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BioRxiv : the Preprint Server For... Jun 2024Mitochondrial creatine kinases are key players in maintaining energy homeostasis in cells by working in conjunction with cytosolic creatine kinases for energy transport...
Mitochondrial creatine kinases are key players in maintaining energy homeostasis in cells by working in conjunction with cytosolic creatine kinases for energy transport from mitochondria to cytoplasm. High levels of MtCK observed in Her2+ breast cancer and inhibition of breast cancer cell growth by substrate analog, cyclocreatine, indicate dependence of cancer cells on the 'energy shuttle' for cell growth and survival. Hence, understanding the key mechanistic features of creatine kinases and their inhibition plays an important role in the development of cancer therapeutics. Herein, we present the mutational and structural investigation on understudied ubiquitous mitochondrial creatine kinase (uMtCK). Our cryo-EM structures and biochemical data on uMtCK showed closure of the loop comprising residue His61 is specific to and relies on creatine binding and the reaction mechanism of phosphoryl transfer depends on electrostatics in the active site. In addition, the previously identified covalent inhibitor CKi showed inhibition in breast cancer BT474 cells, however our biochemical and structural data indicated that CKi is not a potent inhibitor for breast cancer due to strong dependency on the covalent link formation and inability to induce conformational changes upon binding.
PubMed: 38948784
DOI: 10.1101/2024.06.18.598884 -
BioRxiv : the Preprint Server For... Jun 2024The protein alpha-synuclein (αSyn) plays a critical role in the pathogenesis of synucleinopathy, which includes Parkinson's disease and multiple system atrophy, and...
UNLABELLED
The protein alpha-synuclein (αSyn) plays a critical role in the pathogenesis of synucleinopathy, which includes Parkinson's disease and multiple system atrophy, and mounting evidence suggests that lipid dyshomeostasis is a critical phenotype in these neurodegenerative conditions. Previously, we identified that αSyn localizes to mitochondria-associated endoplasmic reticulum membranes (MAMs), temporary functional domains containing proteins that regulate lipid metabolism, including the de novo synthesis of phosphatidylserine. In the present study, we have analyzed the lipid composition of postmortem human samples, focusing on the substantia nigra pars compacta of Parkinson's disease and controls, as well as three less affected brain regions of Parkinson's donors. To further assess synucleinopathy-related lipidome alterations, similar analyses were performed on the striatum of multiple system atrophy cases. Our data show region-and disease-specific changes in the levels of lipid species. Specifically, our data revealed alterations in the levels of specific phosphatidylserine species in brain areas most affected in Parkinson's disease. Some of these alterations, albeit to a lesser degree, are also observed multiples system atrophy. Using induced pluripotent stem cell-derived neurons, we show that αSyn contributes to regulating phosphatidylserine metabolism at MAM domains, and that αSyn dosage parallels the perturbation in phosphatidylserine levels. Our results support the notion that αSyn pathophysiology is linked to the dysregulation of lipid homeostasis, which may contribute to the vulnerability of specific brain regions in synucleinopathy. These findings have significant therapeutic implications.
SIGNIFICANCE STATEMENT
Synucleinopathy is a complex group of neurodegenerative disorders whose causes and underlying mechanisms remain unknown. In this work, we examined synucleinopathy postmortem brain samples and patient-derived neuron models and identified the functional impairment of the mitochondrial-associated endoplasmic reticulum membrane (MAM) domain, which facilitates lipid regulation. The protein alpha-synuclein is associated with synucleinopathy and increasing levels result in the mislocalization of this protein and the disruption of MAM domains, which, in turn, results in lipid and membrane composition alterations. Specifically, we report that increased alpha-synuclein expression impairs the regulation of phosphatidylserine synthase 2 and the levels of phosphatidylserine in cellular membranes from affected cells. Our study offers mechanistic insight tying alpha-synuclein pathology and lipid dysregulation as seminal factors in synucleinopathy, which may have pathogenic and therapeutic implications.
PubMed: 38948777
DOI: 10.1101/2024.06.17.599406 -
BioRxiv : the Preprint Server For... Jun 2024Sjögren's disease (SjD) is a common exocrine disorder typified by chronic inflammation and dryness, but also profound fatigue, suggesting a pathological basis in...
OBJECTIVES
Sjögren's disease (SjD) is a common exocrine disorder typified by chronic inflammation and dryness, but also profound fatigue, suggesting a pathological basis in cellular bioenergetics. In healthy states, damaged or dysfunctional mitochondrial components are broken down and recycled by mitophagy, a specialized form of autophagy. In many autoimmune disorders, however, evidence suggests that dysfunctional mitophagy allows poorly functioning mitochondria to persist and contribute to a cellular milieu with elevated reactive oxygen species. We hypothesized that mitophagic processes are dysregulated in SjD and that dysfunctional mitochondria contribute to overall fatigue. We sought to link fatigue with mitochondrial dysfunction directly in SjD, heretofore unexamined, and further sought to assess the pathogenic extent and implications of dysregulated mitophagy in SjD.
METHODS
We isolated pan T cells via negative selection from the peripheral blood mononuclear cells of 17 SjD and 8 age-matched healthy subjects, all of whom completed fatigue questionnaires prior to phlebotomy. Isolated T cells were analyzed for mitochondrial oxygen consumption rate (OCR) and glycolysis using Seahorse, and linear correlations with fatigue measures were assessed. A mitophagy transcriptional signature in SjD was identified by reanalysis of whole-blood microarray data from 190 SjD and 32 healthy subjects. Differential expression analyses were performed by case/control and subgroup analyses comparing SjD patients by mitophagy transcriptional cluster against healthy subjects followed by bioinformatic interpretation using gene set enrichment analysis.
RESULTS
Basal OCR, ATP-linked respiration, maximal respiration, and reserve capacity were significantly lower in SjD compared to healthy subjects with no observed differences in non-mitochondrial respiration, basal glycolysis, or glycolytic stress. SjD lymphocytic mitochondria show structural alterations compared to healthy subjects. Fatigue scores related to pain/discomfort in SjD correlated with the altered OCR. Results from subgroup analyses by mitophagic SjD clusters revealed highly variable inter-cluster differentially expressed genes (DEGs) and expanded the number of SjD-associated gene targets by tenfold within the same dataset.
CONCLUSION
Mitochondrial dysfunction, associated with fatigue, is a significant problem in SjD and warrants further investigation.
PubMed: 38948768
DOI: 10.1101/2024.06.17.598269 -
BioRxiv : the Preprint Server For... Jun 2024Local metabolic demand within cells varies widely and the extent to which individual mitochondria can be specialized to meet these functional needs is unclear. We...
Local metabolic demand within cells varies widely and the extent to which individual mitochondria can be specialized to meet these functional needs is unclear. We examined the subcellular distribution of MICOS, a spatial and functional organizer of mitochondria, and discovered that it dynamically enriches at the tip of a minor population of mitochondria in the cell periphery that we term METEORs. METEORs have a unique composition; MICOS enrichment sites are depleted of mtDNA and matrix proteins and contain high levels of the Ca2+ uniporter MCU, suggesting a functional specialization. METEORs are also enriched for the myosin MYO19, which promotes their trafficking to a small subset of filopodia. We identify a positive correlation between the length of filopodia and the presence of METEORs and show that elimination of mitochondria from filopodia impairs cellular motility. Our data reveal a novel type of mitochondrial heterogeneity and suggest compositionally specialized mitochondria support cell migration.
PubMed: 38948746
DOI: 10.1101/2024.06.21.600105 -
BioRxiv : the Preprint Server For... Jun 2024Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration,...
Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration, and heritable pathologies such as Barth syndrome. Cardiolipin, the signature phospholipid of the mitochondrion promotes proper cristae morphology, bioenergetic functions, and directly affects metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in the gene are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impact metabolic flux through the tricarboxylic acid cycle and associated pathways in yeast. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of C -pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a wild-type strain to mitochondria from a Δ strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin, and mitochondria from a Δ strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the C-label from the pyruvate substrate was distributed through about twelve metabolites. Several of the identified metabolites were specific to yeast pathways, including branched chain amino acids and fusel alcohol synthesis. Most metabolites showed similar kinetics amongst the different strains but mevalonate and α-ketoglutarate, as well as the NAD+/NADH couple measured in separate nuclear magnetic resonance experiments, showed pronounced differences. Taken together, the results show that cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.
PubMed: 38948727
DOI: 10.1101/2024.06.18.599628 -
BioRxiv : the Preprint Server For... Jun 2024Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL,...
BACKGROUND
Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL, ), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear.
METHODS
We utilized human heart tissue samples from healthy donor hearts and patients with hypertrophic cardiomyopathy. Further, we used CRISPR/Cas9 gene editing to inactivate in cardiomyocytes of MyH6-Cas9 mice. positron emission tomography and stable isotope tracer labeling were used to quantify metabolic flux changes in response to the loss of ACL. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics and proteomics. Experimental data were integrated into computational modeling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level.
RESULTS
Here, we show that in mice, ACL drives metabolic adaptation in the heart to sustain contractile function, histone acetylation, and lipid modulation. Notably, we show that loss of ACL increases glucose oxidation while maintaining fatty acid oxidation. isotope tracing experiments revealed a reduced efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. We demonstrate that YAP inactivation facilitates ACL deficiency. Computational flux analysis and integrative multi-omics analysis indicate that loss of ACL induces alternative isocitrate dehydrogenase 1 flux to compensate.
CONCLUSIONS
This study mechanistically delineates how cardiac metabolism compensates for suppressed citrate metabolism in response to ACL loss and uncovers metabolic vulnerabilities in the heart.
PubMed: 38948703
DOI: 10.1101/2024.06.21.600099 -
BioRxiv : the Preprint Server For... Jun 2024Relevance-based selectivity and high energy cost are two distinct features of long-term memory (LTM) formation that warrant its default inhibition. Spaced repetition of...
Relevance-based selectivity and high energy cost are two distinct features of long-term memory (LTM) formation that warrant its default inhibition. Spaced repetition of learning is a highly conserved cognitive mechanism that can lift this inhibition. Here, we questioned how the spacing effect integrates experience selection and energy efficiency at the cellular and molecular levels. We showed in that spaced training triggers LTM formation by extending over several hours an increased mitochondrial metabolic activity in neurons of the associative memory center, the mushroom bodies (MBs). We found that this effect is mediated by PKCδ, a member of the so-called 'novel PKC' family of enzymes, which uncovers the critical function of PKCδ in neurons as a regulator of mitochondrial metabolism for LTM. Additionally, PKCδ activation and translocation to mitochondria result from LTM-specific dopamine signaling on MB neurons. By bridging experience-dependent neuronal circuit activity with metabolic modulation of memory-encoding neurons, PKCδ signaling binds the cognitive and metabolic constraints underlying LTM formation into a unified gating mechanism.
PubMed: 38948698
DOI: 10.1101/2023.10.06.561186 -
Frontiers in Pharmacology 2024The escalation of global population aging has accentuated the prominence of senile diabetes mellitus (SDM) as a consequential public health concern. Oxidative stress and...
Network analysis combined with experimental assessment to explore the therapeutic mechanisms of New Shenqi Pills formula targeting mitochondria on senile diabetes mellitus.
BACKGROUND
The escalation of global population aging has accentuated the prominence of senile diabetes mellitus (SDM) as a consequential public health concern. Oxidative stress and chronic inflammatory cascades prevalent in individuals with senile diabetes significantly amplify disease progression and complication rates. Traditional Chinese Medicine (TCM) emerges as a pivotal player in enhancing blood sugar homeostasis and retarding complication onset in the clinical management of senile diabetes. Nonetheless, an evident research gap persists regarding the integration of TCM's renal tonification pharmacological mechanisms with experimental validation within the realm of senile diabetes therapeutics.
AIMS
The objective of this study was to investigate the mechanisms of action of New Shenqi Pills (SQP) in the treatment of SDM and make an experimental assessment.
METHODS
Network analysis is used to evaluate target pathways related to SQP and SDM. Mitochondrial-related genes were obtained from the MitoCarta3.0 database and intersected with the common target genes of the disease and drugs, then constructing a protein-protein interaction (PPI) network making use of the GeneMANIA database. Representative compounds in the SQP were quantitatively measured using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to ensure quality control and quantitative analysis of the compounds. A type 2 diabetes mice (C57BL/6) model was used to investigate the pharmacodynamics of SQP. The glucose lowering efficacy of SQP was assessed through various metrics including body weight and fasting blood glucose (FBG). To elucidate the modulatory effects of SQP on pancreatic beta cell function, we measured oral glucose tolerance test (OGTT), insulin histochemical staining and tunel apoptosis detection, then assessed the insulin-mediated phosphoinositide 3-kinase (PI3K)/protein kinase A (Akt)/glycogen synthase kinase-3β (GSK-3β) pathway in diabetic mice via Western blotting. Additionally, we observe the structural changes of the nucleus, cytoplasmic granules and mitochondria of pancreatic islet β cells.
RESULTS
In this investigation, we identified a total of 1876 genes associated with senile diabetes, 278 targets of SQP, and 166 overlapping target genes, primarily enriched in pathways pertinent to oxidative stress response, peptide response, and oxygen level modulation. Moreover, an intersection analysis involving 1,136 human mitochondrial genes and comorbidity targets yielded 15 mitochondria-related therapeutic targets. Quality control assessments and quantitative analyses of SQP revealed the predominant presence of five compounds with elevated concentrations: Catalpol, Cinnamon Aldehyde, Rehmanthin D, Trigonelline, and Paeonol Phenol. Vivo experiments demonstrated notable findings. Relative to the control group, mice in the model group exhibited significant increases in body weight and fasting blood glucose levels, alongside decreased insulin secretion and heightened islet cell apoptosis. Moreover, β-cells nuclear condensation and mitochondrial cristae disappearance were observed, accompanied by reduced expression levels of p-GSK-3β protein in islet cells ( < 0.05 or < 0.01). Conversely, treatment groups administered SQP and Rg displayed augmented expressions of the aforementioned protein markers ( < 0.05 or < 0.01), alongside preserved mitochondrial cristae structure in islet β cells.
CONCLUSION
Our findings suggest that SQP can ameliorate diabetes by reducing islet cell apoptosis and resist oxidative stress. These insulin-mediated PI3K/AKT/GSK-3β pathway plays an important regulatory role in this process.
PubMed: 38948458
DOI: 10.3389/fphar.2024.1339758 -
World Journal of Experimental Medicine Jun 2024Mitochondrial dysfunction is a key driver of cardiovascular disease (CVD) in metabolic syndrome and diabetes. This dysfunction promotes the production of reactive oxygen... (Review)
Review
Mitochondrial dysfunction is a key driver of cardiovascular disease (CVD) in metabolic syndrome and diabetes. This dysfunction promotes the production of reactive oxygen species (ROS), which cause oxidative stress and inflammation. Angiotensin II, the main mediator of the renin-angiotensin-aldosterone system, also contributes to CVD by promoting ROS production. Reduced activity of sirtuins (SIRTs), a family of proteins that regulate cellular metabolism, also worsens oxidative stress. Reduction of energy production by mitochondria is a common feature of all metabolic disorders. High SIRT levels and 5' adenosine monophosphate-activated protein kinase signaling stimulate hypoxia-inducible factor 1 beta, which promotes ketosis. Ketosis, in turn, increases autophagy and mitophagy, processes that clear cells of debris and protect against damage. Sodium-glucose cotransporter-2 inhibitors (SGLT2i), a class of drugs used to treat type 2 diabetes, have a beneficial effect on these mechanisms. Randomized clinical trials have shown that SGLT2i improves cardiac function and reduces the rate of cardiovascular and renal events. SGLT2i also increase mitochondrial efficiency, reduce oxidative stress and inflammation, and strengthen tissues. These findings suggest that SGLT2i hold great potential for the treatment of CVD. Furthermore, they are proposed as anti-aging drugs; however, rigorous research is needed to validate these preliminary findings.
PubMed: 38948421
DOI: 10.5493/wjem.v14.i2.91519 -
Sichuan Da Xue Xue Bao. Yi Xue Ban =... May 2024Female fertility gradually decreases with the increase in women's age. The underlying reasons include the decline in the quantity and quality of oocytes. Oocyte aging is...
OBJECTIVE
Female fertility gradually decreases with the increase in women's age. The underlying reasons include the decline in the quantity and quality of oocytes. Oocyte aging is an important manifestation of the decline in oocyte quality, including oocyte aging before ovulation and oocyte aging after ovulation. Currently, few studies have been done to examine oocyte aging, and the relevant molecular mechanisms are not fully understood. Therefore, we used zebrafish as a model to investigate oocyte aging. Three different age ranges of female zebrafish were selected to mate with male zebrafish of the best breeding age. In this way, we studied the effects of maternal age-related oocyte aging on fertility and investigated the potential molecular mechanisms behind maternal age-related fertility decline.
METHODS
Eight female zebrafish aged between 158 and 195 d were randomly selected for the 6-month age group (180±12) d, 8 female zebrafish aged between 330 and 395 d were randomly selected for the 12-month age group (360±22) d, and 8 female zebrafish aged between 502 and 583 d were randomly selected for the 18-month age group (540±26) d. Male zebrafish of (180±29) d were randomly selected from zebrafish aged between 158 and 195 d and mated with female zebrafish in each group. Each mating experiment included 1 female zebrafish and 1 male zebrafish. Zebrafish embryos produced by the mating experiments were collected and counted. The embryos at 4 hours post-fertilization were observed under the microscope, the total number of embryos and the number of unfertilized embryos were counted, and the fertilization rate was calculated accordingly. The numbers of malformed embryos and dead embryos were counted 24 hours after fertilization, and the rates of embryo malformation and mortality were calculated accordingly. The primary outcome measure was the embryo fertilization rate, and the secondary outcome measures were the number of embryos per spawn (the total number of embryos laid within 1.5 hours after the beginning of mating and reproduction of the zebrafish), embryo mortality, and embryo malformation rate. The outcome measures of each group were compared. The blastocyst embryos of female zebrafish from each group born after mating with male zebrafish in their best breeding period were collected for transcriptomics analysis. Fresh oocytes of female zebrafish in each group were collected for transcriptomics analysis to explore the potential molecular mechanisms of maternal age-related fertility decline.
RESULTS
Compared with that of the 6-month group (94.9%±3.6%), the embryo fertilization rate of the 12-month group (92.3%±4.2%) showed no significant difference, but that of the 18-month group (86.8%±5.5%) decreased significantly (<0.01). In addition, the fertilization rate in the 18-month group was significantly lower than that in the 12-month group (<0.05). Compared with that of the 6-month group, the embryo mortality of the female zebrafish in the 12-month group and that in the 18-month group were significantly higher than that in the 6-month group (<0.000 1, <0.001). There was no significant difference in the number of embryos per spawn or in the embryo malformation rate among the three groups. The results of the transcriptomics analysis of blastocyst embryos showed that some genes, including , , , , a, , , , etc, were differentially expressed in the 12-month group or the 18-month group compared with their expression levels in the 6-month group. According to the KEGG enrichment analysis, these differentially expressed genes (DEGs) were significantly enriched in the MAPK signaling pathway, the phosphatidylinositol signaling system, and the fatty acid degradation and histidine metabolism pathway (<0.05). The analysis of the expression trends of the genes expressed differentially among the three groups (the 6-month group, the 12-month group, and the 18-month group in turn) showed that the gene expression trends of , , , and , which were involved in Fanconi anemia pathway, were statistically significant (<0.05). In the results of oocyte transcriptomics analysis, the genes that were differentially expressed in the 12-month group or the 18-month group compared with the 6-month group were mainly enriched in cell adhesion molecules and the protein digestion and absorption pathway (<0.05). The results of the trends of gene expression in the zebrafish oocytes of the three groups (the 6-month group, the 12-month group, and the 18-month group in turn) showed that three kinds of gene expression trends of declining fertility with growing maternal age had significant differences (<0.05). Further analysis of the three significantly differential expression trends showed 51 DEGs related to mitochondria and 5 DEGs related to telomere maintenance and DNA repair, including , , , , etc.
CONCLUSION
With the increase in the maternal age of the zebrafish, the embryo fertilization rate decreased significantly and the embryo mortality increased significantly. In addition, with the increase in the maternal age of the zebrafish, the expression of mitochondria and telomere-related genes, such as , , , and , in female zebrafish oocytes decreased gradually. Maternal age may be a factor contributing to the decrease in oocyte fertilization ability and the increase in early embryo mortality. Maternal age-related oocyte aging affects the fertility and embryo development of the offspring.
Topics: Animals; Zebrafish; Oocytes; Female; Fertility; Male; Transcriptome; Maternal Age; Aging; Models, Animal
PubMed: 38948296
DOI: 10.12182/20240560205