genes (DEGs) between the PD-affected brain regions.

<p>The 10 key pathways most significantly enriched for DEGs in substantia nigra (<b>a</b>), striatum (<b>b</b>) and cortex (<b>c</b>) of PD brains compared to control as measured by microarray. Enrichments for upregulated genes are shown on the left, for downregulated genes on the right. The numbers of DEGs populating each pathway are denoted in the right columns (#DEGs). (<b>d</b>) Overlap between DEGs in PD cortex and striatum as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right. (<b>e</b>) Overlap between DEGs in striatum and substantia nigra (S.nigra) as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right. (<b>f</b>) Overlap between DEGs in PD cortex and substantia nigra (S. nigra) as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right. (<b>g</b>) Overlap between DEGs in PD cortex, striatum and substantia nigra (S. nigra) as measured by microarrays. The overlap of upregulated genes is shown on the left, the overlap of downregulated genes on the right.</p

Protein alterations in PD brain are dominated by mitochondrial and lipid transport defects, and are largely independent of transcriptional changes.

<p>(<b>a</b>) Overlap between proteins and differentially expressed genes in striatum, as measured by mass spectrometry and microarray technologies. The overlap of upregulated proteins/genes is shown on the left, the overlap of downregulated proteins/genes on the right. (<b>b</b>) Overlap between proteins and differentially expressed genes in PD cortex, as measured by proteomics and microarray technologies. The overlap of upregulated proteins/genes is shown on the left, the overlap of downregulated proteins/genes on the right. (<b>c</b>) Oxidative phosphorylation pathway. The most significantly enriched upregulated pathway for PD cortex based on proteomics data. Red thermometers represent proteins with increased abundance. (<b>d</b>) Regulation of CDK5 in presynaptic signaling. The most significantly enriched downregulated pathway for PD cortex based on proteomics data. Blue thermometers represent proteins with decreased abundance.</p

Overview of brain regions and methodology used in this study.

<p>(<b>a, b</b>) Overview of workflow for functional overview and focused analysis. (<b>a</b>) Expression data for healthy and diseased brain regions are statistically analyzed to obtain differentially expressed genes (DEGs). In the first part, the functional overview, the DEGs are used to identify expression regulators as well as pathways that are significantly enriched with DEGs. (<b>b</b>) In the second part, the focused analysis, pathways that are significantly enriched with expression regulators are combined with pathways that are significantly enriched with DEGs. Combining the two-pathway enrichment results leads to the identification of key pathways, which are the basis for the reconstruction of causal networks. (<b>c</b>) Cartoon representation of different brain regions used in the study, and the associated disease severity of each region denoted by gradations of red. Also shown is connectivity between the substantia nigra, striatum and cortex and the three methods used to interrogate the brain regions (microarray, RNAseq and proteomics).</p

Creation of causal network models for PD brain regions reveals parallel yet distinct dysregulated pathways.

<p>(<b>a</b>) Integrated causal network model for upregulated genes in cortex (1), striatum (2), and substantia nigra (3) based on microarray data. Red thermometers represent upregulated genes. Yellow thermometers correspond to topologically significant genes in cortex (4), striatum (5), and substantia nigra (6). (<b>b)</b> Integrated causal network model for downregulated genes in cortex (1), striatum (2), and substantia nigra (3) based on microarray data. Blue thermometers represent downregulated genes. Yellow thermometers correspond to topologically significant genes in cortex (4), striatum (5), and substantia nigra (6).</p

Elevated Alpha-Synuclein Impairs Innate Immune Cell Function and Provides a Potential Peripheral Biomarker for Parkinson's Disease

<div><p>Alpha-synuclein protein is strongly implicated in the pathogenesis Parkinson's disease. Increased expression of α-synuclein due to genetic multiplication or point mutations leads to early onset disease. While α-synuclein is known to modulate membrane vesicle dynamics, it is not clear if this activity is involved in the pathogenic process or if measurable physiological effects of α-synuclein over-expression or mutation exist <i>in vivo</i>. Macrophages and microglia isolated from BAC α-synuclein transgenic mice, which overexpress α-synuclein under regulation of its own promoter, express α-synuclein and exhibit impaired cytokine release and phagocytosis. These processes were affected <i>in vivo</i> as well, both in peritoneal macrophages and microglia in the CNS. Extending these findings to humans, we found similar results with monocytes and fibroblasts isolated from idiopathic or familial Parkinson's disease patients compared to age-matched controls. In summary, this paper provides 1) a new animal model to measure α-synuclein dysfunction; 2) a cellular system to measure synchronized mobilization of α-synuclein and its functional interactions; 3) observations regarding a potential role for innate immune cell function in the development and progression of Parkinson's disease and other human synucleinopathies; 4) putative peripheral biomarkers to study and track these processes in human subjects. While altered neuronal function is a primary issue in PD, the widespread consequence of abnormal α-synuclein expression in other cell types, including immune cells, could play an important role in the neurodegenerative progression of PD and other synucleinopathies. Moreover, increased α-synuclein and altered phagocytosis may provide a useful biomarker for human PD.</p></div

Alteration in cytokine secretion in BAC transgenic mice.

<p>In all graphs, each dot represents measurements from cells isolated from an individual pup or animal (<b>A</b>) Microglia from line 26 and non TG littermates were stimulated with LPS and TNFα and IL6 measured by ELISA (n = 2; 7 pups/GT/expt +/− s.e.m *P≤0.005 when α-syn TG samples were compared with non-TG). (<b>B</b>) Microglia isolated from three independent human α-syn BAC TG mouse lines, (line 422; 26; and 3) and their corresponding non-TG littermate controls. Cells were stimulated as above and measurements were made for TNFα by ELISA (n = 2; 3 pups/GT/expt +/− s.e.m *P≤0.05 when α-syn TG samples were compared with non-TG). (<b>C</b>) Line 26 TG mice and their non-TG littermates received an injection of low dose LPS for 6 months and serum inflammatory cytokines (TNFα and IL6) were measured by luminex multiplex analysis (n = 1; 7–10 mice/GT/expt +/− s.e.m* P≤0.05 when α-syn TG samples were compared with non-TG). (<b>D</b>) Microglia from line 26/syn ^null or α-syn ^null littermates mice were stimulated with LPS and cytokine expression for IL6 and TNFα assessed at the mRNA level by multiplex analysis (n = 2; 5 pups/GT/expt +/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG). (<b>E+F</b>) Microglia isolated from line 26/syn ^null or α-syn ^null littermates were stimulated with LPS in the presence or absence of Brefeldin A. Tissue culture supernatant (<b>E</b>) or cell lysate (<b>F</b>) was assessed for TNFα production by ELISA (n = 2; 5 pups/GT/expt +/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG).</p

Increased levels of α-syn affect microglial phagocytosis.

<p>(<b>A</b>) Microglia from line 26 TG or non-TG littermates were incubated with 10 µ beads or apoptotic Jurkat T-cells for 90 minutes. A phagocytic index was calculated by microscopic visualization (n = 3; 3 pups/GT/expt +/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG). (<b>B</b>) Microglia from line 26/syn ^null or α-syn ^null littermates were incubated with 10µ beads and a phagocytic index calculated (n = 3; 3 pups/GT/expt +/− s.e.m *p≤0.002 when α-syn TG samples were compared with non-TG). (<b>C</b>) Peritoneal macrophages isolated from line 26 TG or non-TG littermates were cultured with apoptotic Jurkat T-cells and a phagocytic index was calculated (n = 2; 5 pups/GT/expt +/− s.e.m *p≤0.001 when α-syn TG samples were compared with non-TG). (<b>D</b>) Microglia isolated from line 26 TG or non-TG littermates were left unfed or fed beads followed by FM1-43 addition on ice for 10 minutes, fluorescence was assessed by flow cytometry under resting and stimulated (plus bead) conditions, (histogram is representative of 3 independent expts). (<b>E</b>) Geometric mean fluorescence of FM1-43 incorporation in resting and stimulated cell was calculated (n = 3; 3 pups/GT/expt +/− s.e.m *p≤0.001 when FM1-43 incorporation between wild type and line 3 microglia stimulated with beads was compared).</p

Alpha-synuclein drives decreased phagocytosis in α-syn BAC transgenic mice.

<p>(<b>A</b>) Peritoneal macrophages from line 26 mice were cultured in Accell media+/− human α-syn siRNA, or non- targeting siRNA. Human α-syn mRNA and protein levels were assessed by RT-PCR and immunoblot analysis (n = 2; 4 pups/GT/expt +/−s.e.m *p<0.01 when α-syn TG samples treated with α-syn siRNA were compared with α-syn TG or NT siRNA). (<b>B</b>) Following siRNA treatment macrophages were fed 10µ beads and a phagocytic index calculated (n = 2; 4 animals/GT/expt +/− s.e.m *p≤0.001 when the phagocytic index between α-syn TG microglia treated with α-syn siRNA and α-syn TG microglia alone or treated with NT siRNA were compared). (<b>C</b>) H4 cells were transfected with α- or β-syn expression vectors followed by addition of 4 µ beads and a phagocytic index calculated (n = 4 +/− s.e.m *p≤0.001 when the phagocytic index of α-syn transfected H4 cells was compared with vector of β-syn transfected cells). (<b>D</b>) α-syn or vector transfected H4 cells were fed beads followed by FM1-43 labeling and FACS analysis. Data is presented at geometric mean fluorescence (n = 4 +/− s.e.m *p≤0.001 when FM1-43 incorporation was compared between vector treated cells fed beads and cells transfected with α-syn fed beads). (<b>E</b>) H4 cells were transfected with wild type, A53T, A30P, or E46K α-syn expression vectors. After 2 days 4 µ beads were added for and the phagocytic index was measured (n = 4 +/− s.e.m *p≤0.05 when vector transfected cells were compared with cells transfected with wild type or the various familial mutations of α-syn).</p