Deletion of EMBO J︱ neuron Miro1 protein disrupts mitochondrial autophagy and over-activates the integrated stress response

Written by Tang Jiahui, edited by Wang Sizhen, Wang Sizhen Mitochondria (mitochondria) are the main source of ATP, responsible for maintaining the homeostasis of Ca2+, as well as the place for lipid synthesis
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However, when the quality of mitochondria decreases due to factors such as damage or aging, the "work efficiency" of mitochondria becomes worse, and a large amount of reactive oxygen species (ROS) are produced, leading to cell damage
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Therefore, strict control of mitochondrial quality is essential to the normal function of cells
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Mitochondrial quality control is highly dependent on the mechanism of mitochondrial autophagy to remove damaged mitochondrial components [1]
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 PINK1 (PTEN-induced putative kinase 1) is a mitochondrial silk/threonine kinase, and Parkin is an E3 ubiquitin ligase
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PINK1 works with Parkin to promote the selective clearance of damaged mitochondria through mitochondrial autophagy (mitophagy)
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When mitochondria are damaged, PINK1 continues to accumulate on the outer mitochondrial membrane, recruiting Parkin to the outer mitochondrial membrane, and then ubiquitinating the substrate, recruiting autophagy linker molecules, thereby eliminating this part of the damaged mitochondria through the autophagy pathway [2-4 】
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The functional defects of PINK1 and Parkin are related to the rare recessive Parkinson's disease (PD) [5], suggesting that mitochondrial autophagy can affect the survival of neurons and is closely related to the pathogenesis of neurodegenerative diseases
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 Miro (mitochondrial Rho) protein family is mainly divided into Miro1 and Miro2 in mammals.
It has always been considered as a key regulator of mitochondrial transport and distribution, as well as a key component of mitochondrial-endoplasmic reticulum coupling, maintaining intracellular Ca2+ homeostasis 【6, 7】
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When mitochondria are damaged, the Miro protein is rapidly ubiquitinated and degraded through the PINK1/Parkin-dependent autophagy mechanism [8]
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Some scholars believe that this PINK1/Parkin transport regulation mechanism for Miro protein may help separate damaged mitochondria from tubulin and actin, and promote the separation of damaged organelles from normal functional organelles [6]
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 Recent studies have found that Miro protein can directly act as a receptor for Parkin on the outer mitochondrial membrane (OMM) to promote Parkin recruitment to mediate mitochondrial autophagy [9]
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In addition, the degradation of Miro was found to be blocked in fibroblasts derived from PD patients, and multiple Miro1 mutations have been identified as risk factors for PD, which further supports the involvement of Miro1 in the pathogenesis of PD [10]
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Although more and more studies have pointed out the new role of Miro protein in regulating mitochondrial autophagy, and the connection between Miro1 and PD pathology is getting closer, how Miro1 destroys mitochondria and neuron homeostasis, and its long-term consequences, Little is still known
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 Recently, in a research paper published in The EMBO Journal entitled Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response, Josef T Kittler's laboratory at University College London, UK, revealed the ubiquitin of Miro1 after mitochondrial damage.
Chemical and degradation are necessary for Parkin's recruitment to the outer mitochondrial membrane and mitochondrial autophagy.
Conditional knockout of Miro1 in vivo can lead to excessive fusion of neuronal mitochondria and continuous activation of integrated stress response (ISR)
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Therefore, the study provides new insights into the effect of Miro1 on the regulation of PINK1/Parkin-dependent mitochondrial autophagy and its imbalance on neurological diseases
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First, the authors found in mouse embryonic fibroblasts (MEF) that the double knockout of genes Miro1 and Miro2 can delay the process of Parkin being recruited to the damaged mitochondrial OMM during the process of damaged mitochondrial autophagy (Figure 1); Miro1 (non-replenishing Miro2) can restore normal mitochondrial autophagy levels in cells, that is, the presence of Miro on OMM is essential for Parkin to be effectively recruited to damaged mitochondria and mitochondrial autophagy
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At the same time, the authors also found that the decrease in the ubiquitination level of Miro1 protein may affect the turnover rate of damaged mitochondria; the ubiquitination and degradation of Miro may not only be necessary to stop the transport of damaged mitochondria, but also may directly participate in the process of mitochondrial phagocytosis.

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In other words, the ubiquitination and degradation of Miro1 are necessary for the elimination of damaged mitochondria through mitochondrial autophagy
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Figure 1 During the process of mitochondrial autophagy, Parkin is recruited to the outer mitochondrial membrane (A), and the double knockout of Miro1/2 delays the process of mitochondrial autophagy (B)
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(Picture quoted from: López-Doménech G, et al.
, EMBO J 2021; 40: e100715) Miro1 knockout can also block valinomycin-mediated mitochondrial autonomy in mouse primary cortical neurons Phagocytosis and remodeling (Figure 2)
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In the experiment of replenishing different forms of Miro1 (wild-type, lysine mutant, etc.
), it was found that in the process of primary neuronal mitochondrial damage and damaged mitochondrial autophagy (clearance), Miro1 relied on its pan The method of vegetarianization recruits and stabilizes Parkin on the mitochondrial membrane
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Figure 2 Miro1 knockout inhibits valinomycin-induced neuronal mitochondrial autophagy
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(Picture quoted from: López-Doménech G, et al.
, EMBO J 2021; 40: e100715) Mitophagy defects can lead to age-dependent accumulation of mitochondrial dysfunction in neurological diseases
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Subsequently, the authors constructed a mouse model that specifically knocked out Miro1 and Miro2 in hippocampal and cortical neurons, respectively, and found that the substrate of Parkin, mitochondrial fusion protein 1 and 2 (ie The protein level of MFN1, MFN2) increased, and the level of ubiquitination decreased
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This indicates that the absence of Miro1 in the mature neurons of the mouse brain will cause damaged mitochondria to accumulate with age, and at the same time lead to an increase in the mechanism of mitochondrial autophagy (Figure 3)
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Figure 3 Loss of neuron Miro1 up-regulates the level of MFN1/2 (A, B), and the elevated MFN1/2 are localized in mitochondria (C, D)
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(Picture quoted from: López-Doménech G, et al.
, EMBO J 2021; 40: e100715) The above results also imply the significant upregulation of Mfn1 and Mfn2 in Miro1 knockout mice, which may be due to PINK1/parkin mediation The destruction of the mitochondrial autophagy mechanism leads to changes in the dynamic balance of mitochondrial fission/fusion, resulting in a pathological reconstruction of the mitochondrial network
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Indeed, experiments have found that knockout of Miro1 in mouse hippocampal neurons severely damages the morphology and ultrastructure of mitochondria, including mitochondrial cell remodeling, as well as mitochondrial swelling, rounding, and decreased internal electron density, which suggests that Miro deletion can destroy mitochondria Homeostasis; and the appearance of megamitochondria (megamitochondria) due to the imbalance of mitochondrial fusion/division homeostasis
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Experiments have also found that long-term absence of Miro1 in the mouse brain can also trigger excessive activation of the integrated stress response (ISR) and affect the survival of neurons (Figure 4)
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Generally speaking, ISR is a protective way to reduce the overall rate of protein synthesis
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 However, the continuous activation of this pathway will result in a slow reduction in the translation of important proteins, which may lead to neuronal death [11,12]
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Figure 4 Loss of neuron Miro1 causes excessive mitochondrial fusion (A, B) and triggers an integrated stress response (C)
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(Picture quoted from: López-Doménech G, et al.
, EMBO J 2021; 40: e100715) Figure 5 Work summary diagram: Miro1 deletion leads to long-term destruction of mitochondrial homeostasis and mitochondrial fusion, mitochondrial remodeling and integrated stress response ( ISR) continues to be activated
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(Picture quoted from: López-Doménech G, et al.
, EMBO J 2021; 40: e100715) Conclusion and discussion of the article, enlightenment and outlook This discovery provides a new role for Miro1 and Miro2 in mitochondrial autophagy after mitochondrial injury And emphasized their importance in mitochondrial homeostasis in vitro and in vivo
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The study also discovered the role of Miro1 as part of the Parkin receptor complex of the mitochondrial external model, and its potential role in regulating Parkin-mediated mitochondrial quality control
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In addition, the long-term deletion of Miro1 in neurons in mice resulted in up-regulation of Mfn1/2, remodeling of mitochondrial network, and induction of ISR
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Therefore, this research provides a new strategy for targeted therapy of mitochondrial dysfunction in the pathogenesis of neurodegenerative diseases (such as PD) and other related diseases
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References (slide up and down to view) [1] Pickles S , Vigie P,