SIRT2 regulates aSyn aggregation and toxicity.

<p><b>(A)</b> H4 cells were infected with lentiviruses encoding shRNAs against SIRT2 (T2.KD) or scramble shRNA (Ctrl) and selected with puromycin. Cells were then cotransfected with SynT and synphilin-1 (Synph1). SIRT2, synphilin-1, aSyn, and GAPDH levels were assessed by immunoblot analyses. <b>(B)</b> Ctrl and T2.KD cells transiently expressing SynT and synphilin-1 for 48 h were processed for immunocytochemistry (ICC) (aSyn, green). Data show percentage of cells with aSyn inclusions (<i>n</i> = 3). Scale bar 15 μm. <b>(C)</b> Triton X-100 insoluble and total fractions of cells as in (B) probed for aSyn and GAPDH. <b>(D)</b> Native protein extracts from H4 cells as in (B) were separated on a sucrose gradient. Fractions were immunoblotted and probed for aSyn. <b>(E)</b> Anti-aSyn IP from cells as in (B). Fractions were immunoblotted and probed for acetyl-lysine and aSyn. <b>(F)</b> Toxicity of Ctrl and T2.KD measured by lactate dehydrogenase (LDH) release assay (<i>n</i> = 3). Data in all panels are average ± SD, ** <i>p</i> < 0.01, **** <i>p</i> < 0.0001. For (B) and (F), unpaired, two-tailed <i>t</i> test with equal SD. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p

SIRT2 interacts with aSyn and deacetylates lysine 6 and 10.

<p><b>(A)</b> Detection of aSyn acetylation on lysine 6 (K6) and lysine 10 (K10) by peptide mass fingerprinting analysis in total mouse brain lysates. Spectrum shows acetylated peptides in red and aSyn peptides in blue. The corresponding peptide sequences are shown (red residues are acetylated and green residues are oxidated). <b>(B)</b> Human HEK293T cells expressing the indicated proteins were lysed and immunoprecipitated (IP) with anti-aSyn (left panels) or anti-SIRT2 polyclonal antibodies (right panel). The whole-cell lysates (WCL) and immunoprecipitation samples were probed for GFP (left panels) or aSyn (right panels). <b>(C)</b> Mouse whole-brain lysates (WBL) were lysed and IP with anti-aSyn polyclonal antibody. The IP sample was probed with anti-SIRT2 and anti-aSyn. Rabbit IgG was used as a negative control for the IP sample. <b>(D)</b> Immunoprecipitations were probed with an anti–acetyl-lysine antibody in cells expressing either SIRT2 or the SIRT2-H187Y inactive mutant. <b>(E)</b> Immunoblot of thermoenriched aSyn from mouse-brain lysate probed for acetyl-lysine and aSyn. <b>(F)</b> Brain protein extracts from WT and SIRT2 knockout (T2.KO) mice were separated by SDS-PAGE and immunoblotted with antibodies against acetyl-lysine and aSyn (<i>n</i> = 3). The ratio of acetyl-lysine to aSyn is presented. *<i>p</i> < 0.05, unpaired <i>t</i> test with equal standard deviation (SD). <b>(G)</b> Overlay of the deconvoluted intact protein mass spectra obtained from chemically acetylated aSyn (theoretical mass 14,460 Da) in buffer (green) and treated with SIRT2 (purple). The observed masses of the different species correlate with the presence of multiple acetyl modifications (+42 Da), ranging from 2 to 8 before treatment with SIRT2 and from 0 to 6 after deacetylation with SIRT2. <b>(H)</b> Mass spectrometry fragmentation analysis of a peptide from aSyn carrying acetylations at K6 and K10. Red peaks correspond to y-ion series, green peaks to b-ion series, and purple peaks to a-ion series. Corresponding amino acids to mass intervals of y-ion and b-ion series are represented (red and green, respectively). <b>(I)</b> Semisynthetic aSyn acetylated at K6 and K10 were incubated with increasing amounts of recombinant SIRT2 in the presence or absence of NAD at 37°C for 3 h. Proteins were probed for acetyl-lysine residues, aSyn, and SIRT2. All images are representative out of three independent experiments. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p

aSyn acetylation-resistant mutant induces nigral dopaminergic neuronal loss in vivo.

<p><b>(A)</b> AAV6-mediated delivery of EGFP and mutant aSyn (KQ or KR) into the SN of the rat brain. TH and GFP or aSyn expression was examined in brain sections 3 wk after injection by immunohistochemistry (TH, red; GFP or aSyn, green; DAPI, blue). Representative sections are shown. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm. <b>(B)</b> Stereological counting of the number of TH-positive neurons in the SN. The contralateral SN of the different groups of animals was used as a control (intact). Data in panels are average ± SD. <b>(C)</b> Brain sections stained for aSyn (green), pS129 aSyn (red), and DAPI (Blue). Representative sections are shown. Dashed square boxes delineate the magnification presented on the right. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm and 50 μm. *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001, one-way ANOVA with Bonferroni correction used for statistical calculations. In (B), GFP was used as a control; <i>n</i> = 6–7 animals per condition; five sections from a one-in-six series were analyzed per brain. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p

aSyn acetylation mimic is neuroprotective in primary cortical neurons.

<p><b>(A)</b> Cultured primary neurons were transduced with AAVs encoding for EGFP, WT aSyn, KQ, or KR-mutant aSyn at day in vitro 3 (DIV3). Whole-cell lysates were analyzed by immunoblotting 7 d postinfection with antibodies against aSyn and β-actin (<i>n</i> = 3). <b>(B)</b> Primary neuronal cells were coinfected with aSyn and EGFP at DIV3 and monitored over time. 16 images per condition were acquired, and the EGFP fluorescence signal was recorded in living neurons at 7, 10, 15, 18, and 21 d posttransduction (<i>n</i> = 3). Representative images are shown. Scale bar 20 μm. <b>(C)</b> Total number of EGFP positive cells normalized to the number of neurons on WT 7 d posttransduction is presented (<i>n</i> = 6). Data in all panels are average ± SD, * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, two-way ANOVA with Bonferroni post-test. <b>(D)</b> Cortical neuronal cells 15 d posttransduction were processed for ICC (aSyn, red; microtubule-associated protein 2 [MAP2], green; nuclei, Hoechst, blue). Representative images are presented. Scale bar 200 μm. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p

aSyn aggregation is modulated by acetylation.

<p><b>(A)</b> H4 cells were cotransfected with WT, acetylation-resistant mutants (K6R, K10R, K6+10R), or acetylation-mimicking mutants (K6Q, K10Q, K6+10Q) of SynT together with synphilin-1. 48 h after transfection, cells were processed for ICC and the percentage of cells with inclusions was determined (<i>n</i> = 3). Data in panels are average ± SD, **** <i>p</i> < 0.0001, ordinary one-way ANOVA with Tukey’s multiple comparisons test. <b>(B)</b> Oligomerization kinetics of recombinant aSyn assessed by Thioflavin-T reaction. WT, K6+10Q, K6+10R, and a mixture of 1:1 of WT with K6+10Q or K6+10R were evaluated. <b>(C)</b> Superposition of 2D ¹H-¹⁵N HSQC nuclear magnetic resonance (NMR) spectra of recombinant ¹⁵N-labelled aSyn WT (black), K6+10Q (green). <b>(D)</b> Residue-specific changes in ¹H-¹⁵N HSQC signal intensities of aSyn WT (black) and aSyn K6+10Q (green) upon addition of small unilamellar vesicles (SUVs) formed by POPC:POPA (1:1 molar ratio). The aSyn-to-lipid molar ratio was 1:100. <b>(E)</b> Estimation of the binding affinity of aSyn to POPC:POPA SUVs from circular dichroism. Variations in absorption at 222 nm are plotted as a function of lipid:protein molar ratio. Calculated affinities are 57 ± 13 and 71 ± 16 μM for aSyn WT (black) and aSyn K6+10Q (green), respectively. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p

Knockout of SIRT2 protects from aSyn or MPTP toxicity in mice.

<p><b>(A)</b> AAV6-mediated delivery of GFP or WT aSyn into the SN of WT or T2.KO mice brains. TH and GFP or aSyn expression was examined in brain sections 2 wk postinjection by immunohistochemistry (TH, red; GFP or aSyn, green; DAPI, blue). Representative sections are shown. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm. <b>(B)</b> Stereological counting of the number of TH-positive neurons in the SN. The number of TH-positive neurons in the SN of GFP-injected mice was used as control (at least <i>n</i> = 3 per group). Data, presented as percentage of TH-neurons, are average ± SD. *** <i>p</i> < 0.001, unpaired <i>t</i> test with equal SD. Chronic MPTP treatment of WT or T2.KO mice brain. TH <b>(C)</b> or neurons (Nissl) <b>(D)</b> were examined in brain sections 2 wk postinjection by immunohistochemistry (TH, DAB; Neurons, Nissl; DAPI, blue). Representative sections are shown. Scale bar 200 μm. Stereological counting of the number of TH-positive <b>(E)</b> or Nissl-positive neurons <b>(F)</b> in the SN (at least <i>n</i> = 4 per group). Neuron numbers in NaCl-injected mice were used as normalization controls, and data are expressed as percentage of the corresponding NaCl-injected control animals. For (E) and (F), data (presented as percentage of neurons), are average ± SD. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, two-way ANOVA followed by Tukey’s multiple comparisons test. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p

Acetylation of aSyn enhances the clearance of aSyn inclusions by autophagy.

<p><b>(A)</b> H4 cells (control and T2.KD) transiently expressing SynT and synphilin-1 were treated with cycloheximide (CHX) for 4, 8, and 12 h. Protein extracts were then separated on SDS-PAGE and probed for aSyn and GAPDH for normalization (at least <i>n</i> = 3). <b>(B)</b> Cells as in (A) were treated with bafilomycin A1 (BafA) for 2 and 4 h. Protein extracts were probed for LC3 and β-actin. LC3-II levels were normalized to β-actin, and the difference between BafA treatment for 2 h and vehicle (0 h) treatment was calculated (at least <i>n</i> = 3). <b>(C)</b> Cells as in (A) were processed for ICC (LC3, green). Number of LC3 puncta per cell were counted (<i>n</i> = 3). Data in all panels are average ± SD, * <i>p</i> < 0.05, ** <i>p</i> < 0.01. For (B) and (C), ordinary one-way ANOVA with Tukey’s multiple comparisons test. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p