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.

The Alzheimer's disease-associated C99 fragment of APP regulates cellular cholesterol trafficking.

The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid-sensing peptide(s) in the ER that can cluster PM-derived cholesterol into transient detergent-resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol-rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99-aa C-terminal fragment of APP (C99), when delivered to the ER for cleavage by γ-secretase, acts as a lipid-sensing peptide that forms regulatory DRMs in the ER, called mitochondria-associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease.

MAM and C99, key players in the pathogenesis of Alzheimer's disease.

Inter-organelle communication is a rapidly-expanding field that has transformed our understanding of cell biology and pathology. Organelle-organelle contact sites can generate transient functional domains that act as enzymatic hubs involved in the regulation of cellular metabolism and intracellular signaling. One of these hubs is located in areas of the endoplasmic reticulum (ER) connected to mitochondria, called mitochondria-associated ER membranes (MAM). These MAM are transient lipid rafts intimately involved in cholesterol and phospholipid metabolism, calcium homeostasis, and mitochondrial function and dynamics. In addition, γ-secretase-mediated proteolysis of the amyloid precursor protein 99-aa C-terminal fragment (C99) to form amyloid ß also occurs at the MAM. Our most recent data indicates that in Alzheimer's disease, increases in uncleaved C99 levels at the MAM provoke the upregulation of MAM-resident functions, resulting in the loss of lipid homeostasis, and mitochondrial dysfunction. Here, we discuss the relevance of these findings in the field, and the contribution of C99 and MAM dysfunction to Alzheimer's disease neuropathology.

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.

Distinct Neuronal Projections From the Hypothalamic Ventromedial Nucleus Mediate Glycemic and Behavioral Effects.

The hypothalamic ventromedial nucleus (VMN) is implicated both in autonomic control of blood glucose and in behaviors including fear and aggression, but whether these divergent effects involve the same or distinct neuronal subsets and their projections is unknown. To address this question, we used an optogenetic approach to selectively activate the subset of VMN neurons that express neuronal nitric oxide synthase 1 (VMNNOS1 neurons) implicated in glucose counterregulation. We found that photoactivation of these neurons elicits 1) robust hyperglycemia achieved by activation of counterregulatory responses usually reserved for the physiological response to hypoglycemia and 2) defensive immobility behavior. Moreover, we show that the glucagon, but not corticosterone, response to insulin-induced hypoglycemia is blunted by photoinhibition of the same neurons. To investigate the neurocircuitry by which VMNNOS1 neurons mediate these effects, and to determine whether these diverse effects are dissociable from one another, we activated downstream VMNNOS1 projections in either the anterior bed nucleus of the stria terminalis (aBNST) or the periaqueductal gray (PAG). Whereas glycemic responses are fully recapitulated by activation of VMNNOS1 projections to the aBNST, freezing immobility occurred only upon activation of VMNNOS1 terminals in the PAG. These findings support previous evidence of a VMN→aBNST neurocircuit involved in glucose counterregulation and demonstrate that activation of VMNNOS1 neuronal projections supplying the PAG robustly elicits defensive behaviors.

Leptin regulation of core body temperature involves mechanisms independent of the thyroid axis.

The ability to maintain core temperature within a narrow range despite rapid and dramatic changes in environmental temperature is essential for the survival of free-living mammals, and growing evidence implicates an important role for the hormone leptin. Given that thyroid hormone plays a major role in thermogenesis and that circulating thyroid hormone levels are reduced in leptin-deficient states (an effect partially restored by leptin replacement), we sought to determine the extent to which leptin's role in thermogenesis is mediated by raising thyroid hormone levels. To this end, we 1) quantified the effect of physiological leptin replacement on circulating levels of thyroid hormone in leptin-deficient ob/ob mice, and 2) determined if the effect of leptin to prevent the fall in core temperature in these animals during cold exposure is mimicked by administration of a physiological replacement dose of triiodothyronine (T3). We report that, as with leptin, normalization of circulating T3 levels is sufficient both to increase energy expenditure, respiratory quotient, and ambulatory activity and to reduce torpor in ob/ob mice. Yet, unlike leptin, infusing T3 at a dose that normalizes plasma T3 levels fails to prevent the fall of core temperature during mild cold exposure. Because thermal conductance (e.g., heat loss to the environment) was reduced by administration of leptin but not T3, leptin regulation of heat dissipation is implicated as playing a uniquely important role in thermoregulation. Together, these findings identify a key role in thermoregulation for leptin-mediated suppression of thermal conduction via a mechanism that is independent of the thyroid axis.

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.

Aminopeptidase activities as prospective urinary biomarkers for bladder cancer.

PURPOSE: Proteases have been implicated in cancer progression and invasiveness. We have investigated the activities, as opposed to simple protein levels, of selected aminopeptidases in urine specimens to serve as potential novel biomarkers for urothelial cancer. EXPERIMENTAL DESIGN: The unique urinary proteomes of males and females were profiled to establish the presence of a gender-independent set of aminopeptidases. Samples were also collected from patients with urothelial cancer and matched controls. A SOP for urine processing was developed taking into account hydration variation. Five specific aminopeptidase activity assays, using fluorophoric substrates, were optimized for evaluation of marker potential. RESULTS: Nineteen exopeptidases and 21 other proteases were identified in urine and the top-five most abundant aminopeptidases, identical in both genders, selected for functional studies. Depending on the enzyme, activities were consistently lower (p ≤ 0.05), higher or unchanged in the cancer samples as compared to controls. Two selected aminopeptidase activities used as a binary classifier resulted in a ROC curve with an AUC = 0.898. CONCLUSION AND CLINICAL RELEVANCE: We have developed functional assays that characterize aminopeptidase activities in urine specimens with adequate technical and intraindividual reproducibility. With further testing, it could yield a reliable biomarker test for bladder cancer detection or prognostication.

Bromodomain protein 7 interacts with PRMT5 and PRC2, and is involved in transcriptional repression of their target genes.

Histone modification regulates gene expression, and one major regulatory step in this process is the ability of proteins that recognize epigenetic marks to recruit enzymes required to specify transcriptional outcome. Here we show that BRD7 is a component of hSWI-SNF complexes that interacts with PRMT5 and PRC2. Recruitment studies revealed that BRD7 co-localizes with PRMT5 and PRC2 on 'suppressor of tumorigenecity 7' (ST7) and retinoblastoma-like protein 2 (RBL2) promoters in patient-derived B cell lines, and that its association with these target genes correlates with hypermethylation of H3R8, H4R3 and H3K27. Furthermore, inhibition of BRD7 expression reduces PRMT5 and PRC2 recruitment to ST7 and RBL2 promoters; however, only ST7 becomes transcriptionally derepressed. Evaluation of the PRMT5- and PRC2-induced epigenetic marks revealed that while H3(Me(2))R8, H4(Me(2))R3 and H3(Me(3))K27 marks are erased from the ST7 promoter, demethylation of RBL2 promoter histones is incomplete. We also show that the arginine demethylase (RDM) JMJD6, which can erase PRMT5-induced H4R3 methylation, and the H3K27-lysine-specific demethylases, KDM6A/UTX and KDM6B/JMJD3, are differentially recruited to ST7 and RBL2. These findings highlight the role played by BRD7 in PRMT5- and PRC2-induced transcriptional silencing, and indicate that recruitment of specific RDMs and KDMs is required for efficient transcriptional derepression.