Mitochondrial Rho GTPase Research Articles

Miro2 sulfhydration by CBS/H2S promotes human trophoblast invasion and migration via regulating mitochondria dynamics

Insufficient cytotrophoblast (CTB) migration and invasion into the maternal myometrium leads to pregnancy related complications like Intra-uterus Growth Restriction (IUGR), and pre-eclampsia (PE). We previously found that hydrogen sulfide (H2S) enhanced CTB migration without knowing the mechanism(s) and the pathophysiological significance. By studying human samples and cell line, we found that H2S levels were lower in PE patients’ plasma; H2S synthetic enzyme cystathionine β-synthetase (CBS) was reduced in PE extravillious invasive trophoblasts. GYY4137 (H2S donor, 1 µM) promoted CBS/H2S translocation onto mitochondria, preserved mitochondria functions, enhanced cell invasion and migration. CBS knockdown hindered the above functions which were rescued by GYY4137, indicating the vital roles of CBS/H2S signal. Disturbance of mitochondria dynamics inhibited cell invasion and migration. The 185 and 504 cysteines of Mitochondrial Rho GTPase 2 (Miro2C185/C504) were highly sulfhydrated by H2S. Knockdown Miro2 or double mutation of Miro2C185/C504 to serine fragmented mitochondria, and inhibited cell invasion and migration which can’t be rescued by H2S. The present study showed that human cytotrophoblast receives low dose H2S regulation; CBS/H2S sustained mitochondria functions via Miro2C185/C504 sulfhydration to enhance cytotrophoblast mobility. These findings established a new regulatory pathway for cytotrophoblast functions, and provided new targets for IUGR and PE.

LFHP-1c improves cognitive function after TBI in mice by reducing oxidative stress through the PGAM5-NRF2-KEAP1 ternary complex

Traumatic brain injury (TBI) is a leading cause of disability and death. Thus, timely and effective secondary brain injury intervention is crucial, with potential to improve the prognosis of TBI. Oxidative stress contributes to post-traumatic secondary cognitive impairment, and the reduction of post-traumatic oxidative stress effectively enhances cognitive function. Phosphoglycerate-mutating enzyme 5 (PGAM5), a member of the phosphoglycerate transporter enzyme family, is upregulated in TBI and induces mitochondrial autophagy. This further exacerbates damage following TBI. The present study focused on the small molecule drug, LFHP-1c, which is a novel inhibitor of PGAM5. The present study used an in vivo mouse model incorporating a controlled cortical impact-induced TBI, to examine the impact of LFHP-1c on oxidative stress and cognitive function. The present study aimed to determine the impact of LFHP-1c on the PGAM5-Kelch-like ECH-associated protein 1 (KEAP1)- nuclear factor erythroid 2-related factor 2 (NRF2) ternary complex within the TBI context. Results of the present study indicated that LFHP-1c suppresses PGAM5 expression and inhibits the development of the PGAM5-KEAP1-NRF2 ternary complex, thereby promoting the release of NRF2 and KEAP1. This in turn promotes the entry of NRF2 into the nucleus following TBI, leading to increased expression of anti-oxidative stress downstream factors, such as heme oxygenase-1, glutathione peroxidase 1 and superoxide dismutase 1. In addition, LFHP-1c also released KEAP1, leading to mitochondrial Rho GTPase 2 degradation and reducing perinuclear aggregation of mitochondria in the cell, which reduced oxidative stress and ultimately improved cognitive function after TBI.

MIRO1 controls energy production and proliferation of smooth muscle cells.

The outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles invascular diseases, such as neointima formation following vascular injury are widely unknown. An in vivo model of selective Miro1 deletion in VSMCs was generated, and the animals were subjected to carotid artery ligation. The molecular mechanisms relevant to VSMC proliferation were then explored in explanted VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function and interactions with components of the electron transport chain (ETC). MIRO1 was robustly expressed in VSMCs within human atherosclerotic plaques and promoted VSMC proliferation and neointima formation in mice by blocking cell-cycle progression at G1/S, mitochondrial positioning, and PDGF-induced ATP production and respiration; overexpression of a MIRO1 mutant lacking the EF hands that are required for mitochondrial mobility did not fully rescue these effects. At the ultrastructural level, Miro1 deletion distorted the mitochondrial cristae and reduced the formation of super complexes and the activity of ETC complex I. Mitochondrial motility is essential for VSMC proliferation and relies on MIRO1. The EF-hands of MIRO1 regulate the intracellular positioning of mitochondria. Additionally, the absence of MIRO1 leads to distorted mitochondrial cristae and reduced ATP generation. Our findings demonstrate that motility is linked to mitochondrial ATP production. We elucidated two unrecognized mechanisms through which MIRO1 influences cell proliferation by modulating mitochondria: first, by managing mitochondrial placement via Ca2+-dependent EF hands, and second, by affecting cristae structure and ATP synthesis.

Abstract 2770: A novel MIRO2/MYO9B/RhoA signaling axis controls tumor cell invasion and metastasis

Abstract Metastasis of cancer cells to vital organs remains the leading cause of cancer related deaths, emphasizing a strong need for actionable targets in advanced stage cancer. To address this, we study novel dysregulated mitochondrial signaling mechanisms that cells utilize to metastasize. Here, we focus on how outer mitochondrial membrane protein—Mitochondrial Rho GTPase 2 (MIRO2)—promotes tumor cell invasion and metastasis. Our previous work identified higher MIRO2 mRNA expression in cancer vs. normal patient samples in a multitude of cancer types, which correlated with worse patient outcomes. Furthermore, we demonstrated that MIRO2 was critical for prostate cancer cell growth and survival in vitro and in vivo. However, it remains unknown if MIRO2 only affects primary tumor growth or if this protein is important throughout tumor progression. Using siRNA mediated knockdown (KD) of MIRO2 we find MIRO2 KD ubiquitously reduces tumor cell invasion in breast, melanoma, pancreatic, and prostate cancer cells. Utilizing metastatic prostate and breast cancer models, we demonstrate that mice injected with MIRO2 shRNA cells have significantly lower metastatic burden compared to mice injected with control shRNA cells. To determine the mechanism that MIRO2 controls invasion and metastasis, we looked at which of the top protein-binding partners for MIRO2 may regulate tumor cell invasion. Of our top hits, we found that depletion of atypical myosin IXB (MYO9B) by siRNA most significantly reduced tumor cell invasion and phenocopied MIRO2 depletion. MYO9B has a well-defined role in controlling cell motility via inactivation of RhoA. Excitingly, we have found 1) MIRO2 KD results in increased active RhoA, phenocopying MYO9B KD, 2) dual ablation of MIRO2 and RhoA fully rescues tumor cell invasion, and 3) MIRO2 is required for MYO9B driven invasion. Taken together, we propose a novel signaling mechanism by which MIRO2 broadly promotes invasion and metastasis through MYO9B dependent inactivation of RhoA. Citation Format: Dillon P. Boulton, Connor J. Hughes, Valentina Vaira, Ajita Jindal, Masoom Raza, Stephen Connor Purdy, Shilpa Sant, Heide L. Ford, M. Cecilia Caino. A novel MIRO2/MYO9B/RhoA signaling axis controls tumor cell invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2770.

Osteocyte mitochondria inhibit tumor development via STING-dependent antitumor immunity.

Bone is one of the most common sites of tumor metastases. During the last step of bone metastasis, cancer cells colonize and disrupt the bone matrix, which is maintained mainly by osteocytes, the most abundant cells in the bone microenvironment. However, the role of osteocytes in bone metastasis is still unclear. Here, we demonstrated that osteocytes transfer mitochondria to metastatic cancer cells and trigger the cGAS/STING-mediated antitumor response. Blocking the transfer of mitochondria by specifically knocking out mitochondrial Rho GTPase 1 (Rhot1) or mitochondrial mitofusin 2 (Mfn2) in osteocytes impaired tumor immunogenicity and consequently resulted in the progression of metastatic cancer toward the bone matrix. These findings reveal the protective role of osteocytes against cancer metastasis by transferring mitochondria to cancer cells and potentially offer a valuable therapeutic strategy for preventing bone metastasis.

Presenilin2 D439A Mutation Induces Dysfunction of Mitochondrial Fusion/Fission Dynamics and Abnormal Regulation of GTPase Activity

Alzheimer’s disease (AD) is an age-related progressive neurodegenerative disease, and approximately 10% of AD cases are early-onset familial AD (EOFAD), which is mainly linked to point mutations in genes encoding presenilins (PS1 and PS2). Mutations in PS2 are extremely rare and have not received enough attention. Recently, studies have found that Rho GTPase activity is closely related to the pathogenesis of AD. In this study, we used transcriptome sequencing in PS2 siRNA-transfected SH-SY5Y cells and found a group of differentially expressed genes (DEGs) related to the regulation of GTPase activity. Among those DEGs, the most significantly downregulated was Rho guanine nucleotide exchange factor 5 (ARHGEF5). GTPase activity in PS2 siRNA-transfected cells was significantly decreased. Then, we found that the expression of ARHGEF5 and the GTPase activity of Mitochondrial Rho GTPase 2 (Miro2) in PS2 D439A mutant SH-SY5Y cells were significantly decreased. We found for the first time that PS2 can bind to Miro2, and the PS2 D439A mutation reduced the binding between PS2 and Miro2, reduced the expression of Miro2, and resulted in an imbalance in mitochondrial fusion/fission dynamics. In conclusion, PS2 gene knockdown may participate in the pathogenesis of AD through the regulation of GTPase activity. The imbalance in mitochondrial dynamics mediated by the PS2 D439A mutation through regulation of the expression and GTPase activity of Miro2 may be a potential pathogenic mechanism of AD.

Altered anterograde axonal transport of mitochondria in cultured striatal neurons of a knock-in mouse model of Huntington's disease

Huntington's disease (HD) is a progressive genetic neurodegenerative disease caused by an abnormal expansion of a cytosine-adenine-guanine trinucleotide repeat in the huntingtin gene. One pathological feature of HD is neuronal loss in the striatum. Despite many efforts, mechanisms underlying neuronal loss in HD striatum remain elusive. It was suggested that the mutant huntingtin protein interacts mitochondrial proteins and causes mitochondrial dysfunction in striatal neurons. However, whether axonal transport of mitochondria is altered in HD striatal neurons remains controversial. Here, we examined axonal transport of single mitochondria labelled with Mito-DsRed2 in cultured striatal neurons of zQ175 knock-in mice (a knock-in mouse model of HD). We observed decreased anterograde axonal transport of proximal mitochondria in HD striatal neurons compared with wild-type (WT) striatal neurons. Decreased anterograde transport in HD striatal neurons was prevented by overexpressing mitochondrial Rho GTPase 1 (Miro1). Our results offer a new insight into mechanisms underlying neuronal loss in the striatum in HD.

Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK on the mitochondrial membrane is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding to MIRO1. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1’s EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394-431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1’s EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 μM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.

Lipotoxicity-polarised macrophage-derived exosomes regulate mitochondrial fitness through Miro1-mediated mitophagy inhibition and contribute to type 2 diabetes development in mice.

Insulin resistance is a major pathophysiological defect in type 2 diabetes and obesity. Numerous experimental and clinical studies have provided evidence that sustained lipotoxicity-induced mitophagy deficiency can exacerbate insulin resistance, leading to a vicious cycle between mitophagy dysfunction and insulin resistance, and thereby the onset of type 2 diabetes. Emerging evidence suggests that exosomes (Exos) from M2 macrophages play an essential role in modulating metabolic homeostasis. However, how macrophages are affected by lipotoxicity and the role of lipotoxicity in promoting macrophage activation to the M1 state have not been determined. The objective of this study was to determine whether M1 macrophage-derived Exos polarised by lipopolysaccharide (LPS) + palmitic acid (PA)-induced lipotoxicity contribute to metabolic homeostasis and impact the development of insulin resistance in type 2 diabetes. Lipotoxicity-polarised macrophage-derived M1 Exos were isolated from bone marrow (C57BL/6J mouse)-derived macrophages treated with LPS+PA. Exos were characterised by transmission electron microscopy, nanoparticle tracking analysis and western blotting. Flow cytometry, H&E staining, quantitative real-time PCR, immunofluorescence, glucose uptake and output assays, confocal microscopy imaging, western blotting, GTTs and ITTs were conducted to investigate tissue inflammation, mitochondrial function and insulin resistance in vitro and in vivo. The roles of miR-27-3p and its target gene Miro1 (also known as Rhot1, encoding mitochondrial rho GTPase 1) and relevant pathways were predicted and assessed in vitro and in vivo using specific miRNA mimic, miRNA inhibitor, miRNA antagomir and siRNA. miR-27-3p was highly expressed in M1 Exos and functioned as a Miro1-inactivating miRNA through the miR-27-3p-Miro1 axis, leading to mitochondria fission rather than fusion as well as mitophagy impairment, resulting in NOD-like receptor 3 inflammatory activation and development of insulin resistance both in vivo and in vitro. Inactivation of miR-27-3p induced by M1 Exos prevented type 2 diabetes development in high-fat-diet-fed mice. These findings suggest that the miR-27-3p-Miro1 axis, as a novel regulatory mechanism for mitophagy, could be considered as a new therapeutic target for lipotoxicity-related type 2 diabetes disease development.

Inhibition of Mitochondrial Fission Alleviates Zearalenone-Induced Mitochondria-Associated Endoplasmic Reticulum Membrane Dysfunction in Piglet Sertoli Cells.

This study aimed to investigate the effects of zearalenone (ZEA) on piglet Sertoli cell (SC)-mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) based on mitochondrial fission, and to explore the molecular mechanism of ZEA-induced cell damage. After the SCs were exposed to the ZEA, the cell viability decreased, the Ca2+ levels increased, and the MAM showed structural damage. Moreover, glucose-regulated protein 75 (Grp75) and mitochondrial Rho-GTPase 1 (Miro1) were upregulated at the mRNA and protein levels. However, phosphofurin acidic cluster protein 2 (PACS2), mitofusin2 (Mfn2), voltage-dependent anion channel 1 (VDAC1), and inositol 1,4,5-trisphosphate receptor (IP3R) were downregulated at the mRNA and protein levels. A pretreatment with mitochondrial division inhibitor 1 (Mdivi-1) decreased the ZEA-induced cytotoxicity toward the SCs. In the ZEA + Mdivi-1 group, the cell viability increased, the Ca2+ levels decreased, the MAM damage was repaired, and the expression levels of Grp75 and Miro1 decreased, while those of PACS2, Mfn2, VDAC1, and IP3R increased compared with those in the ZEA-only group. Thus, ZEA causes MAM dysfunction in piglet SCs through mitochondrial fission, and mitochondria can regulate the ER via MAM.

Zearalenone induces mitochondria-associated endoplasmic reticulum membranes dysfunction in piglet Sertoli cells based on endoplasmic reticulum stress

Zearalenone (ZEA) is an estrogen-like mycotoxin, which mainly led to reproductive toxicity. The study aimed to investigate the molecular mechanism of ZEA-induced dysfunction of mitochondria-associated endoplasmic reticulum membranes (MAM) in piglet Sertoli cells (SCs) via the endoplasmic reticulum stress (ERS) pathway. In this study, SCs were used as a research object that was exposed to ZEA, and ERS inhibitor 4-Phenylbutyrate acid (4-PBA) was used as a reference. The results showed that ZEA damaged cell viability and increased Ca2+ levels; damaged the structure of MAM; up-regulated the relative mRNA and protein expression of glucose-regulated protein 75 (Grp75) and mitochondrial Rho-GTPase 1 (Miro1), while inositol 1,4,5-trisphosphate receptor (IP3R), voltage-dependent anion channel 1 (VDAC1), mitofusin2 (Mfn2) and phosphofurin acidic cluster protein 2 (PACS2) were down-regulated. After a 3 h 4-PBA-pretreatment, ZEA was added for mixed culture. The results of 4-PBA pretreatment showed that inhibition of ERS reduced the cytotoxicity of ZEA against piglet SCs. Compared with the ZEA group, inhibition of ERS increased cell viability and decreased Ca2+ levels; restored the structural damage of MAM; down-regulated the relative mRNA and protein expression of Grp75 and Miro1; and up-regulated the relative mRNA and protein expression of IP3R, VDAC1, Mfn2, and PACS2. In conclusion, ZEA can induce MAM dysfunction in piglet SCs via the ERS pathway, whereas ER can regulate mitochondria through MAM.

Trak adaptors regulate the recruitment and activation of dynein and kinesin in mitochondrial transport.

Mitochondria are cellular power plants that generate ATP and buffer cytosolic Ca2+. In neurons, mitochondria are distributed along microtubules where energy and Ca2+ is necessary and damaged mitochondria are transported back to the cell body for degradation. Deficiencies in mitochondrial transport are associated with neurodegenerative disorders. Mitochondrial transport is mediated by mitochondrial Rho GTPases Miro1 and TRAK adaptors that recruit kinesin-1 and dynein-dynactin. To understand how these opposing motors are regulated during mitochondrial transport, we reconstituted the bidirectional transport of Miro1/TRAK along microtubules in vitro.Motor proteins kinesin-1 and dynein, cargo adaptors TRAK1/2, MIRO1, and SNPH were recombinantly expressed and fluorescently labeled after purification. Using in vitro reconstitution, the velocity, run length, and landing rate of the motor proteins in the presence of TRAK1/2 were analyzed. In vitro pull-down assays revealed the interactions between motor proteins and TRAK. Force generation of motor-cargo adaptor complexes was measured by optical trapping. We show that the coiled-coil domain of TRAK activates dynein-dynactin and enhances the motility of kinesin-1 activated by its cofactor MAP7. TRAK adaptors that recruit both motors moved towards kinesin-1's direction, whereas kinesin-1 was excluded from binding TRAK transported by dynein-dynactin, demonstrating that TRAK coordinates the activity of these opposing motors to avoid tug-of-war. Using the reconstitution assay, we also tested whether disruption of the transport machinery or passive anchoring to microtubules stalls mitochondrial transport in regions with elevated Ca2+. Transport of Miro1/TRAK by kinesin-1 was not affected by Ca2+. Instead, the microtubule docking protein syntaphilin induced resistive forces that stall kinesin-1 and dynein-driven motility in vitro, supporting the model that mitochondrial transport stalls by Ca2+-mediated recruitment of syntaphilin to the mitochondrial membrane.

Abstract A043: A novel role for Mitochondrial Rho GTPase 2 in tumor cell invasion and metastasis

Abstract Metastasis of cancer cells to secondary sites remains the leading cause of cancer related deaths, emphasizing a strong need for actionable targets in advanced stage cancer. To address this, we study novel dysregulated signaling mechanisms that cells utilize to metastasize to distal sites. Here, we focus on how outer mitochondrial membrane protein—Mitochondrial Rho GTPase 2 (MIRO2)—promotes tumor cell invasion and metastasis through a negative regulation of RhoA. Our previous work identified higher MIRO2 mRNA expression in cancer vs. normal patient samples, which correlated with worse patient outcomes. Furthermore, in the context of prostate cancer we demonstrated that MIRO2 was critical for 2D cell growth, anchorage-dependent and -independent colony formation, and tumor growth in vivo. However, it remains completely unknown if MIRO2 affects primary tumor growth or if this protein is important throughout tumor progression. Using siRNA mediated knockdown (KD) of MIRO2 we clearly show that MIRO2 KD ubiquitously reduces tumor cell invasion in breast, melanoma, pancreatic, and prostate cancer cell lines. Preliminary experiments modeling late-stage metastasis showed that tail vein injection of PC3 cells stably expressing MIRO2 targeting shRNA had reduced kidney and liver metastatic burden in comparison to PC3 cells with control shRNA. In further mechanistic studies, we identified novel MIRO2 binding partners and used siRNA to KD the top hits and observe changes in invasive capacity. We found that atypical myosin IX B (MYO9B) reduced the invasion of cells to the largest extent. MYO9B is a plus-end direct atypical myosin known to control cell motility through spatially inactivating RhoA at the leading edge of migrating cells. Excitingly, MIRO2 KD showed an increase in active RhoA phenocopying MYO9B KD. Lastly, in pilot experiments we found that dual KD of MIRO2 and RhoA rescued invasive capacity in comparison to MIRO2 KD alone. Taken together, we propose a novel signaling mechanism by which MIRO2 broadly promotes invasion and metastasis through MYO9B dependent inactivation of RhoA. Citation Format: Dillon P. Boulton, Madison Furnish, M. Cecilia Caino. A novel role for Mitochondrial Rho GTPase 2 in tumor cell invasion and metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A043.

Abstract 2980: MIRO2/GCN1/ATF4 retrograde signaling modulates prostate cancer growth and invasion

Abstract Our understanding of the mechanisms that drive aggressiveness of metastatic prostate cancer (mPC) are limited. In this context, we aim to identify novel targets in mPC by focusing on key mitochondrial signaling pathways exploited by mPC. Here we identify a novel mitochondrial signaling pathway centered on mitochondrial Rho GTPase 2 (MIRO2). MIRO2 mRNA is upregulated in mPC compared to localized tumors, and higher MIRO2 levels are correlated with poor patient survival. Despite this association, the importance of MIRO2 in tumor cell biology has been overlooked. To address this, we examined MIRO2’s role in tumor cell-intrinsic phenotypes of relevance to metastatic potential. Using human cell lines that represent androgen-independent and -sensitive mPC, we showed that MIRO2 depletion impaired cell growth, colony formation, tumor cell invasion and migration, and tumor growth in mice. Next we reasoned that identifying MIRO2’s protein binding partners would shed light on the molecular function of MIRO2 in mPC. Network analysis of MIRO2’s binding partners identified metabolism, cell cycle, and cellular responses to extracellular stimuli amongst the top over-represented pathways. Interestingly, the top hit on our screen was General Control Non-derepressible 1 (GCN1). GCN1 was an essential gene for 90% of the cell lines in a large pan-cancer screen for gene essentiality. GCN1 is the upstream activator of the kinase GCN2, which senses amino acid availability, actin dynamics cues in cells, and regulates cell cycle progression. Our results showed that MIRO2 is necessary for efficient GCN2 signaling, activation of the integrated stress response, and downstream translation of the transcription factor ATF4 in culture and tumors in mice. Further, exogenous expression of ATF4 restored clonogenic growth of MIRO2 depleted cells. MIRO2 and GCN1 expression were positively correlated in mPC from patient cohorts, and GCN1 was overexpressed in mPC compared to primary PC. Further, MIRO2-GCN1 interacted in mPC cell lines and in primary PC cells. We showed activated GCN2 and HIF1α were positively correlated in tumors grown in mice. Together, the data suggest our pathway is important in nutrient limited regions of tumors. In conclusion, MIRO2 belongs to a novel protein network that controls mPC cellular responses and adaptation to extracellular stimuli. Overall, we propose a new mechanism by which a mitochondrial retrograde signal can drive prostate cancer cell growth. Citation Format: Madison Furnish, Dillon Boulton, Victoria Genther, Denisa Grofova, Mitchell L. Ellinwood, Lina Romero, M. Scott Lucia, Scott D. Cramer, M. Cecilia Caino. MIRO2/GCN1/ATF4 retrograde signaling modulates prostate cancer growth and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2980.

Selected Articles from This Issue

Screening and biomarker identification have improved prostate cancer outcomes, but prostate cancer progression remains a critical challenge. Mitochondria have garnered increasing attention in prostate and other cancers, and metabolic and non-metabolic mitochondrial contributions to cancer merit further investigation. In their study, Furnish and colleagues found that mitochondrial Rho GTPase 2 (MIRO2) expression is upregulated in recurrent and progressive prostate cancer, and that MIRO2 depletion via shRNA abrogates prostate cancer cell growth in vitro and in vivo. The authors identified MIRO2 protein interactors using MIRO2 immunoprecipitation and mass spectrometry and revealed general control nonderepressible (GCN1) is a MIRO2 binding partner. Mechanistically, the authors showed that MIRO2 facilitates GCN1-mediated activation of GCN2, which results in eukaryotic translation initiation factor 2 alpha (eIF2α) phosphorylation and transcriptional upregulation of transcriptional activator 4 (ATF4). Immunohistochemistry data from prostate cancer xenografts implicated HIF1α as an oncogenic MIRO2 regulator. Overall, this work presents a previously unknown mechanism by which mitochondria promote prostate cancer progression and suggests that MIRO2 could be a useful therapeutic target for recurrent and progressive prostate cancer.Few therapeutic options exist for patients with metastatic prostate cancer. Furthermore, longitudinal study challenges and therapeutic effects on tumor biology render elucidating mechanisms that promote prostate cancer metastasis difficult. To address these issues and interrogate molecular drivers of prostate cancer metastasis, Van den Broeck and colleagues performed genomic analysis using samples from patients with localized or locally advanced prostate cancer who participated in longitudinal studies. The authors demonstrated that copy numbers of the gene encoding antizyme inhibitor 1 (AZIN1) are often amplified in samples that progressed to metastasis. Furthermore, abrogating AZIN1 expression using siRNA in prostate cancer cells decreased cell growth and migration in vitro and lung metastasis in vivo. By surveying transcriptomic changes induced by AZIN1 depletion, the authors noted that AZIN1 expression negatively correlates with expression of several collagens that can be processed into matrikines. Accordingly, culture media from AZIN1-deficient and collagen-overexpressing cells both decreased invasion and migration of prostate cancer cells in vitro. Altogether, this study presents a novel molecular facet of prostate cancer metastasis that could inform future therapeutic strategies.The MUC1 C-terminal subunit (MUC1-C) is known to promote cancer stem cell (CSC)-associated dedifferentiation, but whether MUC1-C contributes to dedifferentiation-associated transcription factor transactivation and chromatin remodeling is not clear. In their study, Bhattacharya and colleagues abrogate MUC1-C expression in prostate and breast cancer cells using shRNA and utilize Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq) to demonstrate that MUC1-C influences chromatin remodeling in cancer cells. Integrative analysis using ATAC-Seq, RNA-Seq, and ChIP-Seq revealed that genes whose chromatin accessibility is modified by MUC1-C and that are differentially expressed upon MUC1-C depletion are enriched with FOS, JUN, and NEF2 transcription factor binding motifs. The authors went on to demonstrate that MUC1-C interacts with JUN, and that a MUC1-C/JUN/ARID1A complex increases chromatin accessibility and expression of critical CSC genes, including NOTCH1, EGR1, and LY6E, in both prostate and breast cancer cells. Overall, in addition to documenting a previously unrecognized interaction between MUC1-C and JUN, this study furthers the understanding of how MUC1-C contributes to transcription factor transactivation, chromatin remodeling, and CSC biology.Inhibiting DNA double-strand break (DSB) repair augments DNA damage-inducing therapeutic efficacy, but mechanisms underlying this therapeutic potentiation have not been fully elucidated. In their study, Carr and colleagues used DNA-PK inhibitor peposertib and DNA damage-inducing radiation to demonstrate that DNA DSB repair inhibition enhances both radiation-mediated cell death and inflammation. The authors found that peposertib augments radiation-induced chromosomal abnormalities, causing distorted chromosomal segregation and cell death in cancer cells in vitro. Immunofluorescent imaging revealed that cancer cells treated with radiation and peposertib harbor numerous micronuclei that associate with inflammatory cytosolic DNA sensor cGAS. Accordingly, treatment with radiation and peposertib induced expression of inflammatory signaling mediators and cytokines, as well as immunoregulatory programmed cell death ligand 1 (PD-L1). The authors used bintrafusp alfa in vivo to demonstrate that PD-L1 blockade enhances CD8+ T cell recruitment to tumors and therapeutic efficacy of combination radiation–peposertib treatment. Taken together, this study reveals a previously unappreciated mechanism by which DNA DSB repair inhibition augments DNA damage-inducing therapy and demonstrates how that mechanism can be leveraged in therapeutic strategies.

Neurons Release Injured Mitochondria as "Help-Me" Signaling After Ischemic Stroke.

Mitochondrial dysfunction has been regarded as one of the major contributors of ischemic neuronal death after stroke. Recently, intercellular mitochondrial transfer between different cell types has been widely studied and suggested as a potential therapeutic approach. However, whether mitochondria are involved in the neuron-glia cross-talk following ischemic stroke and the underlying mechanisms have not been explored yet. In this study, we demonstrated that under physiological condition, neurons release few mitochondria into the extracellular space, and the mitochondrial release increased when subjected to the challenges of acidosis, hydrogen peroxide (H2O2), N-methyl-D-aspartate (NMDA), or glutamate. Acidosis reduced the mitochondrial basal respiration and lowered the membrane potential in primary-cultured mouse cortical neurons. These defective mitochondria were prone to be expelled to the extracellular space by the injured neurons, and were engulfed by adjacent astrocytes, leading to increased astrocytic expressions of mitochondrial Rho GTPase 1 (Miro 1) and mitochondrial transcription factor A (TFAM) at mRNA level. In mice subjected to transient focal cerebral ischemia, the number of defective mitochondria in the cerebrospinal fluid increased. Our results suggested that the neuron-derived mitochondria may serve as a “help-me” signaling and mediate the neuron-astrocyte cross-talk following ischemic stroke. Promoting the intercellular mitochondrial transfer by accelerating the neuronal releasing or astrocytic engulfing might be a potential and attractive therapeutic strategy for the treatment of ischemic stroke in the future.

Broad activation of the Parkin pathway induces synaptic mitochondrial deficits in early tauopathy.

Mitochondrial defects are a hallmark of early pathophysiology in Alzheimer's disease, with pathologically phosphorylated tau reported to induce mitochondrial toxicity. Mitophagy constitutes a key pathway in mitochondrial quality control by which damaged mitochondria are targeted for autophagy. However, few details are known regarding the intersection of mitophagy and pathologies in tauopathy. Here, by applying biochemical and cell biological approaches including time-lapse confocal imaging in live tauopathy neurons, combined with gene rescue experiments via stereotactic injections of adeno-associated virus particles into tauopathy mouse brains, electrophysiological recordings and behavioural tests, we demonstrate for the first time that mitochondrial distribution deficits at presynaptic terminals are an early pathological feature in tauopathy brains. Furthermore, Parkin-mediated mitophagy is extensively activated in tauopathy neurons, which accelerates mitochondrial Rho GTPase 1 (Miro1) turnover and consequently halts Miro1-mediated mitochondrial anterograde movement towards synaptic terminals. As a result, mitochondrial supply at tauopathy synapses is disrupted, impairing synaptic function. Strikingly, increasing Miro1 levels restores the synaptic mitochondrial population by enhancing mitochondrial anterograde movement and thus reverses tauopathy-associated synaptic failure. In tauopathy mouse brains, overexpression of Miro1 markedly elevates synaptic distribution of mitochondria and protects against synaptic damage and neurodegeneration, thereby counteracting impairments in learning and memory as well as synaptic plasticity. Taken together, our study reveals that activation of the Parkin pathway triggers an unexpected effect-depletion of mitochondria from synaptic terminals, a characteristic feature of early tauopathy. We further provide new mechanistic insights into how parkin activation-enhanced Miro1 degradation and impaired mitochondrial anterograde transport drive tauopathy-linked synaptic pathogenesis and establish a foundation for future investigations into new therapeutic strategies to prevent synaptic deterioration in Alzheimer's disease and other tauopathies.

MIRO2 Regulates Prostate Cancer Cell Growth via GCN1-Dependent Stress Signaling.

MIRO2/GCN1/GCN2 constitute a novel mitochondrial signaling pathway that controls androgen-independent and androgen-sensitive prostate cancer cell growth.

Miro1 provides neuroprotection via the mitochondrial trafficking pathway in a rat model of traumatic brain injury

The outer mitochondrial membrane protein mitochondrial Rho-GTPase 1 (Miro1) is known to be involved in the regulation of mitochondrial transport required for neuronal protection. Previous reports established that disruption of Miro1-dependent mitochondrial movement could result in nervous system diseases such as Parkinson’s disease and Alzheimer’s disease. This study was designed to explore the expression and mechanisms of Miro1 in secondary brain injury after traumatic brain injury (TBI). A total of 115 male Sprague Dawley rats were used in the weight-drop TBI rat model, and Miro1 in vivo knockdown was performed 24 h before TBI modeling by treatment with Miro1 short-interfering RNA. Real-time polymerase chain reaction, western blot, immunofluorescence, adenosine triphosphate (ATP) level assay, neuronal apoptosis, brain water content measurement, and neurological score analyses were carried out. Our results showed that the mRNA and protein levels of Miro1 were increased after TBI and co-localized with neurons and astrocytes in the peri-injury cortex. Moreover, Miro1 knockdown further exacerbated neuronal apoptosis, brain edema, and neurological deficits at 48 h after TBI, accompanied by impaired mitochondrial transport, reduction of mitochondria number and energy deficiency. Additionally, the apoptosis-related factors Bax upregulation and Bcl-2 downregulation as Miro1 knockdown after TBI implied that antiapoptotic effects on neuroprotection of Miro1, which were verified by the Fluoro-Jade C (FJC) staining and TUNEL staining. In conclusion, these findings suggest that Miro1 probably plays a neuroprotective role against secondary brain injury through the mitochondria trafficking pathway, suggesting that enhancing Miro1 might be a new strategy for the treatment of TBI.

Miro1 functions as an inhibitory regulator of MFN at elevated mitochondrial Ca2+ levels.

Mitochondria function as an integrated network that moves along the microtubules within cells and changes the morphology through membrane fusion and fission events. Mitofusin (MFN) mediates membrane tethering and subsequent fusion of the mitochondrial outer membrane. Understanding the regulatory mechanisms of MFN function is critical to tackling the pathology related to mitochondrial network imbalance. Here, we reveala novel inhibitory mechanism of MFN-mediated fusion by mitochondrial Rho GTPase (Miro1) in response to elevated mitochondrial Ca2+ concentration ([Ca2+ ]m ). We showed that elevated [Ca2+ ]m prevents the fusion between mitochondria forming the outer membrane tether by ectopically expressing MFN. Lowering [Ca2+ ]m by treating cells with an inhibitor of mitochondrial calcium uniporter or knocking down Miro1/2 induces more fused networks. Miro1 interacts with MFN as supported by co-immunoprecipitation and protein association identified by proximity labeling proteomics. It suggests that Miro1 functions as a Ca2+ -sensor and inhibits MFN function at elevated [Ca2+ ]m. Miro1 EF-hand mutant has a compromised inhibitory effect, which reiterates Ca2+ -modulated regulation. Dysregulated Ca2+ -handling and mitochondrial network imbalance are highly relevant in the pathology of cancers, cardiovascular, and neurodegenerative diseases. Miro1 functions as a coordinated Ca2+ -responder by pausing mitochondrial transport while reducing network fusion and cooperating with Drp1-mediated fission. It likely prevents the detrimental effect of Ca2+m overload and facilitates mitophagy. Our finding reveals a novel regulation of mitochondrial network dynamics responding to [Ca2+ ]m through the interplay of Miro1 and MFN. Modulation of Miro1 and MFN interaction is a potential intervention to promote network homeostasis.