Robert H. Cichewicz

Neuroprotective role of Sirt1 in mammalian models of Huntington's disease through activation of multiple Sirt1 targets

Huntingtons disease is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in huntingtin (HTT) protein. We previously showed that calorie restriction ameliorated Huntingtons disease pathogenesis and slowed disease progression in mice that model Huntingtons disease (Huntingtons disease mice). We now report that overexpression of sirtuin 1 (Sirt1), a mediator of the beneficial metabolic effects of calorie restriction, protects neurons against mutant HTT toxicity, whereas reduction of Sirt1 exacerbates mutant HTT toxicity. Overexpression of Sirt1 improves motor function, reduces brain atrophy and attenuates mutant-HTT–mediated metabolic abnormalities in Huntingtons disease mice. Further mechanistic studies suggested that Sirt1 prevents the mutant-HTT–induced decline in brain-derived neurotrophic factor (BDNF) concentrations and the signaling of its receptor, TrkB, and restores dopamine- and cAMP-regulated phosphoprotein, 32 kDa (DARPP32) concentrations in the striatum. Sirt1 deacetylase activity is required for Sirt1-mediated neuroprotection in Huntingtons disease cell models. Notably, we show that mutant HTT interacts with Sirt1 and inhibits Sirt1 deacetylase activity, which results in hyperacetylation of Sirt1 substrates such as forkhead box O3A (Foxo3a), thereby inhibiting its pro-survival function. Overexpression of Sirt1 counteracts the mutant-HTT–induced deacetylase deficit, enhances the deacetylation of Foxo3a and facilitates cell survival. These findings show a neuroprotective role for Sirt1 in mammalian Huntingtons disease models and open new avenues for the development of neuroprotective strategies in Huntingtons disease.

Chemical induction of silent biosynthetic pathway transcription in Aspergillus niger

Manipulation of the fungal epigenome is hypothesized to be an effective method for accessing natural products from silent biosynthetic pathways. A library of epigenetic modifiers was tested using the fungus Aspergillus niger to determine the impact of small-molecule inhibitors on reversing the transcriptional suppression of biosynthetic genes involved in polyketide (PKS), non-ribosomal peptide (NRPS), and hybrid PKS-NRPS (HPN) production. Examination of expressed sequence tag libraries from A. niger demonstrated that >70% of its PKS-, NRPS-, and HPN-encoding gene clusters were transcriptionally suppressed under standard laboratory culture conditions. Using a chemical epigenetic methodology, we showed that treatment of A. niger with suberoylanilide hydroxamic acid and 5-azacytidine led to the transcriptional upregulation of many secondary-metabolite-encoding biosynthetic gene clusters. Chemical epigenetic modifiers exhibited positional biases for upregulating chromosomally distal gene clusters. In addition, a phylogenetic-based preference was noted in the upregulation of reducing clade I PKS gene clusters, while reducing clade IV PKS gene clusters were largely unaffected. Manipulating epigenetic features in fungi is a powerful method for accessing the products of silent biosynthetic pathways. Moreover, this approach can be readily incorporated into modern microbial screening operations.

Chemical epigenetics alters the secondary metabolite composition of guttate excreted by an atlantic-forest-soil-derived Penicillium citreonigrum.

Chemical epigenetic manipulation of Penicillium citreonigrum led to profound changes in the secondary metabolite profile of its guttate. While guttate from control cultures exhibited a relatively simple assemblage of secondary metabolites, the guttate collected from cultures treated with 50 muM 5-azacytidine (a DNA methyltransferase inhibitor) was highly enriched in compounds representing at least three distinct biosynthetic families. The metabolites obtained from the fungus included six azaphilones (sclerotiorin (1), sclerotioramine (6), ochrephilone (2), dechloroisochromophilone III (3), dechloroisochromophilone IV (4), and 6-((3E,5E)-5,7-dimethyl-2-methylenenona-3,5-dienyl)-2,4-dihydroxy-3-methylbenzaldehyde (5)), pencolide (7), and two new meroterpenes (atlantinones A and B (9 and 10, respectively)). While pencolide was detected in the exudates of both control and 5-azacytidine-treated cultures, all of the other natural products were found exclusively in the guttates of the epigenetically modified fungus. All of the metabolites from the P. citreonigrum guttate were tested for antimicrobial activity in a disk diffusion assay. Both sclerotiorin and sclerotioramine caused modest inhibition of Staphylococcus epidermidis growth; however, only sclerotioramine was active against a panel of Candida strains.

trans-(-)-ε-Viniferin increases mitochondrial sirtuin 3 (SIRT3), activates AMP-activated protein kinase (AMPK), and protects cells in models of Huntington Disease.

Background: Mitochondrial dysfunction is a key event mediating mutant Htt-induced neurotoxicity. Results: trans-(−)-ϵ-Viniferin attenuates mutant Htt-induced SIRT3 depletion, activates AMPK, and preserves mitochondrial function. Conclusion: Increasing SIRT3 protects cells in HD. Significance: The result suggests a promising new target for development of HD therapeutics. Huntington disease (HD) is an inherited neurodegenerative disorder caused by an abnormal polyglutamine expansion in the protein Huntingtin (Htt). Currently, no cure is available for HD. The mechanisms by which mutant Htt causes neuronal dysfunction and degeneration remain to be fully elucidated. Nevertheless, mitochondrial dysfunction has been suggested as a key event mediating mutant Htt-induced neurotoxicity because neurons are energy-demanding and particularly susceptible to energy deficits and oxidative stress. SIRT3, a member of sirtuin family, is localized to mitochondria and has been implicated in energy metabolism. Notably, we found that cells expressing mutant Htt displayed reduced SIRT3 levels. trans-(−)-ϵ-Viniferin (viniferin), a natural product among our 22 collected naturally occurring and semisynthetic stilbenic compounds, significantly attenuated mutant Htt-induced depletion of SIRT3 and protected cells from mutant Htt. We demonstrate that viniferin decreases levels of reactive oxygen species and prevents loss of mitochondrial membrane potential in cells expressing mutant Htt. Expression of mutant Htt results in decreased deacetylase activity of SIRT3 and further leads to reduction in cellular NAD+ levels and mitochondrial biogenesis in cells. Viniferin activates AMP-activated kinase and enhances mitochondrial biogenesis. Knockdown of SIRT3 significantly inhibited viniferin-mediated AMP-activated kinase activation and diminished the neuroprotective effects of viniferin, suggesting that SIRT3 mediates the neuroprotection of viniferin. In conclusion, we establish a novel role for mitochondrial SIRT3 in HD pathogenesis and discovered a natural product that has potent neuroprotection in HD models. Our results suggest that increasing mitochondrial SIRT3 might be considered as a new therapeutic approach to counteract HD, as well as other neurodegenerative diseases with similar mechanisms.

Mutanobactin A from the human oral pathogen Streptococcus mutans is a cross-kingdom regulator of the yeast-mycelium transition

The recent investigation of a gene cluster encoding for a hybrid PKS-NRPS metabolite in the oral pathogen Streptococcus mutans UA159 yielded evidence that this natural product might play an important role regulating a range of stress tolerance factors. We have now characterized the major compound generated from this gene cluster, mutanobactin A, and demonstrated that this secondary metabolite is also capable of influencing the yeast-mycelium transition of Candida albicans.

Reassessing the ichthyotoxin profile of cultured Prymnesium parvum (golden algae) and comparing it to samples collected from recent freshwater bloom and fish kill events in North America

Within the last two decades, Prymnesium parvum (golden algae) has rapidly spread into inland waterways across the southern portion of North America and this organism has now appeared in more northerly distributed watersheds. In its wake, golden algae blooms have left an alarming trail of ecological devastation, namely massive fish kills, which are threatening the economic and recreational value of freshwater systems throughout the United States. To further understand the nature of this emerging crisis, our group investigated the chemical nature of the toxin(s) produced by P. parvum. We approached the problem using a two-pronged strategy that included analyzing both laboratory-grown golden algae and field-collected samples of P. parvum. Our results demonstrate that there is a striking difference in the toxin profiles for these two systems. An assemblage of potently ichthyotoxic fatty acids consisting primarily of stearidonic acid was identified in P. parvum cultures. While the concentration of the fatty acids alone was sufficient to account for the rapid-onset ichthyotoxic properties of cultured P. parvum, we also detected a second type of highly labile ichthyotoxic substance(s) in laboratory-grown golden algae that remains uncharacterized. In contrast, the amounts of stearidonic acid and its related congeners present in samples from recent bloom and fish kill sites fell well below the limits necessary to induce acute toxicity in fish. However, a highly labile ichthyotoxic substance, which is similar to the one found in laboratory-grown P. parvum cultures, was also detected. We propose that the uncharacterized labile metabolite produced by P. parvum is responsible for golden algaes devastating fish killing effects. Moreover, we have determined that the biologically-relevant ichthyotoxins produced by P. parvum are not the prymnesins as is widely believed. Our results suggest that further intensive efforts will be required to chemically define P. parvums ichthyotoxins under natural bloom conditions.

Secondary Metabolites from an Algicolous Aspergillus versicolor Strain

Two new compounds, asperversin A (1) and 9ξ-O-2(2,3-dimethylbut-3-enyl)brevianamide Q (2), and nine known compounds, brevianamide K (3), brevianamide M (4), aversin (5), 6,8-di-O-methylnidurufin (6), 6,8-di-O-methylaverufin (7), 6-O-methylaverufin (8), 5α,8α-epidioxyergosta-6,22-dien-3β-ol (9), ergosta-7,22-diene-3β,5α,6β-triol (10), and 6β-methoxyergosta-7,22-diene-3β,5α-diol (11), were obtained from the culture of Aspergillus versicolor, an endophytic fungus isolated from the marine brown alga Sargassum thunbergii. The structures of these compounds were established by spectroscopic techniques. Compounds 4, 7 and 8 exhibited antibacterial activities against Escherichia coli and Staphyloccocus aureus, and 7 also showed lethality against brine shrimp (Artemia salina) with an LC50 value of 0.5 μg/mL.

Genomic Island TnSmu2 of Streptococcus mutans Harbors a Nonribosomal Peptide Synthetase-Polyketide Synthase Gene Cluster Responsible for the Biosynthesis of Pigments Involved in Oxygen and H2O2 Tolerance

ABSTRACT The oral biofilm community consists of >800 microbial species, among which Streptococcus mutans is considered a primary pathogen for dental caries. The genomic island TnSmu2 of S. mutans comprises >2% of the genome. In this study, we demonstrate that TnSmu2 harbors a gene cluster encoding nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and accessory proteins and regulators involved in nonribosomal peptide (NRP) and polyketide (PK) biosynthesis. Interestingly, the sequences of these genes and their genomic organizations and locations are highly divergent among different S. mutans strains, yet each TnSmu2 region encodes NRPS/PKS and accessory proteins. Mutagenesis of the structural genes and putative regulatory genes in strains UA159, UA140, and MT4653 resulted in colonies that were devoid of their yellow pigmentation (for strains UA140 and MT4653). In addition, these mutant strains also displayed retarded growth under aerobic conditions and in the presence of H2O2. High-performance liquid chromatography profiling of cell surface extracts identified unique peaks that were missing in the mutant strains, and partial characterization of the purified product from UA159 demonstrated that it is indeed a hybrid NRP/PK, as predicted. A genomic survey of 94 clinical S. mutans isolates suggests that the TnSmu2 gene cluster may be more prevalent than previously recognized.

Resveratrol oligomers: Structure, chemistry, and biological activity

Abstract Both plants and fungi have been reported to be capable of polymerizing the natural bioactive trihydroxystilbene resveratrol into a host of complex oligomers. In recent years, these resveratrol oligomers have received the attention of natural products researchers worldwide as scientists have come to appreciate their pharmacological potential. During this time, the number of known resveratrol oligomers has swelled with scores of new compounds reported. In this chapter, the structure, chemistry, and biological activity of the resveratrol oligomers will be reviewed. This review consists of a brief assessment of previous resveratrol research followed by a thorough examination of the resveratrol oligomers including their roles as phytoalexins, detoxification products of the fungal metabolism of resveratrol, and potential medicinal agents for humans. A detailed analysis of the methods used for the isolation and structure elucidation of these compounds is provided.

Crowdsourcing Natural Products Discovery to Access Uncharted Dimensions of Fungal Metabolite Diversity

A fundamental component for success in drug discovery is the ability to assemble and screen compounds that encompass a broad swath of biologically relevant chemical-diversity space. Achieving this goal in a natural-products-based setting requires access to a wide range of biologically diverse specimens. For this reason, we introduced a crowdsourcing program in which citizen scientists furnish soil samples from which new microbial isolates are procured. Illustrating the strength of this approach, we obtained a unique fungal metabolite, maximiscin, from a crowdsourced Alaskan soil sample. Maximiscin, which exhibits a putative combination of polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), and shikimate pathway components, was identified as an inhibitor of UACC-62 melanoma cells (LC50=0.93 μM). The metabolite also exhibited efficacy in a xenograft mouse model. These results underscore the value of building cooperative relationships between research teams and citizen scientists to enrich drug discovery efforts.