Mitochondrial Quality Control Pathways Research Articles

N-degron-mediated degradation and regulation of mitochondrial PINK1 kinase.

Parkinson's disease (PD) is a progressive neurodegenerative condition characterized by a gradual loss of a specific group of dopaminergic neurons in the substantia nigra. Importantly, current treatments only address the symptoms of PD, yet not the underlying molecular causes. Concomitantly, the function of genes that cause inherited forms of PD point to mitochondrial dysfunction as a major contributor in the etiology of PD. An inherent challenge that mitochondria face is the continuous exposure to diverse stresses including high levels of reactive oxygen species and protein misfolding, which increase their likelihood of dysregulation. In response, eukaryotic cells have evolved sophisticated quality control mechanisms to identify, repair and/or eliminate abnormal dysfunctional mitochondria. One such mechanism is mitophagy, a process which involves PTEN-induced putative kinase 1 (PINK1), a mitochondrial Ser/Thr kinase and Parkin, an E3 ubiquitin ligase, each encoded by genes responsible for early-onset autosomal recessive familial PD. Over 100 loss-of-function mutations in the PTEN-induced putative kinase 1 (PINK1) gene have been reported to cause autosomal recessive early-onset PD. PINK1 acts upstream of Parkin and is essential for the mitochondrial localization and activation of Parkin. Upon mitochondrial damage, PINK1 builds up on the outer mitochondrial membrane (OMM) and mediates the activation of Parkin. Activated Parkin then ubiquitinates numerous OMM proteins, eliciting mitochondrial autophagy (mitophagy). As a result, damaged mitochondrial components can be selectively eliminated. Thus, PINK1 acts a sensor of damage via fine-tuning of its levels on mitochondria, where it activates Parkin to orchestrate the clearance of unhealthy mitochondria. Previous work has unveiled that the Arg-N-end rule degradation pathway (Arg-N-degron pathway) mediates the degradation of PINK1, and thus fine-tune PINK1-dependent mitochondrial quality control pathway. Herein, we briefly discuss the interconnection between N-end rule degradation pathways and mitophagy in the context of N-degron mediated degradation of mitochondrial kinase PINK1 and highlight some of the future prospects.

Inter-Organelle Membrane Contact Sites and Mitochondrial Quality Control during Aging: A Geroscience View.

Mitochondrial dysfunction and failing mitochondrial quality control (MQC) are major determinants of aging. Far from being standalone organelles, mitochondria are intricately related with cellular other compartments, including lysosomes. The intimate relationship between mitochondria and lysosomes is reflected by the fact that lysosomal degradation of dysfunctional mitochondria is the final step of mitophagy. Inter-organelle membrane contact sites also allow bidirectional communication between mitochondria and lysosomes as part of nondegradative pathways. This interaction establishes a functional unit that regulates metabolic signaling, mitochondrial dynamics, and, hence, MQC. Contacts of mitochondria with the endoplasmic reticulum (ER) have also been described. ER-mitochondrial interactions are relevant to Ca2+ homeostasis, transfer of phospholipid precursors to mitochondria, and integration of apoptotic signaling. Many proteins involved in mitochondrial contact sites with other organelles also participate to degradative MQC pathways. Hence, a comprehensive assessment of mitochondrial dysfunction during aging requires a thorough evaluation of degradative and nondegradative inter-organelle pathways. Here, we present a geroscience overview on (1) degradative MQC pathways, (2) nondegradative processes involving inter-organelle tethering, (3) age-related changes in inter-organelle degradative and nondegradative pathways, and (4) relevance of MQC failure to inflammaging and age-related conditions, with a focus on Parkinson’s disease as a prototypical geroscience condition.

Current Progress of Mitochondrial Quality Control Pathways Underlying the Pathogenesis of Parkinson's Disease.

Parkinson's disease (PD), clinically characterized by motor and nonmotor symptoms, is a common progressive and multisystem neurodegenerative disorder, which is caused by both genetic and environmental risk factors. The main pathological features of PD are the loss of dopaminergic (DA) neurons and the accumulation of alpha-synuclein (α-syn) in the residual DA neurons in the substantia nigra pars compacta (SNpc). In recent years, substantial progress has been made in discovering the genetic factors of PD. In particular, a total of 19 PD-causing genes have been unraveled, among which some members have been regarded to be related to mitochondrial dysfunction. Mitochondria are key regulators of cellular metabolic activity and are critical for many important cellular processes including energy metabolism and even cell death. Their normal function is basically maintained by the mitochondrial quality control (MQC) mechanism. Accordingly, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a kind of neurotoxin, exerts its neurotoxic effects at least partially by producing its toxic metabolite, namely, 1-methyl-4-phenylpyridine (MPP+), which in turn causes mitochondrial dysfunction by inhibiting complex I and mimicking the key features of PD pathogenesis. This review focused on three main aspects of the MQC signaling pathways, that is, mitochondrial biogenesis, mitochondrial dynamics, and mitochondrial autophagy; hence, it demonstrates in detail how genetic and environmental factors result in PD pathogenesis by interfering with MQC pathways, thereby hopefully contributing to the discovery of novel potential therapeutic targets for PD.

Mechanisms of PINK1, ubiquitin and Parkin interactions in mitochondrial quality control and beyond.

Parkinson's disease (PD) is a degenerative movement disorder resulting from the loss of specific neuron types in the midbrain. Early environmental and pathophysiological studies implicated mitochondrial damage and protein aggregation as the main causes of PD. These findings are now vindicated by the characterization of more than 20 genes implicated in rare familial forms of the disease. In particular, two proteins encoded by the Parkin and PINK1 genes, whose mutations cause early-onset autosomal recessive PD, function together in a mitochondrial quality control pathway. In this review, we will describe recent development in our understanding of their mechanisms of action, structure, and function. We explain how PINK1 acts as a mitochondrial damage sensor via the regulated proteolysis of its N-terminus and the phosphorylation of ubiquitin tethered to outer mitochondrial membrane proteins. In turn, phospho-ubiquitin recruits and activates Parkin via conformational changes that increase its ubiquitin ligase activity. We then describe how the formation of polyubiquitin chains on mitochondria triggers the recruitment of the autophagy machinery or the formation of mitochondria-derived vesicles. Finally, we discuss the evidence for the involvement of these mechanisms in physiological processes such as immunity and inflammation, as well as the links to other PD genes.

Neuroprotective effect of anodal transcranial direct current stimulation on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice through modulating mitochondrial dynamics

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of protein inclusions and the loss of dopaminergic neurons. Abnormal mitochondrial homeostasis is thought to be important for the pathogenesis of PD. Transcranial direct current stimulation (tDCS), a noninvasive brain stimulation technique, constitutes a promising approach for promoting recovery of various neurological conditions. However, little is known about its mechanism of action. The present study elucidated the neuroprotective effects of tDCS on the mitochondrial quality control pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We used the MPTP-induced neurotoxicity in vivo model. Mice were stimulated for 5 consecutive days with MPTP treatment. After observation of behavioral alteration using the rotarod test, mice were sacrificed for the measurement of the PD- and mitochondrial quality control-related protein levels in the substantia nigra. tDCS improved the behavioral alterations and changes in tyrosine hydroxylase levels in MPTP-treated mice. Furthermore, tDCS attenuated mitochondrial damage, as indicated by diminished mitochondrial swelling and mitochondrial glutamate dehydrogenase activity in the MPTP-induced PD mouse model. MPTP significantly increased mitophagy and decreased mitochondrial biogenesis-related proteins. These changes were attenuated by tDCS. Furthermore, MPTP significantly increased fission-related protein dynamin-related protein 1 with no effect on fusion-related protein mitofusin-2, and tDCS attenuated these changes. Our findings demonstrated the neuroprotective effect of anodal tDCS on the MPTP-induced neurotoxic mouse model through suppressing excessive mitophagy and balancing mitochondrial dynamics. The neuroprotective effect of anodal tDCS with modulation of mitochondrial dynamics provides a new therapeutic strategy for the treatment of PD.

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human isletsamples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

The plant triterpenoid celastrol blocks PINK1-dependent mitophagy by disrupting PINK1's association with the mitochondrial protein TOM20

A critical function of the PTEN-induced kinase 1 (PINK1)-Parkin pathway is to mediate the clearing of unhealthy or damaged mitochondria via mitophagy. Loss of either PINK1 or Parkin protein expression is associated with Parkinson's disease. Here, using a high-throughput screening approach along with recombinant protein expression and kinase, immunoblotting, and immunofluorescence live-cell imaging assays, we report that celastrol, a pentacyclic triterpenoid isolated from extracts of the medicinal plant Tripterygium wilfordii, blocks recruitment pof Parkin to mitochondria, preventing mitophagy in response to mitochondrial depolarization induced by carbonyl cyanide m-chlorophenylhydrazone or to gamitrinib-induced inhibition of mitochondrial heat shock protein 90 (HSP90). Celastrol's effect on mitophagy was independent of its known role in microtubule disruption. Instead, we show that celastrol suppresses Parkin recruitment by inactivating PINK1 and preventing it from phosphorylating Parkin and also ubiquitin. We also observed that PINK1 directly and strongly associates with TOM20, a component of the translocase of outer mitochondrial membrane (TOM) machinery and relatively weak binding to another TOM subunit, TOM70. Moreover, celastrol disrupted binding between PINK1 and TOM20 both in vitro and in vivo but did not affect binding between TOM20 and TOM70. Using native gel analysis, we also show that celastrol disrupts PINK1 complex formation upon mitochondrial depolarization and sequesters PINK1 to high-molecular-weight protein aggregates. These results reveal that celastrol regulates the mitochondrial quality control pathway by interfering with PINK1-TOM20 binding.

Abstract P3-09-04: Possible role of p53/Mieap-regulated mitochondrial quality control as a tumor suppressor in human breast cancer

Abstract [Background] In sporadic breast cancers, the most important gene is p53, given that it is a tumor suppressor gene and mutated in greater than 50% of human cancers. According to previous reports, p53 is also mutated in approximately 20–40% of breast cancers. Recent data from the cancer genome atlas (TCGA) revealed that 37% of breast cancer specimens had alterations in p53-particularly, 72% in (human epidermal growth factor) HER2-rich and 80% of basal-like breast cancer cases—indicating that it is a critical driver of tumor development even in breast cancer. p53 is clinically very important not only because of its high mutation rate but also because mutation is associated with more aggressive disease and worse overall survival. Mitochondria-eating protein (Mieap) is a p53-target gene that plays an important role in mitochondrial quality control. Mieap has been reported to have a critical role in tumorsuppression of colorectal cancer. Here, we investigated the role of Mieap as a tumorsuppressor in breast cancer. [Marerial and methods] We overexpressed Mieap using the constructed adenovirus in breast cancer cell lines such as MCF-7, SK-BR-3, and MDA-MB-231 cells. The percentages of cells in different cell cycle phases (subG1, G1, S, and G2/M) were determined using FACS analysis and also caspase activities (Caspase, 3/7, 9) were measured. Cleaved PARP, which is a marker of cells undergoing apoptosis, was detected by western blot. For in vivo experiments, we examined the expression of Mieap using surgical specimens (invasive ductal carcinomas (IDCs): 75, ductal carcinoma in situ (DCIS): 27, fibroadenomas (FAs): 18) by immunohistochemistry. Next, we performed methylation-specific PCR (MSP) for Mieap, NIX, and BNIP3 promoters and p53-mutation search using 46 samples that were cryopreserved, among 75 IDC cases used for immunohistochemistry. These studies were approved by the central ethics committee of Gifu University.[Results] The enforced-expression of exogenous Mieap in breast cancer cells induced caspase-dependent apoptosis, with activation of both caspase-3/7 and caspase-9. Immunohistochemistry revealed endogenous Mieap in the cytoplasm in 24/75 (32%) invasive ductal carcinomas (IDCs), 15/27 (55.6%) cases of ductal carcinoma in situ (DCIS), and 16/18 (88.9%) fibroadenomas (FAs) (IDC vs DCIS; p = 0.0389, DCIS vs FA; p = 0.0234, IDC vs FA; p < 0.0001). In IDCs, the Mieap promoter was methylated in 6/46 (13%) cases whereas p53 was mutated in 6/46 (13%) cases. Therefore, the p53/Mieap-regulated mitochondrial quality control pathway was inactivated in 12/46 IDCs (26.1%). Interestingly, all of the tumors derived from the 12 patients with the Mieap-promoter methylation or p53 mutation pathologically exhibited more aggressive and malignant phenotype of breast cancers, resulting in significantly shorter disease-free survival (DFS) (p = 0.021). [Conclusion]These results indicate that p53/Mieap-regulated mitochondrial quality control has a critical role in tumor suppression of breast cancer, possibly in part, through mitochondrial apoptotic pathway. Citation Format: Futamura M, Gaowa S, Arakawa H, Yoshida K. Possible role of p53/Mieap-regulated mitochondrial quality control as a tumor suppressor in human breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-09-04.

Potential Mechanisms Driving Mitochondrial Motility Impairments in Developing Iron-Deficient Neurons.

Brain development is highly demanding energetically, requiring neurons to have tightly regulated and highly dynamic metabolic machinery to achieve their ultimately complex cellular architecture. Mitochondria are the main source of neuronal adenosine 5′-triphosphate (ATP) and regulate critical neurodevelopmental processes including calcium signaling, iron homeostasis, oxidative stress, and apoptosis. Metabolic perturbations during critical neurodevelopmental windows impair neurological function not only acutely during the period of rapid growth/development, but also in adulthood long after the early-life insult has been rectified. Our laboratory uses iron deficiency (ID), the most common nutrient deficiency, as a model of early-life metabolic disruptions of neuronal metabolism because iron has a central role in mitochondrial function. Recently, we published that ID reduces hippocampal neuronal dendritic mitochondrial motility and size. In this commentary, we delve deeper into speculation about potential cellular mechanisms that drive the effects of neuronal ID on mitochondrial dynamics and quality control pathways. We propose that understanding the basic cellular biology of how mitochondria respond and adapt to ID and other metabolic perturbations during brain development may be a key factor in designing strategies to reduce the risk of later-life psychiatric, cognitive, and neurodegenerative disorders associated with early-life ID.

Possible role of p53/Mieap-regulated mitochondrial quality control as a tumor suppressor in human breast cancer.

Mitochondria‐eating protein (Mieap), encoded by a p53‐target gene, plays an important role in mitochondrial quality control (MQC). Mieap has been reported to have a critical role in tumor suppression in colorectal cancer. Here, we investigated its role as a tumor suppressor in breast cancer. The enforced expression of exogenous Mieap in breast cancer cells induced caspase‐dependent apoptosis, with activation of both caspase‐3/7 and caspase‐9. Immunohistochemistry revealed endogenous Mieap in the cytoplasm in 24/75 (32%) invasive ductal carcinomas (IDC), 15/27 (55.6%) cases of ductal carcinoma in situ (DCIS) and 16/18 (88.9%) fibroadenomas (FA) (IDC vs DCIS; P = 0.0389, DCIS vs FA; P = 0.0234, IDC vs FA; P < 0.0001). In IDC, the Mieap promoter was methylated in 6/46 (13%) cases, whereas p53 was mutated in 6/46 (13%) cases. Therefore, the p53/Mieap‐regulated MQC pathway was inactivated in 12/46 IDC (26.1%). Interestingly, all tumors derived from the 12 patients with Mieap promoter methylation or p53 mutations pathologically exhibited more aggressive and malignant breast cancer phenotypes. Impairment of p53/Mieap‐regulated MQC pathway resulted in significantly shorter disease‐free survival (DFS) (P = 0.021), although p53 status is more prognostic in DFS than Mieap promoter methylation. These results indicate that p53/Mieap‐regulated MQC has a critical role in tumor suppression in breast cancer, possibly in part through mitochondrial apoptotic pathway.

Evolving Concepts of Mitochondrial Dynamics.

The concept that mitochondria are highly dynamic is as widely accepted as it is untrue for a number of important contexts. Healthy mitochondria of the most energy-dependent and mitochondrial-rich mammalian organ, the heart, only rarely undergo fusion or fission and are seemingly static within cardiac myocytes. Here, we revisit mitochondrial dynamism with a fresh perspective developed from the recently discovered multifunctionality of mitochondrial fusion proteins and newly defined mechanisms for direct cross talk between mitochondrial dynamics, biogenesis, quality control, and trafficking pathways. Insights gained from comparing static mitochondrial biology in cardiac myocytes and dynamic mitochondrial biology in neurons are reviewed with the goal of understanding contextual fallacies of overly generalized characterizations of these essential and intriguing organelles.

Age- and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease

ABSTRACTAlthough exact causes of Parkinson disease (PD) remain enigmatic, mitochondrial dysfunction is increasingly appreciated as a key determinant of dopaminergic neuron susceptibility in both familial and sporadic PD. Two genes associated with recessive, early-onset PD encode the ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase PRKN/PARK2/Parkin, which together orchestrate a protective mitochondrial quality control (mitoQC) pathway. Upon stress, both enzymes cooperatively identify and decorate damaged mitochondria with phosphorylated poly-Ub (p-S65-Ub) chains. This specific label is subsequently recognized by autophagy receptors that further facilitate mitochondrial degradation in lysosomes (mitophagy). Here, we analyzed human post-mortem brain specimens and identified distinct pools of p-S65-Ub-positive structures that partially colocalized with markers of mitochondria, autophagy, lysosomes and/or granulovacuolar degeneration bodies. We further quantified levels and distribution of the ‘mitophagy tag’ in 2 large cohorts of brain samples from normal aging and Lewy body disease (LBD) cases using unbiased digital pathology. Somatic p-S65-Ub structures independently increased with age and disease in distinct brain regions and enhanced levels in LBD brain were age- and Braak tangle stage-dependent. Additionally, we observed significant correlations of p-S65-Ub with LBs and neurofibrillary tangle levels in disease. The degree of co-existing p-S65-Ub signals and pathological PD hallmarks increased in the pre-mature stage, but decreased in the late stage of LB or tangle aggregation. Altogether, our study provides further evidence for a potential pathogenic overlap among different forms of PD and suggests that p-S65-Ub can serve as a biomarker for mitochondrial damage in aging and disease.Abbreviations: BLBD: brainstem predominant Lewy body disease; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DLB: dementia with Lewy bodies; DLBD: diffuse neocortical Lewy body disease; EOPD: early-onset Parkinson disease; GVB: granulovacuolar degeneration body; LB: Lewy body; LBD: Lewy body disease; mitoQC: mitochondrial quality control; nbM: nucleus basalis of Meynert; PD: Parkinson disease; PDD: Parkinson disease with dementia; p-S65-Ub: PINK1-phosphorylated serine 65 ubiquitin; SN: substantia nigra; TLBD: transitional Lewy body disease; Ub: ubiquitin

Mitochondrial Quality Control in Neurodegenerative Diseases: Focus on Parkinson's Disease and Huntington's Disease.

In recent years, several important advances have been made in our understanding of the pathways that lead to cell dysfunction and death in Parkinson's disease (PD) and Huntington's disease (HD). Despite distinct clinical and pathological features, these two neurodegenerative diseases share critical processes, such as the presence of misfolded and/or aggregated proteins, oxidative stress, and mitochondrial anomalies. Even though the mitochondria are commonly regarded as the “powerhouses” of the cell, they are involved in a multitude of cellular events such as heme metabolism, calcium homeostasis, and apoptosis. Disruption of mitochondrial homeostasis and subsequent mitochondrial dysfunction play a key role in the pathophysiology of neurodegenerative diseases, further highlighting the importance of these organelles, especially in neurons. The maintenance of mitochondrial integrity through different surveillance mechanisms is thus critical for neuron survival. Mitochondria display a wide range of quality control mechanisms, from the molecular to the organellar level. Interestingly, many of these lines of defense have been found to be altered in neurodegenerative diseases such as PD and HD. Current knowledge and further elucidation of the novel pathways that protect the cell through mitochondrial quality control may offer unique opportunities for disease therapy in situations where ongoing mitochondrial damage occurs. In this review, we discuss the involvement of mitochondrial dysfunction in neurodegeneration with a special focus on the recent findings regarding mitochondrial quality control pathways, beyond the classical effects of increased production of reactive oxygen species (ROS) and bioenergetic alterations. We also discuss how disturbances in these processes underlie the pathophysiology of neurodegenerative disorders such as PD and HD.

Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy.

Despite their importance as signaling hubs, the function of mitochondria-ER contact sites in mitochondrial quality control pathways remains unexplored. Here we describe a mechanism by which Mfn2, a mitochondria-ER tether, gates the autophagic turnover of mitochondria by PINK1 and parkin. Mitochondria-ER appositions are destroyed during mitophagy, and reducing mitochondria-ER contacts increases the rate of mitochondrial degradation. Mechanistically, parkin/PINK1 catalyze a rapid burst of Mfn2 phosphoubiquitination to trigger p97-dependent disassembly of Mfn2 complexes from the outer mitochondrial membrane, dissociating mitochondria from the ER. We additionally demonstrate that a major portion of the facilitatory effect of p97 on mitophagy is epistatic to Mfn2 and promotes the availability of other parkin substrates such as VDAC1. Finally, we reconstitute the action of these factors on Mfn2 and VDAC1 ubiquitination in a cell-free assay. We show that mitochondria-ER tethering suppresses mitophagy and describe a parkin-/PINK1-dependent mechanism that regulates the destruction of mitochondria-ER contact sites.

Regulation of Mitophagy by the Parkin Ubiquitin Ligase and PINK1 Ubiquitin Kinase

Familial forms of Parkinson's disease arise from loss of the mitochondrial quality control pathway mediated by PINK1, a protein kinase, and Parkin, an E3 ubiquitin ligase. Together, they regulate the disposal of damaged mitochondria by autophagy (mitophagy). When damaged, mitochondria accumulate PINK1, which phosphorylates ubiquitin to recruit cytosolic Parkin. Normally quiescent due to autoinhibitory interactions, Parkin is activated by both phosphorylation by PINK1 and binding phosphorylated ubiquitin. The molecular mechanisms that activate activate Parkin have been the focus of much speculation. Here, I describe the crystal structure of the active conformation of Parkin that shows phosphorylation of parkin by PINK1 leads to a dramatic conformational change to derepress its E2‐binding and catalytic sites. Using a variety of experimental approaches, the activation pathways mediated by phospho‐ubiquitin binding and Parkin phosphorylation are compared. The study deepens our understanding of the mechanisms of Parkin activation and suggests new approaches for the treatment of Parkinson's disease by activating Parkin with small molecules.Support or Funding InformationCanadian Institutes of Health Research and the Michael J Fox FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterizing the Spatial Variance of Autophagic Cargo Receptor, OPTN

The mitochondria are required for various cellular processes spanning from cellular energy generation to signal transduction, growth and survival. Not surprisingly, cumulative mitochondrial damage is tightly linked to the development of multiple human diseases including various neurodegenerative diseases such as Parkinson's and Huntington's disease. In order to maintain functional mitochondria, cells have coped with multiple mitochondrial quality control pathways such as mitochondrial Unfolded Protein Response (mt‐UPR), the ubiquitin proteasome system, and mitochondrial autophagy (mitophagy). The most well‐characterized mitophagy is the PINK1‐PARKIN pathway, where damaged mitochondria are selectively marked with ubiquitin chains by the PINK1 kinase and PARKIN ubiquitin E3 ligase, engulfed by autophagosome, and cleared by the lysosome. While we now have a better understanding on this pathway, it is still unclear how autophagic cargo adaptor proteins, such as optineurin (OPTN), get recruited to damaged mitochondria, synthesized or recycled throughout the process. We hypothesized that the availability of OPTN would be the rate‐limiting factor to achieve complete removal of damaged mitochondria and this would require synthesis of new OPTN molecule. To test this hypothesis, we generated a cell line containing a genomic Halo tag at OPTN N‐terminus by utilizing CRISPR/cas9, where differentially fluorescent labeled Halo tag can distinguish older versus newer OPTN populations over time. After successfully generating a CRISPR/Cas9 knock‐in cell line for OPTN, single cell imaging was performed to determine the spatial variance and emergence of OPTN recruited to mitochondria. We were able to conclude that OPTN localizes to a fraction of damaged mitochondria despite the fact that the entire mitochondria are coated with ubiquitin chains, suggesting specificity of this recruitment. Additionally, our preliminary results indicated that newly synthesized OPTN is recruited to mitochondria for the continuation of mitophagy. Together, these observations suggest that OPTN is the rate‐limiting factor and that in order to completely remove all dysfunctional mitochondria, cells need to synthesize new OPTN. In the future, we would like to further explore the underlying mechanism of OPTN's binding specificity and determine what drives this selective recruitment of OPTN to mitochondria.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The PERK Arm of the Unfolded Protein Response Regulates Mitochondrial Morphology during Acute Endoplasmic Reticulum Stress

SUMMARYEndoplasmic reticulum (ER) stress is transmitted to mitochondria and is associated with pathologic mitochondrial dysfunction in diverse diseases. The PERK arm of the unfolded protein response (UPR) protects mitochondria during ER stress through the transcriptional and translational remodeling of mitochondrial molecular quality control pathways. Here, we show that ER stress also induces dynamic remodeling of mitochondrial morphology by promoting protective stress-induced mitochondrial hyperfusion (SIMH). ER-stress-associated SIMH is regulated by the PERK arm of the UPR and activated by eIF2α phosphorylation-dependent translation attenuation. We show that PERK-regulated SIMH is a protective mechanism to prevent pathologic mitochondrial fragmentation and promote mitochondrial metabolism in response to ER stress. These results identify PERK-dependent SIMH as a protective stress-responsive mechanism that regulates mitochondrial morphology during ER stress. Furthermore, our results show that PERK integrates transcriptional and translational signaling to coordinate mitochondrial molecular and organellar quality control in response to pathologic ER insults.

Neurodegenerative Loss of Mitochondrial Quality via the 18kDa Protein TSPO

The 18kDa Translocator Protein (TSPO), is a nuclear-encoded protein residing on the outer mitochondrial membrane and linked to a variety of pathological conditions. Traditionally a target of pharmacological agents, its ability to efficiently signal cellular stress has been employed in diagnostic protocols of neuroinflammation1. Recently, breakthroughs on its role in mitochondrial physiology and metabolism, including the negative regulation of mitophagy, have provided evidences for its mechanistic role as stress-response adaptive element in pathophyioslogy. In previous studies, we demonstrated that TSPO is able to impair Parkin-mediated mitophagy via a Reactive Oxygen Species (ROS)-dependent mechanism2 and exploited in glutamate-induced excitotoxicity3. Using ex-vivo and in vitro models of Sporadic Parkinsonism, we now find that TSPO plays part in chronic damage associated with dopaminergic neurons’ demise. Our results suggest that, downstream of the ERK1/2 pathway, Parkinson's disease (PD) toxins induce a sustained upregulation of TSPO, which enacts a programme of mitochondrial signalling adaptation and impairment of mitochondria-targeted autophagy pivotal to cellular degeneration. All this makes of TSPO a pro-pathological candidate protein in PD, by impairing both mitochondrial quality control and signalling pathways of retrograde communications. The molecular determinants of this will be discussed and so the pharmacologically means4 to revert the phenotype by targeting TSPO. 1. Gatliff J and Campanella M. Biochem J. 2016 Jan 15;473(2):107-2. 2. Gatliff, et al. Autophagy 2014 Dec 2;10(12):2279-96. 3. Gatliff J, East D, et al. Cell Death Dis. 2017 Jun 22;8(6):e2896. 4. Georgakopoulos ND et al. Nat Chem Biol. 2017 Jan 19;13(2):136-146.

The Mitochondrial Basis of Aging and Age-Related Disorders.

Aging is a natural phenomenon characterized by progressive decline in tissue and organ function leading to increased risk of disease and mortality. Among diverse factors that contribute to human aging, the mitochondrial dysfunction has emerged as one of the key hallmarks of aging process and is linked to the development of numerous age-related pathologies including metabolic syndrome, neurodegenerative disorders, cardiovascular diseases and cancer. Mitochondria are central in the regulation of energy and metabolic homeostasis, and harbor a complex quality control system that limits mitochondrial damage to ensure mitochondrial integrity and function. The intricate regulatory network that balances the generation of new and removal of damaged mitochondria forms the basis of aging and longevity. Here, I will review our current understanding on how mitochondrial functional decline contributes to aging, including the role of somatic mitochondrial DNA (mtDNA) mutations, reactive oxygen species (ROS), mitochondrial dynamics and quality control pathways. I will further discuss the emerging evidence on how dysregulated mitochondrial dynamics, mitochondrial biogenesis and turnover mechanisms contribute to the pathogenesis of age-related disorders. Strategies aimed to enhance mitochondrial function by targeting mitochondrial dynamics, quality control, and mitohormesis pathways might promote healthy aging, protect against age-related diseases, and mediate longevity.

Systems Phytohormone Responses to Mitochondrial Proteotoxic Stress

Mitochondrial function is controlled by two separate genomes. This feature makes mitochondria prone to proteotoxic stress when a stoichiometric imbalance occurs in the protein complexes that perform oxidative phosphorylation, which consist of both nuclear- and mitochondrial-encoded proteins. Such a proteotoxic stress is known to induce the mitochondrial unfolded protein response (UPRmt) in animals. It is unknown whether UPRmt occurs in plants. Here, we induced a mitonuclear protein imbalance in Arabidopsis through chemical or genetic interference. Mitochondrial proteotoxic stress activated a plant-specific UPRmt and impaired plant growth and development. The plant UPRmt pathway is triggered by a transient oxidative burst, activating MAPK and hormonal (involving ethylene and auxin) signaling, which are all geared to repair proteostasis. This also establishes phytohormones as bona fide plant mitokines. Our data ascertain that mitochondrial protein quality control pathways, such as the UPRmt, are conserved in plants and that hormone signaling is an essential mediator that regulates mitochondrial proteostasis.