Dataset Information

Proteolysis of mitochondrial calpain-13 in cerebral ischemia-reperfusion injury.

ABSTRACT: Calpains are calcium-dependent cysteine proteases activated by intracellular Ca²⁺. Although calpains mainly exist in the cytosol, calpain-13 is present in the mitochondria in mouse brains; however, the enzymatic properties and physiological functions of calpain-13 remain unknown. Hence, in this study, we predicted and evaluated the enzymatic properties of calpain-13. Based on our bioinformatic approaches, calpain-13 possessed a catalytic triad and EF-hand domain, similar to calpain-1, a well-studied calpain. Therefore, we hypothesized that calpain-13 had calpain-1-like enzymatic properties; however, calpain-13 was not proteolyzed in C57BL/6J mouse brains. Subsequently, cerebral ischemia/reperfusion (I/R) injury caused proteolysis of mitochondrial calpain-13. Thus, our study showed that mitochondrial calpain-13 was proteolyzed in the mitochondria of the I/R injured mouse brain. This finding could be valuable in further research elucidating the involvement of calpain-13 in cell survival or death in brain diseases, such as cerebral infarction.

SUBMITTER: Chukai Y

PROVIDER: S-EPMC11267081 | biostudies-literature | 2024 Sep

REPOSITORIES: biostudies-literature

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Proteolysis of mitochondrial calpain-13 in cerebral ischemia-reperfusion injury.

Chukai Yusaku Y Sudo Toru T Fukuda Tomokazu T Tomita Hiroshi H Sugano Eriko E Ozaki Taku T

Biochemistry and biophysics reports 20240701

Calpains are calcium-dependent cysteine proteases activated by intracellular Ca<sup>2+</sup>. Although calpains mainly exist in the cytosol, calpain-13 is present in the mitochondria in mouse brains; however, the enzymatic properties and physiological functions of calpain-13 remain unknown. Hence, in this study, we predicted and evaluated the enzymatic properties of calpain-13. Based on our bioinformatic approaches, calpain-13 possessed a catalytic triad and EF-hand domain, similar to calpain-1, ...[more]

PMID: 39050013

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Project description:BackgroundElectroacupuncture (EA) has been shown to promote functional recovery after cerebral ischemia-reperfusion (I/R) injury. However, the contribution of mitochondrial dynamics to recovery remains unclear. The aim of this study was to investigate whether mitochondrial dynamics are involved in the effects of EA on cerebral I/R injury.MethodsThe rats with cerebral I/R injury were established by the middle cerebral artery occlusion/reperfusion. Subsequently, EA was applied to Baihui (GV20) and Dazhui (GV14) acupoints, with 2 Hz/5 Hz in frequency, 1.0 mA in intensity, 20 min each time, once a day for seven consecutive days. The therapeutic outcomes were assessed by modified neurological severity score (mNSS), 2,3,5-Triphenyte-trazolium chloride (TTC) staining, and hematoxylin-eosin (HE) staining. Mitochondrial morphology was observed under transmission electron microscopy. Adenosine triphosphate (ATP) content and ATP synthases (ATPases) activity were evaluated to measure mitochondrial function using ELISA. Finally, mitochondrial dynamics-related molecules, including dynamin-related protein 1 (Drp1), fission 1 (Fis1), mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and optic atrophy 1 (OPA1), were detected by Western blot and immunofluorescence staining.ResultsCerebral I/R injury induced neurological dysfunction, cerebral infarction and neuronal injury, all of which were ameliorated by EA. And EA improved mitochondrial morphology and function. Moreover, EA altered the balance of mitochondrial dynamics. Specifically, the data showed a significant decrease in the expression of Drp1 and Fis1, leading to the inhibition of mitochondrial fission. Additionally, Mfn1, Mfn2 and Opa1, which are related to mitochondrial fusion, were effectively promoted after EA treatment. However, sham EA did not show any neuroprotective effects in rats with cerebral I/R injury.ConclusionsIn summary, our study indicates that the balance of mitochondrial dynamics is crucial for EA therapy to treat cerebral I/R injury.

Project description:BackgroundMitochondrial dysfunctions play a pivotal role in cerebral ischemia-reperfusion (I/R) injury. Although mitochondrial transplantation has been recently explored for the treatment of cerebral I/R injury, the underlying mechanisms and fate of transplanted mitochondria are still poorly understood.MethodsMitochondrial morphology and function were assessed by fluorescent staining, electron microscopy, JC-1, PCR, mitochondrial stress testing, and metabolomics. Therapeutic effects of mitochondria were evaluated by cell viability, reactive oxygen species (ROS), and apoptosis levels in a cellular hypoxia-reoxygenation model. Rat middle cerebral artery occlusion model was applied to assess the mitochondrial therapy in vivo. Transcriptomics was performed to explore the underlying mechanisms. Mitochondrial fate tracking was implemented by a variety of fluorescent labeling methods.ResultsNeuro-2a (N2a) cell-derived mitochondria had higher mitochondrial membrane potential, more active oxidative respiration capacity, and less mitochondrial DNA copy number. Exogenous mitochondrial transplantation increased cellular viability in an oxygen-dependent manner, decreased ROS and apoptosis levels, improved neurobehavioral deficits, and reduced infarct size. Transcriptomic data showed that the differential gene enrichment pathways are associated with metabolism, especially lipid metabolism. Mitochondrial tracking indicated specific parts of the exogenous mitochondria fused with the mitochondria of the host cell, and others were incorporated into lysosomes. This process occurred at the beginning of internalization and its efficiency is related to intercellular connection.ConclusionsMitochondrial transplantation may attenuate cerebral I/R injury. The mechanism may be related to mitochondrial component separation, altering cellular metabolism, reducing ROS, and apoptosis in an oxygen-dependent manner. The way of isolated mitochondrial transfer into the cell may be related to intercellular connection.

Dataset Information

Proteolysis of mitochondrial calpain-13 in cerebral ischemia-reperfusion injury.

Publications

Proteolysis of mitochondrial calpain-13 in cerebral ischemia-reperfusion injury.

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