Beneficial Effects of Sideritis clandestina Extracts and Sideridiol against Amyloid ß Toxicity.

Alzheimer's disease (AD) is the most common form of dementia. Given the link between oxidative stress and AD, many studies focus on the identification of natural antioxidants against AD. Although their antioxidant capacity is important, increasing data suggest that additional activities are related to their beneficial effects, including properties against amyloid beta (Aß) aggregation. Sideritis spp. (mountain tea) extracts possess not only antioxidant activity but also other bioactivities that confer neuroprotection. Although various Sideritis spp. extracts have been extensively studied, there are scarce data on S. clandestina subsp. peloponnesiaca (SCP) phytochemical composition and neuroprotective potential, while nothing is known of the responsible compounds. Given that SCP is a weaker antioxidant compared to other Sideritis spp., here, we investigated its potential beneficial properties against Aß aggregation. We characterized different SCP extracts and revealed their anti-aggregation activity by taking advantage of established C. elegans AD models. Importantly, we identified two pure compounds, namely, sideridiol and verbascoside, being responsible for the beneficial effects. Furthermore, we have revealed a potential anti-Aß aggregation mechanism for sideridiol. Our results support the use of mountain tea in the elderly against dementia and demonstrate the activity of sideridiol against Aß aggregation that could be exploited for drug development.

Neuron-specific proteasome activation exerts cell non-autonomous protection against amyloid-beta (Aß) proteotoxicity in Caenorhabditis elegans.

Proteostasis reinforcement is a promising approach in the design of therapeutic interventions against proteinopathies, including Alzheimer's disease. Understanding how and which parts of the proteostasis network should be enhanced is crucial in developing efficient therapeutic strategies. The ability of specific tissues to induce proteostatic responses in distal ones (cell non-autonomous regulation of proteostasis) is attracting interest. Although the proteasome is a major protein degradation node, nothing is known on its cell non-autonomous regulation. We show that proteasome activation in the nervous system can enhance the proteasome activity in the muscle of Caenorhabditis elegans. Mechanistically, this communication depends on Small Clear Vesicles, with glutamate as one of the neurotransmitters required for the distal regulation. More importantly, we demonstrate that this cell non-autonomous proteasome activation is translated into efficient prevention of amyloid-beta (Αß)-mediated proteotoxic effects in the muscle of C. elegans but notably not to resistance against oxidative stress. Our in vivo data establish a mechanistic link between neuronal proteasome reinforcement and decreased Aß proteotoxicity in the muscle. The identified distal communication may have serious implications in the design of therapeutic strategies based on tissue-specific proteasome manipulation.

A Multilevel Study of Eupatorin and Scutellarein as Anti-Amyloid Agents in Alzheimer's Disease.

Today, Alzheimer's disease (AD)-the most common neurodegenerative disorder, which affects 50 million people-remains incurable. Several studies suggest that one of the main pathological hallmarks of AD is the accumulation of abnormal amyloid beta (Aß) aggregates; therefore, many therapeutic approaches focus on anti-Aß aggregation inhibitors. Taking into consideration that plant-derived secondary metabolites seem to have neuroprotective effects, we attempted to assess the effects of two flavones-eupatorin and scutellarein-on the amyloidogenesis of Aß peptides. Biophysical experimental methods were employed to inspect the aggregation process of Aß after its incubation with each natural product, while we monitored their interactions with the oligomerized Aß through molecular dynamics simulations. More importantly, we validated our in vitro and in silico results in a multicellular organismal model-namely, Caenorhabditis elegans-and we concluded that eupatorin is indeed able to delay the amyloidogenesis of Aß peptides in a concentration-dependent manner. Finally, we propose that further investigation could lead to the exploitation of eupatorin or its analogues as potential drug candidates.

In-Plate and In-Gel Assays for the Assessment of Proteasome Activity in Caenorhabditis elegans.

This chapter describes two methods for the study of proteasome function in Caenorhabditis elegans (C. elegans). The first method, referred to as "in-plate activities," provides a quantitative measurement of proteasome activities in C. elegans lysates by means of a kinetic reaction in a 96-well plate. The second one, referred to as "in-gel activities," involves the separation of C. elegans protein lysates in a native polyacrylamide gel and the assessment of the activity of each proteasome form. Downstream immunoblotting also allows the semi-quantitative assessment of proteasome assembly. This chapter outlines two detailed protocols along with helpful schematics and representative results that will facilitate researchers to replicate both protocols accurately and reproducibly.

Healthspan improvement and anti-aggregation effects induced by a marine-derived structural proteasome activator.

Proteasome activation has been shown to promote cellular and organismal healthspan and to protect against aggregation-related conditions, such as Alzheimer's disease (AD). Various natural compounds have been described for their proteasome activating properties but scarce data exist on marine metabolites that often possess unique chemical structures, exhibiting pronounced bioactivities with novel mechanisms of action. In this study, we have identified for the first time a marine structural proteasome activator, namely (1R,3E,6R,7Z,11S,12S)-dolabella-3,7,18-trien-6,17-olide (DBTO). DBTO activates the 20S proteasome complex in cell-free assays but also in cellulo. Continuous supplementation of human primary fibroblasts with DBTO throughout their cellular lifespan confers an improved healthspan while ameliorated health status is also observed in wild type (wt) Caenorhabditis elegans (C. elegans) nematodes supplemented with DBTO. Furthermore, treatment of various AD nematode models, as well as of human cells of neuronal origin challenged with exogenously added Aß peptide, with DBTO results in enhanced protection against Aß-induced proteotoxicity. In total, our results reveal the first structural proteasome activator derived from the marine ecosystem and highlight its potential as a compound that might be used for healthspan maintenance and preventive strategies against proteinopathies, such as AD.

Low-Grade Systemic Inflammation Interferes with Anabolic and Catabolic Characteristics of the Aged Human Skeletal Muscle.

Aging is associated with the development of chronic low-grade systemic inflammation (LGSI) characterized by increased circulating levels of proinflammatory cytokines and acute phase proteins such as C-reactive protein (CRP). Collective evidence suggests that elevated levels of inflammatory mediators such as CRP, interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) are correlated with deteriorated skeletal muscle mass and function, though the molecular footprint of this observation in the aged human skeletal muscle remains obscure. Based on animal models showing impaired protein synthesis and enhanced degradation in response to LGSI, we compared here the response of proteolysis- and protein synthesis-related signaling proteins as well as the satellite cell and amino acid transporter protein content between healthy older adults with increased versus physiological blood hs-CRP levels in the fasted (basal) state and after an anabolic stimulus comprised of acute resistance exercise (RE) and protein feeding. Our main findings indicate that older adults with increased hs-CRP levels demonstrate (i) increased proteasome activity, accompanied by increased protein carbonylation and IKKα/ß phosphorylation; (ii) reduced Pax7+ satellite cells; (iii) increased insulin resistance, at the basal state; and (iv) impaired S6 ribosomal protein phosphorylation accompanied by hyperinsulinemia following an acute RE bout combined with protein ingestion. Collectively, these data provide support to the concept that age-related chronic LGSI may upregulate proteasome activity via induction of the NF-κB signaling and protein oxidation and impair the insulin-dependent anabolic potential of human skeletal muscle.

Oxidative Stress and Neurodegeneration: Interconnected Processes in PolyQ Diseases.

Neurodegenerative polyglutamine (polyQ) disorders are caused by trinucleotide repeat expansions within the coding region of disease-causing genes. PolyQ-expanded proteins undergo conformational changes leading to the formation of protein inclusions which are associated with selective neuronal degeneration. Several lines of evidence indicate that these mutant proteins are associated with oxidative stress, proteasome impairment and microglia activation. These events may correlate with the induction of inflammation in the nervous system and disease progression. Here, we review the effect of polyQ-induced oxidative stress in cellular and animal models of polyQ diseases. Furthermore, we discuss the interplay between oxidative stress, neurodegeneration and neuroinflammation using as an example the well-known neuroinflammatory disease, Multiple Sclerosis. Finally, we review some of the pharmaceutical interventions which may delay the onset and progression of polyQ disorders by targeting disease-associated mechanisms.

Senolytics and senomorphics: Natural and synthetic therapeutics in the treatment of aging and chronic diseases.

Cellular senescence is a heterogeneous process guided by genetic, epigenetic and environmental factors, characterizing many types of somatic cells. It has been suggested as an aging hallmark that is believed to contribute to aging and chronic diseases. Senescent cells (SC) exhibit a specific senescence-associated secretory phenotype (SASP), mainly characterized by the production of proinflammatory and matrix-degrading molecules. When SC accumulate, a chronic, systemic, low-grade inflammation, known as inflammaging, is induced. In turn, this chronic immune system activation results in reduced SC clearance thus establishing a vicious circle that fuels inflammaging. SC accumulation represents a causal factor for various age-related pathologies. Targeting of several aging hallmarks has been suggested as a strategy to ameliorate healthspan and possibly lifespan. Consequently, SC and SASP are viewed as potential therapeutic targets either through the selective killing of SC or the selective SASP blockage, through natural or synthetic compounds. These compounds are members of a family of agents called senotherapeutics divided into senolytics and senomorphics. Few of them are already in clinical trials, possibly representing a future treatment of age-related pathologies including diseases such as atherosclerosis, osteoarthritis, osteoporosis, cancer, diabetes, neurodegenerative diseases such as Alzheimer's disease, cardiovascular diseases, hepatic steatosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and age-related macular degeneration. In this review, we present the already identified senolytics and senomorphics focusing on their redox-sensitive properties. We describe the studies that revealed their effects on cellular senescence and enabled their nomination as novel anti-aging agents. We refer to the senolytics that are already in clinical trials and we present various adverse effects exhibited by senotherapeutics so far. Finally, we discuss aspects of the senotherapeutics that need improvement and we suggest the design of future senotherapeutics to target specific redox-regulated signaling pathways implicated either in the regulation of SASP or in the elimination of SC.

Network analysis in aged C. elegans reveals candidate regulatory genes of ageing.

Ageing is a biological process guided by genetic and environmental factors that ultimately lead to adverse outcomes for organismal lifespan and healthspan. Determination of molecular pathways that are affected with age and increase disease susceptibility is crucial. The gene expression profile of the ideal ageing model, namely the nematode Caenorhabditis elegans mapped with the microarray technology initially led to the identification of age-dependent gene expression alterations that characterize the nematode's ageing process. The list of differentially expressed genes was then utilized to construct a network of molecular interactions with their first neighbors/interactors using the interactions listed in the WormBase database. The subsequent network analysis resulted in the unbiased selection of 110 candidate genes, among which well-known ageing regulators appeared. More importantly, our approach revealed candidates that have never been linked to ageing before, thus suggesting promising potential targets/ageing regulators.

Modulation of the ubiquitin-proteasome system by marine natural products.

The ubiquitin-proteasome system (UPS) is a key player in the maintenance of cellular protein homeostasis (proteostasis). Since proteasome function declines upon aging leading to the acceleration of its progression and the manifestation of age-related pathologies, many attempts have been performed towards proteasome activation as a strategy to promote healthspan and longevity. The marine environment hosts a plethora of organisms that produce a vast array of primary and secondary metabolites, the majority of which are unique, exhibiting a wide spectrum of biological activities. The fact that these biologically important compounds are also present in edible marine organisms has sparked the interest for elucidating their potential health-related applications. In this review, we focus on the antioxidant, anti-aging, anti-aggregation and anti-photoaging properties of various marine constituents. We further discuss representatives of marine compounds classes with regard to their potential (direct or indirect) action on UPS components that could serve as UPS modulators and exert beneficial effects on conditions such as oxidative stress, aging and age-related diseases.

Synthesis, biological evaluation and QSAR studies of new thieno[2,3-d]pyrimidin-4(3H)-one derivatives as antimicrobial and antifungal agents.

A series of new thieno[2,3-d]pyrimidin-4(3H)-one derivatives were synthesized and evaluated for their activity against four gram-positive and four gram-negative bacterial and eight fungal species. The majority of the compounds exhibited excellent antimicrobial and antifungal activity, being more potent than the control compounds. Compound 22, bearing a m-methoxyphenyl group and an ethylenediamine side chain anchored at C-2 of the thienopyrimidinone core, is the most potent antibacterial compound with broad antimicrobial activity with MIC values in the range of 0.05-0.13 mM, being 6 to 15 fold more potent than the controls, streptomycin and ampicillin. Furthermore, compounds 14 and 15 which bear a p-chlorophenyl and m-methoxyphenyl group, respectively, and share a 2-(2-mercaptoethoxy)ethan-1-ol side chain showed the best antifungal activity, being 10-15 times more potent than ketoconazole or bifonazole with MIC values 0.013-0.026 and 0.027 mM, respectively. Especially in the case of compound 15 the low MIC values were accompanied by excellent MFC values ranging from 0.056 to 0.058 mM. Evaluation of toxicity in vitro on HFL-1 human embryonic primary cells and in vivo in the nematode C. elegans revealed no toxic effects for both compounds 15 and 22 tested at the MIC concentrations. Ligand-based similarity search and molecular docking predicted that the antibacterial activity of analogue 22 is related to inhibition of the topoisomerase II DNA gyrase enzyme and the antifungal activity of compound 15 to CYP51 lanosterol demethylase enzyme. R-Group analysis as a means of computational structure activity relationship tool, highlighted the compounds' crucial pharmacophore features and their impact on the antibacterial and antifungal activity. The presence of a N-methyl piperidine ring fused to the thienopyrimidinone core plays an important role in both activities.

Mitochondria (cross)talk with proteostatic mechanisms: Focusing on ageing and neurodegenerative diseases.

Perturbations of proteostatic mechanisms and mitochondrial decline during ageing and neurodegenerative diseases are well-established. Nevertheless, only a handful of interventions boosting proteostasis and mitochondrial function have been shown to delay ageing while therapies against neurodegeneration are still unavailable. Increasing evidence links the function of proteostatic mechanisms with each other and with the mitochondrial network. Tracing of this complex crosstalking network might lead to effective anti-ageing or neurodegenerative disease-modifying approaches. In this review we present evidence on the crosstalk of proteostatic mechanisms with mitochondria and discuss how incorporating this knowledge in future studies may help us develop more efficacious interventions against ageing and neurodegeneration.

Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation.

A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short-lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post-transcriptional mechanisms that account for over 40% of the age-regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly/disassembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is a major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age-related neurodegeneration.

We Are What We Eat: Ubiquitin-Proteasome System (UPS) Modulation Through Dietary Products.

During lifetime, the molecular mechanisms that are responsible for cellular defense against adverse conditions such as oxidative and heat stress tend to be less efficient, thus gradually leading to the natural phenomenon of aging. Aging is linked to increased oxidative stress and is characterized by the accumulation of damaged macromolecules. The accumulation of oxidized and misfolded proteins is also accusable for various neurodegenerative pathologies that are linked to aging. Among self-defense mechanisms of cells, proteostasis network is responsible for the proper biogenesis/folding/trafficking of proteins and their elimination through proteolysis. The ubiquitin-proteasome system (UPS) is the major proteolytic mechanism that has attracted the interest of many researchers as an antiaging target. Interestingly, many natural compounds have been identified as potent UPS activators. Given that diet is a manageable environmental factor that affects aging, consumption of natural dietary products that may potentially enhance the UPS function, would contribute to increased health span and delayed onset or progression of age-related disorders. Herein, we summarize natural compounds and extracts derived from edible products that have exhibited antiaging and anti-aggregation properties and the beneficial properties have been linked to the UPS modulation.

Nuclear inclusions of pathogenic ataxin-1 induce oxidative stress and perturb the protein synthesis machinery.

Spinocerebellar ataxia type-1 (SCA1) is caused by an abnormally expanded polyglutamine (polyQ) tract in ataxin-1. These expansions are responsible for protein misfolding and self-assembly into intranuclear inclusion bodies (IIBs) that are somehow linked to neuronal death. However, owing to lack of a suitable cellular model, the downstream consequences of IIB formation are yet to be resolved. Here, we describe a nuclear protein aggregation model of pathogenic human ataxin-1 and characterize IIB effects. Using an inducible Sleeping Beauty transposon system, we overexpressed the ATXN1(Q82) gene in human mesenchymal stem cells that are resistant to the early cytotoxic effects caused by the expression of the mutant protein. We characterized the structure and the protein composition of insoluble polyQ IIBs which gradually occupy the nuclei and are responsible for the generation of reactive oxygen species. In response to their formation, our transcriptome analysis reveals a cerebellum-specific perturbed protein interaction network, primarily affecting protein synthesis. We propose that insoluble polyQ IIBs cause oxidative and nucleolar stress and affect the assembly of the ribosome by capturing or down-regulating essential components. The inducible cell system can be utilized to decipher the cellular consequences of polyQ protein aggregation. Our strategy provides a broadly applicable methodology for studying polyQ diseases.

Bioinformatic framework for analysis of transcription factor changes as the molecular link between replicative cellular senescence signaling pathways and carcinogenesis.

Cellular senescence is a natural condition of irreversible cell cycle arrest and apoptotic resistance that occurs in cells exposed to various stress factors, such as replicative stress or overexpression of oncogenes. Unraveling the complex regulation of senescence in cells is essential to strengthen senescence-related therapeutic approaches in cancer, as cellular senescence plays a dual role in tumorigenesis, having both anti- and pro-tumorigenic effects. In our study we created a model of replicative cellular senescence, based on transcriptomic data, including an extra intermediate time-point prior to cells entering senescence, to elucidate the interplay of networks governing cellular senescence with networks involved in tumorigenesis. We reveal specific changes that occur in transcription factor activity at different timepoints before and after cells entering senescence and model the signaling networks that govern these changes.

The dietary triterpenoid 18α-Glycyrrhetinic acid protects from MMC-induced genotoxicity through the ERK/Nrf2 pathway.

18α-Glycyrrhetinic acid (18α-GA) is a bioactive triterpenoid that has been shown to activate the nuclear factor (erythroid-derived-2)-like 2 (Nrf2), the main transcription factor that orchestrates the cellular antioxidant response, in both cellular and organismal context. Although various beneficial properties of 18α-GA have been revealed, including its anti-oxidation and anti-aging activity, its possible protective effect against DNA damage has never been addressed. In this study, we investigated the potential beneficial properties of 18α-GA against DNA damage induced by mitomycin C (MMC) treatment. Using human primary fibroblasts exposed to MMC following pre-treatment with 18α-GA, we reveal an Nrf2-mediated protective effect against MMC-induced cell death that depends on extracellular signal-regulated kinase (ERK) signaling. In total, our results reveal an additional beneficial effect of the Nrf2 activator 18α-GA, suggesting that this important phytochemical compound is a potential candidate in preventive and/or therapeutic schemes against conditions (such as aging) or diseases that are characterized by both oxidative stress and DNA damage.

Bacterial production and direct functional screening of expanded molecular libraries for discovering inhibitors of protein aggregation.

Protein misfolding and aggregation are associated with a many human disorders, including Alzheimer's and Parkinson's diseases. Toward increasing the effectiveness of early-stage drug discovery for these conditions, we report a bacterial platform that enables the biosynthesis of molecular libraries with expanded diversities and their direct functional screening for discovering protein aggregation inhibitors. We illustrate this approach by performing, what is to our knowledge, the largest functional screen of small-size molecular entities described to date. We generated a combinatorial library of ~200 million drug-like, cyclic peptides and rapidly screened it for aggregation inhibitors against the amyloid-ß peptide (Aß42), linked to Alzheimer's disease. Through this procedure, we identified more than 400 macrocyclic compounds that efficiently reduce Aß42 aggregation and toxicity in vitro and in vivo. Finally, we applied a combination of deep sequencing and mutagenesis analyses to demonstrate how this system can rapidly determine structure-activity relationships and define consensus motifs required for bioactivity.

Sugar-derived AGEs accelerate pharyngeal pumping rate and increase the lifespan of Caenorhabditis elegans.

All living organisms are normally undergoing aging. Dietary habits constitute the main environmental factor that may accelerate or decelerate this process. Advanced glycation end products (AGEs) are constituents of dietary products that are consumed daily, such as bread and milk. Although AGEs have been widely regarded as toxic agents, recent studies seem to contradict this view: they either find no adverse effects of AGEs or even attribute beneficial properties to them. The aim of our study was to investigate the effects of sugar-derived AGEs on organismal lifespan using as a model the nematode Caenorhabditis elegans. Exposure to sugar-derived AGEs prolonged the lifespan of wild type animals; this lifespan extension was accompanied by an enhanced pharyngeal pumping rate. We demonstrate that elevation of the pharyngeal pumping rate depends on W06A7.4 and eat-4 expression, as well as on daf-16, which encodes a FOXO family transcription factor. Our results suggest that sugar-derived AGEs modulate the lifespan of C. elegans at least in part through transcriptional regulation of pharyngeal pumping throughout the animals' lifespan.