Aberrant ER-mitochondria communication is a common pathomechanism in mitochondrial disease.

Genetic mutations causing primary mitochondrial disease (i.e those compromising oxidative phosphorylation [OxPhos]) resulting in reduced bioenergetic output display great variability in their clinical features, but the reason for this is unknown. We hypothesized that disruption of the communication between endoplasmic reticulum (ER) and mitochondria at mitochondria-associated ER membranes (MAM) might play a role in this variability. To test this, we assayed MAM function and ER-mitochondrial communication in OxPhos-deficient cells, including cybrids from patients with selected pathogenic mtDNA mutations. Our results show that each of the various mutations studied indeed altered MAM functions, but notably, each disorder presented with a different MAM "signature". We also found that mitochondrial membrane potential is a key driver of ER-mitochondrial connectivity. Moreover, our findings demonstrate that disruption in ER-mitochondrial communication has consequences for cell survivability that go well beyond that of reduced ATP output. The findings of a "MAM-OxPhos" axis, the role of mitochondrial membrane potential in controlling this process, and the contribution of MAM dysfunction to cell death, reveal a new relationship between mitochondria and the rest of the cell, as well as providing new insights into the diagnosis and treatment of these devastating disorders.

Towards a Unitary Hypothesis of Alzheimer's Disease Pathogenesis.

The "amyloid cascade" hypothesis of Alzheimer's disease (AD) pathogenesis invokes the accumulation in the brain of plaques (containing the amyloid-ß protein precursor [AßPP] cleavage product amyloid-ß [Aß]) and tangles (containing hyperphosphorylated tau) as drivers of pathogenesis. However, the poor track record of clinical trials based on this hypothesis suggests that the accumulation of these peptides is not the only cause of AD. Here, an alternative hypothesis is proposed in which the AßPP cleavage product C99, not Aß, is the main culprit, via its role as a regulator of cholesterol metabolism. C99, which is a cholesterol sensor, promotes the formation of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a cholesterol-rich lipid raft-like subdomain of the ER that communicates, both physically and biochemically, with mitochondria. We propose that in early-onset AD (EOAD), MAM-localized C99 is elevated above normal levels, resulting in increased transport of cholesterol from the plasma membrane to membranes of intracellular organelles, such as ER/endosomes, thereby upregulating MAM function and driving pathology. By the same token, late-onset AD (LOAD) is triggered by any genetic variant that increases the accumulation of intracellular cholesterol that, in turn, boosts the levels of C99 and again upregulates MAM function. Thus, the functional cause of AD is upregulated MAM function that, in turn, causes the hallmark disease phenotypes, including the plaques and tangles. Accordingly, the MAM hypothesis invokes two key interrelated elements, C99 and cholesterol, that converge at the MAM to drive AD pathogenesis. From this perspective, AD is, at bottom, a lipid disorder.

Mitochondrial disease: Replace or edit?

Questions over mitochondrial replacement suggest a role for mitochondrial DNA editing.

Mitochondria, OxPhos, and neurodegeneration: cells are not just running out of gas.

Mitochondrial respiratory deficiencies have been observed in numerous neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. For decades, these reductions in oxidative phosphorylation (OxPhos) have been presumed to trigger an overall bioenergetic crisis in the neuron, resulting in cell death. While the connection between respiratory defects and neuronal death has never been proven, this hypothesis has been supported by the detection of nonspecific mitochondrial DNA mutations in these disorders. These findings led to the notion that mitochondrial respiratory defects could be initiators of these common neurodegenerative disorders, instead of being consequences of a prior insult, a theory we believe to be misconstrued. Herein, we review the roots of this mitochondrial hypothesis and offer a new perspective wherein mitochondria are analyzed not only from the OxPhos point of view, but also as a complex organelle residing at the epicenter of many metabolic pathways.

MFN2 mutations in Charcot-Marie-Tooth disease alter mitochondria-associated ER membrane function but do not impair bioenergetics.

Charcot-Marie-Tooth disease (CMT) type 2A is a form of peripheral neuropathy, due almost exclusively to dominant mutations in the nuclear gene encoding the mitochondrial protein mitofusin-2 (MFN2). However, there is no understanding of the relationship of clinical phenotype to genotype. MFN2 has two functions: it promotes inter-mitochondrial fusion and mediates endoplasmic reticulum (ER)-mitochondrial tethering at mitochondria-associated ER membranes (MAM). MAM regulates a number of key cellular functions, including lipid and calcium homeostasis, and mitochondrial behavior. To date, no studies have been performed to address whether mutations in MFN2 in CMT2A patient cells affect MAM function, which might provide insight into pathogenesis. Using fibroblasts from three CMT2AMFN2 patients with different mutations in MFN2, we found that some, but not all, examined aspects of ER-mitochondrial connectivity and of MAM function were indeed altered, and correlated with disease severity. Notably, however, respiratory chain function in those cells was unimpaired. Our results suggest that CMT2AMFN2 is a MAM-related disorder but is not a respiratory chain-deficiency disease. The alterations in MAM function described here could also provide insight into the pathogenesis of other forms of CMT.

Three-Dimensional Analysis of Mitochondrial Crista Ultrastructure in a Patient with Leigh Syndrome by In Situ Cryoelectron Tomography.

Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryoelectron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from a patient with LS and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization due to the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture in the context of human disease.

A key role for MAM in mediating mitochondrial dysfunction in Alzheimer disease.

In the last few years, increased emphasis has been devoted to understanding the contribution of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) to human pathology in general, and neurodegenerative diseases in particular. A major reason for this is the central role that this subdomain of the ER plays in metabolic regulation and in mitochondrial biology. As such, aberrant MAM function may help explain the seemingly unrelated metabolic abnormalities often seen in neurodegeneration. In the specific case of Alzheimer disease (AD), besides perturbations in calcium and lipid homeostasis, there are numerous documented alterations in mitochondrial behavior and function, including reduced respiratory chain activity and oxidative phosphorylation, increased free radical production, and altered organellar morphology, dynamics, and positioning (especially perinuclear mitochondria). However, whether these alterations are primary events causative of the disease, or are secondary downstream events that are the result of some other, more fundamental problem, is still unclear. In support of the former possibility, we recently reported that C99, the C-terminal processing product of the amyloid precursor protein (APP) derived from its cleavage by ß-secretase, is present in MAM, that its level is increased in AD, and that this increase reduces mitochondrial respiration, likely via a C99-induced alteration in cellular sphingolipid homeostasis. Thus, the metabolic disturbances seen in AD likely arise from increased ER-mitochondrial communication that is driven by an increase in the levels of C99 at the MAM.

SCO2 mutations cause early-onset axonal Charcot-Marie-Tooth disease associated with cellular copper deficiency.

Recessive mutations in the mitochondrial copper-binding protein SCO2, cytochrome c oxidase (COX) assembly protein, have been reported in several cases with fatal infantile cardioencephalomyopathy with COX deficiency. Significantly expanding the known phenotypic spectrum, we identified compound heterozygous variants in SCO2 in two unrelated patients with axonal polyneuropathy, also known as Charcot-Marie-Tooth disease type 4. Different from previously described cases, our patients developed predominantly motor neuropathy, they survived infancy, and they have not yet developed the cardiomyopathy that causes death in early infancy in reported patients. Both of our patients harbour missense mutations near the conserved copper-binding motif (CXXXC), including the common pathogenic variant E140K and a novel change D135G. In addition, each patient carries a second mutation located at the same loop region, resulting in compound heterozygote changes E140K/P169T and D135G/R171Q. Patient fibroblasts showed reduced levels of SCO2, decreased copper levels and COX deficiency. Given that another Charcot-Marie-Tooth disease gene, ATP7A, is a known copper transporter, our findings further underline the relevance of copper metabolism in Charcot-Marie-Tooth disease.

Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease.

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by ß-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by γ-secretase to generate the ß-amyloid (Aß) found in senile plaques. In previous reports, we and others have shown that γ-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.

Low-dose rapamycin extends lifespan in a mouse model of mtDNA depletion syndrome.

Mitochondrial disorders affecting oxidative phosphorylation (OxPhos) are caused by mutations in both the nuclear and mitochondrial genomes. One promising candidate for treatment is the drug rapamycin, which has been shown to extend lifespan in multiple animal models, and which was previously shown to ameliorate mitochondrial disease in a knock-out mouse model lacking a nuclear-encoded gene specifying an OxPhos structural subunit (Ndufs4). In that model, relatively high-dose intraperitoneal rapamycin extended lifespan and improved markers of neurological disease, via an unknown mechanism. Here, we administered low-dose oral rapamycin to a knock-in (KI) mouse model of authentic mtDNA disease, specifically, progressive mtDNA depletion syndrome, resulting from a mutation in the mitochondrial nucleotide salvage enzyme thymidine kinase 2 (TK2). Importantly, low-dose oral rapamycin was sufficient to extend Tk2KI/KI mouse lifespan significantly, and did so in the absence of detectable improvements in mitochondrial dysfunction. We found no evidence that rapamycin increased survival by acting through canonical pathways, including mitochondrial autophagy. However, transcriptomics and metabolomics analyses uncovered systemic metabolic changes pointing to a potential 'rapamycin metabolic signature.' These changes also implied that rapamycin may have enabled the Tk2KI/KI mice to utilize alternative energy reserves, and possibly triggered indirect signaling events that modified mortality through developmental reprogramming. From a therapeutic standpoint, our results support the possibility that low-dose rapamycin, while not targeting the underlying mtDNA defect, could represent a crucial therapy for the treatment of mtDNA-driven, and some nuclear DNA-driven, mitochondrial diseases.

Alzheimer Disease.

The most widely accepted hypothesis to explain the pathogenesis of Alzheimer disease (AD) is the amyloid cascade, in which the accumulation of extraneuritic plaques and intracellular tangles plays a key role in driving the course and progression of the disease. However, there are other biochemical and morphological features of AD, including altered calcium, phospholipid, and cholesterol metabolism and altered mitochondrial dynamics and function that often appear early in the course of the disease, prior to plaque and tangle accumulation. Interestingly, these other functions are associated with a subdomain of the endoplasmic reticulum (ER) called mitochondria-associated ER membranes (MAM). MAM, which is an intracellular lipid raft-like domain, is closely apposed to mitochondria, both physically and biochemically. These MAM-localized functions are, in fact, increased significantly in various cellular and animal models of AD and in cells from AD patients, which could help explain the biochemical and morphological alterations seen in the disease. Based on these and other observations, a strong argument can be made that increased ER-mitochondria connectivity and increased MAM function are fundamental to AD pathogenesis.

On the Pathogenesis of Alzheimer's Disease: The MAM Hypothesis.

The pathogenesis of Alzheimer's disease (AD) is currently unclear and is the subject of much debate. The most widely accepted hypothesis designed to explain AD pathogenesis is the amyloid cascade, which invokes the accumulation of extracellular plaques and intracellular tangles as playing a fundamental role in the course and progression of the disease. However, besides plaques and tangles, other biochemical and morphological features are also present in AD, often manifesting early in the course of the disease before the accumulation of plaques and tangles. These include altered calcium, cholesterol, and phospholipid metabolism; altered mitochondrial dynamics; and reduced bioenergetic function. Notably, these other features of AD are associated with functions localized to a subdomain of the endoplasmic reticulum (ER), known as mitochondria-associated ER membranes (MAMs). The MAM region of the ER is a lipid raft-like domain closely apposed to mitochondria in such a way that the 2 organelles are able to communicate with each other, both physically and biochemically, thereby facilitating the functions of this region. We have found that MAM-localized functions are increased significantly in cellular and animal models of AD and in cells from patients with AD in a manner consistent with the biochemical findings noted above. Based on these and other observations, we propose that increased ER-mitochondrial apposition and perturbed MAM function lie at the heart of AD pathogenesis.-Area-Gomez, E., Schon, E. A. On the pathogenesis of Alzheimer's disease: the MAM hypothesis.

Natural underlying mtDNA heteroplasmy as a potential source of intra-person hiPSC variability.

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high-quality biorepositories. Beyond inter-person variability, the root cause of intra-person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next-generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non-mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC-derived cardiomyocytes with expanded mtDNA mutations non-related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra-person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.

Mitochondria-associated ER membranes and Alzheimer disease.

The series of events underlying the pathogenesis of Alzheimer disease (AD) in unknown. The most widely accepted hypothesis is called the amyloid cascade, based on the observation that the brains of AD patients contain high levels of extracellular plaques, composed mainly of ß-amyloid (Aß), and intracellular tangles, composed of hyperphosphorylated forms of the microtubule-associated protein tau. However, AD is also characterized by other features, including aberrant cholesterol, phospholipid, and calcium metabolism, and mitochondrial dysfunction, all ostensibly unrelated to plaque and tangle formation. Notably, these 'other' aspects of AD pathology are functions related to mitochondria-associated ER membranes (MAM), a subdomain of the endoplasmic reticulum (ER) that is apposed to, and communicates with, mitochondria. Given the potential relationship between MAM and AD, we explored the possibility that perturbed MAM function might play a role in AD pathogenesis. We found that γ-secretase activity, which processes the amyloid precursor protein to generate Aß, is located predominantly in the MAM, and that ER-mitochondrial apposition and MAM function are increased significantly in cells from AD patients. These observations may help explain not only the aberrant Aß production, but also many of the 'other' biochemical and morphological features of the disease. Based on these, and other, data we propose that AD is fundamentally a disorder of ER-mitochondrial hyperconnectivity.

Mitochondrial iron chelation ameliorates cigarette smoke-induced bronchitis and emphysema in mice.

Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element-binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.

ApoE4 upregulates the activity of mitochondria-associated ER membranes.

In addition to the appearance of senile plaques and neurofibrillary tangles, Alzheimer's disease (AD) is characterized by aberrant lipid metabolism and early mitochondrial dysfunction. We recently showed that there was increased functionality of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a subdomain of the ER involved in lipid and cholesterol homeostasis, in presenilin-deficient cells and in fibroblasts from familial and sporadic AD patients. Individuals carrying the ε4 allele of apolipoprotein E (ApoE4) are at increased risk for developing AD compared to those carrying ApoE3. While the reason for this increased risk is unknown, we hypothesized that it might be associated with elevated MAM function. Using an astrocyte-conditioned media (ACM) model, we now show that ER-mitochondrial communication and MAM function-as measured by the synthesis of phospholipids and of cholesteryl esters, respectively-are increased significantly in cells treated with ApoE4-containing ACM as compared to those treated with ApoE3-containing ACM. Notably, this effect was seen with lipoprotein-enriched preparations, but not with lipid-free ApoE protein. These data are consistent with a role of upregulated MAM function in the pathogenesis of AD and may help explain, in part, the contribution of ApoE4 as a risk factor in the disease.

No Evidence for Association of SCO2 Heterozygosity with High-Grade Myopia or Other Diseases with Possible Mitochondrial Dysfunction.

SCO2 mutations cause recessively inherited cytochrome c oxidase deficiency. Recently Tran-Viet et al. proposed that heterozygosity for pathogenic SCO2 variants, including the common E140K variant, causes high-grade myopia. To investigate the association of SCO2 mutations with myopia, ophthalmic examinations were performed on 35 E140K carriers, one homozygous infant, and on a mouse model of Sco2 deficiency. Additionally, a screen for other putative effects of SCO2 heterozygosity was carried out by comparing the prevalence of the common E140K variant in a population of patients with undiagnosed diseases compatible with SCO2-related pathogenesis to that in a general population sample. High-grade myopia was not identified in any of the studied individuals. Of the carriers, 17 were emmetropic, and 18 possessed refractive errors. Additionally, no significant axial elongation indicative of high-grade myopia was found in mice carrying E129K (corresponding to E140K in humans) knock-in mutations. The prevalence of E140K carriers in the symptomatic cohort was evaluated as 1:103 (CI: 0.44-2.09) and did not differ significantly from the population prevalence (1:147, CI: 0.45-1.04).Our study demonstrates that heterozygosity for pathogenic SCO2 variants is not associated with high-grade myopia in either human patients or in mice.

ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.