Barry E. Kennedy

MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein

Niemann-Pick Type C (NPC) disease is a fatal, neurodegenerative disorder, caused in most cases by mutations in the late endosomal protein NPC1. A hallmark of NPC disease is endosomal cholesterol accumulation and an impaired cholesterol homeostatic response, which might affect cholesterol transport to mitochondria and, thus, mitochondrial and cellular function. This study aimed to characterize mitochondrial cholesterol homeostasis in NPC disease. Using wild-type and NPC1-deficient Chinese hamster ovary cells, stably transfected with a CYP11A1 complex to assess mitochondrial cholesterol import by pregnenolone production, we show that cholesterol transport to the mitochondrial inner membrane is not affected by loss of NPC1. However, mitochondrial cholesterol content was higher in NPC1-deficient than in wild-type cells. Cholesterol transport to the mitochondrial inner membrane increased markedly upon exposure of cholesterol-deprived cells to lipoproteins, indicating transport of endosomal cholesterol to mitochondria. Reduction of endosomal metastatic lymph node protein 64 (MLN64) by RNA interference decreased cholesterol transport to the mitochondrial inner membrane and reduced mitochondrial cholesterol levels in NPC1-deficient cells, suggesting that MLN64 transported cholesterol to mitochondria even in the absence of NPC1. In summary, this study describes a transport pathway for endosomal cholesterol to mitochondria that requires MLN64, but not NPC1, and that may be responsible for increased mitochondrial cholesterol in NPC disease.

Niemann-Pick Type C2 protein contributes to the transport of endosomal cholesterol to mitochondria without interacting with NPC1

Mitochondrial cholesterol is maintained within a narrow range to regulate steroid and oxysterol synthesis and to ensure mitochondrial function. Mitochondria acquire cholesterol through several pathways from different cellular pools. Here we have characterized mitochondrial import of endosomal cholesterol using Chinese hamster ovary cells expressing a CYP11A1 fusion protein that converts cholesterol to pregnenolone at the mitochondrial inner membrane. RNA interference-mediated depletion of the voltage-dependent anion channel 1 in the mitochondrial outer membrane or of Niemann-Pick Type C2 (NPC2) in the endosome lumen decreased arrival of cholesterol at the mitochondrial inner membrane. Expression of NPC2 mutants unable to transfer cholesterol to NPC1 still restored mitochondrial cholesterol import in NPC2-depleted cells. Transport assays in semi-permeabilized cells showed nonvesicular cholesterol trafficking directly from endosomes to mitochondria that did not require cytosolic transport proteins but that was reduced in the absence of NPC2. Our findings indicate that NPC2 delivers cholesterol to the perimeter membrane of late endosomes, where it becomes available for transport to mitochondria without requiring NPC1.

Pre-Symptomatic Activation of Antioxidant Responses and Alterations in Glucose and Pyruvate Metabolism in Niemann-Pick Type C1-Deficient Murine Brain

Niemann-Pick Type C (NPC) disease is an autosomal recessive neurodegenerative disorder caused in most cases by mutations in the NPC1 gene. NPC1-deficiency is characterized by late endosomal accumulation of cholesterol, impaired cholesterol homeostasis, and a broad range of other cellular abnormalities. Although neuronal abnormalities and glial activation are observed in nearly all areas of the brain, the most severe consequence of NPC1-deficiency is a near complete loss of Purkinje neurons in the cerebellum. The link between cholesterol trafficking and NPC pathogenesis is not yet clear; however, increased oxidative stress in symptomatic NPC disease, increases in mitochondrial cholesterol, and alterations in autophagy/mitophagy suggest that mitochondria play a role in NPC disease pathology. Alterations in mitochondrial function affect energy and neurotransmitter metabolism, and are particularly harmful to the central nervous system. To investigate early metabolic alterations that could affect NPC disease progression, we performed metabolomics analyses of different brain regions from age-matched wildtype and Npc1 -/- mice at pre-symptomatic, early symptomatic and late stage disease by 1H-NMR spectroscopy. Metabolic profiling revealed markedly increased lactate and decreased acetate/acetyl-CoA levels in Npc1 -/- cerebellum and cerebral cortex at all ages. Protein and gene expression analyses indicated a pre-symptomatic deficiency in the oxidative decarboxylation of pyruvate to acetyl-CoA, and an upregulation of glycolytic gene expression at the early symptomatic stage. We also observed a pre-symptomatic increase in several indicators of oxidative stress and antioxidant response systems in Npc1 -/- cerebellum. Our findings suggest that energy metabolism and oxidative stress may present additional therapeutic targets in NPC disease, especially if intervention can be started at an early stage of the disease.

Adaptations of Energy Metabolism Associated with Increased Levels of Mitochondrial Cholesterol in Niemann-Pick Type C1-deficient Cells

Background: Endosomal cholesterol storage in Niemann-Pick type C1 deficiency is associated with elevated mitochondrial cholesterol and alterations in energy metabolism. Results: Blocking endosomal cholesterol transport to mitochondria prevented the metabolic alterations in NPC1-deficient cells. Conclusion: Mitochondrial cholesterol accumulation increases oxidative stress and alters energy metabolism. Significance: Mitochondrial cholesterol is a regulator of energy metabolism and mitochondrial function. Niemann-Pick type C1 (NPC1) is a late endosomal transmembrane protein, which, together with NPC2 in the endosome lumen, mediates the transport of endosomal cholesterol to the plasma membrane and endoplasmic reticulum. Loss of function of NPC1 or NPC2 leads to cholesterol accumulation in late endosomes and causes neuronal dysfunction and neurodegeneration. Recent studies indicate that cholesterol also accumulates in mitochondria of NPC1-deficient cells and brain tissue and that NPC1 deficiency leads to alterations in mitochondrial function and energy metabolism. Here, we have investigated the effects of increased mitochondrial cholesterol levels on energy metabolism, using RNA interference to deplete Chinese hamster ovary cells of NPC1 alone or in combination with MLN64, which mediates endosomal cholesterol transport to mitochondria. Mitochondrial cholesterol levels were also altered by depletion of NPC2 in combination with the expression of NPC2 mutants. We found that the depletion of NPC1 increased lactate secretion, decreased glutamine-dependent mitochondrial respiration, and decreased ATP transport across mitochondrial membranes. These metabolic alterations did not occur when transport of endosomal cholesterol to mitochondria was blocked. In addition, the elevated mitochondrial cholesterol levels in NPC1-depleted cells and in NPC2-depleted cells expressing mutant NPC2 that allows endosomal cholesterol trafficking to mitochondria were associated with increased expression of the antioxidant response factor Nrf2. Antioxidant treatment not only prevented the increase in Nrf2 mRNA levels but also prevented the increased lactate secretion in NPC1-depleted cells. These results suggest that mitochondrial cholesterol accumulation can increase oxidative stress and in turn cause increased glycolysis to lactate and other metabolic alterations.

NAD+ salvage pathway in cancer metabolism and therapy

Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme for various physiological processes including energy metabolism, DNA repair, cell growth, and cell death. Many of these pathways are typically dysregulated in cancer cells, making NAD+ an intriguing target for cancer therapeutics. NAD+ is mainly synthesized by the NAD+ salvage pathway in cancer cells, and not surprisingly, the pharmacological targeting of the NAD+ salvage pathway causes cancer cell cytotoxicity in vitro and in vivo. Several studies have described the precise consequences of NAD+ depletion on cancer biology, and have demonstrated that NAD+ depletion results in depletion of energy levels through lowered rates of glycolysis, reduced citric acid cycle activity, and decreased oxidative phosphorylation. Additionally, depletion of NAD+ causes sensitization of cancer cells to oxidative damage by disruption of the anti-oxidant defense system, decreased cell proliferation, and initiation of cell death through manipulation of cell signaling pathways (e.g., SIRT1 and p53). Recently, studies have explored the effect of well-known cancer therapeutics in combination with pharmacological depletion of NAD+ levels, and found in many cases a synergistic effect on cancer cell cytotoxicity. In this context, we will discuss the effects of NAD+ salvage pathway inhibition on cancer cell biology and provide insight on this pathway as a novel anti-cancer therapeutic target.

Human Brain Chemokine and Cytokine Expression in Sepsis: A Report of Three Cases.

BACKGROUND Sepsis is a systemic response to infection that can affect brain function by inducing resident cells (including astrocytes and microglia) to generate brain chemokines and cytokines. However, there are few studies on the human brain. Since this information may shed further light on pathogenesis, our study objective was to measure the expression of 36 chemokines and cytokines in autopsied brain from 3 cases of sepsis and 10 controls, and to relate this to astrocyte and microglial activation. METHODS The right frontal pole was removed at autopsy and chemokine and cytokine expression measured by multiplexed enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction (qPCR). Immunohistochemistry and image analysis were carried out to determine the expression of glial fibrillary acidic protein (GFAP), a marker of activated astrocytes, and CD68 and CD45, markers of activated microglial cells. RESULTS Concentrations of the chemokines CXCL8, CXCL10, CXCL12, CCL13 and CCL22 were increased in pooled data from the three cases of sepsis (p<0.05); however, their messenger RNA (mRNA) expression was unaltered. CXCL13, CXCL1, CXCL2, CCL1, CCL2, CCL8, CCL20, (interleukin) IL-16, IL-1β and (tumour necrosis factor) TNF concentrations showed increases in two of three sepsis cases. Additionally, individual sepsis cases showed increases in mRNA expression for HDAC (histone deacetylase) 6 and EIF (eukaryotic translation initiation factor) 4A2. Brain GFAP expression was significantly increased (p<0.05) in pooled data from the three sepsis cases. Individual sepsis cases showed increases in CD68 or CD45 expression. CONCLUSIONS These expression patterns add to our understanding of the pathogenesis of sepsis and its effects on the brain.

Autophagic homeostasis is required for the pluripotency of cancer stem cells

ABSTRACT Pluripotency is an important feature of cancer stem cells (CSCs) that contributes to self-renewal and chemoresistance. The maintenance of pluripotency of CSCs under various pathophysiological conditions requires a complex interaction between various cellular pathways including those involved in homeostasis and energy metabolism. However, the exact mechanisms that maintain the CSC pluripotency remain poorly understood. In this report, using both human and murine models of CSCs, we demonstrate that basal levels of autophagy are required to maintain the pluripotency of CSCs, and that this process is differentially regulated by the rate-limiting enzyme in the NAD+ synthesis pathway NAMPT (nicotinamide phosphoribosyltransferase) and the transcription factor POU5F1/OCT4 (POU class 5 homeobox 1). First, our data show that the pharmacological inhibition and knockdown (KD) of NAMPT or the KD of POU5F1 in human CSCs significantly decreased the expression of pluripotency markers POU5F1, NANOG (Nanog homeobox) and SOX2 (SRY-box 2), and upregulated the differentiation markers TUBB3 (tubulin β 3 class III), CSN2 (casein β), SPP1 (secreted phosphoprotein 1), GATA6 (GATA binding protein 6), T (T brachyury transcription factor) and CDX2 (caudal type homeobox 2). Interestingly, these pluripotency-regulating effects of NAMPT and POU5F1 were accompanied by contrasting levels of autophagy, wherein NAMPT KD promoted while POU5F1 KD inhibited the autophagy machinery. Most importantly, any deviation from the basal level of autophagy, either increase (via rapamycin, serum starvation or Tat-beclin 1 [Tat-BECN1] peptide) or decrease (via ATG7 or ATG12 KD), strongly decreased the pluripotency and promoted the differentiation and/or senescence of CSCs. Collectively, these results uncover the link between the NAD+ biosynthesis pathway, CSC transcription factor POU5F1 and pluripotency, and further identify autophagy as a novel regulator of pluripotency of CSCs.

TRPM2 channel-mediated regulation of autophagy maintains mitochondrial function and promotes gastric cancer cell survival via the JNK signaling pathway

A lack of effective treatment is one of the main factors contributing to gastric cancer–related death. Discovering effective targets and understanding their underlying anti-cancer mechanism are key to achieving the best response to treatment and to limiting side effects. Although recent studies have shown that the cation channel transient receptor potential melastatin-2 (TRPM2) is crucial for cancer cell survival, the exact mechanism remains unclear, limiting its therapeutic potential. Here, using molecular and functional assays, we investigated the role of TRPM2 in survival of gastric cancer cells. Our results indicated that TRPM2 knockdown in AGS and MKN-45 cells decreases cell proliferation and enhances apoptosis. We also observed that the TRPM2 knockdown impairs mitochondrial metabolism, indicated by a decrease in basal and maximal mitochondrial oxygen consumption rates and ATP production. These mitochondrial defects coincided with a decrease in autophagy and mitophagy, indicated by reduced levels of autophagy- and mitophagy-associated proteins (i.e. ATGs, LC3A/B II, and BNIP3). Moreover, we found that TRPM2 modulates autophagy through a c-Jun N-terminal kinase (JNK)-dependent and mechanistic target of rapamycin-independent pathway. We conclude that in the absence of TRPM2, down-regulation of the JNK-signaling pathway impairs autophagy, ultimately causing the accumulation of damaged mitochondria and death of gastric cancer cells. Of note, by inhibiting cell proliferation and promoting apoptosis, the TRPM2 down-regulation enhanced the efficacy of paclitaxel and doxorubicin in gastric cancer cells. Collectively, we provide compelling evidence that TRPM2 inhibition may benefit therapeutic approaches for managing gastric cancer.

Quantitative Temporal in Vivo Proteomics Deciphers the Transition of Virus-Driven Myeloid Cells into M2 Macrophages

Myeloid cells play a central role in the context of viral eradication, yet precisely how these cells differentiate throughout the course of acute infections is poorly understood. In this study, we have developed a novel quantitative temporal in vivo proteomics (QTiPs) platform to capture proteomic signatures of temporally transitioning virus-driven myeloid cells directly in situ, thus taking into consideration host–virus interactions throughout the course of an infection. QTiPs, in combination with phenotypic, functional, and metabolic analyses, elucidated a pivotal role for inflammatory CD11b+, Ly6G–, Ly6Chigh-low cells in antiviral immune response and viral clearance. Most importantly, the time-resolved QTiPs data set showed the transition of CD11b+, Ly6G–, Ly6Chigh-low cells into M2-like macrophages, which displayed increased antigen-presentation capacities and bioenergetic demands late in infection. We elucidated the pivotal role of myeloid cells in virus clearance and show how these cells phenotypically, functionally, and metabolically undergo a timely transition from inflammatory to M2-like macrophages in vivo. With respect to the growing appreciation for in vivo examination of viral–host interactions and for the role of myeloid cells, this study elucidates the use of quantitative proteomics to reveal the role and response of distinct immune cell populations throughout the course of virus infection.

Measurement of Mitochondrial Cholesterol Import Using a Mitochondria-Targeted CYP11A1 Fusion Construct

All animal membranes require cholesterol as an essential regulator of biophysical properties and function, but the levels of cholesterol vary widely among different subcellular compartments. Mitochondria, and in particular the inner mitochondrial membrane, have the lowest levels of cholesterol in the cell. Nevertheless, mitochondria need cholesterol for membrane maintenance and biogenesis, as well as oxysterol, steroid, and hepatic bile acid production. Alterations in mitochondrial cholesterol have been associated with a range of pathological conditions, including cancer, hepatosteatosis, cardiac ischemia, Alzheimers, and Niemann-Pick Type C Disease. The mechanisms of mitochondrial cholesterol import are not fully elucidated yet, and may vary in different cell types and environmental conditions. Measuring cholesterol trafficking to the mitochondrial membranes is technically challenging because of its low abundance; for example, traditional pulse-chase experiments with isotope-labeled cholesterol are not feasible. Here, we describe improvements to a method first developed by the Miller group at the University of California to measure cholesterol trafficking to the inner mitochondrial membrane (IMM) through the conversion of cholesterol to pregnenolone. This method uses a mitochondria-targeted, ectopically expressed fusion construct of CYP11A1, ferredoxin reductase and ferredoxin. Pregnenolone is formed exclusively from cholesterol at the IMM, and can be analyzed with high sensitivity and specificity through ELISA or radioimmunoassay of the medium/buffer to reflect mitochondrial cholesterol import. This assay can be used to investigate the effects of genetic or pharmacological interventions on mitochondrial cholesterol import in cultured cells or isolated mitochondria.