DataSheet1.pdf

<p>Alpha-synuclein (non A4 component of amyloid precursor, SNCA, NM_000345.3) plays a central role in the pathogenesis of Parkinson's disease (PD) and related Lewy body disorders such as Parkinson's disease dementia, Lewy body dementia, and multiple system atrophy. Since its discovery as a disease-causing gene in 1997, alpha-synuclein has been a central point of scientific interest both at the protein and gene level. Mutations, including copy number variants, missense mutations, short structural variants, and single nucleotide polymorphisms, can be causative for PD and affect conformational changes of the protein, can contribute to changes in expression of alpha-synuclein and its isoforms, and can influence regulation of temporal as well as spatial levels of alpha-synuclein in different tissues and cell types. A lot of progress has been made to understand both the physiological transcriptional and epigenetic regulation of the alpha-synuclein gene and whether changes in transcriptional regulation could lead to disease and neurodegeneration in PD and related alpha-synucleinopathies. Although the histopathological changes in these neurodegenerative disorders are similar, the temporal and spatial presentation and progression distinguishes them which could be in part due to changes or disruption of transcriptional regulation of alpha-synuclein. In this review, we describe different genetic alterations that contribute to PD and neurodegenerative conditions and review aspects of transcriptional regulation of the alpha-synuclein gene in the context of the development of PD. New technologies, advanced gene engineering and stem cell modeling, are on the horizon to shed further light on a better understanding of gene regulatory processes and exploit them for therapeutic developments.</p

Mitochondrial membrane potential (MMP) and energy balance.

<p><b>A) Fluorescence microscopy</b> of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 µM Rotenone (HG+R) or with 1 µM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 µm). <b>B)</b><b>Plate reader based high throughput screen (HTS) of MMP</b> in live NPCs loaded with 20 µM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD for HG+R: 202/29/194 (xE04), *<i>p</i>≤0.05; for NG: 92/30/118 (xE03) **<i>p</i>≤0.006). <b>C) Plate reader based HTS for MMP loss</b> in live NPCs prepared and analyzed as under B). Fluorescence measurements were acquired as under B) every 5 min for 10 cycles and loss of MMP with time graphed as ΔRFU/min. (n = 8, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: −0.02/−0.06/−0.01, *p≤0.05; HG+R: −0.17/−0.70/−0.22 ***p<0.001, NG: −0.08/−0.33/−0.04, *p≤0.05). <b>D)</b><b>Luminescence plate reader based HTS</b><b>of ATP levels</b> in Ctrl, SNCA-Tri and SNCA-Tri KD NPCs under the above growth conditions (HG, HG+R, NG) assayed by a coupled luciferin/luciferase assay. Depicted are ATP contents in cells treated with 20 µM rotenone (R) for 18 hrs. (n = 8, mean ± SD nMATP/ug protein in: Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 1.66/0.75/1.37, **<i>p</i> = 0.003; NG: 0.69/0.45/0.51, *<i>p</i> = 0.04). <b>E and F) Mitochondrial metabolic activity</b> studied by Seahorse XF24 analysis. <b>E)</b> Oxygen Consumption Rate (OCR) and <b>F)</b> Extracellular Acidification Rate (ECAR). Shown are relative OCR compared to basal values as a function of the sequential addition of mitochondrial inhibitors Oligomycin (1 µM), CCCP (1.5 µM) and Rotenone (Rot, 5 µM) + Antimycin A (Ant, 1 µM). Significant changes compared to basal OCR rates (*p<0.05) and differences between lines treated with and without 6-OHDA (250 µM) for 1 hr are indicated by # (#p<0.05, mean ± SEM, n≥17; from five independent experiments).</p

Mitochondrial integrity, MPT opening, and apoptosis.

<p><b>A) Mitochondrial calcein loading</b> by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl₂ were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 µM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean ± SD, Ctrl/SNCA-Tri: 3.4/4.9, *<i>p</i> = 0.039). <b>B)</b><b>MPT-induced mitochondrial calcein loss</b> in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl₂ were assayed 1 hr. after treatment with 4 µM staurosporine under NG conditions. MPT opening results in entry of CoCl₂ into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 µm). <b>Inserts:</b> Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 µm). <b>C) HCI automated fluorescence microscopy analysis</b> of MPT in NPCs treated with 4 µM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean ± SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *<i>p</i>≤0.01). <b>D)</b><b>Kinetic evaluation of MPT opening</b> and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS analysis. NPCs treated and prepared as under B) were loaded with 4 µM stauropsporine and changes in calcein signal normalized to cell number and mitochondrial content (Δ Norm. RFU) were recorded every 1 min for 20 min (n = 8, mean ± SD, Ctrl/SNCA-Tri, HG: −0.06/−0.12, HG+R: −0.17/−0.28, HG+Iono: −0.03/−0.04, *<i>p</i>≤0.01). <b>E)</b><b>Cytochrome c immuno-cytochemistry</b> in Ctrl and SNCA-tri NPCs challenged with 200 µM paraquat (PQ) 15 min. before fixation. Shown are permeabilized cells probed with cytochrome c antibody, detected by an Alexa-488 nm labeled secondary antibody (green). Cells were counter stained with Hoechst 33342 (blue) (Scale bar: 100 µm, insert: 10 µm). <b>F)</b><b>Immunoblot analysis of cytochrome c levels</b> in sub-cellular fractions containing either cellular organelles (containing bound cytochrome c) or cytosolic proteins (with soluble cytochrome c) from NPC cell lysates (Ctrl and SNCA-Tri) treated with paraquat (PQ) as under E). Cytochrome c (14 kDa) and GAPDH (40 kDa) specific antibodies were detected by a secondary IR-dye conjugate.</p

NPC viability.

<p><b>A) Cell cycle analysis</b> by propidium-iodine (PI) staining and flow cytometry analysis of Ctrl and SNCA-Tri NPCs with staining grouped by cell cycle phase (G0/1, S and G2/M), showing a reduced percentage of SNCA-Tri NPCs in the S phase (n = 3, mean ± SD, *<i>p</i> = 0.047). <b>B)</b><b>Survival under nutritional and toxicant stress.</b> NPCs propagated in medium without glucose (NG) untreated or treated with 20 µM rotenone (R) or 20 µM paraquat (PQ). Survival curves (every 12 hours) for the Ctrl, SNCA-Tri and SNCA-Tri KD cell lines after analysis of adherent cell count (ImageJ). Percentage of surviving cells with time (hrs) (n = 3, mean ± SEM). <b>C)</b><b>Cell viability assayed by plate reader based high throughput screen (HTS)</b> of NPCs untreated (HG), treated with 20 µM rotenone (HG+R) or without glucose (NG) for 18 hrs. Live cells were stained with 1 µM of the RedOx indicator C₁₂-Resazurin/Alamar Blue for 15 min before analysis. Graphed are endpoint fluorescence units (RFU) normalized to total cellular protein/well (ug protein) (n = 3, mean ± SEM, *<i>p</i>≤0.05). <b>D)</b><b>Cell viability assayed by</b> flow cytometry evaluation of apoptosis and cell death in live NPCs treated as under A). Cells stained with C₁₂-Resazurin for cell viability and with Sytox-Green. Graphed are percentages of metabolic active NPCs, determined by Resarufin (Ex./Em. 563/587 nm) fluorescence (viable), apoptotic cells (cell membrane asymmetry detected by an Annexin-V Alexa-660 nm conjugated antibody) (n = 3, mean ± SD, Ctrl/SNCA-Tri: 5.3%/24.4%, *<i>p</i> = 0.027) or cell death (nuclear fragmentation, detected by Sytox-Green, Ex./Em. 488/530 nm) (n = 3, mean ± SD, Ctrl/SNCA-Tri: 5.3%/24.4%, **<i>p</i> = 0.004).</p

Apoptosis sensitivity and caspase activation.

<p><b>A) Caspase 3 activity</b> in cell lysates from adherent NPCs either left untreated or treated with 20 µM rotenone (R) for 18 hrs and then exposed to 1 uM staurosporine for 120 min before analysis. HTS analysis for caspase 3 activity from cell lysates was by activation of the fluorescent caspase substrate 7-amino-4-methylcoumarin (AMC) (Ex./Em. 340/440 nm) (n = 9, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD, HG: 33/69/42, HG+R: 42/129/87, NG: 55/138/85, *p≤0.050, **p≤0.0035; from three independent experiments). <b>B)</b><b>Kinetics of caspase 3/7</b><b>activity</b> in permeabilized NPCs pretreated as described under B) and assayed 15 min after staurosporine treatment. Changes in caspase 3 activity are depicted as ΔµM AMC fluorescence/min + mg cellular protein (detected by Bradford protein assay) (n = 9, mean ± SEM).</p

Protein biosynthesis and proteasome function.

<p><b>A) Mitochondrial protein biosynthesis and protein import.</b> Fluorescent protein expression patterns in confluent adherent NPC cultures (PC: Phase Contrast) transduced with two baculoviral vectors expressing fluorescent proteins targeted to either the peroxisomal (Perox.; Green) or the mitochondrial (Mito.; Red) compartment. Shown are fluorescent protein expression patterns in live confluent Ctrl and SNCA-Tri cell lines grown under normal growth conditions (HG) and evaluated 20 hrs post transduction (Scale bar: 200 µm, 5 µm). <b>B) Time resolved peroxisomal and mitochondrial protein biosynthesis</b>. Fluorescent protein expression patterns as under A), but imaged at 8 and 18 hrs post viral transduction. <b>C)</b><b>Proteasome activity measured by fluorescence microscopy</b> of adherent NPCs cultured with 20 µM rotenone alone or with 10 µM of the proteasome inhibitor MG132. Depicted are fixed cells stained with 5 µM of the aggresome/proteasome specific dye Bodipy TMR-AHX3L3VS (red). Hoechst 33342 was used as nuclear counter stain (blue) (Scale bar: 20 µm). <b>D)</b><b>Proteasome activity measured by flow cytometry</b> evaluation of cells treated and stained as under B). Charted are the aggresome propensity factors (APF) of NPCs calculated from the mean RFU (MRFU) of Bodipy-TMR fluorescence (APF = 100×[MRFU MG132 treated−MRFU untreated]/MRFU MG132 treated (n = 3, mean ± SD, APF Ctrl/SNCA-Tri: 51/120, *<i>p</i> = 0.041).</p

Reactive oxygen species (ROS) production.

<p><b>A) Fluorescence microscopy</b> of live adherent NPCs untreated (HG) or treated with 100 µM TBHP (HG+TBHP), loaded with CM-H₂DCFDA and imaged under controlled exposure conditions (10 sec fluorescent light exposure before image acquisition). Hoechst 33342 was used as counter stain (Scale bar: 20 µm). <b>B) Plate reader based HTS</b> of ROS levels in adherent NPC in 96-well plates and treated as under A). Relative CM-H₂DCFDA fluorescence intensities (RFU) were normalized to Hoechst 33342 (H33342) (n = 12, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.5/1/0.75, HG+R: 0.7/1.3/0.6, NG: 0.4/1.1/0.7, *<i>p</i>≤0.046, **p≤0.009, ***≤0.001). <b>C) ROS production rates</b> by HTS plate reader analysis of CM-H₂DCFDA fluorescence development over time (Δ RFU CM-H₂DCFDA/sec + H33342) in cells exposed to TBHP as under A), measured with normal medium (HG) with or without rotenone (R) and in medium without glucose (NG) (n = 12, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 22/75/68, HG+R: 177/367/178, NG: 80/353/184, *<i>p</i>≤0.010, **<i>p</i>≤0.007, ***<i>p</i>≤0.001). <b>D) Mitochondrial superoxide production</b><b>rates</b> assayed by HTS plate reader analysis of the mitochondrial targeted fluorescent superoxide indicator MitoSOX. Depicted are changes in relative fluorescence units normalized to H33342) (Δ RFU MitoSOX/min + H33342) (n = 4, mean ± SD, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.28/1.2/0.3, HG+R: 2.1/5.5/3.7, NG: 2.3/5.2/0.8,*<i>p</i>≤0.038, **<i>p</i>≤0.007).</p

NPC characterization.

<p><b>A) Phase contrast microscopy</b> of α-synuclein gene triplication (SNCA-Tri), control (Ctrl) and α-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 µm) shows normal cell morphology. <b>B)</b><b>Mitochondrial and nuclear morphology</b> of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 µm). <b>C)</b><b>Stem cell marker expression</b>. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 µm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by a secondary Alexa-488 conjugated antibody (green) (Scale bar: 20 µm). Nuclear counter stain by Hoechst 33342 (blue). <b>D) Representative α-synuclein protein expression</b> patterns (left) by immunoblot of protein lysates from a control line (Ctrl), the SNCA-Tri NPC line and the corresponding α-synuclein knock down line (SNCA-Tri KD) with β-actin serving as loading control. Right: Quantification of β-actin normalized α-synuclein expression levels (n = 4, mean ± SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: 12.4/5.9/8.3, ***<i>p≤</i>0.001, t-test; from two independent experiments). <b>E) ICC of α-synuclein protein expression in adherent NPCs</b> detected by a polyclonal α-syn antibody and visualized by Alexa-488 conjugated secondary antibody (green). DAPI nuclear counterstain (blue); (Scale bar 20 µm). Insert: Higher magnification image (Scale bar: 10 µm). <b>F)</b><b>Colocalization of subcellular</b><b>α-synuclein distribution with mitochondria</b> in adherent NPCs labeled with Mitotracker Red CMX Ros (red) and probed for α-syn as under E) (Scale bar: 5 µm).</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

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