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The eukaryotic glyoxalase system consists of two enzymatic components, glyoxalase I (lactoylglutathionelyase) and glyoxalase II (hydroxyacylglutathione hydrolase). These enzymes are dedicated to the removal of toxic alpha-oxoaldehydes like methylglyoxal (MG). MG is formed as a by-product of glycolysis and MG toxicity results from its damaging capability leading to modifications of proteins, lipids and nucleic acids. An efficient removal of MG appears to be essential to ensure cellular functionality and viability. Here we study the effects of the genetic modulation of genes encoding the components of the glyoxalase system in the filamentous ascomycete and aging model Podospora anserina. Overexpression of PaGlo1 leads to a lifespan reduction on glucose rich medium, probably due to depletion of reduced glutathione. Deletion of PaGlo1 leads to hypersensitivity against MG added to the growth medium. A beneficial effect on lifespan is observed when both PaGlo1 and PaGlo2 are overexpressed and the corresponding strains are grown on media containing increased glucose concentrations. Notably, the double mutant has a ‘healthy’ phenotype without physiological impairments. Moreover, PaGlo1/PaGlo2_OEx strains are not long-lived on media containing standard glucose concentrations suggesting a tight correlation between the efficiency and capacity to remove MG within the cell, the level of available glucose and lifespan. Overall, our results identify the up-regulation of both components of the glyoxalase system as an effective intervention to increase lifespan in P. anserina. Key words: Podospora anserina, aging, lifespan, glycation, glucose, methylglyoxal, advanced glycation end products
Mitochondrial dysfunction is well documented in presymptomatic brain tissue with Parkinson's disease (PD). Identification of the autosomal recessive variant PARK6 caused by loss-of-function mutations in the mitochondrial kinase PINK1 provides an opportunity to dissect pathogenesis. Although PARK6 shows clinical differences to PD, the induction of alpha-synuclein "Lewy" pathology by PINK1-deficiency proves that mitochondrial pathomechanisms are relevant for old-age PD. Mitochondrial dysfunction is induced by PINK1 deficiency even in peripheral tissues unaffected by disease, consistent with the ubiquitous expression of PINK1. It remains unclear whether this dysfunction is due to PINK1-mediated phosphorylation of proteins inside or outside mitochondria. Although PINK1 deficiency affects the mitochondrial fission/fusion balance, cell stress is required in mammals to alter mitochondrial dynamics and provoke apoptosis. Clearance of damaged mitochondria depends on pathways including PINK1 and Parkin and is critical for postmitotic neurons with high energy demand and cumulative stress, providing a mechanistic concept for the tissue specificity of disease.
A recent report showed PINK1 transcript levels to be up- or down-regulated by the gain or loss of Ataxin-2 function, respectively, in human blood, in a human neural cell line and in mouse tissues. These observations may have profound implications for the regulation of cell growth and may be medically exploited for the treatment of cancer and neural atrophy...
Ataxin-2 (Atxn2)-knock-out mice show branched chain amino acids and fatty acids pathway alterations
(2016)
Human Ataxin-2 (ATXN2) gene locus variants have been associated with obesity, diabetes mellitus type 1,and hypertension in genome-wide association studies, whereas mouse studies showed the knock-out of Atxn2 to lead to obesity, insulin resistance, and dyslipidemia. Intriguingly, the deficiency of ATXN2 protein orthologs in yeast and flies rescues the neurodegeneration process triggered by TDP-43 and Ataxin-1 toxicity. To understand the molecular effects of ATXN2 deficiency by unbiased approaches, we quantified the global proteome and metabolome of Atxn2-knock-out mice with label-free mass spectrometry. In liver tissue, significant downregulations of the proteins ACADS, ALDH6A1, ALDH7A1, IVD, MCCC2, PCCA, OTC, together with bioinformatic enrichment of downregulated pathways for branched chain and other amino acid metabolism, fatty acids, and citric acid cycle were observed. Statistical trends in the cerebellar proteome and in the metabolomic profiles supported these findings. They are in good agreement with recent claims that PBP1, the yeast ortholog of ATXN2, sequestrates the nutrient sensor TORC1 in periods of cell stress. Overall, ATXN2 appears to modulate nutrition and metabolism, and its activity changes are determinants of growth excess or cell atrophy.
Ataxin-2 (human gene symbol ATXN2) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide–sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters.
Parkinson’s disease (PD) is characterized by distinct motor and non-motor symptoms. Sleep disorders are the most frequent and challenging non-motor symptoms in PD patients, and there is growing evidence that they are a consequence of disruptions within the circadian system. PD is characterized by a progressive degeneration of the dorsal vagal nucleus and midbrain dopaminergic neurons together with an imbalance of many other neurotransmitters. Mutations in α-synuclein (SNCA), a protein modulating SNARE complex-dependent neurotransmission, trigger dominantly inherited PD variants and sporadic cases of PD. The A53T SNCA missense mutation is associated with an autosomal dominant early-onset familial PD. To test whether this missense mutation affects the circadian system, we analyzed the spontaneous locomotor behavior of non-transgenic wildtype mice and transgenic mice overexpressing mutant human A53T α-synuclein (A53T). The mice were subjected to entrained- and free-running conditions as well as to experimental jet lag. Furthermore, the vesicular glutamate transporter 2 (VGLUT2) in the suprachiasmatic nucleus (SCN) was analyzed by immunohistochemistry. Free-running circadian rhythm and, thus, circadian rhythm generation, were not affected in A53T mice. A53T mice entrained to the light–dark cycle, however, with an advanced phase angle of 2.65 ± 0.5 h before lights off. Moreover, re-entrainment after experimental jet lag was impaired in A53T mice. Finally, VGLUT2 immunoreaction was reduced in the SCN of A53T mice. These data suggest an impaired light entrainment of the circadian system in A53T mice.
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.
Parkinson's disease (PD) is a frequent neurodegenerative process in old age. Accumulation and aggregation of the lipid-binding SNARE complex component α-synuclein (SNCA) underlies this vulnerability and defines stages of disease progression. Determinants of SNCA levels and mechanisms of SNCA neurotoxicity have been intensely investigated. In view of the physiological roles of SNCA in blood to modulate vesicle release, we studied blood samples from a new large pedigree with SNCA gene duplication (PARK4 mutation) to identify effects of SNCA gain of function as potential disease biomarkers. Downregulation of complexin 1 (CPLX1) mRNA was correlated with genotype, but the expression of other Parkinson's disease genes was not. In global RNA-seq profiling of blood from presymptomatic PARK4 indviduals, bioinformatics detected significant upregulations for platelet activation, hemostasis, lipoproteins, endocytosis, lysosome, cytokine, Toll-like receptor signaling and extracellular pathways. In PARK4 platelets, stimulus-triggered degranulation was impaired. Strong SPP1, GZMH and PLTP mRNA upregulations were validated in PARK4. When analysing individuals with rapid eye movement sleep behavior disorder, the most specific known prodromal stage of general PD, only blood CPLX1 levels were altered. Validation experiments confirmed an inverse mutual regulation of SNCA and CPLX1 mRNA levels. In the 3′-UTR of the CPLX1 gene we identified a single nucleotide polymorphism that is significantly associated with PD risk. In summary, our data define CPLX1 as a PD risk factor and provide functional insights into the role and regulation of blood SNCA levels. The new blood biomarkers of PARK4 in this Turkish family might become useful for PD prediction.
Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)31. This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissuespecific effects, which may be partially alleviated by the induction of FBXW8.
The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5′-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.