Prodromal Markers in Parkinson's Disease:Limitations in Longitudinal Studies and Lessons Learned

A growing body of evidence supports a prodromal neurodegenerative process preceding the clinical onset of Parkinson's disease (PD). Studies have identified several different prodromal markers that may have the potential to predict the conversion from healthy to clinical PD but use considerably different approaches. We systematically reviewed 35 longitudinal studies reporting prodromal PD features and evaluated the methodological quality across 10 different predefined domains. We found limitations in the following domains: PD diagnosis (57% of studies), prodromal marker assessments (51%), temporal information on prodromal markers or PD diagnosis (34%), generalizability of results (17%), statistical methods (accounting for at least age as confounder; 17%), study design (14%), and sample size (9%). However, no limitations regarding drop-out (or bias investigation), or report of inclusion/exclusion criteria or prodromal marker associations were revealed. Lessons learned from these limitations and additional aspects of current prodromal marker studies in PD are discussed to provide a basis for the evaluation of findings and the improvement of future research in prodromal PD. The observed heterogeneity of studies, limitations and analyses might be addressed in future longitudinal studies using a, yet to be established, modular minimal set of assessments improving comparability of findings and enabling data sharing and combined analyses across studies

Sirt5 Deacylation Activities Show Differential Sensitivities to Nicotinamide Inhibition

<div><p>Sirtuins are protein deacylases regulating metabolism and aging processes, and the seven human isoforms are considered attractive therapeutic targets. Sirtuins transfer acyl groups from lysine sidechains to ADP-ribose, formed from the cosubstrate NAD⁺ by release of nicotinamide, which in turn is assumed to be a general Sirtuin inhibitor. Studies on Sirtuin regulation have been hampered, however, by shortcomings of available assays. Here, we describe a mass spectrometry–based, quantitative deacylation assay not requiring any substrate labeling. Using this assay, we show that the deacetylation activity of human Sirt5 features an unusual insensitivity to nicotinamide inhibition. In contrast, we find similar values for Sirt5 and Sirt3 for the intrinsic NAD⁺ affinity as well as the apparent NAD⁺ affinity in presence of peptide. Structure comparison and mutagenesis identify an Arg neighboring to the Sirt5 nicotinamide binding pocket as a mediator of nicotinamide resistance, and statistical sequence analyses along with testing further Sirtuins reveal a network of coevolved residues likely defining a nicotinamide-insensitive Sirtuin deacetylase family. The same Arg was recently reported to render Sirt5 a preferential desuccinylase, and we find that this Sirt5 activity is highly sensitive to nicotinamide inhibition. Analysis of Sirt5 structures and activity data suggest that an Arg/succinate interaction is the molecular basis of the differential nicotinamide sensitivities of the two Sirt5 activities. Our results thus indicate a Sirtuin subfamily with nicotinamide-insensitive deacetylase activity and suggest that the molecular features determining nicotinamide sensitivity overlap with those dominating deacylation specificity, possibly suggesting that other subfamily members might also prefer other acylations than acetylations.</p> </div

A molecular mechanism for direct sirtuin activation by resveratrol.

Sirtuins are protein deacetylases regulating metabolism, stress responses, and aging processes, and they were suggested to mediate the lifespan extending effect of a low calorie diet. Sirtuin activation by the polyphenol resveratrol can mimic such lifespan extending effects and alleviate metabolic diseases. The mechanism of Sirtuin stimulation is unknown, hindering the development of improved activators. Here we show that resveratrol inhibits human Sirt3 and stimulates Sirt5, in addition to Sirt1, against fluorophore-labeled peptide substrates but also against peptides and proteins lacking the non-physiological fluorophore modification. We further present crystal structures of Sirt3 and Sirt5 in complex with fluorogenic substrate peptide and modulator. The compound acts as a top cover, closing the Sirtuin's polypeptide binding pocket and influencing details of peptide binding by directly interacting with this substrate. Our results provide a mechanism for the direct activation of Sirtuins by small molecules and suggest that activators have to be tailored to a specific Sirtuin/substrate pair

Structure comparison and identification of a Sirtuin sequence motive indicating nicotinamide insensitive deacetylation activity.

<p>(<b>A</b>) Structure comparison of Sirt3 (red) and Sirt5 (blue), overlaid with a Sir2Tm/nicotinamide complex (grey). Nicotinamide and coupled residues (see below) in Sirt3 and Sirt5 are displayed as sticks and colored by atom type for Sirt5 and for nicotinamide. (<b>B</b>) Statistical coupling analysis scores (lower panel) identify residues apparently coevolving (score cutoff used: 1.5) with Sirt5-Arg105: Thr69 and Tyr102 (highlighted in panel A), Trp77, Arg217, and Trp222. Amino acids in other Sirtuin sequences and the corresponding Sirtuin class are shown on top of the scores. (<b>C</b>) Inhibition Sirt5-Arg105Leu by nicotinamide. Activities, determined using our mass spectrometry assay, were normalized against activity in absence of nicotinamide. Data for wildtype Sirt3 and Sirt5 from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045098#pone-0045098-g002" target="_blank">Fig. 2A</a> are shown here for comparison. (<b>D</b>) Comparison of IC₅₀ values, determined using the mass spectrometry assay, for nicotinamide inhibition of class I and III Sirtuins and of the Sirt5-Arg105Leu variant. Error bars represent the standard error of the fit. NAM, nicotinamide.</p

Development of a label-free, quantitative mass spectrometry-based deacylation assay.

<p>(<b>A</b>) Different amounts of acetylated (○) and deacetylated (•) CPS1-Lys527 peptide are plotted against their respective mass spectrometry signal areas. Interpolations (lines) show the linear correlations between peptide amounts and detected signals, and the slightly different slopes for the two peptide species. (<b>B</b>) Ratios of the injected amounts of deacetylated and acetylated peptide plotted against ratios of the measured log₁₀ signal areas (•). Equation and correlation for the linear interpolation (line) are indicated. (<b>C</b>) Scheme for the mass spectrometry-based deacylation assay. Percent deacetylation is calculated by normalizing the product area to the total signal area, and deacetylation rates are determined through analysis of aliquots taken after different incubation times.</p

The mass spectrometry-based deacetylation assay reveals an unusual low Sirt5 sensitivity for nicotinamide inhibition.

<p>(<b>A</b>) Dose-dependent nicotinamide inhibition of the deacetylation of an ACS2 or CPS1 peptide, respectively, by Sirt3 and Sirt5. % activity was determined through relative quantification of reaction product by mass spectrometry and normalization to the respective non inhibited activity (set to 100%). Error bars represent standard errors for three independent measurements. (<b>B</b>) Dissociation constants for the interaction of NAD⁺ with Sirt3 and Sirt5, respectively, in presence of different nicotinamide concentrations. K_d values were determined by microscale thermophoresis measurements. Error bars represent standard errors of three independent measurements. NAM, nicotinamide.</p

Effects of resveratrol and piceatannol on Sirt3 and Sirt5 deacetylation activities against fluorophore-free peptides and proteins.

<p><b>A</b> Overlay of the Sirt5/FdL1/resveratrol (FdL1 in green, activator omitted for clarity) and Sirt5/succinylated H3 peptide/NAD⁺ (H3 peptide in atom type coloring, NAD⁺ omitted for clarity) complexes. The fluorophore occupies the site normally accommodating residues of the substrate polypeptide. <b>B</b> Overlay of Sirt5/FdL1/resveratrol (FdL1 omitted for clarity) and Sirt5/succinylated H3-peptide/NAD⁺ complex. Ligands are colored according to atom types with carbon atoms in green (resveratrol), yellow (H3 peptide), and blue (NAD⁺). <b>C+D</b> Sirt5-dependent deacetylation of Prx1-Lys197 peptide (<b>C</b>) and Prx1 protein specifically acetylated at Lys197 (<b>D</b>) is activated by resveratrol-related compounds. <b>E</b> Sirt5-dependent deacetylation of Cytochrome c determined in an ELISA shows that resveratrol and piceatannol stimulate this activity (which leads to a loss of signal in this assay). <b>F</b> Dose-reponse experiment for the piceatannol-dependent stimulation of Cytochrome c deacetyalation by Sirt5. Shown is the loss in absorption at different piceatannol concentrations relative to untreated Cytochrome c. <b>G</b> Sirt3-dependent GDH deacetylation tested in an ELISA (deacetylation decreases absorbance) shows an inhibitory effect of resveratrol. Error bars represent standard errors of linear fits to time-series experiments (C+D) or standard deviations (E–G), respectively.</p

Crystal structure of human Sirt3 in complex with FdL1-peptide and piceatannol.

<p><b>A</b> Overall structure of the Sirt3/FdL1/piceatannol complex. Secondary structure elements are labeled according to the Sirt5/FdL1/resveratrol complex for comparison. <b>B</b> Closer view of the activator binding site, showing the direct interaction between substrate peptide and piceatannol similar to the arrangement in the Sirt5/FdL1/resveratrol complex structure. The 2F_O-F_C electron density is contoured at 1σ. Residues of the symmetry related Sirt3 monomer (*) contributing to piceatannol binding are shown in yellow. <b>C</b> Interface between Sirt3 (blue) and its symmetry related monomer (orange) showing the binding mode for the FdL1-peptide and piceatannol. Ligands and residues involved in their binding are presented as blue and yellow sticks. <b>D</b> Superimposition of Sirt5/FdL1/resveratrol (blue) and Sirt3/FdL1/piceatannol (yellow). The residues N-terminal to the acetylated lysine are labeled for comparison.</p