LACTB is a tumour suppressor that modulates lipid metabolism and cell state.

Post-mitotic, differentiated cells exhibit a variety of characteristics that contrast with those of actively growing neoplastic cells, such as the expression of cell-cycle inhibitors and differentiation factors. We hypothesized that the gene expression profiles of these differentiated cells could reveal the identities of genes that may function as tumour suppressors. Here we show, using in vitro and in vivo studies in mice and humans, that the mitochondrial protein LACTB potently inhibits the proliferation of breast cancer cells. Its mechanism of action involves alteration of mitochondrial lipid metabolism and differentiation of breast cancer cells. This is achieved, at least in part, through reduction of the levels of mitochondrial phosphatidylserine decarboxylase, which is involved in the synthesis of mitochondrial phosphatidylethanolamine. These observations uncover a novel mitochondrial tumour suppressor and demonstrate a connection between mitochondrial lipid metabolism and the differentiation program of breast cancer cells, thereby revealing a previously undescribed mechanism of tumour suppression.

Historical perspective: phosphatidylserine and phosphatidylethanolamine from the 1800s to the present.

This article provides a historical account of the discovery, chemistry, and biochemistry of two ubiquitous phosphoglycerolipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), including the ether lipids. In addition, the article describes the biosynthetic pathways for these phospholipids and how these pathways were elucidated. Several unique functions of PS and PE in mammalian cells in addition to their ability to define physical properties of membranes are discussed. For example, the translocation of PS from the inner to the outer leaflet of the plasma membrane of cells occurs during apoptosis and during some other specific physiological processes, and this translocation is responsible for profound life-or-death events. Moreover, mitochondrial function is severely impaired when the PE content of mitochondria is reduced below a threshold level. The discovery and implications of the existence of membrane contact sites between the endoplasmic reticulum and mitochondria and their relevance for PS and PE metabolism, as well as for mitochondrial function, are also discussed. Many of the recent advances in these fields are due to the use of isotope labeling for tracing biochemical pathways. In addition, techniques for disruption of specific genes in mice are now widely used and have provided major breakthroughs in understanding the roles and metabolism of PS and PE in vivo.

Phospholipid synthesis and transport in mammalian cells.

Membranes of mammalian subcellular organelles contain defined amounts of specific phospholipids that are required for normal functioning of proteins in the membrane. Despite the wide distribution of most phospholipid classes throughout organelle membranes, the site of synthesis of each phospholipid class is usually restricted to one organelle, commonly the endoplasmic reticulum (ER). Thus, phospholipids must be transported from their sites of synthesis to the membranes of other organelles. In this article, pathways and subcellular sites of phospholipid synthesis in mammalian cells are summarized. A single, unifying mechanism does not explain the inter-organelle transport of all phospholipids. Thus, mechanisms of phospholipid transport between organelles of mammalian cells via spontaneous membrane diffusion, via cytosolic phospholipid transfer proteins, via vesicles and via membrane contact sites are discussed. As an example of the latter mechanism, phosphatidylserine (PS) is synthesized on a region of the ER (mitochondria-associated membranes, MAM) and decarboxylated to phosphatidylethanolamine in mitochondria. Some evidence is presented suggesting that PS import into mitochondria occurs via membrane contact sites between MAM and mitochondria. Recent studies suggest that protein complexes can form tethers that link two types of organelles thereby promoting lipid transfer. However, many questions remain about mechanisms of inter-organelle phospholipid transport in mammalian cells.

Lack of phosphatidylethanolamine N-methyltransferase in mice does not promote fatty acid oxidation in skeletal muscle.

Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt(-/-) mice than in Pemt(+/+) mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt(-/-) mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source.

The critical role of phosphatidylcholine and phosphatidylethanolamine metabolism in health and disease.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in all mammalian cell membranes. In the 1950s, Eugene Kennedy and co-workers performed groundbreaking research that established the general outline of many of the pathways of phospholipid biosynthesis. In recent years, the importance of phospholipid metabolism in regulating lipid, lipoprotein and whole-body energy metabolism has been demonstrated in numerous dietary studies and knockout animal models. The purpose of this review is to highlight the unappreciated impact of phospholipid metabolism on health and disease. Abnormally high, and abnormally low, cellular PC/PE molar ratios in various tissues can influence energy metabolism and have been linked to disease progression. For example, inhibition of hepatic PC synthesis impairs very low density lipoprotein secretion and changes in hepatic phospholipid composition have been linked to fatty liver disease and impaired liver regeneration after surgery. The relative abundance of PC and PE regulates the size and dynamics of lipid droplets. In mitochondria, changes in the PC/PE molar ratio affect energy production. We highlight data showing that changes in the PC and/or PE content of various tissues are implicated in metabolic disorders such as atherosclerosis, insulin resistance and obesity. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

APP overexpression in the absence of NPC1 exacerbates metabolism of amyloidogenic proteins of Alzheimer's disease.

Amyloid-ß (Aß) peptides originating from ß-amyloid precursor protein (APP) are critical in Alzheimer's disease (AD). Cellular cholesterol levels/distribution can regulate production and clearance of Aß peptides, albeit with contradictory outcomes. To better understand the relationship between cholesterol homeostasis and APP/Aß metabolism, we have recently generated a bigenic ANPC mouse line overexpressing mutant human APP in the absence of Niemann-Pick type C-1 protein required for intracellular cholesterol transport. Using this unique bigenic ANPC mice and complementary stable N2a cells, we have examined the functional consequences of cellular cholesterol sequestration in the endosomal-lysosomal system, a major site of Aß production, on APP/Aß metabolism and its relation to neuronal viability. Levels of APP C-terminal fragments (α-CTF/ß-CTF) and Aß peptides, but not APP mRNA/protein or soluble APPα/APPß, were increased in ANPC mouse brains and N2a-ANPC cells. These changes were accompanied by reduced clearance of peptides and an increased level/activity of γ-secretase, suggesting that accumulation of APP-CTFs is due to decreased turnover, whereas increased Aß levels may result from a combination of increased production and decreased turnover. APP-CTFs and Aß peptides were localized primarily in early-/late-endosomes and to some extent in lysosomes/autophagosomes. Cholesterol sequestration impaired endocytic-autophagic-lysosomal, but not proteasomal, clearance of APP-CTFs/Aß peptides. Moreover, markers of oxidative stress were increased in vulnerable brain regions of ANPC mice and enhanced ß-CTF/Aß levels increased susceptibility of N2a-ANPC cells to H2O2-induced toxicity. Collectively, our results show that cellular cholesterol sequestration plays a key role in APP/Aß metabolism and increasing neuronal vulnerability to oxidative stress in AD-related pathology.

Lack of phosphatidylethanolamine N-methyltransferase alters hepatic phospholipid composition and induces endoplasmic reticulum stress.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is associated with development of steatohepatitis. Phosphatidylethanolamine N-methyltransferase (PEMT) is a hepatic enzyme located on the ER and mitochondria-associated membranes and catalyzes phosphatidylcholine (PC) synthesis via methylation of phosphatidylethanolamine (PE). We hypothesized that PEMT deficiency in mice alters ER phospholipid content, thereby inducing ER stress and sensitizing the mice to diet-induced steatohepatitis. METHODS: PC and PE mass were measured in hepatic ER fractions from chow-fed and high fat-fed Pemt(-/-) and Pemt(+/+) mice. Proteins implicated in ER stress and the unfolded protein response (UPR) were assessed in mouse livers and in McArdle-RH7777 hepatoma cells that expressed or lacked PEMT. The chemical chaperone 4-phenyl butyric acid was administered to cells and HF-fed Pemt(-/-) mice to alleviate ER stress. RESULTS: In chow-fed Pemt(-/-) mice, the hepatic PC/PE ratio in the ER was lower than in Pemt(+/+) mice, and levels of ER stress markers, CHOP and BIP, were higher without activation of the UPR. In livers of HF-fed Pemt(-/-) mice the ER had a lower PC/PE ratio, and exhibited more ER stress and UPR activation. Similarly, the UPR was repressed in McArdle cells expressing PEMT compared with those lacking PEMT, with concomitantly lower levels of CHOP and BIP. 4-Phenyl butyric acid attenuated activation of the UPR and ER stress in McArdle cells lacking PEMT, but not the hepatic ER stress in HF-fed Pemt(-/-) mice. CONCLUSION: PEMT deficiency reduces the PC/PE ratio in the ER and induces ER stress, which sensitizes the mice to HF-induced steatohepatitis.

Insufficient glucose supply is linked to hypothermia upon cold exposure in high-fat diet-fed mice lacking PEMT.

Mice that lack phosphatidylethanolamine N-methyltransferase (Pemt(-/-) mice) are protected from high-fat (HF) diet-induced obesity. HF-fed Pemt(-/-) mice show higher oxygen consumption and heat production, indicating that more energy might be utilized for thermogenesis and might account for the resistance to diet-induced weight gain. To test this hypothesis, HF-fed Pemt(-/-) and Pemt(+/+) mice were challenged with acute cold exposure at 4°C. Unexpectedly, HF-fed Pemt(-/-) mice developed hypothermia within 3 h of cold exposure. In contrast, chow-fed Pemt(-/-) mice, possessing similar body mass, maintained body temperature. Lack of PEMT did not impair the capacity for thermogenesis in skeletal muscle or brown adipose tissue. Plasma catecholamines were not altered by Pemt genotype, and stimulation of lipolysis was intact in brown and white adipose tissue of Pemt(-/-) mice. HF-fed Pemt(-/-) mice also developed higher systolic blood pressure, accompanied by reduced cardiac output. Choline supplementation reversed the cold-induced hypothermia in HF-fed Pemt(-/-) mice with no effect on blood pressure. Plasma glucose levels were ∼50% lower in HF-fed Pemt(-/-) mice compared with Pemt(+/+) mice. Choline supplementation normalized plasma hypoglycemia and the expression of proteins involved in gluconeogenesis. We propose that cold-induced hypothermia in HF-fed Pemt(-/-) mice is linked to plasma hypoglycemia due to compromised hepatic glucose production.

MAM (mitochondria-associated membranes) in mammalian cells: lipids and beyond.

One mechanism by which communication between the endoplasmic reticulum (ER) and mitochondria is achieved is by close juxtaposition between these organelles via mitochondria-associated membranes (MAM). The MAM consist of a region of the ER that is enriched in several lipid biosynthetic enzyme activities and becomes reversibly tethered to mitochondria. Specific proteins are localized, sometimes transiently, in the MAM. Several of these proteins have been implicated in tethering the MAM to mitochondria. In mammalian cells, formation of these contact sites between MAM and mitochondria appears to be required for key cellular events including the transport of calcium from the ER to mitochondria, the import of phosphatidylserine into mitochondria from the ER for decarboxylation to phosphatidylethanolamine, the formation of autophagosomes, regulation of the morphology, dynamics and functions of mitochondria, and cell survival. This review focuses on the functions proposed for MAM in mediating these events in mammalian cells. In light of the apparent involvement of MAM in multiple fundamental cellular processes, recent studies indicate that impaired contact between MAM and mitochondria might underlie the pathology of several human neurodegenerative diseases, including Alzheimer's disease. Moreover, MAM has been implicated in modulating glucose homeostasis and insulin resistance, as well as in some viral infections.

Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin.

Niemann-Pick type C (NPC) disease is a lysosomal storage disease in which endocytosed cholesterol becomes sequestered in late endosomes/lysosomes (LEs/Ls) because of mutations in either the NPC1 or NPC2 gene. Mutations in either of these genes can lead to impaired functions of the NPC1 or NPC2 proteins and progressive neurodegeneration as well as liver and lung disease. NPC1 is a polytopic protein of the LE/L limiting membrane, whereas NPC2 is a soluble protein in the LE/L lumen. These two proteins act in tandem and promote the export of cholesterol from LEs/Ls. Consequently, a defect in either NPC1 or NPC2 causes cholesterol accumulation in LEs/Ls. In this review, we summarize the molecular mechanisms leading to NPC disease, particularly in the CNS. Recent exciting data on the mechanism by which the cholesterol-sequestering agent cyclodextrin can bypass the functions of NPC1 and NPC2 in the LEs/Ls, and mobilize cholesterol from LEs/Ls, will be highlighted. Moreover, the possible use of cyclodextrin as a valuable therapeutic agent for treatment of NPC patients will be considered.

Phosphatidylethanolamine deficiency in Mammalian mitochondria impairs oxidative phosphorylation and alters mitochondrial morphology.

Mitochondrial dysfunction is implicated in neurodegenerative, cardiovascular, and metabolic disorders, but the role of phospholipids, particularly the nonbilayer-forming lipid phosphatidylethanolamine (PE), in mitochondrial function is poorly understood. Elimination of mitochondrial PE (mtPE) synthesis via phosphatidylserine decarboxylase in mice profoundly alters mitochondrial morphology and is embryonic lethal (Steenbergen, R., Nanowski, T. S., Beigneux, A., Kulinski, A., Young, S. G., and Vance, J. E. (2005) J. Biol. Chem. 280, 40032-40040). We now report that moderate <30% depletion of mtPE alters mitochondrial morphology and function and impairs cell growth. Acute reduction of mtPE by RNAi silencing of phosphatidylserine decarboxylase and chronic reduction of mtPE in PSB-2 cells that have only 5% of normal phosphatidylserine synthesis decreased respiratory capacity, ATP production, and activities of electron transport chain complexes (C) I and CIV but not CV. Blue native-PAGE analysis revealed defects in the organization of CI and CIV into supercomplexes in PE-deficient mitochondria, correlated with reduced amounts of CI and CIV proteins. Thus, mtPE deficiency impairs formation and/or membrane integration of respiratory supercomplexes. Despite normal or increased levels of mitochondrial fusion proteins in mtPE-deficient cells, and no reduction in mitochondrial membrane potential, mitochondria were extensively fragmented, and mitochondrial ultrastructure was grossly aberrant. In general, chronic reduction of mtPE caused more pronounced mitochondrial defects than did acute mtPE depletion. The functional and morphological changes in PSB-2 cells were largely reversed by normalization of mtPE content by supplementation with lyso-PE, a mtPE precursor. These studies demonstrate that even a modest reduction of mtPE in mammalian cells profoundly alters mitochondrial functions.

Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells.

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Normalization of cholesterol homeostasis by 2-hydroxypropyl-ß-cyclodextrin in neurons and glia from Niemann-Pick C1 (NPC1)-deficient mice.

Niemann-Pick C (NPC) disease is an inherited, progressive neurodegenerative disorder caused by mutations in the NPC1 or NPC2 gene that result in an accumulation of unesterified cholesterol in late endosomes/lysosomes (LE/L) and impaired export of cholesterol from LE/L to the endoplasmic reticulum (ER). Recent studies demonstrate that administration of cyclodextrin (CD) to Npc1(-/-) mice eliminates cholesterol sequestration in LE/L of many tissues, including the brain, delays neurodegeneration, and increases lifespan of the mice. We have now investigated cholesterol homeostasis in NPC1-deficient cells of the brain in response to CD. Primary cultures of neurons and glial cells from Npc1(-/-) mice were incubated for 24 h with 0.1 to 10 mm CD after which survival and cholesterol homeostasis were monitored. Although 10 mm CD was profoundly neurotoxic, and altered astrocyte morphology, 0.1 and 1 mm CD were not toxic but effectively mobilized stored cholesterol from the LE/L as indicated by filipin staining. However, 0.1 and 1 mm CD altered cholesterol homeostasis in opposite directions. The data suggest that 0.1 mm CD releases cholesterol trapped in LE/L of neurons and astrocytes and increases cholesterol availability at the ER, whereas 1 mm CD primarily extracts cholesterol from the plasma membrane and reduces ER cholesterol. These studies in Npc1(-/-) neurons and astrocytes establish a dose of CD (0.1 mm) that would likely be beneficial in NPC disease. The findings are timely because treatment of NPC disease patients with CD is currently being initiated.

A potential neuroprotective role of apolipoprotein E-containing lipoproteins through low density lipoprotein receptor-related protein 1 in normal tension glaucoma.

Glaucoma is an optic neuropathy and the second major cause of blindness worldwide next to cataracts. The protection from retinal ganglion cell (RGC) loss, one of the main characteristics of glaucoma, would be a straightforward treatment for this disorder. However, the clinical application of neuroprotection has not, so far, been successful. Here, we report that apolipoprotein E-containing lipoproteins (E-LPs) protect primary cultured RGCs from Ca(2+)-dependent, and mitochondrion-mediated, apoptosis induced by glutamate. Binding of E-LPs to the low density lipoprotein receptor-related protein 1 recruited the N-methyl-d-aspartate receptor, blocked intracellular Ca(2+) elevation, and inactivated glycogen synthase kinase 3ß, thereby inhibiting apoptosis. When compared with contralateral eyes treated with phosphate-buffered saline, intravitreal administration of E-LPs protected against RGC loss in glutamate aspartate transporter-deficient mice, a model of normal tension glaucoma that causes glaucomatous optic neuropathy without elevation of intraocular pressure. Although the presence of α2-macroglobulin, another ligand of the low density lipoprotein receptor-related protein 1, interfered with the neuroprotective effect of E-LPs against glutamate-induced neurotoxicity, the addition of E-LPs overcame the inhibitory effect of α2-macroglobulin. These findings may provide a potential therapeutic strategy for normal tension glaucoma by an LRP1-mediated pathway.

Phosphatidylcholine biosynthesis and lipoprotein metabolism.

Phosphatidylcholine (PC) is the major phospholipid component of all plasma lipoprotein classes. PC is the only phospholipid which is currently known to be required for lipoprotein assembly and secretion. Impaired hepatic PC biosynthesis significantly reduces the levels of circulating very low density lipoproteins (VLDLs) and high density lipoproteins (HDLs). The reduction in plasma VLDLs is due in part to impaired hepatic secretion of VLDLs. Less PC within the hepatic secretory pathway results in nascent VLDL particles with reduced levels of PC. These particles are recognized as being defective and are degraded within the secretory system by an incompletely defined process that occurs in a post-endoplasmic reticulum compartment, consistent with degradation directed by the low-density lipoprotein receptor and/or autophagy. Moreover, VLDL particles are taken up more readily from the circulation when the PC content of the VLDLs is reduced, likely due to a preference of cell surface receptors and/or enzymes for lipoproteins that contain less PC. Impaired PC biosynthesis also reduces plasma HDLs by inhibiting hepatic HDL formation and by increasing HDL uptake from the circulation. These effects are mediated by elevated expression of ATP-binding cassette transporter A1 and hepatic scavenger receptor class B type 1, respectively. Hepatic PC availability has recently been linked to the progression of liver and heart disease. These findings demonstrate that hepatic PC biosynthesis can regulate the amount of circulating lipoproteins and suggest that hepatic PC biosynthesis may represent an important pharmaceutical target. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

N-Myc and SP regulate phosphatidylserine synthase-1 expression in brain and glial cells.

Phosphatidylserine (PS) is an essential constituent of biological membranes and plays critical roles in apoptosis and cell signaling. Because no information was available on transcriptional mechanisms that regulate PS biosynthesis in mammalian cells, we investigated the regulation of expression of the mouse PS synthase-1 (Pss1) gene. The Pss1 core promoter was characterized in vitro and in vivo through gel shift and chromatin immunoprecipitation assays. Transcription factor-binding sites, such as a GC-box cluster that binds Sp1/Sp3/Sp4 and N-Myc, and a degenerate E-box motif that interacts with Tal1 and E47, were identified. Pss1 transactivation was higher in brain of neonatal mice than in other tissues, consistent with brain being a major site of expression of Pss1 mRNA and PSS1 activity. Enzymatic assays revealed that PSS1 activity is enriched in primary cortical astrocytes compared with primary cortical neurons. Site-directed mutagenesis of binding sites within the Pss1 promoter demonstrated that Sp and N-Myc synergistically activate Pss1 expression in astrocytes. Chromatin immunoprecipitation indicated that Sp1, Sp3, and Sp4 interact with a common DNA binding site on the promoter. Reduction in levels of Sp1, Sp3, or N-Myc proteins by RNA interference decreased promoter activity. In addition, disruption of Sp/DNA binding with mithramycin significantly reduced Pss1 expression and PSS1 enzymatic activity, underscoring the essential contribution of Sp factors in regulating PSS1 activity. These studies provide the first analysis of mechanisms that regulate expression of a mammalian Pss gene in brain.

Niemann-Pick Type C1 deficiency in microglia does not cause neuron death in vitro.

Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder that results in accumulation of cholesterol and other lipids in late endosomes/lysosomes and leads to progressive neurodegeneration and premature death. The mechanism by which lipid accumulation causes neurodegeneration remains unclear. Inappropriate activation of microglia, the resident immune cells of the central nervous system, has been implicated in several neurodegenerative disorders including NPC disease. Immunohistochemical analysis demonstrates that NPC1 deficiency in mouse brains alters microglial morphology and increases the number of microglia. In primary cultures of microglia from Npc1(-/-) mice cholesterol is sequestered intracellularly, as occurs in other NPC-deficient cells. Activated microglia secrete potentially neurotoxic molecules such as tumor necrosis factor-α (TNFα). However, NPC1 deficiency in isolated microglia did not increase TNFα mRNA or TNFα secretion in vitro. In addition, qPCR analysis shows that expression of pro-inflammatory and oxidative stress genes is the same in Npc1(+/+) and Npc1(-/-) microglia, whereas the mRNA encoding the anti-inflammatory cytokine, interleukin-10 in Npc1(-/-) microglia is ~60% lower than in Npc1(+/+) microglia. The survival of cultured neurons was not impaired by NPC1 deficiency, nor was death of Npc1(-/-) and Npc1(+/+) neurons in microglia-neuron co-cultures increased by NPC1 deficiency in microglia. However, a high concentration of Npc1(-/-) microglia appeared to promote neuron survival. Thus, although microglia exhibit an active morphology in NPC1-deficient brains, lack of NPC1 in microglia does not promote neuron death in vitro in microglia-neuron co-cultures, supporting the view that microglial NPC1 deficiency is not the primary cause of neuron death in NPC disease.

Involvement of low-density lipoprotein receptor-related protein and ABCG1 in stimulation of axonal extension by apoE-containing lipoproteins.

Apolipoprotein E (apoE)-containing lipoproteins (LpE) are produced by glial cells in the central nervous system (CNS). When LpE are supplied to distal axons, but not cell bodies, of CNS neurons (retinal ganglion cells) the rate of axonal extension is increased. In this study we have investigated the molecular requirements underlying the stimulatory effect of LpE on axonal extension. We show that enhancement of axonal growth by LpE requires the presence of the low-density lipoprotein receptor-related protein-1 (LRP1) in neurons since RNA silencing of LRP1 in neurons, or antibodies directed against LRP, suppressed the LpE-induced axonal extension. In contrast, an alternative LRP1 ligand, α2-macroglobulin, failed to stimulate axonal extension, suggesting that LpE do not exert their growth-stimulatory effect solely by activation of a LRP1-mediated signaling pathway. In addition, although apoE3-containing LpE enhanced axonal extension, apoE4-containing LpE did not. Over-expression of ABCG1 in rat cortical glial cells resulted in production of LpE that increased the rate of axonal extension to a greater extent than did expression of an inactive, mutant form of ABGC1. Furthermore, reconstituted lipoprotein particles containing apoE3, phosphatidylcholine and sphingomyelin, but not cholesterol, stimulated axonal extension, suggesting that sphingomyelin, but not cholesterol, is involved in the stimulatory effect of LpE. These observations demonstrate that LpE and LRP1 promote axonal extension, and suggest that lipids exported to LpE by ABCG1 are important for the enhancement of axonal extension mediated by LpE.

Involvement of CTP:phosphocholine cytidylyltransferase-ß2 in axonal phosphatidylcholine synthesis and branching of neurons.

In the brain, phosphatidylcholine (PC) is synthesized by the CDP-choline pathway in which the rate-limiting step is catalyzed by two isoforms of CTP:phosphocholine cytidylyltransferase (CT): CTα and CTß2. In mice, CTß2 mRNA is more highly expressed in the brain than in other tissues, and several observations suggest that CTß2 plays an important role in the nervous system. We, therefore, investigated the importance of CTß2 for PC synthesis as well as for axon formation, growth and branching of primary sympathetic neurons. We show that in cultured primary neurons nerve growth factor increases the amount of CTß2, but not CTα, mRNA and protein. The brains of mice lacking CTß2 had normal PC content despite having 35% lower CT activity than wild-type brains. CTß2 mRNA and protein are abundant in distal axons of mouse sympathetic neurons whereas CTα mRNA and protein were not detected. Moreover, CTß2 deficiency in distal axons reduced the incorporation of [(3)H]choline into PC by 95% whereas PC synthesis in cell bodies/proximal axons was unaltered. These data suggest that CTß2 is the major CT isoform involved in PC synthesis in axons. Axons of CTß2-deficient sympathetic neurons contained 32% fewer branch points than did wild-type neurons although the number of axons/neuron and the rate of axon extension were the same as in wild-type neurons. We conclude that in distal axons of primary sympathetic neurons CTß2 is a major contributor to PC synthesis and promotes axon branching, whereas CTα appears to be the major CT isoform involved in PC synthesis in cell bodies/proximal axons.