Resveratrol supplementation at old age reverts changes associated with aging in inflammatory, oxidative and apoptotic markers in rat heart

Purpose Aging is known to play a critical role in the etiopathogenesis of several diseases. Among them, cardiovascular disorders are especially relevant since they are becoming the first cause of death in western countries. Resveratrol is a polyphenolic compound that has been shown to exert beneficial effects at different levels, including neuronal and cardiovascular protection. Those effects of resveratrol are related, at least in part, to its antioxidant and anti-inflammatory properties. In the current investigation we were interested in exploring whether the positive effects of resveratrol at cardiac level were taking place even when the supplementation started in already old animals. Methods Old male rats were supplemented with resveratrol during 10 weeks. Using RT-PCR, we analyzed the effects of resveratrol supplementation on the expression of different genes related to inflammation, oxidative stress and apoptosis in rat heart. Results Resveratrol reverted age-related changes in inflammatory, oxidative and apoptotic markers in the rat heart. Among others, the expression of two major inflammatory markers, INF-gamma and TNF-alpha and two oxidative markers, heme oxygenase-1 and nitric oxide synthase, were increased with aging, and resveratrol supplementation reduced the level of some of these to those observed in the heart of young animals. Moreover, age-related changes in apoptotic markers in rat heart tend to be also reverted by resveratrol treatment. Conclusion Our results suggest that resveratrol might exert beneficial effects as an anti-aging compound to revert age-related changes in cardiac function.Peer reviewe

Replication fork rescue in mammalian mitochondria

Replication stalling has been associated with the formation of pathological mitochondrial DNA (mtDNA) rearrangements. Yet, almost nothing is known about the fate of stalled replication intermediates in mitochondria. We show here that replication stalling in mitochondria leads to replication fork regression and mtDNA double-strand breaks. The resulting mtDNA fragments are normally degraded by a mechanism involving the mitochondrial exonuclease MGME1, and the loss of this enzyme results in accumulation of linear and recombining mtDNA species. Additionally, replication stress promotes the initiation of alternative replication origins as an apparent means of rescue by fork convergence. Besides demonstrating an interplay between two major mechanisms rescuing stalled replication forks - mtDNA degradation and homology-dependent repair - our data provide evidence that mitochondria employ similar mechanisms to cope with replication stress as known from other genetic systems.Peer reviewe

ALS and Parkinson's disease genes CHCHD10 and CHCHD2 modify synaptic transcriptomes in human iPSC-derived motor neurons

Mitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson's disease. They form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Mitochondria in knockout iPSC and motor neurons sustain ultrastructure but show increased proton leakage and respiration, and reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that the absence of either CHCHD2 or CHCHD10 alters mitochondrial respiration in human motor neurons, inducing similar compensatory responses. Thus, pathogenic mechanisms may involve loss of synaptic function resulting from defective energy metabolism.Peer reviewe

Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons

Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot-Marie-Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.Peer reviewe

Human IP3 receptor triple knockout stem cells remain pluripotent despite altered mitochondrial metabolism

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ER Ca2+-release channels that control a broad set of cellular processes. Animal models lacking IP3Rs in different combinations display severe developmental phenotypes. Given the importance of IP3Rs in human diseases, we investigated their role in human induced pluripotent stem cells (hiPSC) by developing single IP3R and triple IP3R knockouts (TKO). Genome edited TKO-hiPSC lacking all three IP3R isoforms, IP3R1, IP3R2, IP3R3, failed to generate Ca2+ signals in response to agonists activating GPCRs, but retained stemness and pluripotency. Steady state metabolite profiling and flux analysis of TKO-hiPSC indicated distinct alterations in tricarboxylic acid cycle metabolites consistent with a deficiency in their pyruvate utilization via pyruvate dehydrogenase, shifting towards pyruvate carboxylase pathway. These results demonstrate that IP3Rs are not essential for hiPSC identity and pluripotency but regulate mitochondrial metabolism. This set of knockout hiPSC is a valuable resource for investigating IP3Rs in human cell types of interest.Peer reviewe

Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism

Tausta: Mitokondriaalisen ATP6-geenin koodaama proteiini on yksi ATP-syntaasin alayksiköistä. Tämän geenin mutaatiot aiheuttavat useita neurologisia sairauksia vaihtelevin fenotyypein. Näitä ovat esimerkiksi Leighn syndrooma ja NARP sekä erilaiset ataksiat ja neuropatiat. ATP-syntaasi on avainasemassa solujen ATP:n tuotannossa ja lisäksi sen dimerisaatio on oleellista mitokondrioiden kristojen muodostumisen kannalta. Tavoitteet: Tutkimme uuden heteroplasmisen MT-ATP6-geenin mutaation (m.9154C>T) toiminnallisia vaikutuksia solutasolla. Metodit: Mutaatiota kantavan potilaan fenotyyppiin kuuluivat perifeerinen neuropatia, ataksia, sekä IgA-nefropatia. Keräsimme potilaalta solunäytteitä ja mallinsimme mutaatiota erilaistamalla potilaalle spesifejä indusoituja pluripotentteja kantasoluja motoneuroneiksi. Lisäksi arvioimme mutaation vaikutuksia molekyylirakenteeseen ja -stabiliteettiin simulaatiomenetelmin. Tulokset: MT-ATP6-geenin mutaation seurauksena ATP-syntaasi ei muodostunut normaalisti ja aiheutti muutoksia mitokondriaalisten kristojen morfologiassa. Tutkimuksessamme havaittiin useita heteroplasmian raja-arvoja, joiden ylittyessä ilmeni muutoksia solujen uudelleen ohjelmoinnissa, neurogeneesissä Notch-geenin yliaktivaatioon liittyen sekä kypsien motoneuronien laktaattimetaboliassa. Johtopäätökset: Nämä tulokset viittaavat siihen, että ATP-syntaasin säätelemällä mitokondrioiden morfologialla on kriittinen rooli ihmisen solujen erilaistumisprosessissa, sekä motoneuronien metaboliassa. Tämä saattaa olla oleellista mitokondrioiden roolin ja mekanismien ymmärtämisessä perifeeristen neuropatioiden yhteydessä

Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism

Tausta: Mitokondriaalisen ATP6-geenin koodaama proteiini on yksi ATP-syntaasin alayksiköistä. Tämän geenin mutaatiot aiheuttavat useita neurologisia sairauksia vaihtelevin fenotyypein. Näitä ovat esimerkiksi Leighn syndrooma ja NARP sekä erilaiset ataksiat ja neuropatiat. ATP-syntaasi on avainasemassa solujen ATP:n tuotannossa ja lisäksi sen dimerisaatio on oleellista mitokondrioiden kristojen muodostumisen kannalta. Tavoitteet: Tutkimme uuden heteroplasmisen MT-ATP6-geenin mutaation (m.9154C>T) toiminnallisia vaikutuksia solutasolla. Metodit: Mutaatiota kantavan potilaan fenotyyppiin kuuluivat perifeerinen neuropatia, ataksia, sekä IgA-nefropatia. Keräsimme potilaalta solunäytteitä ja mallinsimme mutaatiota erilaistamalla potilaalle spesifejä indusoituja pluripotentteja kantasoluja motoneuroneiksi. Lisäksi arvioimme mutaation vaikutuksia molekyylirakenteeseen ja -stabiliteettiin simulaatiomenetelmin. Tulokset: MT-ATP6-geenin mutaation seurauksena ATP-syntaasi ei muodostunut normaalisti ja aiheutti muutoksia mitokondriaalisten kristojen morfologiassa. Tutkimuksessamme havaittiin useita heteroplasmian raja-arvoja, joiden ylittyessä ilmeni muutoksia solujen uudelleen ohjelmoinnissa, neurogeneesissä Notch-geenin yliaktivaatioon liittyen sekä kypsien motoneuronien laktaattimetaboliassa. Johtopäätökset: Nämä tulokset viittaavat siihen, että ATP-syntaasin säätelemällä mitokondrioiden morfologialla on kriittinen rooli ihmisen solujen erilaistumisprosessissa, sekä motoneuronien metaboliassa. Tämä saattaa olla oleellista mitokondrioiden roolin ja mekanismien ymmärtämisessä perifeeristen neuropatioiden yhteydessä