Drug synergy as a strategy for compression of morbidity in a Caenorhabditis elegans model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. AD is a multifactorial disease with simultaneous occurrence of several connected pathological processes including mitochondrial dysfunction and impaired proteostasis. Most of these are also implicated in organismal aging per se. The presence of separable pathological conditions poses the opportunity to try combination treatments that target these different processes separately. This approach may provide an effective strategy to target AD; therefore, we investigated whether a combination of metformin (targeting mitochondria and energy metabolism) and lithium (targeting proteostasis) could result in synergistic benefits. In this perspective paper, we looked for benefits in lifespan and healthspan using a transgenic nematode strain, GRU102, which expresses pan-neuronal human amyloid-beta (Aß). Individually, metformin and lithium extended the lifespan of both non-transgenic GRU101 controls and GRU102. Combination treatment using metformin and lithium did not result in any synergistic increase in GRU102 lifespan, but this treatment did result in a significant compression of morbidity when compared with each individual drug, resulting in relative and absolute extension of healthspan. Despite over-expressing pathogenic human Aß in their neurons, GRU102 worms treated with the combination treatment enjoyed longer lifespans and significantly compressed morbidity, even compared with untreated non-transgenic animals. These findings suggest combination treatment as a strategy to compress morbidity, and highlight the distinction between healthspan and lifespan.

Metabolic stress is a primary pathogenic event in transgenic Caenorhabditis elegans expressing pan-neuronal human amyloid beta.

Alzheimer's disease (AD) is the most common neurodegenerative disease affecting the elderly worldwide. Mitochondrial dysfunction has been proposed as a key event in the etiology of AD. We have previously modeled amyloid-beta (Aß)-induced mitochondrial dysfunction in a transgenic Caenorhabditis elegans strain by expressing human Aß peptide specifically in neurons (GRU102). Here, we focus on the deeper metabolic changes associated with this Aß-induced mitochondrial dysfunction. Integrating metabolomics, transcriptomics and computational modeling, we identify alterations in Tricarboxylic Acid (TCA) cycle metabolism following even low-level Aß expression. In particular, GRU102 showed reduced activity of a rate-limiting TCA cycle enzyme, alpha-ketoglutarate dehydrogenase. These defects were associated with elevation of protein carbonyl content specifically in mitochondria. Importantly, metabolic failure occurred before any significant increase in global protein aggregate was detectable. Treatment with an anti-diabetes drug, Metformin, reversed Aß-induced metabolic defects, reduced protein aggregation and normalized lifespan of GRU102. Our results point to metabolic dysfunction as an early and causative event in Aß-induced pathology and a promising target for intervention.

Identification of a previously undetected metabolic defect in the Complex II Caenorhabditis elegans mev-1 mutant strain using respiratory control analysis.

Hypometabolism may play an important role in the pathogenesis of ageing and ageing-related diseases. The nematode Caenorhabditis elegans offers the opportunity to study "living mitochondria" in a small (~1 mm) animal replete with a highly stereotypical, yet complex, anatomy and physiology. Basal oxygen consumption rate is often employed as a proxy for energy metabolism in this context. This parameter is traditionally measured using single-chamber Clark electrodes without the addition of metabolic modulators. Recently, multi-well oxygen electrodes, facilitating addition of metabolic modulators and hence study of respiratory control during different mitochondrial respiration states, have been developed. However, only limited official protocols exist for C. elegans, and key limitations of these techniques are therefore unclear. Following modification and testing of some of the existing protocols, we used these methods to explore mitochondrial bioenergetics in live nematodes of an electron transfer chain Complex II mutant strain, mev-1, and identified a previously undetected metabolic defect. We find that mev-1 mutants cannot respond adequately to increased energy demands, suggesting that oxidative phosphorylation is more severely impaired in these animals than has previously been appreciated.

Energy crisis precedes global metabolic failure in a novel Caenorhabditis elegans Alzheimer Disease model.

Alzheimer Disease (AD) is a progressive neurological disorder characterized by the deposition of amyloid beta (Aß), predominantly the Aß1-42 form, in the brain. Mitochondrial dysfunction and impaired energy metabolism are important components of AD pathogenesis. However, the causal and temporal relationships between them and AD pathology remain unclear. Using a novel C. elegans AD strain with constitutive neuronal Aß1-42 expression that displays neuromuscular defects and age-dependent behavioural dysfunction reminiscent of AD, we have shown that mitochondrial bioenergetic deficit is an early event in AD pathogenesis, preceding dysfunction of mitochondrial electron transfer chain (ETC) complexes and the onset of global metabolic failure. These results are consistent with an emerging view that AD may be a metabolic neurodegenerative disease, and also confirm that Aß-driven metabolic and mitochondrial effects can be reproduced in organisms separated by large evolutionary distances.

Caenorhabditis elegans: What We Can and Cannot Learn from Aging Worms.

SIGNIFICANCE: The nematode Caenorhabditis elegans is a widely used model organism for research into aging. However, nematodes diverged from other animals between 600 and 1300 million years ago. Beyond the intuitive impression that some aspects of aging appear to be universal, is there evidence that insights into the aging process of nematodes may be applicable to humans? RECENT ADVANCES: There have been a number of results in nematodes that appear to contradict long-held beliefs about mechanisms and causes of aging. For example, ablation of several key antioxidant systems has often failed to result in lifespan shortening in C. elegans. CRITICAL ISSUES: While it is clear that some central signaling pathways controlling lifespan are broadly conserved across large evolutionary distances, it is less clear to what extent downstream molecular mechanisms of aging are conserved. In this review we discuss the biology of C. elegans and mammals in the context of aging and age-dependent diseases. We consider evidence from studies that attempt to investigate basic, possibly conserved mechanisms of aging especially in the context of the free radical theory of aging. Practical points, such as the need for blinding of lifespan studies and for appropriate biomarkers, are also considered. FUTURE DIRECTIONS: As data on the aging process(es) in different organisms increase, it is becoming increasingly clear that there are both conserved (public) and private aspects to aging. It is important to explore the dividing lines between these two aspects and to be aware of the large gray areas in-between.

In vivo evaluation of resorbable bone graft substitutes in beagles: histological properties.

Calcium phosphate cement (CPC) is a promising material for use in minimally invasive surgery for bone defect repairs due to its bone-like apatitic final setting product, biocompatibility, bioactivity, self-setting characteristics, low setting temperature, adequate stiffness, and easy shaping into complicated geometrics. However, even though CPC is stable in vivo, the resorption rate of this bone cement is very slow and its long setting time poses difficulties for clinical use. Calcium sulfate dehydrate (CSD) has been used as a filler material and/or as a replacement for cancellous bone grafts due to its biocompatibility. However, it is resorbed too quickly to be optimal for bone regeneration. This study examines the invivo response of a hydroxyapatite (HA), [apatitic phase (AP)]/calcium sulfate (CSD) composite using different ratios in the mandibular premolar sockets of beagles. The HA (AP)/CSD composite materials were prepared in the ratios of 30/70, 50/50, and 70/30 and then implanted into the mandibular premolar sockets for terms of 5 and 10 weeks. The control socket was left empty. The study shows better new bone morphology and more new bone area in the histological and the histomorphometric study of the HA (AP)/CSD in the 50/50 ratio.

Mitochondria-targeted antioxidants and metabolic modulators as pharmacological interventions to slow ageing.

Populations in many nations today are rapidly ageing. This unprecedented demographic change represents one of the main challenges of our time. A defining property of the ageing process is a marked increase in the risk of mortality and morbidity with age. The incidence of cancer, cardiovascular and neurodegenerative diseases increases non-linearly, sometimes exponentially with age. One of the most important tasks in biogerontology is to develop interventions leading to an increase in healthy lifespan (health span), and a better understanding of basic mechanisms underlying the ageing process itself may lead to interventions able to delay or prevent many or even all age-dependent conditions. One of the putative basic mechanisms of ageing is age-dependent mitochondrial deterioration, closely associated with damage mediated by reactive oxygen species (ROS). Given the central role that mitochondria and mitochondrial dysfunction play not only in ageing but also in apoptosis, cancer, neurodegeneration and other age-related diseases there is great interest in approaches to protect mitochondria from ROS-mediated damage. In this review, we explore strategies of targeting mitochondria to reduce mitochondrial oxidative damage with the aim of preventing or delaying age-dependent decline in mitochondrial function and some of the resulting pathologies. We discuss mitochondria-targeted and -localized antioxidants (e.g.: MitoQ, SkQ, ergothioneine), mitochondrial metabolic modulators (e.g. dichloroacetic acid), and uncouplers (e.g.: uncoupling proteins, dinitrophenol) as well as some alternative future approaches for targeting compounds to the mitochondria, including advances from nanotechnology.

Mitochondrial changes in ageing Caenorhabditis elegans--what do we learn from superoxide dismutase knockouts?

One of the most popular damage accumulation theories of ageing is the mitochondrial free radical theory of ageing (mFRTA). The mFRTA proposes that ageing is due to the accumulation of unrepaired oxidative damage, in particular damage to mitochondrial DNA (mtDNA). Within the mFRTA, the "vicious cycle" theory further proposes that reactive oxygen species (ROS) promote mtDNA mutations, which then lead to a further increase in ROS production. Recently, data have been published on Caenorhabditis elegans mutants deficient in one or both forms of mitochondrial superoxide dismutase (SOD). Surprisingly, even double mutants, lacking both mitochondrial forms of SOD, show no reduction in lifespan. This has been interpreted as evidence against the mFRTA because it is assumed that these mutants suffer from significantly elevated oxidative damage to their mitochondria. Here, using a novel mtDNA damage assay in conjunction with related, well established damage and metabolic markers, we first investigate the age-dependent mitochondrial decline in a cohort of ageing wild-type nematodes, in particular testing the plausibility of the "vicious cycle" theory. We then apply the methods and insights gained from this investigation to a mutant strain for C. elegans that lacks both forms of mitochondrial SOD. While we show a clear age-dependent, linear increase in oxidative damage in WT nematodes, we find no evidence for autocatalytic damage amplification as proposed by the "vicious cycle" theory. Comparing the SOD mutants with wild-type animals, we further show that oxidative damage levels in the mtDNA of SOD mutants are not significantly different from those in wild-type animals, i.e. even the total loss of mitochondrial SOD did not significantly increase oxidative damage to mtDNA. Possible reasons for this unexpected result and some implications for the mFRTA are discussed.

Ageing in nematodes: do antioxidants extend lifespan in Caenorhabditis elegans?

Antioxidants are often investigated as a promising strategy for extending lifespan. Accordingly, there is significant interest in novel antioxidant compounds derived from natural sources such as plant extracts. However, because lifespan studies are laborious and expensive to conduct, candidate compounds are frequently selected based simply on their in vitro antioxidant efficacy, with the implicit assumption that in vitro antioxidants are also in vivo antioxidants, and that in vivo antioxidants will decrease functionally relevant oxidative damage and thereby extend lifespan. We investigated the validity of these assumptions in the model organism, Caenorhabditis elegans. Nematodes were exposed to 6 plant extracts, selected out of a total of 34 based on a simple in vitro antioxidant assay. We found no correlation between in vitro and in vivo antioxidant capacities. Antioxidant efficacies were also not predictive of lifespan benefits. Further studies into those extracts that produced significant lifespan extension indicated that a direct antioxidant effect is unlikely to be the main factor responsible for the modulation of nematode lifespan.