PINK1 Inhibits Local Protein Synthesis to Limit Transmission of Deleterious Mitochondrial DNA Mutations.

We have previously proposed that selective inheritance, the limited transmission of damaging mtDNA mutations from mother to offspring, is based on replication competition in Drosophila melanogaster. This model, which stems from our observation that wild-type mitochondria propagate much more vigorously in the fly ovary than mitochondria carrying fitness-impairing mutations, implies that germ cells recognize the fitness of individual mitochondria and selectively boost the propagation of healthy ones. Here, we demonstrate that the protein kinase PINK1 preferentially accumulates on mitochondria enriched for a deleterious mtDNA mutation. PINK1 phosphorylates Larp to inhibit protein synthesis on the mitochondrial outer membrane. Impaired local translation on defective mitochondria in turn limits the replication of their mtDNA and hence the transmission of deleterious mutations to the offspring. Our work confirms that selective inheritance occurs at the organelle level during Drosophila oogenesis and provides molecular entry points to test this model in other systems.

Targeting metabolic sensing switch GPR84 on macrophages for cancer immunotherapy.

INTRODUCTION: As one of the major components of the tumor microenvironment, tumor-associated macrophages (TAMs) possess profound inhibitory activity against T cells and facilitate tumor escape from immune checkpoint blockade therapy. Converting this pro-tumorigenic toward the anti-tumorigenic phenotype thus is an important strategy for enhancing adaptive immunity against cancer. However, a plethora of mechanisms have been described for pro-tumorigenic differentiation in cancer, metabolic switches to program the anti-tumorigenic property of TAMs are elusive. MATERIALS AND METHODS: From an unbiased analysis of single-cell transcriptome data from multiple tumor models, we discovered that anti-tumorigenic TAMs uniquely express elevated levels of a specific fatty acid receptor, G-protein-coupled receptor 84 (GPR84). Genetic ablation of GPR84 in mice leads to impaired pro-inflammatory polarization of macrophages, while enhancing their anti-inflammatory phenotype. By contrast, GPR84 activation by its agonist, 6-n-octylaminouracil (6-OAU), potentiates pro-inflammatory phenotype via the enhanced STAT1 pathway. Moreover, 6-OAU treatment significantly retards tumor growth and increases the anti-tumor efficacy of anti-PD-1 therapy. CONCLUSION: Overall, we report a previously unappreciated fatty acid receptor, GPR84, that serves as an important metabolic sensing switch for orchestrating anti-tumorigenic macrophage polarization. Pharmacological agonists of GPR84 hold promise to reshape and reverse the immunosuppressive TME, and thereby restore responsiveness of cancer to overcome resistance to immune checkpoint blockade.

Parkin and PINK1 mitigate STING-induced inflammation.

Although serum from patients with Parkinson's disease contains elevated levels of numerous pro-inflammatory cytokines including IL-6, TNF, IL-1ß, and IFNγ, whether inflammation contributes to or is a consequence of neuronal loss remains unknown1. Mutations in parkin, an E3 ubiquitin ligase, and PINK1, a ubiquitin kinase, cause early onset Parkinson's disease2,3. Both PINK1 and parkin function within the same biochemical pathway and remove damaged mitochondria from cells in culture and in animal models via mitophagy, a selective form of autophagy4. The in vivo role of mitophagy, however, is unclear, partly because mice that lack either PINK1 or parkin have no substantial Parkinson's-disease-relevant phenotypes5-7. Mitochondrial stress can lead to the release of damage-associated molecular patterns (DAMPs) that can activate innate immunity8-12, suggesting that mitophagy may mitigate inflammation. Here we report a strong inflammatory phenotype in both Prkn-/- and Pink1-/- mice following exhaustive exercise and in Prkn-/-;mutator mice, which accumulate mutations in mitochondrial DNA (mtDNA)13,14. Inflammation resulting from either exhaustive exercise or mtDNA mutation is completely rescued by concurrent loss of STING, a central regulator of the type I interferon response to cytosolic DNA15,16. The loss of dopaminergic neurons from the substantia nigra pars compacta and the motor defect observed in aged Prkn-/-;mutator mice are also rescued by loss of STING, suggesting that inflammation facilitates this phenotype. Humans with mono- and biallelic PRKN mutations also display elevated cytokines. These results support a role for PINK1- and parkin-mediated mitophagy in restraining innate immunity.

The Mitochondrial Basis of Aging.

A decline in mitochondrial quality and activity has been associated with normal aging and correlated with the development of a wide range of age-related diseases. Here, we review the evidence that a decline in mitochondria function contributes to aging. In particular, we discuss how mitochondria contribute to specific aspects of the aging process, including cellular senescence, chronic inflammation, and the age-dependent decline in stem cell activity. Signaling pathways regulating the mitochondrial unfolded protein response and mitophagy are also reviewed, with particular emphasis placed on how these pathways might, in turn, regulate longevity. Taken together, these observations suggest that mitochondria influence or regulate a number of key aspects of aging and suggest that strategies directed at improving mitochondrial quality and function might have far-reaching beneficial effects.

Measuring In Vivo Mitophagy.

Alterations in mitophagy have been increasingly linked to aging and age-related diseases. There are, however, no convenient methods to analyze mitophagy in vivo. Here, we describe a transgenic mouse model in which we expressed a mitochondrial-targeted form of the fluorescent reporter Keima (mt-Keima). Keima is a coral-derived protein that exhibits both pH-dependent excitation and resistance to lysosomal proteases. Comparison of a wide range of primary cells and tissues generated from the mt-Keima mouse revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima mice to analyze how mitophagy is altered by conditions including diet, oxygen availability, Huntingtin transgene expression, the absence of macroautophagy (ATG5 or ATG7 expression), an increase in mitochondrial mutational load, the presence of metastatic tumors, and normal aging. The ability to assess mitophagy under a host of varying environmental and genetic perturbations suggests that the mt-Keima mouse should be a valuable resource.

The Rbf1, Hfl1 and Dbp4 of Candida albicans regulate common as well as transcription factor-specific mitochondrial and other cell activities.

BACKGROUND: Our interest in Candida albicans mitochondria began with the identification of GOA1. We demonstrated its role in cell energy production, cross-talk among mitochondria and peroxisomes, non-glucose energy metabolism, maintenance of stationary phase growth, and prevention of premature apoptosis. Its absence results in avirulence. However, what regulated transcription of GOA1 was unknown. RESULTS: To identify transcriptional regulators (TRs) of GOA1, we screened a C. albicans TF knockout library (TRKO) and identified Rbf1p, Hfl1p, and Dpb4p as positive TRs of GOA1. The phenotypes of each mutant (reduced respiration, inability to grow on glycerol, reduced ETC CI and CIV activities) are reasonable evidence for their required roles especially in mitochondrial functions. While the integration of mitochondria with cell metabolic activities is presumed to occur, there is minimal information on this subject at the genome level. Therefore, microarray analysis was used to provide this information for each TR mutant. Transcriptional profiles of Rbf1p and Hfl1p are more similar than that of Dpn4p. Our data demonstrate common and also gene-specific regulatory functions for each TR. We establish their roles in carbon metabolism, stress adaptation, cell wall synthesis, transporter efflux, peroxisomal metabolism, phospholipid synthesis, rRNA processing, and nuclear/mtDNA replication. CONCLUSIONS: The TRs regulate a number of common genes but each also regulates specific gene transcription. These data for the first time create a genome roadmap that can be used to integrate mitochondria with other cell processes. Of interest, the TRs are fungal-specific, warranting consideration as antifungal drug targets.

A self-activating electron transfer antibacterial strategy: Co3O4/TiO2 P-N heterojunctions combined with photothermal therapy.

Implant-associated infections are significant impediments to successful surgical outcomes, often resulting from persistent bacterial contamination. It has been hypothesized that bacteria can transfer electrons to semiconductors with comparable potential to the biological redox potential (BRP). Building on this concept, we developed an antibiotic-free bactericidal system, Co3O4/TiO2-Ti, capable of achieving real-time and sustainable bactericidal effects. Our study demonstrated that Co3O4/TiO2-Ti, possessing an appropriately set valence band, initiated charge transfer, reactive oxygen species (ROS) production, and membrane damage in adherent Staphylococcus aureus (S. aureus). Notably, in vivo experiments illustrated the remarkable antibacterial activity of Co3O4/TiO2-Ti, while promoting soft-tissue reconstruction and demonstrating excellent cytocompatibility. Transcriptomic analysis further revealed a down-regulation of aerobic respiration-associated genes and an up-regulation of ROS-associated genes in S. aureus in the presence of Co3O4/TiO2-Ti compared to Ti. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA) identified alterations in respiratory metabolism, oxidative phosphorylation, and the synthesis of amino acid in S. aureus cultured on Co3O4/TiO2-Ti. Furthermore, when combined with near-infrared (NIR) irradiation and photothermal therapy (PTT), Co3O4/TiO2-Ti eliminated 95.71% of floating and adherent S. aureus in vitro. The findings suggest that this antibiotic-free strategy holds substantial promise in enhancing implant sterilization capabilities, thereby contributing to the prevention and treatment of bacterial infections through bandgap engineering of implants and NIR irradiation.

A Biomimetic Nanogenerator to Enhance Bone Regeneration by Restoring Electric Microenvironments.

Piezoelectric materials have received increasing attention in bone regeneration due to their prominent role in bioelectricity in bone homeostasis. This study aimed to develop bioactive barium titanate-chitosan-graphene oxide piezoelectric nanoparticles (BCG-NPs) to improve biocompatibility and stimulate bone repair. Butterfly loops, hysteresis loops, and in vitro microcurrent studies on BCG-NPs confirmed their good piezoelectric properties. BCG-NPs exhibited enhanced alkaline phosphatase activity, mineralized nodule formation, and expression of osteogenic-associated proteins and genes in human umbilical cord Wharton's jelly-derived mesenchymal stem cells by creating microelectric environments in response to noninvasive ultrasound stimulation. Further, BCG-NPs upregulated intracellular calcium ions via electrical stimulation. They acted synergistically with piezo-type mechanosensitive ion channel component 1 and calcium-permeable cation channel transient receptor potential vanilloid 4 to activate osteogenic differentiation. In conclusion, ultrasound-assisted BCG-NPs created a microelectric environment that putatively promoted bone repair in a noninvasive manner.

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in the development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health, and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from patients with PAH. Endothelial haploinsufficiency of RAB7 caused spontaneous pulmonary hypertension (PH) in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA-Seq, and RAB7-silenced ECs showed impaired angiogenesis and expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, suggesting inhibition of autophagy at the predegradation level. Furthermore, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in rats with chronic hypoxia/SU5416. In conclusion, we demonstrate for the first time to our knowledge the fundamental impairment of EC function by loss of RAB7, causing PH, and show RAB7 activation to be a potential therapeutic strategy in a preclinical model of PH.

Multidrug-resistant transporter mdr1p-mediated uptake of a novel antifungal compound.

The activity of many anti-infectious drugs has been compromised by the evolution of multidrug-resistant (MDR) pathogens. For life-threatening fungal infections, such as those caused by Candida albicans, overexpression of MDR1, which encodes an MDR efflux pump of the major facilitator superfamily (MFS), often confers resistance to chemically unrelated substances, including the most commonly used azole antifungals. As the development of new and efficacious antifungals has lagged far behind the growing emergence of resistant strains, it is imperative to develop strategies to overcome multidrug resistance. Previous advances have been mainly to deploy combinational therapy to restore azole susceptibility, which, however, requires coordination of two or more compounds. We observed a unique phenotype in which Mdr1p facilitates the uptake of a specific class of compounds. Among them, we describe a novel antifungal small molecule, bis[1,6-a:5',6'-g]quinolizinium 8-methyl-salt (BQM) (U.S. patent application no. 61/793,090,2013), that has potent and broad antifungal activity. Notably, BQM exploits the MDR phenotype in C. albicans to promote the inhibitory effect. Rather than causing an antagonism of MDR strains, it exhibits a highly potentiated activity against a collection of clinical isolates and lab strains that overexpress MDR1. The activity of BQM against MDR1-overexpressing isolates is due to its facilitated intracellular accumulation. Microarray comparisons showed an extensive upregulation of MDR1 as well as polyamine transporter genes in a fluconazole-resistant strain. We then demonstrated that the polyamine transporters augment the accumulation of BQM. Importantly, BQM had greater activity than fluconazole and itraconazole against various fungal pathogens, including MDR Aspergillus fumigatus. Thus, our findings offer a paradigm shift to overcome MDR and the promise of improving antifungal treatment, especially in MDR pathogens.

Azole susceptibility and transcriptome profiling in Candida albicans mitochondrial electron transport chain complex I mutants.

Mitochondrial dysfunction in pathogenic fungi or model yeast causes altered susceptibilities to antifungal drugs. Here we have characterized the role of mitochondrial complex I (CI) of Candida albicans in antifungal susceptibility. Inhibitors of CI to CV, except for CII, increased the susceptibility of both patient and lab isolates, even those with a resistance phenotype. In addition, in a C. albicans library of 12 CI null mutants, 10 displayed hypersusceptibility to fluconazole and were severely growth inhibited on glycerol, implying a role for each gene in cell respiration. We chose two other hypersusceptible null mutants of C. albicans, the goa1Δ and ndh51Δ mutants, for transcriptional profiling by RNA-Seq. Goa1p is required for CI activity, while Ndh51p is a CI subunit. RNA-Seq revealed that both the ndh51Δ mutant and especially the goa1Δ mutant had significant downregulation of transporter genes, including CDR1 and CDR2, which encode efflux proteins. In the goa1Δ mutant, we noted the downregulation of genes required for the biogenesis and replication of peroxisomes, as well as metabolic pathways assigned to peroxisomes such as ß-oxidation of fatty acids, glyoxylate bypass, and acetyl coenzyme A (acetyl-CoA) transferases that are known to shuttle acetyl-CoA between peroxisomes and mitochondria. The transcriptome profile of the ndh51Δ mutant did not include downregulation of peroxisome genes but had, instead, extensive downregulation of the ergosterol synthesis gene family. Our data establish that cell energy is required for azole susceptibility and that downregulation of efflux genes may be an outcome of that dysfunction. However, there are mutant-specific changes that may also increase the susceptibility of both of these C. albicans mutants to azoles.

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from PAH patients. Endothelial haploinsufficiency of RAB7 caused spontaneous PH in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA sequencing and RAB7 silenced ECs showed impaired angiogenesis, expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, which suggests inhibition of autophagy at the pre-degradation level. Further, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in chronic hypoxia/SU5416 rats. In conclusion, we demonstrate here for the first time the fundamental impairment of EC function by loss of RAB7 that leads to PH and show RAB7 activation as a potential therapeutic strategy in a preclinical model of PH.

Caloric restriction restores the chronological life span of the Goa1 null mutant of Candida albicans in spite of high cell levels of ROS.

The Candida albicans Goa1p is required for mitochondrial functions. In a strain lacking GOA1 (GOA31), respiration, mitochondrial membrane potential, complex I (CI) activity of the electron transport chain, and ATP synthesis are significantly decreased. A shortened chronological life span (CLS) of GOA31 occurs in 2% glucose that is associated with an increase in cell reactive oxidant species (ROS) and apoptosis. We now show that caloric restriction (CR) in media containing 0.5% glucose instead of 2% glucose-SC extends the CLS to the level of parental and gene-reconstituted strains. Paradoxically, ROS levels in GOA31 far exceed those of control strains in 0.5% glucose and, as a consequence, increased lipid peroxidation occurs even though CLS is restored. Microarray analysis was used to characterize transcriptional changes during CR in GOA31. We found that CR shifts cells of all strains to a non-glucose carbon metabolism (ß-oxidation). Our model of ROS formation in GOA31 follows the paradigm that the generation of oxygen radicals from ß-oxidation of cell lipids via FADH(2) (CII) and NADH (CI) creates an unfavorable cellular FADH(2)/NADH ratio that causes a transient overload in CII activity resulting in excess free cell radicals. In GOA31 the CI and peroxisomal dysfunctions increase the levels of ROS compared to control strains. Recovery from high levels of ROS may be associated with an increase in iron and sugar transporters, as well as an anti-stress response that includes the SOD1 and GPX1. Thus, CR creates a favorable growth environment, but cells of GOA31 must overcome a high but transient ROS production.

Significance of mitochondrial activity in neurogenesis and neurodegenerative diseases.

Mitochondria play a multidimensional role in the function and the vitality of the neurological system. From the generation of neural stem cells to the maintenance of neurons and their ultimate demise, mitochondria play a critical role in regulating our neural pathways' homeostasis, a task that is critical to our cognitive health and neurological well-being. Mitochondria provide energy via oxidative phosphorylation for the neurotransmission and generation of an action potential along the neuron's axon. This paper will first review and examine the molecular subtleties of the mitochondria's role in neurogenesis and neuron vitality, as well as outlining the impact of defective mitochondria in neural aging. The authors will then summarize neurodegenerative diseases related to either neurogenesis or homeostatic dysfunction. Because of the significant detriment neurodegenerative diseases have on the quality of life, it is essential to understand their etiology and ongoing molecular mechanics. The mitochondrial role in neurogenesis and neuron vitality is essential. Dissecting and understanding this organelle's role in the genesis and homeostasis of neurons should assist in finding pharmaceutical targets for neurodegenerative diseases.

The Mitochondrial Deubiquitinase USP30 Regulates AKT/mTOR Signaling.

Mitophagy is an intracellular mechanism to maintain mitochondrial health by removing dysfunctional mitochondria. The E3 ligase Parkin ubiquitinates the membrane proteins on targeted mitochondria to initiate mitophagy, whereas USP30 antagonizes Parkin-dependent mitophagy by removing ubiquitin from Parkin substrates. The AKT/mTOR signaling is a master regulator of cell proliferation, differentiation, apoptosis, and autophagy. Although mounting evidence suggests that perturbations in the AKT/mTOR signaling pathway may contribute to mitophagy regulation, the specific mechanisms between Parkin/USP30 and AKT/mTOR signaling have not been elucidated. In this study, we employ a set of genetic reagents to investigate the role of Parkin and USP30 in regulating the AKT/mTOR signaling during mitophagy. We demonstrated that, in the setting of mitochondrial stress, the AKT/mTOR signaling is regulated, at least in part, by the activity of Parkin and USP30. Parkin inhibits AKT/mTOR signaling following an in vitro mitochondrial stress, thereby promoting apoptosis. However, USP30 overexpression antagonizes the activity of Parkin to sustain AKT/mTOR activity and inhibit apoptosis. These findings provide new insights into Parkin and USP30's role in apoptosis and suggest that inhibiting USP30 might provide a specific strategy to synergize with AKT/mTOR inhibitors in cancer treatment.

Assessment of mitophagy in human iPSC-derived cardiomyocytes.

Defective mitophagy contributes to normal aging and various neurodegenerative and cardiovascular diseases. The newly developed methodologies to visualize and quantify mitophagy allow for additional progress in defining the pathophysiological significance of mitophagy in various model organisms. However, current knowledge regarding mitophagy relevant to human physiology is still limited. Model organisms such as mice might not be optimal models to recapitulate all the key aspects of human disease phenotypes. The development of the human-induced pluripotent stem cells (hiPSCs) may provide an exquisite approach to bridge the gap between animal mitophagy models and human physiology. To explore this premise, we take advantage of the pH-dependent fluorescent mitophagy reporter, mt-Keima, to assess mitophagy in hiPSCs and hiPSC-derived cardiomyocytes (hiPSC-CMs). We demonstrate that mt-Keima expression does not affect mitochondrial function or cardiomyocytes contractility. Comparison of hiPSCs and hiPSC-CMs during different stages of differentiation revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima hiPSC-CMs to analyze how mitophagy is altered under certain pathological conditions including treating the hiPSC-CMs with doxorubicin, a chemotherapeutic drug well known to cause life-threatening cardiotoxicity, and hypoxia that stimulates ischemia injury. We have further developed a chemical screening to identify compounds that modulate mitophagy in hiPSC-CMs. The ability to assess mitophagy in hiPSC-CMs suggests that the mt-Keima hiPSCs should be a valuable resource in determining the role mitophagy plays in human physiology and hiPSC-based disease models. The mt-Keima hiPSCs could prove a tremendous asset in the search for pharmacological interventions that promote mitophagy as a therapeutic target.Abbreviations: AAVS1: adeno-associated virus integration site 1; AKT/protein kinase B: AKT serine/threonine kinase; CAG promoter: cytomegalovirus early enhancer, chicken ACTB/ß-actin promoter; CIS: cisplatin; CRISPR: clustered regularly interspaced short palindromic repeats; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide p-trifluoromethoxyphenylhydrazone; hiPSC: human induced pluripotent stem cell; hiPSC-CMs: human induced pluripotent stem cell-derived cardiomyocytes; ISO: isoproterenol; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RT: room temperature; SB: SBI-0206965; ULK1: unc-51 like autophagy activating kinase 1.

Molecular signature of cardiac remodeling associated with Polymerase Gamma mutation.

AIMS: Metabolic function/dysfunction is central to aging biology. This is well illustrated by the Polymerase Gamma (POLG) mutant mouse where a key residue of the mitochondrial DNA polymerase is mutated (D257A), causing loss of mitochondrial DNA stability and dramatically accelerated aging processes. Given known cardiac phenotypes in the POLG mutant, we sought to characterize the course of cardiac dysfunction in the POLG mutant to guide future intervention studies. MATERIALS AND METHODS: Cardiac echocardiography and terminal hemodynamic analyses were used to define the course of dysfunction in the right and left cardiac ventricles in the POLG mutant. We also conducted RNA-seq analysis on cardiac right ventricles to identify mechanisms engaged by severe metabolic dysfunction and compared this analysis to several publically available datasets. KEY FINDINGS: Interesting sex differences were noted as female POLG mutants died earlier than male POLG mutants and LV chamber diameters were impacted earlier in females than males. Moreover, male mutants showed LV wall thinning while female mutant LV walls were thicker. Both males and females displayed significant RV hypertrophy. POLG mutants displayed a gene expression pattern associated with inflammation, fibrosis, and heart failure. Finally, comparative omics analyses of publically available data provide additional mechanistic and therapeutic insights. SIGNIFICANCE: Aging-associated cardiac dysfunction is a growing clinical problem. This work uncovers sex-specific cardiac responses to severe metabolic dysfunction that are reminiscent of patterns seen in human heart failure and provides insights to the molecular mechanisms engaged downstream of severe metabolic dysfunction that warrant further investigation.

Pharmacological inhibition of USP30 activates tissue-specific mitophagy.

AIM: Mitophagy is the regulated process that targets damaged or dysfunctional mitochondria for lysosomal-mediated removal. This process is an essential element of mitochondrial quality control, and dysregulation of mitophagy may contribute to a host of diseases, most notably neurodegenerative conditions such as Parkinson's disease. Mitochondria targeted for mitophagic destruction are molecularly marked by the ubiquitination of several outer mitochondrial membrane (OMM) proteins. This ubiquitination is positively regulated, in part, by the mitochondrial-targeted kinase PINK1 and the E3 ubiquitin ligase Parkin. In contrast, the reverse phenomenon, deubiquitination, removes ubiquitin from Parkin substrates embedded in the OMM proteins, antagonizing mitophagy. Recent evidence suggests that the mitochondrial deubiquitinase USP30 negatively regulates Parkin-mediated mitophagy, providing opportunities to identify USP30 inhibitors and test for their effects in augmenting mitophagy. Here we will characterize a USP30 inhibitor and demonstrate how the pharmacological inhibition of USP30 can augment stress-induced mitophagic flux. METHODS: We have conducted mitophagy and mitochondrial analyses in cultured cells. We have determined the plasma pharmacokinetics of the USP30 inhibitor in mice and conducted analyses using the mt-Keima mice to measure in vivo mitophagy directly. RESULTS: The compound has minimal mitochondrial toxicity in cultured cells and is tolerated well in mice. Interestingly, we demonstrated tissue-specific induction of mitophagy following USP30 pharmacological inhibition. In particular, pharmacological inhibition of USP30 induces a significant increase in cardiac mitophagy without detriment to cardiac function. CONCLUSION: Our data support the evidence that USP30 inhibition may serve as a specific strategy to selectively increase mitophagic flux, allowing for the development of novel therapeutic approaches.

Hyper-Synergistic Antifungal Activity of Rapamycin and Peptide-Like Compounds against Candida albicans Orthogonally via Tor1 Kinase.

Candida albicans is a life-threatening, opportunistic fungal pathogen with a high mortality rate, especially within the immunocompromised populations. Multidrug resistance combined with limited antifungal drugs even worsens the situation. Given the facts that the current drug discovery strategies fail to deliver sufficient antifungals for the emerging multidrug resistance, we urgently need to develop novel approaches. By systematically investigating what caused the different antifungal activity of rapamycin in RPMI 1640 and YPD, we discovered that peptide-like compounds can generate a hyper-synergistic antifungal effect with rapamycin on both azole-resistant and sensitive clinical C. albicans isolates. The minimum inhibitory concentration (MIC) of rapamycin reaches as low as 2.14 nM (2-9 µg/mL), distinguishing this drug combination as a hyper-synergism by having a fractional inhibitory concentration (FIC) index ≤ 0.05 from the traditional defined synergism with an FIC index < 0.5. Further studies reveal that this hyper-synergism orthogonally targets the protein Tor1 and affects the TOR signaling pathway in C. albicans, very likely without crosstalk to the stress response, Ras/cAMP/PKA, or calcineurin signaling pathways. These results lead to a novel strategy of controlling drug resistant C. albicans infection in the immunocompromised populations. Instead of prophylactically administering other antifungals with undesirable side-effects for extended durations, we now only need to coadminister some nontoxic peptide additives. The novel antifungal strategy approached in this study not only provides a new therapeutic method to control fungal infections in rapamycin-taking immunocompromised patients but also mitigates the immunosuppressive side-effects of rapamycin, repurposing rapamycin as an antifungal agent with wide applications.

Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p.

Clinical use of antimicrobials faces great challenges from the emergence of multidrug-resistant pathogens. The overexpression of drug efflux pumps is one of the major contributors to multidrug resistance (MDR). Reversing the function of drug efflux pumps is a promising approach to overcome MDR. In the life-threatening fungal pathogen Candida albicans, the major facilitator superfamily (MFS) transporter Mdr1p can excrete many structurally unrelated antifungals, leading to MDR. Here we report a counterintuitive case of reversing MDR in C. albicans by using a natural product berberine to hijack the overexpressed Mdr1p for its own importation. Moreover, we illustrate that the imported berberine accumulates in mitochondria and compromises the mitochondrial function by impairing mitochondrial membrane potential and mitochondrial Complex I. This results in the selective elimination of Mdr1p overexpressed C. albicans cells. Furthermore, we show that berberine treatment can prolong the mean survival time of mice with blood-borne dissemination of Mdr1p overexpressed multidrug-resistant candidiasis. This study provides a potential direction of novel anti-MDR drug discovery by screening for multidrug efflux pump converters.