Mitochondrial Protein Import Research Articles

Cardiac-specific deletion of heat shock protein 60 induces mitochondrial stress and disrupts heart development in mice

Congenital heart diseases are the most common birth defects around the world. Emerging evidence suggests that mitochondrial homeostasis is required for normal heart development. In mitochondria, a series of molecular chaperones including heat shock protein 60 (HSP60) are engaged in assisting the import and folding of mitochondrial proteins. However, it remains largely obscure whether and how these mitochondrial chaperones regulate cardiac development. Here, we generated a cardiac-specific Hspd1 deletion mouse model by αMHC-Cre and investigated the role of HSP60 in cardiac development. We observed that deletion of HSP60 in embryonic cardiomyocytes resulted in abnormal heart development and embryonic lethality, characterized by reduced cardiac cell proliferation and thinner ventricular walls, highlighting an essential role of cardiac HSP60 in embryonic heart development and survival. Our results also demonstrated that HSP60 deficiency caused significant downregulation of mitochondrial ETC subunits and induced mitochondrial stress. Analysis of gene expression revealed that P21 that negatively regulates cell proliferation is significantly upregulated in HSP60 knockout hearts. Moreover, HSP60 deficiency induced activation of eIF2α-ATF4 pathway, further indicating the underlying mitochondrial stress in cardiomyocytes after HSP60 deletion. Taken together, our study demonstrated that regular function of mitochondrial chaperones is pivotal for maintaining normal mitochondrial homeostasis and embryonic heart development.

Cytosolic retention of HtrA2 during mitochondrial protein import stress triggers the DELE1-HRI pathway

Mitochondrial stress inducers such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and oligomycin trigger the DELE1-HRI branch of the integrated stress response (ISR) pathway. Previous studies performed using epitope-tagged DELE1 showed that these stresses induced the cleavage of DELE1 to DELE1-S, which stimulates HRI. Here, we report that mitochondrial protein import stress (MPIS) is an overarching stress that triggers the DELE1-HRI pathway, and that endogenous DELE1 could be cleaved into two forms, DELE1-S and DELE1-VS, the latter accumulating only upon non-depolarizing MPIS. Surprisingly, while the mitochondrial protease OMA1 was crucial for DELE1 cleavage in HeLa cells, it was dispensable in HEK293T cells, suggesting that multiple proteases may be involved in DELE1 cleavage. In support, we identified a role for the mitochondrial protease, HtrA2, in mediating DELE1 cleavage into DELE1-VS, and showed that a Parkinson’s disease (PD)-associated HtrA2 mutant displayed reduced DELE1 processing ability, suggesting a novel mechanism linking PD pathogenesis to mitochondrial stress. Our data further suggest that DELE1 is likely cleaved into DELE1-S in the cytosol, while the DELE1-VS form might be generated during halted translocation into mitochondria. Together, this study identifies MPIS as the overarching stress detected by DELE1 and identifies a novel role for HtrA2 in DELE1 processing.

Abstract 5898: Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells

Abstract Compared to normal hematopoietic cells, AML cells exhibit a heightened reliance on mitochondrial metabolism for survival and proliferation. To support this unique phenotype, AML cells increase import of nuclear-encoded mitochondrial proteins which must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins. Failure to properly fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on maintaining mitochondrial proteostasis, we assessed gene dependency datasets (eg: depmap.org) and identified the mitochondrial protease LONP1, as the top essential gene. LONP1 is a matrix-localized nuclear-encoded AAA+ protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. By immunoblotting, LONP1 protein was increased in 16/30 primary AML samples compared to bulk (n=8) and CD34+ blood cells (n=3). LONP1 genetic depletion reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX leukemia cells as well as primary AML cells. LONP1 depletion increased aggregated mitochondrial proteins and reactive oxygen species (ROS) production while reducing mitochondrial respiration and membrane potential. To determine the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival, we over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3’UTR of the endogenous gene. Wild type or proteolytically dead (S855A) but not the ATPase mutant (E591A) rescued mitochondrial protein solubility, mitochondrial respiration, ROS generation, membrane potential, and cell viability. Thus, the ATPase domain but not the proteolytic activity of LONP1 is necessary for mitochondrial proteostasis. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that allosterically inhibits the LONP1 ATPase site. CDDO-Me killed OCI-AML2 and NB4 cells with IC50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200 nM) killed 3 out of 4 primary AML samples with high LONP1 expression but none of the 10 primary AML samples with low LONP1. Likewise, CDDO-Me induced mitochondrial protein aggregation in AML cell lines and primary AML patient samples with high LONP1 expression but not AML cells or primary AML patient samples with low LONP1. In summary, LONP1 is over-expressed in a subset of AML cells and primary samples where it maintains mitochondrial protein solubility. Selective inhibition of the LONP1 ATPase domain leads to mitochondrial protein aggregation and AML cell death, representing a novel therapeutic strategy for AML. Citation Format: Matthew Tcheng, Marcela Gronda, Rose Hurren, Lan Xin Zhang, Chaitra Sarathy, Yongran Yan, Andrea Arruda, Mark D. Minden, Aaron D. Schimmer. Inhibition of the LONP1 ATPase domain induces mitochondrial proteotoxic stress and is cytotoxic to acute myeloid leukemia cells [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 5898.

Abstract 350: Targeting prohibitins by fluorizoline leads to the activation of ISR and apoptosis through DELE1 and HRI by an impairment of the mitochondrial protein import machinery

Abstract Prohibitins are ubiquitously expressed within the cell, but they are mainly located at the inner mitochondrial membrane. These proteins are closely associated with tumour progression, invasion, and apoptotic resistance, being overexpressed in various primary tumours such as lymphoma and endometrial cancer. Notably, we have developed a novel synthetic molecule, fluorizoline, which induces apoptosis by selectively targeting PHBs in different cancer cell lines and primary samples from various haematological neoplasias. We have previously described that fluorizoline-induced apoptosis is mediated through the activation of the pro-apoptotic branch of the integrated stress response (ISR) pathway in HeLa and HAP1 cells, concretely, by the ATF4-CHOP-NOXA axis. In a recent study, we identified compensatory mechanisms for four ISR-related kinases. Importantly, HRI emerged as the primary kinase responsible for activating this pathway and promoting apoptosis, suggesting that mitochondrial stress could lead to this activation of the ISR.Here, we investigate the OMA1-DELE1-HRI molecular pathway responsible for sensing mitochondrial stress and activating the ISR cancer cell lines. Firstly, we demonstrate that fluorizoline treatment triggers the cleavage of large DELE1 (L-DELE1) to its cleaved form (S-DELE1) through western blot analysis, and its subsequent sorting to the cytosol as confirmed by confocal analysis. Using DELE1 KO lines generated by CRISPR/Cas9 technology, we demonstrate that DELE1 is involved in the activation of the ISR and apoptosis induced by fluorizoline. However, although we cannot rule out OMA1 involvement, fluorizoline-induced ISR activation does not strongly depend on OMA1. Interestingly, our data suggests that targeting PHBs, either by fluorizoline or by their downregulation, could result in mitochondrial protein import machinery impairment. This impairment may potentially be the cause of DELE1 accumulation in the cytosol, leading to the subsequent activation of the ISR by HRI upon fluorizoline treatment. Citation Format: Ismael Sánchez-Vera, Nekane Maritorena-Hualde, Max-Hinderk Schuler, Ana M- Cosialls, Rodolfo Lavilla, Gabriel Pons, Lucas T Jae, Daniel Iglesias-Serret, Joan Gil. Targeting prohibitins by fluorizoline leads to the activation of ISR and apoptosis through DELE1 and HRI by an impairment of the mitochondrial protein import machinery [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 350.

Neuroprotective mushrooms

Abstract Objectives Alternative medicines commonly supplement or, at times, replace standard medical treatment. One area of increasing attention is disease-modifying medicines for neurodegenerative diseases. However, few such alternatives have been investigated thoroughly with an eye towards understanding mechanisms of action for clinical use. Medicinal mushrooms have important health benefits and pharmacological activities with anti-inflammatory, antioxidant, antibacterial, antiviral, immunomodulatory, digestive, cytoprotective, homeostatic, and neuroprotective activities. Edible mushrooms are known to play roles in preventing age-related diseases. Several studies have revealed that polysaccharides, terpenes, and phenolic compounds are chemical components derived from mushrooms with pharmacological activities. Due to limited effective protocols for mushroom protein extraction for proteomic studies, information about these medicinally related proteins and their biological functions remains enigmatic. Methods Herein, we have performed proteomic studies of two mushroom species Laricifomes officinalis (agarikon) and Grifola frondosa (maitake). Results These studies serve to uncover a foundation for putative proteome-associated neuroprotective processes. The recovered proteins from both species show multiple cell-specific signaling pathways including unfolded protein response, and mitochondrial protein import as well as those linked to BAG2, ubiquitination, apoptosis, microautophagy, glycolysis, SNARE, and immunogenic cell signaling pathways. Conclusions This study uncovered mushroom proteome-associated proteins which serve to better understand the structural and functional properties of mushrooms used as alternative medicines for broad potential health benefits.

Defining the molecular basis and regulation of mitochondrial co-translational protein import

Defining the molecular basis and regulation of mitochondrial co-translational protein import

Stress response silencing by an E3 ligase mutated in neurodegeneration

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1–3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Phosphate starvation signaling increases mitochondrial membrane potential through respiration-independent mechanisms.

Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.

Quantifying mitochondrial protein import by mePRODmt proteomics.

Mitochondrial protein import is crucial for maintaining cellular health and homeostasis. Disruptions in this process have been linked to various diseases. Traditional methods for studying mitochondrial protein import predominantly focus on individual proteins and lack the dynamic resolution needed to fully appreciate the complexity of mitochondrial proteostasis and protein trafficking. To address these limitations, we developed a technique called mitochondria-specific multiplexed enhanced protein dynamics (mePRODmt). This method is a novel application of the mePROD methodology and utilizes pulsed stable isotope labeling with amino acids in cell culture (pSILAC)-based proteomics approach to study transient mitochondrial protein import. This chapter outlines the mePRODmt protocol, which includes the preparation of heavy SILAC-labeled peptides for boosting overall mitochondrial peptide signals (booster), SILAC labeling of cultured cells under experimental conditions, mitochondria isolation, sample preparation for multiplex proteomics using tandem mass tags (TMT) for isobaric labeling, recommended liquid chromatography-mass spectrometry (LC-MS) settings for reporter ion quantitation and a data analysis pipeline to analyze pSILAC-TMT data.

Monitoring mitochondrial protein import by live cell imaging.

The majority of mitochondrial proteins are synthesized in the cytosol and must be imported into mitochondria to attain their mature forms and execute their functions. Disruption of mitochondrial functions, whether caused by external or internal stress, may compromise mitochondrial protein import. Therefore, monitoring mitochondrial protein import has become a standard approach to assess mitochondrial health and gain insights into mitochondrial biology, especially during stress. This chapter describes a detailed protocol for monitoring mitochondrial import in live cells using microscopy. Co-localization between mitochondria and a genetic reporter of mitochondrially targeted enhanced GFP (eGFP) is employed to evaluate mitochondrial protein import efficiency under different physiological conditions. Overall, this technique provides a simple and robust approach to assess mitochondrial protein import efficiency within its native cellular environment.

Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.

Mitochondrial protein import is a complex process governing the delivery of the organelle's proteome. This process, in turn, is essential for maintaining mitochondrial function and cellular homeostasis. Initiated by protein synthesis in the cytoplasm, precursor proteins destined for the mitochondria possess targeting signals that guide them to the mitochondrial surface. At mitochondria, the translocation of proteins across the mitochondrial membranes involves an intricate interplay between translocases, chaperones, and receptors. The mitochondrial import assay offers researchers the opportunity to recapitulate the process of protein import in vitro. The assay has served as an indispensable tool in helping decipher the intricacies of protein translocation into mitochondria, first in fungal models, and subsequently in higher eukaryotic models. In this chapter, we will describe how protein import can be assayed using mammalian mitochondria and provide insight into the types of questions that can be addressed in mammalian mitochondrial biology using this experimental approach.

The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.

The NanoLuc split luciferase assay has proven to be a powerful tool for the analysis of protein translocation. Its flexibility has enabled in vivo, ex vivo, and in vitro studies-including systems reconstituting protein transport from pure components. The assay has been particularly useful in the characterization of bacterial secretion and mitochondrial protein import. In the latter case, MitoLuc has been developed for the investigation of the TIM23-pathway via import into the matrix of isolated yeast mitochondria. Subsequent analysis identified three distinct phases of import, rather than in a single continuous step. The assay has also been developed to monitor import into the mitochondrial matrix of intact cultured cells. This latter innovation has laid the foundations for further analysis of the import process in humans, including the consequences of interactions with cytosolic factors and neighboring organelles. The versatility of the MitoLuc assay is conducive for its adaptation to also monitor import into the inter-membrane space (MIA-pathway), and into the inner-membrane via the TIM22- and TIM23-complexes. Here, we present detailed protocols for the application of MitoLuc to mitochondria isolated from yeast and to those within cultured human cells.

Import of mitochondrial precursor proteins into mitochondria of semi-intact yeast cells.

Mitochondria import hundreds of different precursor proteins from the cytosol and direct each of these to its specific mitochondrial subcompartment. The import routes and mechanisms by which precursors are transported into the outer membrane, the intermembrane space (IMS), the inner membrane and the matrix have been characterized in depth by use of very powerful in vitro assays. In the 'classical' import assays, radiolabeled precursor proteins are incubated with isolated mitochondria and the protein uptake is monitored by one or more of the following observations: intramitochondrial processing, resistance to externally added proteases, or the formation of disulfide bonds. In this chapter, we describe an alternative import assay which employs semi-intact yeast cells. This assay uses spheroplasts from which the cell wall had been removed by enzymatic digestion before the plasma membrane was partially permeabilized by a freeze-thawing step. Since the organellar architecture is largely maintained in semi-intact cells, this in vitro import assay allows to elucidate the targeting of precursor proteins from the cytoplasm to the mitochondrial surface. Thereby the contribution of other compartments such as the endoplasmic reticulum (ER) can be assessed. Here we describe how semi-intact cells are prepared and used in the in vitro import assay and discuss the pros and cons of this approach.

In vitro import of mitochondrial precursor proteins into yeast mitochondria.

Mitochondria contain about 1000 different proteins, only a handful of which are encoded in the mitochondrial genome. The remaining c. 99% of mitochondrial proteins are encoded in the nuclear genome, synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals and are subsequently imported into the organelle. Mitochondrial targeting signals are very diverse and mitochondria therefore also have a number of very sophisticated molecular machines that recognize, import and sort mitochondrial precursor proteins to the different mitochondrial subcompartments. The ability to synthesize mitochondrial precursor proteins in vitro and subsequently import them into isolated mitochondria has revolutionized our understanding of mitochondrial protein import pathways. Here, we describe the basic protocol for synthesis of mitochondrial precursor proteins in vitro and their subsequent import into isolated mitochondria from yeast Saccharomyces cerevisiae, the method which was used to elucidate and characterize the vast majority of mitochondrial protein import pathways.

Role of mitochondrial DNA in yeast replicative aging

Despite the variety of manifestations of aging, there are some common features and underlying mechanisms. In particular, mitochondria appears to be one of the most vulnerable systems in both metazoa and fungi. In this review, we discuss how mitochondrial dysfunction is related to replicative aging in the simplest eukaryotic model, the baker’s yeast Saccharomyces cerevisiae. We discuss a chain of events that starts from asymmetric inheritance of mitochondria by mother and daughter cells. With age, yeast mother cells start to experience a decrease in mitochondrial transmembrane potential and, consequently, a decrease in mitochondrial protein import efficiency. This induces mitochondrial protein precursors accumulation in the cytoplasm, the loss of mitochondrial DNA, and at the later stages - cell death. Interestingly, yeast strains without mitochondrial DNA can have both increased and increased lifespan compared to their counterparts with mtDNA. The direction of the effect depends on their ability to activate compensatory mechanisms preventing or mitigating negative consequences of mitochondrial dysfunction. The central role of mitochondria in yeast aging and death indicates that it is one of the most complex and, therefore, deregulation-prone systems in eukaryotic cells.

Role of Mitochondrial DNA in Yeast Replicative Aging.

Despite the diverse manifestations of aging across different species, some common aging features and underlying mechanisms are shared. In particular, mitochondria appear to be among the most vulnerable systems in both metazoa and fungi. In this review, we discuss how mitochondrial dysfunction is related to replicative aging in the simplest eukaryotic model, the baker's yeast Saccharomyces cerevisiae. We discuss a chain of events that starts from asymmetric distribution of mitochondria between mother and daughter cells. With age, yeast mother cells start to experience a decrease in mitochondrial transmembrane potential and, consequently, a decrease in mitochondrial protein import efficiency. This induces mitochondrial protein precursors in the cytoplasm, the loss of mitochondrial DNA (mtDNA), and at the later stages - cell death. Interestingly, yeast strains without mtDNA can have either increased or decreased lifespan compared to the parental strains with mtDNA. The direction of the effect depends on their ability to activate compensatory mechanisms preventing or mitigating negative consequences of mitochondrial dysfunction. The central role of mitochondria in yeast aging and death indicates that it is one of the most complex and, therefore, deregulation-prone systems in eukaryotic cells.

The Atpase Domain of LONP1 Is Necessary for Mitochondrial Protein Solubility and the Viability of Acute Myeloid Leukemia (AML) Cells

Compared to normal hematopoietic cells, we and others have shown that AML cells have a heightened reliance on oxidative phosphorylation and mitochondrial metabolism for survival and proliferation. To support this unique metabolic phenotype, we previously demonstrated that AML cells have increased import of nuclear-encoded mitochondrial proteins. These newly imported proteins must be properly folded by mitochondrial chaperones, proteases, and heat shock proteins, and failure to properly process and fold these precursors leads to protein aggregation, mitochondrial dysfunction, and cell death. To evaluate the reliance of AML cells on this family of mitochondrial chaperones, proteases, and heat shock proteins, we assessed their dependencies using the gene dependency datasets (eg: depmap.org). From this analysis, we identified the mitochondrial protease, LONP1, as the top hit, and among the top 10% of all essential genes for AML. Localized to the mitochondrial matrix, LONP1 is a nuclear encoded AAA+ serine protease. Proteins are unfolded by its ATPase domain and degraded by its serine-catalyzed proteolytic domain. Compared to normal hematopoietic cells, LONP1 mRNA was overexpressed in AML across three publicly available datasets. Compared to normal hematopoietic cells (n=8) and CD34+ cells (n=3), LONP1 protein was increased in 16/30 primary AML samples by immunoblotting. Using shRNA and CRISPR, LONP1 knockdown and knockout reduced the growth and viability of OCI-AML2, OCI-M2, NB4, and TEX cells. Genetic knockdown or knockout of LONP1 increased levels of insoluble, aggregated mitochondrial proteins as measured by mass spectrometry, proteostat fluorescence and confocal microscopy, and immunoblotting of soluble and insoluble protein fractions. LONP1 knockdown/knockout also reduced mitochondrial respiration, depolarized the mitochondria, and induced AML cell death. We next determined the domain of LONP1 that was necessary for mitochondrial protein solubility, mitochondrial function, and AML survival. We over-expressed wild type, ATPase dead (E591A), or proteolytically dead (S855A) LONP1 cDNA in OCI-AML2 cells and knocked down endogenous LONP1 with shRNA targeting the 3'UTR of the endogenous gene. We then measured mitochondrial protein solubility/aggregation, mitochondrial respiration and cell viability. Wild type LONP1 and the proteolytically dead (S855A) mutant, but not the ATPase mutant (E591A), rescued mitochondrial protein solubility, mitochondrial respiration and cell viability, thus demonstrating that the ATPase domain of LONP1 is necessary for these functions. Bardoxolone methyl (CDDO-Me) is a synthetic triterpenoid that inhibits the ATPase activity of LONP1 by binding to an allosteric site near the ATPase domain of the enzyme. CDDO-Me killed OCI-AML2 and NB4 cells with IC 50 values of 178.5±29.7 and 156.5±39.7 nM, respectively. CDDO-Me (200nM) also killed >50% of cells in 3 out of 4 high-LONP1 expressing primary AML patient samples, but 10 of 10 tested primary AML samples with low levels of LONP1 were insensitive to the drug. Likewise, CDDO-Me induced mitochondrial protein aggregation in OCI-AML2 cells and in a primary AML patient sample with high LONP1 expression, while a primary AML patient sample with undetectable LONP1 expression showed no protein aggregation. In summary, the mitochondrial serine AAA+ protease LONP1 is over-expressed in a subset of AML cells and primary samples. The ATPase domain is necessary for LONP1's function in maintaining mitochondrial protein solubility. Selective inhibition of this domain leads to mitochondrial protein aggregation, impaired mitochondrial respiration and AML cell death. Thus, inhibiting the ATPase domain of LONP1 may be a novel therapeutic strategy for AML.

Integration of FUNDC1-associated mitochondrial protein import and mitochondrial quality control contributes to TDP-43 degradation

Though TDP-43 protein can be translocated into mitochondria and causes mitochondrial damage in TDP-43 proteinopathy, little is known about how TDP-43 is imported into mitochondria. In addition, whether mitochondrial damage is caused by mitochondrial mislocalization of TDP-43 or a side effect of mitochondria-mediated TDP-43 degradation remains to be investigated. Here, our bioinformatical analyses reveal that mitophagy receptor gene FUNDC1 is co-expressed with TDP-43, and both TDP-43 and FUNDC1 expression is correlated with genes associated with mitochondrial protein import pathway in brain samples of patients diagnosed with TDP-43 proteinopathy. FUNDC1 promotes mitochondrial translocation of TDP-43 possibly by promoting TDP-43-TOM70 and DNAJA2-TOM70 interactions, which is independent of the LC3 interacting region of FUNDC1 in cellular experiments. In the transgenic fly model of TDP-43 proteinopathy, overexpressing FUNDC1 enhances TDP-43 induced mitochondrial damage, whereas down-regulating FUNDC1 reverses TDP-43 induced mitochondrial damage. FUNDC1 regulates mitochondria-mediated TDP-43 degradation not only by regulating mitochondrial TDP-43 import, but also by increasing LONP1 level and by activating mitophagy, which plays important roles in cytosolic TDP-43 clearance. Together, this study not only uncovers the mechanism of mitochondrial TDP-43 import, but also unravels the active role played by mitochondria in regulating TDP-43 homeostasis.

Interactions of amyloidogenic proteins with mitochondrial protein import machinery in aging-related neurodegenerative diseases.

Most mitochondrial proteins are targeted to the organelle by N-terminal mitochondrial targeting sequences (MTSs, or "presequences") that are recognized by the import machinery and subsequently cleaved to yield the mature protein. MTSs do not have conserved amino acid compositions, but share common physicochemical properties, including the ability to form amphipathic α-helical structures enriched with basic and hydrophobic residues on alternating faces. The lack of strict sequence conservation implies that some polypeptides can be mistargeted to mitochondria, especially under cellular stress. The pathogenic accumulation of proteins within mitochondria is implicated in many aging-related neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. Mechanistically, these diseases may originate in part from mitochondrial interactions with amyloid-β precursor protein (APP) or its cleavage product amyloid-β (Aβ), α-synuclein (α-syn), and mutant forms of huntingtin (mHtt), respectively, that are mediated in part through their associations with the mitochondrial protein import machinery. Emerging evidence suggests that these amyloidogenic proteins may present cryptic targeting signals that act as MTS mimetics and can be recognized by mitochondrial import receptors and transported into different mitochondrial compartments. Accumulation of these mistargeted proteins could overwhelm the import machinery and its associated quality control mechanisms, thereby contributing to neurological disease progression. Alternatively, the uptake of amyloidogenic proteins into mitochondria may be part of a protein quality control mechanism for clearance of cytotoxic proteins. Here we review the pathomechanisms of these diseases as they relate to mitochondrial protein import and effects on mitochondrial function, what features of APP/Aβ, α-syn and mHtt make them suitable substrates for the import machinery, and how this information can be leveraged for the development of therapeutic interventions.

Decoding mitochondrial-nuclear (epi)genome interactions: the emerging role of ncRNAs.

Bidirectional communication between the mitochondria and the nucleus is required for several physiological processes, and the nuclear epigenome is a key mediator of this relationship. ncRNAs are an emerging area of discussion for their roles in cellular function and regulation. In this review, we highlight the role of mitochondrial-encoded ncRNAs as mediators of communication between the mitochondria and the nuclear genome. We focus primarily on retrograde signaling, a process in which the mitochondrion relays ncRNAs to translate environmental stress signals to changes in nuclear gene expression, with implications on stress responses that may include disease(s). Other biological roles of mitochondrial-encoded ncRNAs, such as mitochondrial import of proteins and regulation of cell signaling, will also be discussed.