Project description:Ischemic stroke induces pathological glycogen deposition in astrocytes, but its role in post-injury neural dysfunction remains undefined. We firstly intend to identify the glycogen granule proteome after ischemic stroke via glycogen pulldown for protein mass spectrometry detection. KEGG pathway analysis of the glycogen granule proteome revealed highly enriched mitochondrial-related functional signals, such as respiratory chain complexes, mitochondrial respirasomes, and mitochondrial membrane protein complexes. GO functional annotation of biological processes revealed enrichment of genes related to mitochondrial electron transport, respiratory chain complexes, and mitochondrial ATP synthesis. To explore detailed mitochondrial biological processes in detail, we summarized mitochondrial function gene sets from the mitoproteome database and intersected them with many glycogen-enriched proteins. The data revealed that glycogen stress granules are related mainly to oxidative phosphorylation subunits, assembly factors and electron carriers; mitochondrial dynamics (such as MFF, DNM1L, and GDAP1), homeostasis, and quality control (such as AFG3L2, CLPB, and ATAD3A). Evidence from our experimental data has shown that ATAD3A oligomerization requires the accumulation of astrocytic glycogen stress granules. Under physiological conditions, ATAD3A does not oligomerize, and mitochondria do not exhibit obvious dysfunction in nonglycogen stress granule-deposited cells. To explore what candidate partners are involved with ATAD3A. The proximity-biotinylating enzyme TurboID was reconstituted with ATAD3A to detect biotinylated proteins adjacent to ATAD3A. We next constructed a stable ATAD3A-TurboID cell line (or primary astrocytes). Pulldown biotinylated proteins were subjected to quantitative high-resolution mass spectrometry. Based on the above mass spectrometry data and a series of experimental results, we found that: glycogen-laden astrocytes in the ischemic penumbra undergo HDAC3-dependent mitochondrial fragmentation via a stress granule-mediated mechanism, exacerbating neuronal injury and hindering functional recovery. Mechanistic studies demonstrate that glycogen aggregates sequester cytoplasmic HDAC3, enabling its translocation to mitochondria. There, HDAC3 deacetylates outer mitochondrial membrane protein ATAD3A, promoting oligomerization-driven mitochondrial fission. Astrocyte-specific ATAD3A ablation prevents stroke-induced synaptic disorganization, neural circuit disruption, and cognitive deficits. Therapeutically, combined administration of cotadutide (a glycogen-depleting GLP-1/GCGR agonist) and HDAC3 inhibitor RGFP966 reverses glycogen accumulation, rescues mitochondrial architecture/function, and restores synaptic plasticity and circuit reorganization, thereby accelerating sensorimotor recovery.

Project description:Background: Heart failure involves metabolic alterations including increased glycolysis despite unchanged or decreased glucose oxidation. The mitochondrial pyruvate carrier (MPC) regulates pyruvate entry into the mitochondrial matrix, and cardiac deletion of the MPC in mice causes heart failure. How MPC deletion results in heart failure is unknown. Methods: We performed targeted metabolomics and isotope tracing in wildtype (fl/fl) and cardiac-specific Mpc2-/- (CS-Mpc2-/-) hearts after in vivo injection of 13C-glucose. Cardiac glycogen was measured biochemically and by transmission electron microscopy. Cardiac glucose uptake of 2-deoxyglucose was measured and western blotting performed to analyze insulin signaling and enzymatic regulators of glycogen synthesis and degradation. Isotope tracing and glycogen analysis was also performed in hearts from mice fed either low-fat diet or a ketogenic diet previously shown to reverse the heart failure in CS-Mpc2-/- mice. Cardiac glycogen was also assessed in mice infused with angiotensin-II that were fed either low-fat or ketogenic diet. Results: Failing CS-Mpc2-/- hearts contained normal levels of ATP and phosphocreatine, suggesting their heart failure is not caused by energetic stress. These hearts displayed increased enrichment from 13C-glucose and increased glycolytic metabolite pool sizes. 13C enrichment and pool size was also increased for the glycogen intermediate UDP-glucose, as well as increased enrichment of the glycogen pool. Glycogen levels were increased ~6-fold in the failing CS-Mpc2-/- hearts, and glycogen granules were easily detected by electron microscopy. This increased glycogen synthesis occurred despite enhanced inhibitory phosphorylation of glycogen synthase and reduced expression of the priming enzyme glycogenin-1. In young, non-failing CS-Mpc2-/- hearts, increased glycolytic 13C enrichment occurred, but glycogen levels remained low and unchanged compared to fl/fl hearts. Feeding a ketogenic diet to CS-Mpc2-/- mice reversed the heart failure and normalized the cardiac glycogen and glycolytic metabolite accumulation. Cardiac glycogen levels were also elevated in mice infused with angiotensin II, and both the cardiac hypertrophy and glycogen levels were improved by ketogenic diet. Conclusions: Our results indicate that loss of MPC in the heart increases glycolytic metabolism and ultimately glycogen accumulation and heart failure, while a ketogenic diet can reverse both the glycogen accumulation and heart failure. We conclude that maintaining mitochondrial pyruvate import and metabolism is critical for the heart, unless cardiac pyruvate metabolism is dramatically reduced by consumption of a ketogenic diet.

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	SRR33367884_1.fastq.gz	Fastqsanger.gz
	SRR33367884_2.fastq.gz	Fastqsanger.gz
	SRR33367885_1.fastq.gz	Fastqsanger.gz
	SRR33367885_2.fastq.gz	Fastqsanger.gz
	SRR33367886_1.fastq.gz	Fastqsanger.gz

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Glycogen stress granules cause astrocytic mitochondrial dysfunction to aggravate ischemic damage and delay recovery after stroke

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