On the relevance of mitochondrial fusions for the accumulation of mitochondrial deletion mutants: A modelling study

The molecular mechanisms underlying the aging process are still unclear, but the clonal accumulation of mitochondrial deletion mutants is one of the prime candidates. An important question for the mitochondrial theory of aging is to discover how defective organelles might be selected at the expense of wild-type mitochondria. We propose that mitochondrial fission and fusion events are of critical importance for resolving this apparent contradiction. We show that the occurrence of fusions removes the problems associated with the idea that smaller DNA molecules accumulate because they replicate in a shorter time – the survival of the tiny (SOT) hypothesis. Furthermore, stochastic simulations of mitochondrial replication, mutation and degradation show that two important experimental findings, namely the overall low mosaic pattern of oxidative phosphorylation (OXPHOS) impaired cells in old organisms and the distribution of deletion sizes, can be reproduced and explained by this hypothesis. Finally, we make predictions that can be tested experimentally to further verify our explanation for the age-related accumulation of mitochondrial deletion mutants

Mech Ageing Dev

Mitochondrial morphology is regulated in many cultured eukaryotic cells by fusion and fission of mitochondria. A tightly controlled balance between fission and fusion events is required to ensure normal mitochondrial and cellular functions. During ageing, mitochondria are undergoing significant changes on the functional and morphological level. The effect of ageing on fusion and fission of mitochondria and consequences of altered fission and fusion activity are still unknown although theoretical models on ageing consider the significance of these processes. Human umbilical vein endothelial cells (HUVECs) have been established as a cell culture model to follow mitochondrial activity and dysfunction during the ageing process. Mitochondria of old and postmitotic HUVECs showed distinct alterations in overall morphology and fine structure, and furthermore, loss of mitochondrial membrane potential. In parallel, a decrease of intact mitochondrial DNA (mtDNA) was observed. Fission and fusion activity of mitochondria were quantified in living cells. Mitochondria of old HUVECs showed a significant and equal decrease of both fusion and fission activity indicating that these processes are sensitive to ageing and could contribute to the accumulation of damaged mitochondria during ageing

Primary skin fibroblasts as a model of Parkinson's disease

Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues

Restriction of trophic factors and nutrients induces PARKIN expression

Parkinson’s disease (PD) is the most frequent neurodegenerative movement disorder and manifests at old age. While many details of its pathogenesis remain to be elucidated, in particular the protein and mitochondrial quality control during stress responses have been implicated in monogenic PD variants. Especially the mitochondrial kinase PINK1 and the ubiquitin ligase PARKIN are known to cooperate in autophagy after mitochondrial damage. As autophagy is also induced by loss of trophic signaling and PINK1 gene expression is modulated after deprivation of cytokines, we analyzed to what extent trophic signals and starvation stress regulate PINK1 and PARKIN expression. Time course experiments with serum deprivation and nutrient starvation of human SH-SY5Y neuroblastoma cells and primary mouse neurons demonstrated phasic induction of PINK1 transcript up to twofold and PARKIN transcript levels up to sixfold. The corresponding threefold starvation induction of PARKIN protein was limited by its translocation to lysosomes. Analysis of primary mouse cells from PINK1-knockout mice indicated that PARKIN induction and lysosomal translocation occurred independent of PINK1. Suppression of the PI3K-Akt-mTOR signaling by pharmacological agents modulated PARKIN expression accordingly. In conclusion, this expression survey demonstrates that PARKIN and PINK1 are coregulated during starvation and suggest a role of both PD genes in response to trophic signals and starvation stress

Mitochondrial fragmentation and superoxide anion production in coronary endothelial cells from a mouse model of type 1 diabetes

Mitochondria frequently change their shapes by fusion and fission and these morphological dynamics play important roles in mitochondrial function and development as well as programmed cell death. The goal of this study is to investigate whether: (1) mitochondria in mouse coronary endothelial cells (MCECs) isolated from diabetic mice exhibit increased fragmentation; and (2) chronic treatment with a superoxide anion (O2 −) scavenger has a beneficial effect on mitochondrial fragmentation in MCECs. MCECs were freshly isolated and lysed for protein measurement, or cultured to determine mitochondrial morphology and O2 − production. For the ex vivo hyperglycaemia experiments, human coronary endothelial cells were used. Elongated mitochondrial tubules were observed in MCECs isolated from control mice, whereas mitochondria in MCECs from diabetic mice exhibited augmented fragmentation. The level of optic atrophy 1 (OPA1) protein, which leads to mitochondrial fusion, was significantly decreased, while dynamin-related protein 1 (DRP1), which leads to mitochondrial fission, was significantly increased in MCECs from diabetic mice. Diabetic MCECs exhibited significantly higher O2 − concentrations in cytosol and mitochondria than control MCECs. Administration of the O2 − scavenger TEMPOL to diabetic mice for 4 weeks led to a significant decrease in mitochondrial fragmentation without altering the levels of OPA1 and DRP1 proteins in MCECs. High-glucose treatment for 24 h significantly induced mitochondrial fragmentation, which was restored by TEMPOL treatment. In addition, excess O2 − production, either in cytosol or in mitochondria, significantly increased mitochondrial fragmentation. These data suggest that lowering the O2 − concentration can restore the morphological change in mitochondria and may help improve mitochondrial function in diabetic MCECs

Anomalous Diffusion Induced by Cristae Geometry in the Inner Mitochondrial Membrane

Diffusion of inner membrane proteins is a prerequisite for correct functionality of mitochondria. The complicated structure of tubular, vesicular or flat cristae and their small connections to the inner boundary membrane impose constraints on the mobility of proteins making their diffusion a very complicated process. Therefore we investigate the molecular transport along the main mitochondrial axis using highly accurate computational methods. Diffusion is modeled on a curvilinear surface reproducing the shape of mitochondrial inner membrane (IM). Monte Carlo simulations are carried out for topologies resembling both tubular and lamellar cristae, for a range of physiologically viable crista sizes and densities. Geometrical confinement induces up to several-fold reduction in apparent mobility. IM surface curvature per se generates transient anomalous diffusion (TAD), while finite and stable values of projected diffusion coefficients are recovered in a quasi-normal regime for short- and long-time limits. In both these cases, a simple area-scaling law is found sufficient to explain limiting diffusion coefficients for permeable cristae junctions, while asymmetric reduction of the junction permeability leads to strong but predictable variations in molecular motion rate. A geometry-based model is given as an illustration for the time-dependence of diffusivity when IM has tubular topology. Implications for experimental observations of diffusion along mitochondria using methods of optical microscopy are drawn out: a non-homogenous power law is proposed as a suitable approach to TAD. The data demonstrate that if not taken into account appropriately, geometrical effects lead to significant misinterpretation of molecular mobility measurements in cellular curvilinear membranes

Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer’s disease mouse model

BACKGROUND: Deposition of amyloid beta (Aß) and hyperphosphorylated tau along with glial cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer's disease (AD). In recent years, impairment of autophagy has been identified as another important feature contributing to AD progression. Therefore, the potential of the autophagy activator spermidine, a small body-endogenous polyamine often used as dietary supplement, was assessed on Aß pathology and glial cell-mediated neuroinflammation. RESULTS: Oral treatment of the amyloid prone AD-like APPPS1 mice with spermidine reduced neurotoxic soluble Aß and decreased AD-associated neuroinflammation. Mechanistically, single nuclei sequencing revealed AD-associated microglia to be the main target of spermidine. This microglia population was characterized by increased AXL levels and expression of genes implicated in cell migration and phagocytosis. A subsequent proteome analysis of isolated microglia confirmed the anti-inflammatory and cytoskeletal effects of spermidine in APPPS1 mice. In primary microglia and astrocytes, spermidine-induced autophagy subsequently affected TLR3- and TLR4-mediated inflammatory processes, phagocytosis of Aß and motility. Interestingly, spermidine regulated the neuroinflammatory response of microglia beyond transcriptional control by interfering with the assembly of the inflammasome. CONCLUSIONS: Our data highlight that the autophagy activator spermidine holds the potential to enhance Aß degradation and to counteract glia-mediated neuroinflammation in AD pathology

Proteomic profiling of mitochondria: what does it tell us about the ageing brain?

Mitochondrial dysfunction is evident in numerous neurodegenerative and age-related disorders. It has also been linked to cellular ageing, however our current understanding of the mitochondrial changes that occur are unclear. Functional studies have made some progress reporting reduced respiration, dynamic structural modifications and loss of membrane potential, though there are conflicts within these findings. Proteomic analyses, together with functional studies, are required in order to profile the mitochondrial changes that occur with age and can contribute to unravelling the complexity of the ageing phenotype. The emergence of improved protein separation techniques, combined with mass spectrometry analyses has allowed the identification of age and cell-type specific mitochondrial changes in energy metabolism, antioxidants, fusion and fission machinery, chaperones, membrane proteins and biosynthesis pathways. Here, we identify and review recent data from the analyses of mitochondria from rodent brains. It is expected that knowledge gained from understanding age-related mitochondrial changes of the brain should lead to improved biomarkers of normal ageing and also age-related disease progression

Parkinson Phenotype in Aged PINK1-Deficient Mice Is Accompanied by Progressive Mitochondrial Dysfunction in Absence of Neurodegeneration

Background Parkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Methodology/Principal Findings Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of alpha-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Conclusion Thus, aging Pink1 -/- mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death