Role of Sirtuins in Modulating Neurodegeneration of the Enteric Nervous System and Central Nervous System.

Neurodegeneration of the central and enteric nervous systems is a common feature of aging and aging-related diseases, and is accelerated in individuals with metabolic dysfunction including obesity and diabetes. The molecular mechanisms of neurodegeneration in both the CNS and ENS are overlapping. Sirtuins are an important family of histone deacetylases that are important for genome stability, cellular response to stress, and nutrient and hormone sensing. They are activated by calorie restriction (CR) and by the coenzyme, nicotinamide adenine dinucleotide (NAD+). Sirtuins, specifically the nuclear SIRT1 and mitochondrial SIRT3, have been shown to have predominantly neuroprotective roles in the CNS while the cytoplasmic sirtuin, SIRT2 is largely associated with neurodegeneration. A systematic study of sirtuins in the ENS and their effect on enteric neuronal growth and survival has not been conducted. Recent studies, however, also link sirtuins with important hormones such as leptin, ghrelin, melatonin, and serotonin which influence many important processes including satiety, mood, circadian rhythm, and gut homeostasis. In this review, we address emerging roles of sirtuins in modulating the metabolic challenges from aging, obesity, and diabetes that lead to neurodegeneration in the ENS and CNS. We also highlight a novel role for sirtuins along the microbiota-gut-brain axis in modulating neurodegeneration

Prion nucleation and propagation by mammalian amyloidogenic proteins in yeast

Cross-β fibrous protein polymers or “amyloids” are associated with a variety of human and animal diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) and are suspected to possess transmissible (prion) properties. However, the molecular mechanisms of amyloid formation and propagation are difficult to investigate in vivo due to complexity of the human organism. While evolutionarily distant from humans, yeast cells carry transmissible amyloids (yeast prions) that can be detected phenotypically. The objectives of the work presented in this dissertation were to understand the molecular mechanisms of initial prion nucleation and propagation by mammalian proteins in yeast. Our model employed chimeric constructs, containing the mammalian amyloidogenic proteins (or domains) fused to various fragments of the yeast prion protein Sup35. Phenotypic and biochemical detection assays, previously developed for the Sup35 prion, enabled us to detect prion nucleation and propagation by mammalian proteins. We have demonstrated that several non-Q/N rich, mammalian amyloidogenic proteins, nucleated a prion in yeast in the absence of pre-existing prions. Sequence alterations antagonizing or enhancing amyloidogenicity of human Aβ (associated with AD) and mouse PrP (associated with prion diseases) respectively antagonized or enhanced nucleation of a yeast prion by these proteins. Mutational dissection of Aβ identified sequences and chemicals that influence initial amyloid nucleation. We have also shown that Aβ and microtubule-associated binding protein tau that is also associated with AD, could propagate a prion state on their own or after transfection with in vitro generated amyloid seeds, in yeast. Aβ- and tau-based chimeric constructs formed distinct variants (“strains”) in the yeast cell. Our data show that prion properties of mammalian proteins detected in the yeast assays correspond with those found in mammals or in vitro, thus making yeast a powerful model for deciphering molecular foundations of amyloid/prion diseases.Ph.D

Polyglutamine toxicity is controlled by prion composition and gene dosage in yeast

© 2012 Gong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.DOI: 10.1371/journal.pgen.1002634Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington’s disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45- bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines

Polyglutamine toxicity and aggregation in the yeast strains with various prion compositions.

<p>A – Polyglutamine constructs used in this work. All constructs were under the control of the galactose-inducible promoter (<i>P_GAL</i>), and contained the FLAG epitope, N-terminal 17 amino acid residues and poly-Q stretch of human Htt, and were fused to the gene coding for green fluorescent protein (GFP) at C-terminus. Numbers indicate length of poly-Q stretch. Poly-QP constructs also contained the proline-rich region of Htt (designated as P), immediately following the poly-Q stretch. B – Expanded poly-Q without a P-rich region (103Q), expressed under the <i>P_GAL</i> promoter on -Ura/Gal medium, is toxic in the presence of either [<i>PIN</i>⁺] or [<i>PSI</i>⁺] (or both), with two prions showing an additive effect. In contrast, expanded poly-Q with a P-rich region (103QP) is toxic only in the presence of [<i>PSI</i>⁺]. The 25Q construct, not exhibiting toxicity under these conditions, is shown as a control. The 25QP construct (not shown) behaved in the same way as 25Q. C – 103Q and 103QP form multiple peripheral aggregates and single aggregate (aggresome), respectively, in cells containing either or both prions ([<i>PIN⁺</i>] and/or [<i>PSI⁺</i>]), as visualized by fluorescence microscopy. Perinuclear location of aggresome (not shown) was confirmed by DAPI staining as described previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002634#pgen.1002634-Wang1" target="_blank">[42]</a>. D – Overexpressed Sup35NM-DsRed (red) forms large clumps in the [<i>PSI⁺</i>] cells, that overlap with the 103QP-GFP aggresome (green), as pointed by arrows. E - Expression of 103Q or 103QP promotes aggregation of Sup35 in the [<i>psi⁻</i>] strain as seen by an increase of pellet (P) versus supernatant (S) fraction, in comparison to the respective strain expressing 25Q. Centrifugation analysis was followed by Western blotting and immunostaining with the Sup35 antibody. F - Expression of the Sup35 derivative, lacking the prion and middle domains (Sup35C), decreases 103Q and 103QP toxicity in the [<i>PIN⁺ PSI⁺</i>] strain but does not influence 103Q toxicity in the [<i>PIN⁺ psi⁻</i>] strain. <i>SUP35C</i> gene was under control of the endogenous <i>SUP35</i> promoter. Serial decimal dilutions were spotted onto -Ura/Gal medium.</p

Isolation and characterization of anti-polyQ toxicity (<i>AQT</i>) derivatives.

<p>A – <i>Ubc4</i>Δ has no significant effect on toxicity of 103Q or 103QP in the [<i>PIN</i>⁺<i>PSI</i>⁺] background. Serial decimal dilutions were spotted onto -Ura/Gal medium. B – Papillae arise spontaneously in the <i>ubc4</i>Δ [<i>PIN</i>⁺<i>PSI</i>⁺] strain expressing 103Q, and are able to stably maintain the anti-polyQ-toxic phenotype after colony purification. These papillae were designated as <i>AQT</i>. C – Comparison of the growth curves of [<i>PIN⁺ PSI⁺</i>] <i>ubc4</i>Δ strains that differ by polyglutamine constructs and by the presence or absence of <i>AQT</i>. Growth was measured by optical density at 600 nm in the liquid –Ura medium with galactose and raffinose instead of glucose. At least 3 independent cultures were characterized per each combination. Error bars represent standard deviations. D – <i>AQT</i> ameliorates 103QP toxicity. E – <i>AQT</i> is dominant (all strains are [<i>PIN⁺ PSI⁺</i>] and <i>ubc4</i>Δ homozygotes). F – Reintroduction of the <i>UBC4</i> gene under galactose-inducible promoter on a multicopy plasmid partly suppresses but does not completely eliminate anti-toxic effect of <i>AQT</i>. -Ura/Gal plates are scored on panels D, E and F.</p

Modulation of polyglutamine toxicity by the plasmid-borne release factor genes.

<p>A - An extra copy of <i>SUP45</i> gene, located on the centromeric plasmid under endogenous promoter, ameliorates toxicity of 103Q in the <i>ubc4</i>Δ [<i>PSI⁺</i>] strain, as seen from serial decimal dilutions plated onto the galactose medium selective for both poly-Q and <i>SUP45</i> (or control) plasmids. B – Amelioration of [<i>PSI⁺</i>]-dependent polyglutamine toxicity by a plasmid-borne extra copy of <i>SUP45</i> gene is detected for both endogenous (<i>CEN-SUP45</i>) galactose-inducible (<i>GAL-SUP45</i>) promoters, for both 103Q and 103QP constructs, and in both <i>ubc4</i>Δ and <i>UBC4⁺</i> strains. Antitoxic effect of the plasmid-borne <i>SUP45</i> gene in the <i>ubc4Δ</i> strain is comparable to antitoxic effect of <i>AQT</i>. Toxicity was scored on the galactose medium selective for both poly-Q and <i>SUP45</i> (or control) plasmids. C and D – Centromeric plasmids with <i>SUP45</i> gene under endogenous (C) or galactose-inducible <i>P_GAL</i> (D) promoters increase levels of Sup45 protein (Sup45p) both [<i>UBC4⁺</i>] and <i>ubc4</i>Δ strains. Cultures were grown in liquid -Ura -Leu glucose (C) or -Ura -Leu galactose/raffinose (D) medium. Ade2 (Ade2p) protein is shown as a loading control. E and F – Plasmids, expressing the <i>SUP45</i> alleles with either C-terminal deletion, <i>SUP45</i>Δ<i>C19</i> (that abolishes Sup45 function and interaction with Sup35) (E) or missense mutation <i>sup45-103</i>, T62C (that impairs Sup45 function) (F) from the endogenous <i>SUP45</i> promoter, do not ameliorate 103Q and 103QP toxicity, as scored on the galactose medium selective for both plasmids.</p

Effects of <i>AQT</i> on polyglutamine aggregation and Sup35 toxicity.

<p>A and B – Typical aggregation patterns of 103Q (multiple dots, A) and 103QP (single clump, B) are not affected by <i>AQT</i>, as confirmed by fluorescence microscopy. C – <i>AQT</i> mutant ameliorates toxicity of excess Sup35 or Sup35N in the [<i>PSI</i>⁺] strain. Sup35 and Sup35N proteins were expressed from centromeric plasmid under control of the galactose-inducible promoter. Cells were grown on the -Ura/glucose medium selective for the plasmid for 1 day. Serial decimal dilutions were plated onto -Ura/Gal medium.</p

<i>AQT</i> derivatives are disomic for chromosome II, and extra-copy of <i>SUP45</i> is responsible for antitoxicity.

<p>A – Tetrad analysis of a diploid obtained from mating of the <i>AQT</i> strain bearing the <i>ubc4</i>Δ<i>::HIS3</i> transplacement, to the strain bearing the <i>ubc4</i>Δ<i>::KanMX</i> transplacement, demonstrates presence of at least 2 copies of the <i>HIS3</i> gene versus one copy of the <i>KanMX</i> gene. This can be concluded from the fact that majority of tetrads produce more than 2 His⁺ spores, in contrast to the typically 2∶2 segregation by G418 resistance caused by KanMX. All <i>AQT</i> spores in this cross were His⁺ (not shown). B – Hybridization of total DNA to a complete DNA microarray of the <i>S. cerevisiae</i> genome confirms that all the coding material of chromosome II is duplicated in the <i>AQT</i> strain. Comparison is performed according to CLAC (CLuster Along Chromosome) consensus plot. For procedure, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002634#pgen.1002634.s008" target="_blank">Text S1</a>. C – Sequential deletion mapping of the chromosome II extra copy in the <i>AQT</i> strain. The <i>AQT#7</i> derivative (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002634#pgen.1002634.s002" target="_blank">Figure S2B</a>) was used in these experiments. Each numbered region corresponds to a respective deletion. Deletions eliminating the antitoxicity phenotype in the [<i>PSI⁺</i>] background are shown as boxes filled in black. All deletions were verified by PCR. Five ORFs located within region 2.1a were each deleted individually; among those deletions, only deletion of <i>SUP45</i> eliminated <i>AQT</i> as shown on panels B and C. D – Elimination of the antitoxic effect on 103Q and 103QP by the <i>sup45</i> deletion in <i>AQT</i> strain. Serial decimal dilutions were spotted onto -Ura/Gal medium. E – Sup45 protein levels are elevated in the <i>AQT</i> strain, more profoundly in <i>ubc4</i>Δ background than in the presence of wild type <i>UBC4</i> gene (<i>UBC4⁺</i>). Sup45p level is shown relative to the isogenic monosomic (non-<i>AQT</i>) control in each case. Ade2 protein was used as the loading control. At least 3 measurements with independent cultures were performed in each case. Error bars correspond to standard deviations. In each case the difference in Sup45 levels between the <i>AQT</i> and non-<i>AQT</i> strain is statistically significant as confidence limits do not overlap, and differences between the <i>UBC4⁺</i> and <i>ubc4Δ</i> strains are statistically significant according to <i>t</i>-criterium (<i>P_Ho</i><0.01).</p