Mitochondrial shape changes: orchestrating cell pathophysiology

Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzodelta yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated.

The field of mitochondrial dynamics has received a great deal of attention as a result of a number of studies linking mitochondrial fission and fusion machinery to apoptosis. Specifically, elevated levels of mitochondrial fission or compromised mitochondrial fusion can sensitize cells to apoptotic stimuli. Conversely, stimulation of mitochondrial fusion can render cells resistant to apoptotic stimuli. In addition, the machinery involved in fission and fusion has been spatially linked to Bax, a pro-apoptotic protein. However, the mechanistic implications of interactions between the machinery of mitochondrial fission and fusion and apoptotic effectors are largely unknown. Our understanding of the pathways of mitochondrial fission and fusion have come from genetic studies coupled with direct observation of both fission and fusion components and mitochondrial organelle morphology and behavior in vivo in Saccharomyces cerevisiae. These approaches have identified the key players in both mitochondrial fission and fusion and have generated good models for their roles in mitochondrial dynamics. However, the lack of in vitro systems for studying these processes has impeded a deeper investigation of the mechanism. We have recapitulated the process of mitochondrial fusion in vitro (5). Using this in vitro fusion assay, we have separated outer mitochondrial membrane fusion from inner and identified the mechanistic requirements for each step.

Mitochondrial dynamics encompass mitochondrial fusion, fission, and movement. Mitochondrial fission and fusion are seemingly ubiquitous, whereas mitochondrial movement is especially important for organelle transport through neuronal axons. Here, we review the roles of different mitochondrial dynamic processes in mitochondrial quantity and quality control, emphasizing their impact on the neurological system in Charcot–Marie–Tooth disease type 2A, amyotrophic lateral sclerosis, Friedrich’s ataxia, dominant optic atrophy, and Alzheimer’s, Huntington’s, and Parkinson’s diseases. In addition to mechanisms and concepts, we explore in detail different technical approaches for measuring mitochondrial dynamic dysfunction in vitro, describe how results from tissue culture studies may be applied to a better understanding of mitochondrial dysdynamism in human neurodegenerative diseases, and suggest how this experimental platform can be used to evaluate candidate therapeutics in different diseases or in individual patients sharing the same clinical diagnosis.

Mitochondria exist as dynamic tubular networks and the morphology of these networks impacts organelle function and cell health. Mitochondrial morphology is maintained in part by the opposing activities of mitochondrial fission and fusion. Mitochondrial fission and fusion are also required to maintain mitochondrial DNA (mtDNA) integrity. In Saccharomyces cerevisiae, the simultaneous inhibition of mitochondrial fission and fusion results in increased mtDNA mutation and the consequent loss of respiratory competence. The mechanism by which fission and fusion maintain mtDNA integrity is not fully understood. Previous work demonstrates that mtDNA is spatially linked to mitochondrial fission sites. Here, we extend this finding using live-cell imaging to localize mtDNA to mitochondrial fusion sites. While mtDNA is present at sites of mitochondrial fission and fusion, mitochondrial fission and fusion rates are not altered in cells lacking mtDNA. Using alleles that alter mitochondrial fission and fusion rates, we find that mtDNA integrity can be maintained in cells with significantly reduced, but balanced, rates of fission and fusion. In addition, we find that increasing mtDNA copy number reduces the loss of respiratory competence in double mitochondrial fission-fusion mutants. Our findings add novel insights into the relationship between mitochondrial dynamics and mtDNA integrity.

Mitochondrial dynamics in heart disease

The mitochondria are involved in active and dynamic processes, such as mitochondrial biogenesis, fission, fusion and mitophagy to maintain mitochondrial and cellular functions. In obesity and type 2 diabetes, impaired oxidation, reduced mitochondrial contents, lowered rates of oxidative phosphorylation and excessive reactive oxygen species (ROS) production have been reported. Mitochondrial biogenesis is regulated by various transcription factors such as peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), peroxisome proliferator-activated receptors (PPARs), estrogen-related receptors (ERRs), and nuclear respiratory factors (NRFs). Mitochondrial fusion is promoted by mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy 1 (OPA1), while fission is governed by the recruitment of dynamin-related protein 1 (DRP1) by adaptor proteins such as mitochondrial fission factor (MFF), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51), and fission 1 (FIS1). Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARKIN promote DRP1-dependent mitochondrial fission, and the outer mitochondrial adaptor MiD51 is required in DRP1 recruitment and PARKIN-dependent mitophagy. This review describes the molecular mechanism of mitochondrial dynamics, its abnormality in diabetes and obesity, and pharmaceuticals targeting mitochondrial biogenesis, fission, fusion and mitophagy.

The dynamin-related GTPase, Dnm1, self-assembles into punctate structures that are targeted to the outer mitochondrial membrane where they mediate mitochondrial division. Post-targeting, Dnm1-dependent division is controlled by the actions of the WD repeat protein, Mdv1, and the mitochondrial tetratricopeptide repeat-like outer membrane protein, Fis1. Our previous studies suggest a model where at this step Mdv1 functions as an adaptor linking Fis1 with Dnm1. To gain insight into the exact role of the Fis1.Mdv1.Dnm1 complex in mitochondrial division, we performed a structure-function analysis of the Mdv1 adaptor. Our analysis suggests that dynamic interactions between Mdv1 and Dnm1 play a key role in division by regulating Dnm1 self-assembly.

Mitochondrial dynamics regulate myocardial contractility and vice versa

Mitochondria provide a myriad of services to the cell, including energy production, calcium buffering and regulation of apoptosis. How these diverse functions are coordinated among the hundreds of mitochondria in a given cell is largely unknown, but is probably dependent on the dynamic nature of mitochondria. In this review, we explore the latest developments in mitochondrial dynamics in mammals. These studies indicate that mitofusins and OPA1 are essential for mitochondrial fusion, whereas Fis1 and Drp1 are essential for mitochondrial fission. The overall morphology of the mitochondrial population depends on the relative activities of these two sets of proteins. In addition to the regulation of mitochondrial shape, these molecules also play important roles in cell and tissue physiology. Perturbation of mitochondrial fusion results in defects in mitochondrial membrane potential and respiration, poor cell growth and increased susceptibility to cell death. These cellular observations may explain why mitochondrial fusion is essential for embryonic development. Two inherited neuropathies, Charcot-Marie-Tooth type 2A and autosomal dominant optic atrophy, are caused by mutations in mitofusin 2 and OPA1, suggesting that proper regulation of mitochondrial dynamics is particularly vital to neurons. Mitochondrial fission accompanies several types of apoptotic cell death and appears important for progression of the apoptotic pathway. These studies provide insight into how mitochondria communicate with one another to coordinate mitochondrial function and morphology.

Mitochondrial dynamics-the balance between fission, fusion, and mitophagy-are essential for maintaining cellular homeostasis and are increasingly implicated in the pathogenesis of Alzheimer's disease (AD). Here, we investigated the effects of targeted modulation of mitochondrial fission and fusion on mitochondrial morphology and metabolic status in primary hippocampal cultures derived from 5xFAD transgenic mice. Mitochondrial dynamics were modulated using the fission inhibitor Mitochondrial Division Inhibitor 1 (Mdivi-1), the fusion promoter mitochondrial fusion promoter M1 (MFP M1), and exogenous zinc as a fission activator. We evaluated mitochondrial morphology, lipofuscin accumulation, beta-amyloid (Aβ42) levels, and reactive oxygen species (ROS). The general condition of the cultures was assessed morphologically using neuronal and astrocytic markers. Modulating mitochondrial dynamics altered mitochondrial morphology, decreased Aβ42, lipofuscin, and ROS levels, and improved cellular organization. Treatments with MFP and Mdivi-1 promoted mitochondrial hyperfusion without complete network integration and were associated with reduced astrogliosis and increased neuronal density. In contrast, zinc induced dose-dependent mitochondrial fragmentation and astrocytic clasmatodendrosis, with lower concentrations enhancing Aβ clearance and higher concentrations inducing toxicity. Mitochondrial fusion and fission significantly influence lipofuscin and amyloid accumulation in 5xFAD cultures, underscoring their potential as therapeutic targets in neurodegenerative diseases. We propose that mitochondrial morphology acts as a key regulator of both cellular homeostasis and disease pathology.

Chapter 15 - The Emerging Role of Mitochondrial Dynamics in Cardiovascular Disease

ATG12–ATG3 and mitochondria

Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that affects approximately 25% of adults globally. Although several pharmacological therapies have been reported to improve NAFLD, the therapeutic ranges of the drugs are limited. Mitochondrial dysfunction could be a key factor involved in NAFLD progression. However, the association between mitochondrial dynamics, which involves mitochondrial fission and fusion, and NAFLD development has not been fully characterized to date. Herein, we examined the effects of alterations of mitochondrial dynamics in hepatocytes and liver sinusoidal endothelial cells (LSECs), the most abundant non‐parenchymal cells in the liver, on NAFLD development and hypothesized that rebalancing of mitochondrial dynamics in these cells could attenuate NAFLD progression. Mice were fed a choline‐deficient, L‐amino acid‐defined diet (CDAA) for 1 or 4 weeks to recapitulate human NAFLD development. Malformed mitochondria, such as megamitochondria and mitochondria with deformed cristae, were substantially increased in both, hepatocytes and LSECs, of mice that were fed CDAA diet, and were associated with NAFLD development. In addition, decreased mitochondrial content and increased mitochondrial number, suggesting excessive mitochondrial fission, were induced in hepatocytes, but not in LSECs, after four weeks of CDAA feeding. These unfavorable changes in the mitochondria of hepatocytes, but not of LSECs, were attenuated by mdivi1, a chemical mitochondrial fission inhibitor. Liver inflammation and fibrosis were also attenuated by mdivi1, as assessed by serum biochemical and histochemical analyses. The expression of several inflammatory and fibrogenic genes (Tnfα, Il6, and Col3α1) tended to decrease with mdivi1 treatment. The expression of genes related to mitochondrial biogenesis, fission, fusion, and mitophagy was also significantly reduced by mdivi1 (p<0.05), suggesting rebalanced mitochondrial dynamics. Ultrastructural analyses revealed that endoplasmic reticulum stress and autophagy in hepatocytes were mitigated by mdivi1 administration. Taken together, these findings suggest that excessive mitochondrial fission in hepatocytes induces alterations in mitochondrial dynamics and other organellar injuries, and subsequently, liver inflammation and fibrogenesis. Therefore, rebalancing mitochondrial dynamics in hepatocytes by inhibiting excessive fission is a promising therapeutic strategy for NAFLD.

Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission and fusion are known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin‐related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause developmental defects, we are using patient‐derived fibroblasts and iPSC‐derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. Using super resolution imaging, we find that patient cells, in addition to displaying elongated mitochondrial and peroxisomal morphology, present with aberrant cristae structure. Given the direct link between cristae morphology and oxidative phosphorylation efficiency, we explored the impact of these mutations on cellular energy production. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and Complex III deficiency. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in hyperpolarized mitochondrial membrane potential. Intriguingly, human fibroblasts are capable of cellular reprogramming into iPSCs and appear to display peroxisome‐mediated mitochondrial adaptations that could help sustain these cell fate transitions. Understanding the mechanism by which DRP1 mutations cause cellular dysfunction will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment.

BACKGROUND: Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in only 45% of patients. TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. Therefore, understanding the mechanisms of resistance to standard chemotherapy is essential. Our previous studies found mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual tumor cells that survived after NACT. Mitochondria are morphologically plastic and dynamic organelles that continuously cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. Mitochondrial fusion is thought to support OXPHOS through maintenance of the mitochondrial genome (mtDNA) which encodes 13 subunits of electron transport chain complexes. On the other hand, mitochondrial fission can be the precursor to mitophagy, the selective degradation of damaged mitochondria. We are investigating how mitochondrial structure dynamics impact metabolic states driving chemoresistance in TNBC. Methods We used a previously characterized orthotopic patient-derived xenograft (PDX) mouse model derived from the primary tumor of a treatment-naïve metastatic TNBC patient. After treatment with a standard NACT regimen, combined Adriamycin (aka doxorubicin) and cyclophosphamide (AC), we observed initial partial response followed by tumor regrowth. We collected PDX tumors at three time points: pre-treatment, post-AC residual (when tumors reached the volume nadir), and post-AC tumors that later regrew to the starting tumor volume. We used transmission electron microscopy (TEM) and immunohistochemical (IHC) analysis of human mitochondria to assess mitochondrial morphology and mass, respectively. For in vitro assays, we assessed mitochondria morphology, mtDNA content, and OXPHOS activity in TNBC cells treated with two different types of chemotherapeutic drugs, DNA-damaging agents (doxorubicin and carboplatin) and a microtubule-stabilizing agent (paclitaxel). To examine metabolic adaptations upon chemotherapy, we conducted 13C-glucose and glutamine flux metabolomics. To pharmaceutically perturb mitochondrial morphology, we used Mdivi-1 and Silibinin, known to induce mitochondrial fusion and fission, respectively. We assessed cell growth by Incucyte real-time imaging. Results: Analyses of residual PDX tumors after in vivo AC treatment revealed increased mitochondrial content, and increased average mitochondrial length in residual tumor cells. Metabolomic analysis of TNBC cells revealed all three chemotherapies induced glucose, but not glutamine, flux through the TCA cycle. Flux through glycolysis was not affected by chemotherapies. Treatment with doxorubicin and carboplatin, increased mitochondrial elongation, mtDNA content, and OXPHOS activity. Conversely, paclitaxel treatment reduced mitochondrial length and OXPHOS activity. We observed Mdivi-1 induced fusion concomitant with increased OXPHOS activity, while treatment with Silibinin reduced fusion and decreased OXPHOS. We next tested if induction of mitochondrial fission enhanced chemosensitivity. Sequential treatment of TNBC cells with DNA-damaging chemotherapeutics followed by Silibinin perturbed OXPHOS and resulted in enhanced cell killing. Conclusion: These findings establish a functional role for mitochondrial structure in chemotherapeutic response and metabolic reprogramming, which may confer a survival advantage to TNBC cells. Given the increased chemosensitivity we observed after pharmacologic inhibition of mitochondrial fusion, our study suggests perturbing mitochondrial structure adaptations may provide an opportunity to overcome TNBC chemoresistance in the neoadjuvant setting. Citation Format: Lily Mokryun Baek, Junegoo Lee, James P. Barrish, Bora Lim, Jeffrey T. Chang, Phillip L. Lorenzi, Weston Porter, Gloria V. Echeverria. Morphological and functional plasticity of mitochondria in chemoresistant triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-08.