The NAD+-dependent deacetylase SIRT2 attenuates oxidative stress and mitochondrial dysfunction and improves insulin sensitivity in hepatocytes

Funding Information: European Regional Development Fund (ERDF), Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000012: HealthyAging2020); COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia (POCI-01-0145-FEDER-007440, SFRH/BPD/109347/ 2015 to R.M.O., SFRH/BD/86655/2012 to L.N. and SFRH/BPD/ 111815/2015 to P.G.); FLAD Life Science 2020 Grant to A.C.R.; European Molecular Biology Organization (EMBO Installation Grant to T.F.O.); DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) to T.F.O.Insulin resistance is a major predictor of the development of metabolic disorders. Sirtuins (SIRTs) have emerged as potential targets that can be manipulated to counteract age-related diseases, including type 2 diabetes. SIRT2 has been recently shown to exert important metabolic effects, but whether SIRT2 regulates insulin sensitivity in hepatocytes is currently unknown. The aim of this study is to investigate this possibility and to elucidate underlying molecular mechanisms. Here, we show that SIRT2 is downregulated in insulin-resistant hepatocytes and livers, and this was accompanied by increased generation of reactive oxygen species, activation of stress-sensitive ERK1/2 kinase, and mitochondrial dysfunction. Conversely, SIRT2 overexpression in insulin-resistant hepatocytes improved insulin sensitivity, mitigated reactive oxygen species production and ameliorated mitochondrial dysfunction. Further analysis revealed a reestablishment of mitochondrial morphology, with a higher number of elongated mitochondria rather than fragmented mitochondria instigated by insulin resistance. Mechanistically, SIRT2 was able to increase fusion-related protein Mfn2 and decrease mitochondrial-associated Drp1. SIRT2 also attenuated the downregulation of TFAM, a key mtDNA-associated protein, contributing to the increase in mitochondrial mass. Importantly, we found that SIRT2 expression in PBMCs of human subjects was negatively correlated with obesity and insulin resistance. These results suggest a novel function for hepatic SIRT2 in the regulation of insulin sensitivity and raise the possibility that SIRT2 activators may offer novel opportunities for preventing or treating insulin resistance and type 2 diabetes.publishersversionpublishe

The NAD+-dependent deacetylase SIRT2 attenuates oxidative stress and mitochondrial dysfunction and improves insulin sensitivity in hepatocytes

Insulin resistance is a major predictor of the development of metabolic disorders. Sirtuins (SIRTs) have emerged as potential targets that can be manipulated to counteract age-related diseases, including type 2 diabetes. SIRT2 has been recently shown to exert important metabolic effects, but whether SIRT2 regulates insulin sensitivity in hepatocytes is currently unknown. The aim of this study is to investigate this possibility and to elucidate underlying molecular mechanisms. Here, we show that SIRT2 is downregulated in insulin-resistant hepatocytes and livers, and this was accompanied by increased generation of reactive oxygen species, activation of stress-sensitive ERK1/2 kinase, and mitochondrial dysfunction. Conversely, SIRT2 overexpression in insulin-resistant hepatocytes improved insulin sensitivity, mitigated reactive oxygen species production and ameliorated mitochondrial dysfunction. Further analysis revealed a reestablishment of mitochondrial morphology, with a higher number of elongated mitochondria rather than fragmented mitochondria instigated by insulin resistance. Mechanistically, SIRT2 was able to increase fusion-related protein Mfn2 and decrease mitochondrial-associated Drp1. SIRT2 also attenuated the downregulation of TFAM, a key mtDNA-associated protein, contributing to the increase in mitochondrial mass. Importantly, we found that SIRT2 expression in PBMCs of human subjects was negatively correlated with obesity and insulin resistance. These results suggest a novel function for hepatic SIRT2 in the regulation of insulin sensitivity and raise the possibility that SIRT2 activators may offer novel opportunities for preventing or treating insulin resistance and type 2 diabetes.European Regional Development Fund (ERDF), Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000012: HealthyAging2020); COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia (POCI-01-0145-FEDER-007440, SFRH/BPD/109347/2015 to R.M.O., SFRH/BD/86655/2012 to L.N. and SFRH/BPD/111815/2015 to P.G.); FLAD Life Science 2020 Grant to A.C.R.; European Molecular Biology Organization (EMBO Installation Grant to T.F.O.); DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) to T.F.O

The Parkinson's Disease-Linked Protein DJ-1 Associates with Cytoplasmic mRNP Granules During Stress and Neurodegeneration.

Mutations in the gene encoding DJ-1 are associated with autosomal recessive forms of Parkinson's disease (PD). DJ-1 plays a role in protection from oxidative stress, but how it functions as an "upstream" oxidative stress sensor and whether this relates to PD is still unclear. Intriguingly, DJ-1 may act as an RNA binding protein associating with specific mRNA transcripts in the human brain. Moreover, we previously reported that the yeast DJ-1 homolog Hsp31 localizes to stress granules (SGs) after glucose starvation, suggesting a role for DJ-1 in RNA dynamics. Here, we report that DJ-1 interacts with several SG components in mammalian cells and localizes to SGs, as well as P-bodies, upon induction of either osmotic or oxidative stress. By purifying the mRNA associated with DJ-1 in mammalian cells, we detected several transcripts and found that subpopulations of these localize to SGs after stress, suggesting that DJ-1 may target specific mRNAs to mRNP granules. Notably, we find that DJ-1 associates with SGs arising from N-methyl-D-aspartate (NMDA) excitotoxicity in primary neurons and parkinsonism-inducing toxins in dopaminergic cell cultures. Thus, our results indicate that DJ-1 is associated with cytoplasmic RNA granules arising during stress and neurodegeneration, providing a possible link between DJ-1 and RNA dynamics which may be relevant for PD pathogenesis

α-synuclein interacts with PrPC to induce cognitive impairment through mGluR5 and NMDAR2B

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.Synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, are neurodegenerative disorders that are characterized by the accumulation of α-synuclein (aSyn) in intracellular inclusions known as Lewy bodies. Prefibrillar soluble aSyn oligomers, rather than larger inclusions, are currently considered to be crucial species underlying synaptic dysfunction. We identified the cellular prion protein (PrPC) as a key mediator in aSyn-induced synaptic impairment. The aSyn-associated impairment of long-term potentiation was blocked in Prnp null mice and rescued following PrPC blockade. We found that extracellular aSyn oligomers formed a complex with PrPC that induced the phosphorylation of Fyn kinase via metabotropic glutamate receptors 5 (mGluR5). aSyn engagement of PrPC and Fyn activated NMDA receptor (NMDAR) and altered calcium homeostasis. Blockade of mGluR5-evoked phosphorylation of NMDAR in aSyn transgenic mice rescued synaptic and cognitive deficits, supporting the hypothesis that a receptor-mediated mechanism, independent of pore formation and membrane leakage, is sufficient to trigger early synaptic damage induced by extracellular aSyn.M.T.F., H.V.M. and J.E.C. were supported by individual grants from Fundação para a Ciência e Tecnologia (FCT) (SFRH/BD/52228/2013; SFRH/BPD/109347/2015; SFRH/BPD/87647/2012); L.V.L. and T.F.O. were supported by a grant from the Fritz Thyssen Stiftung (Az. 10.12.2.165), Germany. L.V.L. received an iMM Lisboa internal fund (BIG – Breakthrough Idea Grant) for part of the project. L.V.L. is an Investigator FCT, Portugal. T.F.O. is supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Germany. LISBOA-01-0145-FEDER-007391, project co-financed by FEDER, POR Lisboa 2020 - Programa Operacional Regional de Lisboa, from PORTUGAL 2020 and by Fundação para a Ciência e a Tecnologia.info:eu-repo/semantics/publishedVersio

Digital activism and Hungarian media reform: The case of Milla

This article examines the rise of the Internet-based opposition group, One Million for the Freedom of the Press in Hungary (or Milla for short), and considers its impact as a form of digital activism in Hungarian political culture. Milla was founded in December 2010 as a Facebook group in response to the newly elected Fidesz government and its fundamental revision of the Hungarian constitution and, in particular, its media laws. Milla is a civil society group, based in Budapest, who saw the Fidesz government as a threat to the democratic freedoms set out in the post-communist settlement in Hungary. It emerged at a time when the mainstream Hungarian opposition parties were in disarray, and it took on the role of challenging the legitimacy of Fidesz actions. Milla is an important example of the idea of digital activism and virtual solidarity, and its experiences serve to illustrate many of the strengths and weaknesses of these notions. The article sets out the ways in which Milla has sought to generate support for itself and opposition to the government, how it has organized its activities and ultimately the specific problems that it faces in Hungarian civil society

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 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