Mitochondrial Dysfunction: A Key Player in Brain Aging and Diseases.

Mitochondria are thought to have become incorporated within the eukaryotic cell approximately 2 billion years ago and play a role in a variety of cellular processes, such as energy production, calcium buffering and homeostasis, steroid synthesis, cell growth, and apoptosis, as well as inflammation and ROS production. Considering that mitochondria are involved in a multitude of cellular processes, mitochondrial dysfunction has been shown to play a role within several age-related diseases, including cancers, diabetes (type 2), and neurodegenerative diseases, although the underlying mechanisms are not entirely understood. The significant increase in lifespan and increased incidence of age-related diseases over recent decades has confirmed the necessity to understand the mechanisms by which mitochondrial dysfunction impacts the process of aging and age-related diseases. In this review, we will offer a brief overview of mitochondria, along with structure and function of this important organelle. We will then discuss the cause and consequence of mitochondrial dysfunction in the aging process, with a particular focus on its role in inflammation, cognitive decline, and neurodegenerative diseases, such as Huntington's disease, Parkinson's disease, and Alzheimer's disease. We will offer insight into therapies and interventions currently used to preserve or restore mitochondrial functioning during aging and neurodegeneration.

Mitochondrial Dysfunction and Protein Homeostasis in Aging: Insights from a Premature-Aging Mouse Model.

Mitochondrial dysfunction has been implicated in aging and age-related disorders. Disturbed-protein homeostasis and clearance of damaged proteins have also been linked to aging, as well as to neurodegenerative diseases, cancers, and metabolic disorders. However, since mitochondrial oxidative phosphorylation, ubiquitin-proteasome, and autophagy-lysosome systems are tightly interdependent, it is not understood whether the facets observed in aging are the causes or consequences of one or all of these failed processes. We therefore used prematurely aging mtDNA-mutator mice and normally aging wild-type littermates to elucidate whether mitochondrial dysfunction per se is sufficient to impair cellular protein homeostasis similarly to that which is observed in aging. We found that both mitochondrial dysfunction and normal aging affect the ubiquitin-proteasome system in a tissue-dependent manner, whereas only normal aging markedly impairs the autophagy-lysosome system. Thus, our data show that the proteostasis network control in the prematurely aging mtDNA-mutator mouse differs in certain aspects from that found in normal aging. Taken together, our findings suggest that severe mitochondrial dysfunction drives an aging phenotype associated with the impairment of certain components of the protein homeostasis machinery, while others, such as the autophagy-lysosome system, are not affected or only minimally affected. Taken together, this shows that aging is a multifactorial process resulting from alterations of several integrated biological processes; thus, manipulating one process at the time might not be sufficient to fully recapitulate all changes associated with normal aging.

Acute Exposure to Microplastics Induced Changes in Behavior and Inflammation in Young and Old Mice.

Environmental pollutants have become quite ubiquitous over the past two centuries; of those, plastics, and in particular, microplastics (<5 mm), are among the most pervasive pollutants. Microplastics (MPs) have found their way into the air, water system, and food chain and are either purposely produced or are derived from the breakdown of larger plastic materials. Despite the societal advancements that plastics have allowed, the mismanagement of plastic waste has become a pressing global issue. Pioneering studies on MPs toxicity have shown that exposure to MPs induces oxidative stress, inflammation, and decreased cell viability in marine organisms. Current research suggests that these MPs are transported throughout the environment and can accumulate in human tissues; however, research on the health effects of MPs, especially in mammals, is still very limited. This has led our group to explore the biological and cognitive consequences of exposure to MPs in a rodent model. Following a three-week exposure to water treated with fluorescently-labeled pristine polystyrene MPs, young and old C57BL/6J mice were assessed using behavioral assays, such as open-field and light-dark preference, followed by tissue analyses using fluorescent immunohistochemistry, Western blot, and qPCR. Data from these assays suggest that short-term exposure to MPs induces both behavioral changes as well as alterations in immune markers in liver and brain tissues. Additionally, we noted that these changes differed depending on age, indicating a possible age-dependent effect. These findings suggest the need for further research to better understand the mechanisms by which microplastics may induce physiological and cognitive changes.

Effect of apparatus characteristics on anxiety-like behavior in young adult and old mice of both sexes assessed by the elevated plus maze assay.

Incidence of anxiety-like disorders in humans has been shown to decrease with aging; however, it is still under debate whether there are similarities in mice, which would support the use of mouse models in understanding the neuronal network changes that regulate anxiety-like behavior in aging. One of the most common tests used to assess anxiety-like behavior in laboratory animals is the elevated plus maze (EPM). Although several variables, such as room brightness and width of the maze arms, have been shown to influence the spontaneous animal behavior during the EPM test, none of these variables have ever been evaluated in aging to understand their possible differential effect on younger and older mice. We therefore decided to investigate the effect of apparatus construction on young adult and old mice of both sexes on EPM test performance. Our results show that distance traveled during the test is the variable that is most affected by apparatus characteristics independent of age and sex. We also found that apparatus construction was key in demonstrating that old mice spent more time and had relatively more entries in the open arms as compared to young mice, suggesting a decrease in anxiety-like behavior with age. Taken together, our data demonstrate that EPM apparatus characteristics dramatically affect test outcome with a wider arm apparatus being more effective in revealing age-dependent changes in anxiety-like behavior, thus, suggesting the use of a wider arm EPM when conducting aging studies in mice.

Mitochondria in Ageing and Diseases: Partie Deux.

The past several decades has seen a huge expansion of the knowledge and research of mitochondrial dysfunction and the role it plays in ageing and age-related diseases [...].

Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Mitochondrial and metabolic dysfunction in ageing and age-related diseases.

Organismal ageing is accompanied by progressive loss of cellular function and systemic deterioration of multiple tissues, leading to impaired function and increased vulnerability to death. Mitochondria have become recognized not merely as being energy suppliers but also as having an essential role in the development of diseases associated with ageing, such as neurodegenerative and cardiovascular diseases. A growing body of evidence suggests that ageing and age-related diseases are tightly related to an energy supply and demand imbalance, which might be alleviated by a variety of interventions, including physical activity and calorie restriction, as well as naturally occurring molecules targeting conserved longevity pathways. Here, we review key historical advances and progress from the past few years in our understanding of the role of mitochondria in ageing and age-related metabolic diseases. We also highlight emerging scientific innovations using mitochondria-targeted therapeutic approaches.

Histological-Based Stainings using Free-Floating Tissue Sections.

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

Voluntary exercise normalizes the proteomic landscape in muscle and brain and improves the phenotype of progeroid mice.

The accumulation of mitochondrial DNA (mtDNA) mutations is a suspected driver of aging and age-related diseases, but forestalling these changes has been a major challenge. One of the best-studied models is the prematurely aging mtDNA mutator mouse, which carries a homozygous knock-in of a proofreading deficient version of the catalytic subunit of mtDNA polymerase-γ (PolgA). We investigated how voluntary exercise affects the progression of aging phenotypes in this mouse, focusing on mitochondrial and protein homeostasis in both brain and peripheral tissues. Voluntary exercise significantly ameliorated several aspects of the premature aging phenotype, including decreased locomotor activity, alopecia, and kyphosis, but did not have major effects on the decreased lifespan of mtDNA mutator mice. Exercise also decreased the mtDNA mutation load. In-depth tissue proteomics revealed that exercise normalized the levels of about half the proteins, with the majority involved in mitochondrial function and nuclear-mitochondrial crosstalk. There was also a specific increase in the nuclear-encoded proteins needed for the tricarboxylic acid cycle and complex II, but not in mitochondrial-encoded oxidative phosphorylation proteins, as well as normalization of enzymes involved in coenzyme Q biosynthesis. Furthermore, we found tissue-specific alterations, with brain coping better as compared to muscle and with motor cortex being better protected than striatum, in response to mitochondrial dysfunction. We conclude that voluntary exercise counteracts aging in mtDNA mutator mice by counteracting protein dysregulation in muscle and brain, decreasing the mtDNA mutation burden in muscle, and delaying overt aging phenotypes.

Thyroid hormone regulates fibronectin expression through the activation of the hypoxia inducible factor 1.

Thyroid hormones regulate gene expression via both canonical and non-canonical signaling. Hyperthyroidism is associated with elevated plasma levels of fibronectin (FN): in this study we elucidate the molecular mechanism through which triiodothyronine (T3) regulates FN and demonstrate that T3 induces FN expression via a non-canonical pathway by activating hypoxia-inducible factor-1 (HIF-1). We found that T3 treatment increased cellular and secreted FN in human hepatoma cells (HepG2) and human dermal fibroblasts (HF) via the PI3K/Akt/HIF-1 pathway. The inhibition of either Akt phosphorylation with wortmannin or HIF-1 with YC1 in both cell types prevented HIF-1α synthesis and FN positive regulation upon T3 treatment. We showed that HIF-1α overexpression per se was sufficient to up-regulate FN in both cell lines as demonstrated by the transient transfection of both the constitutively active and wild-type forms of HIF-1α. Our data demonstrate the involvement of the PI3K/Akt/HIF-1 pathway in mediating T3 induced FN up-regulation.

Mitochondria in Ageing and Diseases: The Super Trouper of the Cell.

The past decade has witnessed an explosion of knowledge regarding how mitochondrial dysfunction may translate into ageing and disease phenotypes, as well as how it is modulated by genetic and lifestyle factors.[...].

Mitochondrial and Ubiquitin Proteasome System Dysfunction in Ageing and Disease: Two Sides of the Same Coin?

Mitochondrial dysfunction and impairment of the ubiquitin proteasome system have been described as two hallmarks of the ageing process. Additionally, both systems have been implicated in the etiopathogenesis of many age-related diseases, particularly neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Interestingly, these two systems are closely interconnected, with the ubiquitin proteasome system maintaining mitochondrial homeostasis by regulating organelle dynamics, the proteome, and mitophagy, and mitochondrial dysfunction impairing cellular protein homeostasis by oxidative damage. Here, we review the current literature and argue that the interplay of the two systems should be considered in order to better understand the cellular dysfunction observed in ageing and age-related diseases. Such an approach may provide valuable insights into molecular mechanisms underlying the ageing process, and further discovery of treatments to counteract ageing and its associated diseases. Furthermore, we provide a hypothetical model for the heterogeneity described among individuals during ageing.

Maternally transmitted mitochondrial DNA mutations can reduce lifespan.

We recently showed that germline transmission of mitochondrial DNA mutations via the oocyte cause aggravation of aging phenotypes in prematurely aging mtDNA mutator (PolgA(mut/mut)) mice. We discovered that 32% of these mice also exhibit stochastic disturbances of brain development, when maternal mtDNA mutations were combined with homozygosity for the PolgA mutation, leading to de novo somatic mtDNA mutations. Surprisingly, we also found that maternally transmitted mtDNA mutations can cause mild premature aging phenotypes also in mice with a wild-type nuclear DNA background. We now report that in addition to the early onset of aging phenotypes, these mice, burdened only by low levels of mtDNA mutations transmitted via the germline, also exhibit reduced longevity. Our data thus demonstrate that low levels of maternally inherited mtDNA mutations when present during development can affect both overall health and lifespan negatively.

Germline mitochondrial DNA mutations aggravate ageing and can impair brain development.

Ageing is due to an accumulation of various types of damage, and mitochondrial dysfunction has long been considered to be important in this process. There is substantial sequence variation in mammalian mitochondrial DNA (mtDNA), and the high mutation rate is counteracted by different mechanisms that decrease maternal transmission of mutated mtDNA. Despite these protective mechanisms, it is becoming increasingly clear that low-level mtDNA heteroplasmy is quite common and often inherited in humans. We designed a series of mouse mutants to investigate the extent to which inherited mtDNA mutations can contribute to ageing. Here we report that maternally transmitted mtDNA mutations can induce mild ageing phenotypes in mice with a wild-type nuclear genome. Furthermore, maternally transmitted mtDNA mutations lead to anticipation of reduced fertility in mice that are heterozygous for the mtDNA mutator allele (PolgA(wt/mut)) and aggravate premature ageing phenotypes in mtDNA mutator mice (PolgA(mut/mut)). Unexpectedly, a combination of maternally transmitted and somatic mtDNA mutations also leads to stochastic brain malformations. Our findings show that a pre-existing mutation load will not only allow somatic mutagenesis to create a critically high total mtDNA mutation load sooner but will also increase clonal expansion of mtDNA mutations to enhance the normally occurring mosaic respiratory chain deficiency in ageing tissues. Our findings suggest that maternally transmitted mtDNA mutations may have a similar role in aggravating aspects of normal human ageing.

Interaction of gamma-herpesvirus genome maintenance proteins with cellular chromatin.

The capacity of gamma-herpesviruses to establish lifelong infections is dependent on the expression of genome maintenance proteins (GMPs) that tether the viral episomes to cellular chromatin and allow their persistence in latently infected proliferating cells. Here we have characterized the chromatin interaction of GMPs encoded by viruses belonging to the genera Lymphocryptovirus (LCV) and Rhadinovirus (RHV). We found that, in addition to a similar diffuse nuclear localization and comparable detergent resistant interaction with chromatin in transfected cells, all GMPs shared the capacity to promote the decondensation of heterochromatin in the A03-1 reporter cell line. They differed, however, in their mobility measured by fluorescence recovery after photobleaching (FRAP), and in the capacity to recruit accessory molecules required for the chromatin remodeling function. While the AT-hook containing GMPs of LCVs were highly mobile, a great variability was observed among GMPs encoded by RHV, ranging from virtually immobile to significantly reduced mobility compared to LCV GMPs. Only the RHV GMPs recruited the bromo- and extra terminal domain (BET) proteins BRD2 and BRD4 to the site of chromatin remodeling. These findings suggest that differences in the mode of interaction with cellular chromatin may underlie different strategies adopted by these viruses for reprogramming of the host cells during latency.

The Epstein-Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins.

Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate-dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation.

The Gly-Ala repeat modulates the interaction of Epstein-Barr virus nuclear antigen-1 with cellular chromatin.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) plays a pivotal role in EBV infection by anchoring the viral episome to cellular DNA, which regulates replication and partitioning in dividing cells. Here, we have used fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques to study the interaction of EBNA1 with cellular chromatin in interphase and mitosis. This analysis revealed that while EBNA1 is highly mobile in both conditions, mobility is significantly reduced in mitosis when an immobile fraction is also detected. The N-terminal chromatin-targeting module of EBNA1 includes two Gly-Arg rich domains (GR1 and GR2) separated by a Gly-Ala repeat (GAr) of variable length. Using a set of deletion mutants and GFP-fusion reporters, we found that the GR domains cooperatively determine the mobility of EBNA1, whereas mobility is increased by the interposed GAr in a length-dependent manner. These findings highlight a previously unrecognized property of the interaction of EBNA1 with cellular chromatin that may fine-tune its function in the maintenance of viral latency.

Oxidized low-density lipoproteins impair endothelial function by inhibiting non-genomic action of thyroid hormone-mediated nitric oxide production in human endothelial cells.

BACKGROUND: Thyroid hormone (TH) plays an important role in the modulation of cardiac function, including contractility and systemic vascular resistance (SVR). 3,5,3'-triiodothyronine (T(3)), the active form of TH, induces the activation of endothelial nitric oxide synthase via PI3K/AKT non-genomic signaling. Hypothyroidism is associated with an increase in SVR and serum low-density lipoproteins (LDL) levels, and accumulation of oxidized LDL (oxLDL) may impair endothelial-dependent vascular relaxation. The aim of this study was to investigate the effects of both native LDL (nLDL) and oxLDL on T(3)-mediated AKT phosphorylation, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) production in human endothelial cells. METHODS: Human umbilical vein endothelial cells were exposed to either nLDL or oxLDL for 3 hours and then stimulated with T(3) (10(-7) M) or pretreated with an antioxidant mixture of vitamins E and C for 12 hours before treatment with LDL. An analysis of AKT phosphorylation was performed by Western blot, and NO production was evaluated by using 4,5-diaminofluorescein diacetate. Intracellular production of cGMP was measured by enzymatic immunoassay. LDL oxidation was carried out by incubating LDL with CuSO(4), and α-tocopherol content of LDL was evaluated by high-performance liquid chromatography. RESULTS: OxLDL impaired T(3)-mediated AKT phosphorylation at serine 473 and significantly decreased the production of both NO (oxLDL+T(3) vs. T(3), 9.79±0.5 AU vs. 80.75±2.8 AU, mean±standard deviation, p<0.0001) and cGMP. Furthermore, pretreatment with the antioxidant mixture obviated the inhibitory effect of LDL on T(3) action. CONCLUSIONS: The results of this study demonstrate that oxLDL may contribute to a blunting of the non-genomic action of T(3) and impair the effect of T(3) on NO and cGMP production in endothelial cells. These data suggest that oxLDL, apart from inducing the atherosclerotic process, may also promote a mechanism of peripheral resistance to T(3,) further amplifying the impact of hypothyroidism on endothelial function by increasing SVR.

Ubiquitin C-terminal hydrolase-L1 interacts with adhesion complexes and promotes cell migration, survival, and anchorage independent growth.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that is highly expressed in neurons and overexpressed in several human cancers. UCH-L1 has been implicated in the regulation of phenotypic properties associated with malignant cell growth but the underlying mechanisms have not been elucidated. By comparing cells expressing catalytically active or inactive versions of UCH-L1, we found that the active enzyme enhances cell adhesion, spreading, and migration; inhibits anoikis; and promotes anchorage independent growth. UCH-L1 accumulates at the motile edge of the cell membrane during the initial phases of adhesion, colocalizes with focal adhesion kinase (FAK), p120-catenin, and vinculin, and enhances the formation of focal adhesions, which correlates with enhanced FAK activation. The involvement of UCH-L1 in the regulation of focal adhesions and adherens junctions is supported by coimmunoprecipitation with key components of these complexes, including FAK, paxillin, p120-catenin, ß-catenin, and vinculin. UCH-L1 stabilizes focal adhesion signaling in the absence of adhesion, as assessed by reduced caspase-dependent cleavage of FAK following cell detachment and sustained activity of the AKT signaling pathway. These findings offer new insights on the molecular interactions through which the deubiquitinating enzyme regulates the survival, proliferation, and metastatic potential of malignant cells.