Multi-omics identify xanthine as a pro-survival metabolite for nematodes with mitochondrial dysfunction.

Aberrant mitochondrial function contributes to the pathogenesis of various metabolic and chronic disorders. Inhibition of insulin/IGF-1 signaling (IIS) represents a promising avenue for the treatment of mitochondrial diseases, although many of the molecular mechanisms underlying this beneficial effect remain elusive. Using an unbiased multi-omics approach, we report here that IIS inhibition reduces protein synthesis and favors catabolism in mitochondrial deficient Caenorhabditis elegans We unveil that the lifespan extension does not occur through the restoration of mitochondrial respiration, but as a consequence of an ATP-saving metabolic rewiring that is associated with an evolutionarily conserved phosphoproteome landscape. Furthermore, we identify xanthine accumulation as a prominent downstream metabolic output of IIS inhibition. We provide evidence that supplementation of FDA-approved xanthine derivatives is sufficient to promote fitness and survival of nematodes carrying mitochondrial lesions. Together, our data describe previously unknown molecular components of a metabolic network that can extend the lifespan of short-lived mitochondrial mutant animals.

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling.

BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.

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.

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.

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.

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.

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.

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.

Systems biology identifies preserved integrity but impaired metabolism of mitochondria due to a glycolytic defect in Alzheimer's disease neurons.

Mitochondrial dysfunction is implicated in most neurodegenerative diseases, including Alzheimer's disease (AD). We here combined experimental and computational approaches to investigate mitochondrial health and bioenergetic function in neurons from a double transgenic animal model of AD (PS2APP/B6.152H). Experiments in primary cortical neurons demonstrated that AD neurons had reduced mitochondrial respiratory capacity. Interestingly, the computational model predicted that this mitochondrial bioenergetic phenotype could not be explained by any defect in the mitochondrial respiratory chain (RC), but could be closely resembled by a simulated impairment in the mitochondrial NADH flux. Further computational analysis predicted that such an impairment would reduce levels of mitochondrial NADH, both in the resting state and following pharmacological manipulation of the RC. To validate these predictions, we utilized fluorescence lifetime imaging microscopy (FLIM) and autofluorescence imaging and confirmed that transgenic AD neurons had reduced mitochondrial NAD(P)H levels at rest, and impaired power of mitochondrial NAD(P)H production. Of note, FLIM measurements also highlighted reduced cytosolic NAD(P)H in these cells, and extracellular acidification experiments showed an impaired glycolytic flux. The impaired glycolytic flux was identified to be responsible for the observed mitochondrial hypometabolism, since bypassing glycolysis with pyruvate restored mitochondrial health. This study highlights the benefits of a systems biology approach when investigating complex, nonintuitive molecular processes such as mitochondrial bioenergetics, and indicates that primary cortical neurons from a transgenic AD model have reduced glycolytic flux, leading to reduced cytosolic and mitochondrial NAD(P)H and reduced mitochondrial respiratory capacity.

A disease-associated Aifm1 variant induces severe myopathy in knockin mice.

OBJECTIVE: Mutations in the AIFM1 gene have been identified in recessive X-linked mitochondrial diseases. Functional and molecular consequences of these pathogenic AIFM1 mutations have been poorly studied in vivo. METHODS/RESULTS: Here we provide evidence that the disease-associated apoptosis-inducing factor (AIF) deletion arginine 201 (R200 in rodents) causes pathology in knockin mice. Within a few months, posttranslational loss of the mutant AIF protein induces severe myopathy associated with a lower number of cytochrome c oxidase-positive muscle fibers. At a later stage, Aifm1 (R200 del) knockin mice manifest peripheral neuropathy, but they do not show neurodegenerative processes in the cerebellum, as observed in age-matched hypomorphic Harlequin (Hq) mutant mice. Quantitative proteomic and biochemical data highlight common molecular signatures of mitochondrial diseases, including aberrant folate-driven one-carbon metabolism and sustained Akt/mTOR signaling. CONCLUSION: Our findings indicate metabolic defects and distinct tissue-specific vulnerability due to a disease-causing AIFM1 mutation, with many pathological hallmarks that resemble those seen in patients.

WAH-1/AIF regulates mitochondrial oxidative phosphorylation in the nematode Caenorhabditis elegans.

Impaired mitochondrial energy metabolism contributes to a wide range of pathologic conditions, including neurodegenerative diseases. Mitochondrial apoptosis-inducing factor (AIF) is required for the correct maintenance of mitochondrial electron transport chain. An emerging body of clinical evidence indicates that several mutations in the AIFM1 gene are causally linked to severe forms of mitochondrial disorders. Here we investigate the consequence of WAH-1/AIF deficiency in the survival of the nematode Caenorhabditis elegans. Moreover, we assess the survival of C. elegans strains expressing a disease-associated WAH-1/AIF variant. We demonstrate that wah-1 downregulation compromises the function of the oxidative phosphorylation system and reduces C. elegans lifespan. Notably, the loss of respiratory subunits induces a nuclear-encoded mitochondrial stress response independently of an evident increase of oxidative stress. Overall, our data pinpoint an evolutionarily conserved role of WAH-1/AIF in the maintenance of proper mitochondrial activity.

Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

The SWI/SNF subunit Bcl7a contributes to motor coordination and Purkinje cell function.

Chromatin remodelers have emerged as prominent regulators of epigenetic processes and potential drivers of various human pathologies. The multi-subunit chromatin-remodeling SWI/SNF complex determines gene expression programs and, consequently, contributes to the differentiation, maturation and plasticity of neurons. Here, we investigate the elusive biological function of Bcl7a and Bcl7b, two newly identified subunits of the SWI/SNF complex that are highly expressed throughout the brain. We generated ubiquitous and neuron-specific Bcl7a and Bcl7b single and double knockout mice. We provide evidence that Bcl7b is dispensable for animal survival as well as behavioral plasticity. Conversely, ubiquitous Bcl7a knockout results in perinatal lethality, while genetic deletion of Bcl7a in postmitotic neurons elicits motor abnormalities and affects dendritic branching of Purkinje cells, with no obvious synergistic relationship with Bcl7b. Collectively, our findings reveal novel insights into the cellular processes linked to BCL7-containing SWI/SNF complexes and their unrecognized roles in the brain.