Galectin-1 Modulates the Fusogenic Activity of Placental Endogenous Retroviral Envelopes.

Syncytin-1 and -2 are glycoproteins encoded by human endogenous retrovirus (hERV) that, through their fusogenic properties, are needed for the formation of the placental syncytiotrophoblast. Previous studies suggested that these proteins, in addition to the EnvP(b) envelope protein, are also involved in other cell fusion events. Since galectin-1 is a ß-galactoside-binding protein associated with cytotrophoblast fusion during placental development, we previously tested its effect on Syncytin-mediated cell fusion and showed that this protein differently modulates the fusogenic potential of Syncytin-1 and -2. Herein, we were interested in comparing the impact of galectin-1 on hERV envelope proteins in different cellular contexts. Using a syncytium assay, we first demonstrated that galectin-1 increased the fusion of Syncytin-2- and EnvP(b)-expressing cells. We then tested the infectivity of Syncytin-1 and -2 vs. VSV-G-pseudotyped viruses toward Cos-7 and various human cell lines. In the presence of galectin-1, infection of Syncytin-2-pseudotyped viruses augmented for all cell lines. In contrast, the impact of galectin-1 on the infectivity of Syncytin-1-pseudotyped viruses varied, being cell- and dose-dependent. In this study, we report the functional associations between three hERV envelope proteins and galectin-1, which should provide information on the fusogenic activity of these proteins in the placenta and other biological and pathological processes.

Apolipoprotein D in Oxidative Stress and Inflammation.

Apolipoprotein D (ApoD) is lipocalin able to bind hydrophobic ligands. The APOD gene is upregulated in a number of pathologies, including Alzheimer's disease, Parkinson's disease, cancer, and hypothyroidism. Upregulation of ApoD is linked to decreased oxidative stress and inflammation in several models, including humans, mice, Drosophila melanogaster and plants. Studies suggest that the mechanism through which ApoD modulates oxidative stress and regulate inflammation is via its capacity to bind arachidonic acid (ARA). This polyunsaturated omega-6 fatty acid can be metabolised to generate large variety of pro-inflammatory mediators. ApoD serves as a sequester, blocking and/or altering arachidonic metabolism. In recent studies of diet-induced obesity, ApoD has been shown to modulate lipid mediators derived from ARA, but also from eicosapentaenoic acid and docosahexaenoic acid in an anti-inflammatory way. High levels of ApoD have also been linked to better metabolic health and inflammatory state in the round ligament of morbidly obese women. Since ApoD expression is upregulated in numerous diseases, it might serve as a therapeutic agent against pathologies aggravated by OS and inflammation such as many obesity comorbidities. This review will present the most recent findings underlying the central role of ApoD in the modulation of both OS and inflammation.

A newly identified interaction between nucleolar NPM1/B23 and the HTLV-I basic leucine zipper factor in HTLV-1 infected cells.

Human T-cell leukemia virus type 1 is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia-lymphoma (ATL). The HTLV-1 basic leucine zipper factor (HBZ) has been associated to the cancer-inducing properties of this virus, although the exact mechanism is unknown. In this study, we identified nucleophosmin (NPM1/B23) as a new interaction partner of HBZ. We show that sHBZ and the less abundant uHBZ isoform interact with nucleolar NPM1/B23 in infected cells and HTLV-1 positive patient cells, unlike equivalent antisense proteins of related non-leukemogenic HTLV-2, -3 and-4 viruses. We further demonstrate that sHBZ association to NPM1/B23 is sensitive to RNase. Interestingly, sHBZ was shown to interact with its own RNA. Through siRNA and overexpression experiments, we further provide evidence that NPM1/B23 acts negatively on viral gene expression with potential impact on cell transformation. Our results hence provide a new insight over HBZ-binding partners in relation to cellular localization and potential function on cell proliferation and should lead to a better understanding of the link between HBZ and ATL development.

Cerebral Apolipoprotein D Exits the Brain and Accumulates in Peripheral Tissues.

Apolipoprotein D (ApoD) is a secreted lipocalin associated with neuroprotection and lipid metabolism. In rodent, the bulk of its expression occurs in the central nervous system. Despite this, ApoD has profound effects in peripheral tissues, indicating that neural ApoD may reach peripheral organs. We endeavor to determine if cerebral ApoD can reach the circulation and accumulate in peripheral tissues. Three hours was necessary for over 40% of all the radiolabeled human ApoD (hApoD), injected bilaterally, to exit the central nervous system (CNS). Once in circulation, hApoD accumulates mostly in the kidneys/urine, liver, and muscles. Accumulation specificity of hApoD in these tissues was strongly correlated with the expression of lowly glycosylated basigin (BSG, CD147). hApoD was observed to pass through bEnd.3 blood brain barrier endothelial cells monolayers. However, cyclophilin A did not impact hApoD internalization rates in bEnd.3, indicating that ApoD exit from the brain is either independent of BSG or relies on additional cell types. Overall, our data showed that ApoD can quickly and efficiently exit the CNS and reach the liver and kidneys/urine, organs linked to the recycling and excretion of lipids and toxins. This indicated that cerebral overexpression during neurodegenerative episodes may serve to evacuate neurotoxic ApoD ligands from the CNS.

Apolipoprotein D.

ApoD is a 25 to 30 kDa glycosylated protein, member of the lipocalin superfamily. As a transporter of several small hydrophobic molecules, its known biological functions are mostly associated to lipid metabolism and neuroprotection. ApoD is a multi-ligand, multi-function protein that is involved lipid trafficking, food intake, inflammation, antioxidative response and development and in different types of cancers. An important aspect of ApoD's role in lipid metabolism appears to involve the transport of arachidonic acid, and the modulation of eicosanoid production and delivery in metabolic tissues. ApoD expression in metabolic tissues has been associated positively and negatively with insulin sensitivity and glucose homeostasis in a tissue dependent manner. ApoD levels rise considerably in association with aging and neuropathologies such as Alzheimer's disease, stroke, meningoencephalitis, moto-neuron disease, multiple sclerosis, schizophrenia and Parkinson's disease. ApoD is also modulated in several animal models of nervous system injury/pathology.

SCD1 activity promotes cell migration via a PLD-mTOR pathway in the MDA-MB-231 triple-negative breast cancer cell line.

BACKGROUND: Breast cancer is the most common cancer in women. Despite high survival rates in Western countries, treatments are less effective in metastatic cases and triple-negative breast cancer (TNBC) patient survival is the shortest across breast cancer subtypes. High expression levels of stearoyl-CoA desaturase-1 (SCD1) have been reported in breast cancer. The SCD1 enzyme catalyzes the formation of oleic acid (OA), a lipid stimulating the migration of metastatic breast cancer cells. Phospholipase activity is also implicated in breast cancer metastasis, notably phospholipase D (PLD). METHODS: Kaplan-Meier survival plots generated from gene expression databases were used to analyze the involvement of SCD1 and PLD in several cancer subtypes. SCD1 enzymatic activity was modulated with a pharmaceutical inhibitor or by OA treatment (to mimic SCD1 over-activity) in three breast cancer cell lines: TNBC-derived MDA-MB-231 cells as well as non-TNBC MCF-7 and T47D cells. Cell morphology and migration properties were characterized by various complementary methods. RESULTS: Our survival analyses suggest that SCD1 and PLD2 expression in the primary tumor are both associated to metastasis-related morbid outcomes in breast cancer patients. We show that modulation of SCD1 activity is associated with the modification of TNBC cell migration properties, including changes in speed, direction and cell morphology. Cell migration properties are regulated by SCD1 activity through a PLD-mTOR/p70S6K signaling pathway. These effects are not observed in non-TNBC cell lines. CONCLUSION: Our results establish a key role for the lipid desaturase SCD1 and delineate an OA-PLD-mTOR/p70S6K signaling pathway in TNBC-derived MDA-MB-231 cell migration.

Endogenous retrovirus-encoded Syncytin-2 contributes to exosome-mediated immunosuppression of T cells†.

Modulation of the activation status of immune cell populations during pregnancy depends on placental villous cytotrophoblast (VCT) cells and the syncytiotrophoblast (STB). Failure in the establishment of this immunoregulatory function leads to pregnancy complications. Our laboratory has been studying Syncytin-2 (Syn-2), an endogenous retroviral protein expressed in placenta and on the surface of placental exosomes. This protein plays an important role not only in STB formation through its fusogenic properties, but also through its immunosuppressive domain (ISD). Considering that Syn-2 expression is importantly reduced in preeclamptic placentas, we were interested in addressing its possible immunoregulatory effects on T cells. Activated Jurkat T cells and peripheral blood mononuclear cells (PBMCs) were treated with monomeric or dimerized version of a control or a Syn-2 ISD peptide. Change in phosphorylation levels of ERK1/2 MAP kinases was selectively noted in Jurkat cells treated with the dimerized ISD peptide. Upon incubation with the dimerized Syn-2 ISD peptide, significant reduction in Th1 cytokine production was further demonstrated by ELISA and Human Th1/Th2 Panel Multi-Analyte Flow Assay. To determine if exosome-associated Syn-2 could also be immunosuppressive placental exosomes were incubated with activated Jurkat and PBMCs. Quantification of Th1 cytokines in the supernatants revealed severe reduction in T cell activation. Interestingly, exosomes from Syn-2-silenced VCT incubated with PBMCs were less suppressive when compared with exosome derived from VCT transfected with control small interfering RNA (siRNA). Our results suggest that Syn-2 is an important immune regulator both locally and systemically, via its association with placental exosomes.

Histone deacetylase 1 interacts with HIV-1 Integrase and modulates viral replication.

BACKGROUND: HIV-1 hijacks the cellular machinery for its own replication through protein-protein interactions between viral and host cell factors. One strategy against HIV-1 infection is thus to target these key protein complexes. As the integration of reverse transcribed viral cDNA into a host cell chromosome is an essential step in the HIV-1 life cycle, catalyzed by the viral integrase and other important host factors, we aimed at identifying new integrase binding partners through a novel approach. METHODS: A LTR-derived biotinylated DNA fragment complexed with the integrase on magnetic beads was incubated with extracts from integrase-expressing 293 T cells. Liquid chromatography-mass spectrometry/mass spectrometry and co-immunoprecipitation/pull-down experiments were used for the identification of binding partners. Transfections of histone deacetylase 1 (HDAC1) expression vectors and/or specific siRNA were conducted in HeLa-CD4 and 293 T cells followed by infection with fully infectious NL4-3 and luciferase-expressing pseudotyped viruses or by proviral DNA transfection. Fully infectious and pseudotyped viruses produced from HDAC1-silenced 293 T cells were tested for their infectivity toward HeLa-CD4 cells, T cell lines and primary CD4+ T cells. Late RT species and integrated viral DNA were quantified by qPCR and infectivity was measured by luciferase activity and p24 ELISA assay. Results were analyzed by the Student's t-test. RESULTS: Using our integrase-LTR bait approach, we successfully identified new potential integrase-binding partners, including HDAC1. We further confirmed that HDAC1 interacted with the HIV-1 integrase in co-immunoprecipitation and pull-down experiments. HDAC1 knockdown in infected HeLa cells was shown to interfere with an early preintegration step of the HIV-1 replication cycle, which possibly involves reverse transcription. We also observed that, while HDAC1 overexpression inhibited HIV-1 expression after integration, HDAC1 knockdown had no effect on this step. In virus producer cells, HDAC1 knockdown had a limited impact on virus infectivity in either cell lines or primary CD4+ T cells. CONCLUSIONS: Our results show that HDAC1 interacts with the HIV-1 integrase and affects virus replication before and after integration. Overall, HDAC1 appears to facilitate HIV-1 replication with a major effect on a preintegration step, which likely occurs at the reverse transcription step.

Galectin-1 interacts with the human endogenous retroviral envelope protein syncytin-2 and potentiates trophoblast fusion in humans.

Syncytin (Syn)-2 is an important fusogenic protein that contributes to the formation of the placental syncytiotrophoblast. Galectin (Gal)-1, a soluble lectin, is also involved in trophoblast cell fusion and modulates the interaction of certain retroviral envelopes with their cellular receptor. This study aimed to investigate the association between Syn-2 and Gal-1 during human trophoblast cell fusion. This association was evaluated in vitro on primary villous cytotrophoblasts (vCTBs) and cell lines using recombinant Gal-1 and Syn-2-pseudotyped viruses. Using lactose, a Gal antagonist, and Gal-1-specific small interfering RNA (siRNA) transfections, we confirmed the implication of Gal-1 in vCTBs and BeWo cell fusion, although RT-PCR and ELISA analyses suggested that Gal-1 alone did not induce syncytialization. Infection assays showed a specific and significant effect of Gal-1 on the infectivity of Syn-2-pseudotyped viruses that depended on the expression of major facilitator superfamily domain-containing 2A (MFSD2a). Moreover, Gal-3, another placental Gal, did not modulate the infectivity of Syn-2-positive viruses, strengthening the specific association between Gal-1 and Syn-2. Interestingly, Gal-1 significantly reduced the infectivity of Syn-1-pseudotyped viruses, suggesting the opposite effects of Gal-1 on Syn-1 and -2. Finally, coimmunoprecipitation experiments showed a glycan-dependent interaction between Syn-2-bearing virions and Gal-1. We conclude that Gal-1 specifically interacts with Syn-2 and possibly regulates Syn-2/MFSD2a interaction during syncytialization of trophoblastic cells.-Toudic, C., Vargas, A., Xiao, Y., St-Pierre, G., Bannert, N., Lafond, J., Rassart, É., Sato, S., Barbeau, B. Galectin-1 interacts with the human endogenous retroviral envelope protein syncytin-2 and potentiates trophoblast fusion in humans.

Apolipoprotein D overexpression alters hepatic prostaglandin and omega fatty acid metabolism during the development of a non-inflammatory hepatic steatosis.

Apolipoprotein D (ApoD) is a secreted lipocalin associated with neuroprotection and lipid metabolism. Overexpression of ApoD in mouse neural tissue induces the development of a non-inflammatory hepatic steatosis in 12-month-old transgenic animals. Previous data indicates that accumulation of arachidonic acid, ApoD's preferential ligand, and overactivation of PPARγ are likely the driving forces in the development of the pathology. However, the lack of inflammation under those conditions is surprising. Hence, we further investigated the apparent repression of inflammation during hepatic steatosis development in aging transgenic animals. The earliest modulation of lipid metabolism and inflammation occurred at 6 months with a transient overexpression of L-PGDS and concomitant overproduction of 15d-PGJ2, a PPARγ agonist. Hepatic lipid accumulation was detectable as soon as 9 months. Inflammatory polarization balance varied in time, with a robust anti-inflammatory profile at 6 months coinciding with 15d-PGJ2 overproduction. Omega-3 and omega-6 fatty acids were preferentially stored in the liver of 12-month-old transgenic mice and resulted in a higher omega-3/omega-6 ratio compared to wild type mice of the same age. Thus, inflammation seems to be controlled by several mechanisms in the liver of transgenic mice: first by an increase in 15d-PGJ2 production and later by a beneficial omega-3/omega-6 ratio. PPARγ seems to play important roles in these processes. The accumulation of several omega fatty acids species in the transgenic mouse liver suggests that ApoD might bind to a broader range of fatty acids than previously thought.

Blood-based redox-signature and their association to the cognitive scores in MCI and Alzheimer's disease patients.

Oxidative stress plays a pivotal and early role in the pathophysiology of Alzheimer's disease (AD). There is convincing evidence that oxidative alterations in AD and in mild cognitive impairment (MCI) patients are not limited to the brain but are extended to the blood compartment. However, the oxidative pattern in plasma is still inconclusive. Moreover, their potential association with the clinical scores MMSE (Mini-Mental State Examination) and MoCA (Montreal Cognitive Assessment) is poorly investigated. The aim of our study was to establish a pattern of blood-based redox alterations in prodromal AD and their evolution during the progression of the disease. Our results showed a reduction in the total antioxidant capacity (TAC) and an increase of the stress-response proteins apolipoprotein J (ApoJ) and Klotho in MCI subjects. For the first time, we evidenced circulating-proteasome activity. We found that the alteration of the circulating-proteasome activity is associated with the accumulation of oxidized proteins in plasma form early AD. Interestingly, the TAC, the levels of vitamin D and the activity of proteasome were positively associated to the clinical scores MMSE and MoCA. The levels of protein carbonyls and of ApoJ were negatively associated to the MMSE and MoCA scores. The levels of apolipoprotein D (ApoD) were not different between groups. Interestingly, the receiver operating characteristic (ROC) curves analysis indicated that these redox markers provide a fair classification of different groups with high accuracy. Overall, our results strengthen the notion that some specific oxidative markers could be considered as non-invasive blood-based biomarkers for an early MCI diagnosis and AD progression.

HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62.

The existence of the antisense transcript-encoded HIV-1 antisense protein (ASP) was recently reinforced by in silico analyses providing evidence for recent appearance of this gene in the viral genome. Our previous studies led to the detection of ASP in various cell lines by Western blotting, flow cytometry, and confocal microscopy analyses and reported that it induced autophagy, potentially through multimer formation. Here, our goals were to assess autophagy induction by ASP from different clades and to identify the implicated autophagy factors. We first demonstrated that ASP formed multimers, partly through its amino-terminal region and cysteine residues. Removal of this region was further associated with lower induction of autophagy, as assessed by autophagosome formation. ASPs from different clades (A, B, C, D, and G) were tested next and were detected in monomeric and multimeric forms at various levels, and all induced autophagy (clade A ASP was less efficient), as determined by LC3-II and p62 (SQSTM1) levels. Furthermore, CRISPR-based knockout of ATG5, ATG7, and p62 genes led to increased ASP levels. Confocal microscopy analyses showed that ASP colocalized with p62 and LC3-II in autophagosome-like structures. Coimmunoprecipitation experiments further demonstrated that p62 associated with ASP through its PB1 domain. Interestingly, immunoprecipitation experiments supported the idea that ASP is ubiquitinated and that ubiquitination was modulating its stability. We are thus suggesting that ASP induces autophagy through p62 interaction and that its abundance is controlled by autophagy, in which ubiquitin plays an important role. Understanding the mechanisms underlying ASP degradation is essential to better assess its function.IMPORTANCE In the present study, we provide the first evidence that a new HIV-1 protein termed ASP derived from different clades acts similarly in inducing autophagy, an important cellular process implicated in the degradation of excess or defective cellular material. We have gained further knowledge on the mechanism mediating the activation of autophagy. Our studies have important ramifications in the understanding of viral replication and the pathogenesis associated with HIV-1 in infected individuals. Indeed, autophagy is implicated in antigen presentation during immune response and could thus be rendered inefficient in infected cells, such as dendritic cells. Furthermore, a possible link with HIV-1-associated neurological disorder (HAND) might also be a possible association with the capacity of ASP to induce autophagy. Our studies hence demonstrate the importance in conducting further studies on this protein as it could represent a new interesting target for antiretroviral therapies and vaccine design.

High ApoD protein level in the round ligament fat depot of severely obese women is associated with an improved inflammatory profile.

PURPOSE: Apolipoprotein D (ApoD) is a lipocalin participating in lipid transport. It binds to a variety of ligands, with a higher affinity for arachidonic acid, and is thought to have a diverse array of functions. We investigated a potential role for ApoD in insulin sensitivity, inflammation, and thrombosis-processes related to lipid metabolism-in severely obese women. METHODS: We measured ApoD expression in a cohort of 44 severely obese women including dysmetabolic and non-dysmetabolic patients. Physical and metabolic characteristics of these women were determined from anthropometric measurements and blood samples. ApoD was quantified at the mRNA and protein levels in samples from three intra-abdominal adipose tissues (AT): omental, mesenteric and round ligament (RL). RESULTS: ApoD protein levels were highly variable between AT of the same individual. High ApoD protein levels, particularly in the RL depot, were linked to lower plasma insulin levels (-40%, p = 0.015) and insulin resistance (-47%, p = 0.022), and increased insulin sensitivity (+10%, p = 0.008). Lower circulating pro-inflammatory PAI-1 (-39%, p = 0.001), and TNF-α (-19%, p = 0.030) levels were also correlated to high ApoD protein in the RL AT. CONCLUSIONS: ApoD variability between AT was consistent with different accumulation efficiencies and/or metabolic functions according to the anatomic location of fat depots. Most statistically significant correlations implicated ApoD protein levels, in agreement with protein accumulation in target tissues. These correlations associated higher ApoD levels in fat depots with improved metabolic health in severely obese women.

[Antisense proteins of HTLV viruses]. / Les protéines antisens des virus HTLV.

There are four human T-lymphotropic viruses (HTLV-1, 2, 3, 4) that have emerged from the transmission of simian viruses. HTLV-1 was the first retrovirus to be shown to be responsible for a human pathology. The expression of retroviral genes depends mostly on their 5'LTR, but it was revealed that HTLV have a promoter in their 3'LTR, capable of transcription from the antisense strand of their genome. These transcripts can be translated into proteins named HBZ, APH-2, APH-3 and APH-4. Antisense transcription in HTLV-1 and its encoded protein HBZ have been thoroughly studied and it has been suggested that HBZ plays an important role in viral replication and the development of ATL. Very few studies have been conducted on antisense transcription from the three other viruses, although it is likely that these genes are also implicated in viral replication.

Implication of Different HIV-1 Genes in the Modulation of Autophagy.

Autophagy is a complex cellular degradation pathway, which plays important roles in the regulation of several developmental processes, cellular stress responses, and immune responses induced by pathogens. A number of studies have previously demonstrated that HIV-1 was capable of altering the regulation of autophagy and that this biological process could be induced in uninfected and infected cells. Furthermore, previous reports have indicated that the involvement of HIV-1 in autophagy regulation is a complex phenomenon and that different viral proteins are contributing in its modulation upon viral infection. Herein, we review the recent literature over the complex crosstalk of the autophagy pathway and HIV-1, with a particular focus on HIV-1 viral proteins, which have been shown to modulate autophagy.

Apolipoprotein D Overexpression Protects Against Kainate-Induced Neurotoxicity in Mice.

Excitotoxicity due to the excessive activation of glutamatergic receptors leads to neuronal dysfunction and death. Excitotoxicity has been implicated in the pathogenesis of a myriad of neurodegenerative diseases with distinct etiologies such as Alzheimer's and Parkinson's. Numerous studies link apolipoprotein D (apoD), a secreted glycoprotein highly expressed in the central nervous system (CNS), to maintain and protect neurons in various mouse models of acute stress and neurodegeneration. Here, we used a mouse model overexpressing human apoD in neurons (H-apoD Tg) to test the neuroprotective effects of apoD in the kainic acid (KA)-lesioned hippocampus. Our results show that apoD overexpression in H-apoD Tg mice induces an increased resistance to KA-induced seizures, significantly attenuates inflammatory responses and confers protection against KA-induced cell apoptosis in the hippocampus. The apoD-mediated protection against KA-induced toxicity is imputable in part to increased plasma membrane Ca2+ ATPase type 2 expression (1.7-fold), decreased N-methyl-D-aspartate receptor (NMDAR) subunit NR2B levels (30 %) and lipid metabolism alterations. Indeed, we demonstrate that apoD can attenuate intracellular cholesterol content in primary hippocampal neurons and in brain of H-apoD Tg mice. In addition, apoD can be internalised by neurons and this internalisation is accentuated in ageing and injury conditions. Our results provide additional mechanistic information on the apoD-mediated neuroprotection in neurodegenerative conditions.

Apolipoprotein D deficiency is associated to high bone turnover, low bone mass and impaired osteoblastic function in aged female mice.

BACKGROUND: Apolipoprotein D (ApoD) is a member of the lipocalin family known to transport small hydrophobic ligands. A major site of ApoD expression in mice is the central nervous system where evidence suggests that it plays a protective role. Gene expression of ApoD was reported in bone-forming osteoblasts but its impact on bone metabolism remains undocumented. METHODS: We compared basic bone parameters of ApoD(-/-) (null) and transgenic (tg) mice to wild-type (wt) littermates through microCT and histochemistry, as well as ApoD expression and secretion in osteoblasts under various culture conditions through real-time PCR and immunoblotting. RESULTS: ApoD-null females displayed progressive bone loss with aging, resulting in a 50% reduction in trabecular bone volume and a 23% reduction in cortical bone volume by 9months of age. Only cortical bone volume was significantly reduced in ApoD-null males by an average of 24%. Histochemistry indicated significantly higher osteoblast surface and number of osteoclasts in femora from ApoD-null females. ApoD gene expression was confirmed in primary cultures of bone marrow mesenchymal cells (MSC), with higher expression levels in MSC from females compared to males. ApoD-null MSC exhibited impaired proliferation and differentiation potentials. Moreover, exogenous ApoD partially rescued the osteogenic potential of null MSC, which were shown to readily uptake the protein from media. ApoD expression was upregulated under low proliferation conditions, by contact inhibition and osteoblastic differentiation in MC3T3-E1 osteoblast-like cells. CONCLUSION: Our results indicate that ApoD influences bone metabolism in mice in a gender-specific manner, potentially through an auto-/paracrine pathway.

Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPARγ and Fatty Acid Uptake.

Transgenic mice (Tg) overexpressing human apolipoprotein D (H-apoD) in the brain are resistant to neurodegeneration. Despite the use of a neuron-specific promoter to generate the Tg mice, they expressed significant levels of H-apoD in both plasma and liver and they slowly develop hepatic steatosis and insulin resistance. We show here that hepatic PPARγ expression in Tg mice is increased by 2-fold compared to wild type (WT) mice. Consequently, PPARγ target genes Plin2 and Cide A/C are overexpressed, leading to increased lipid droplets formation. Expression of the fatty acid transporter CD36, another PPARgamma target, is also increased in Tg mice associated with elevated fatty acid uptake as measured in primary hepatocytes. Elevated expression of AMPK in the liver of Tg leads to phosphorylation of acetyl CoA carboxylase, indicating a decreased activity of the enzyme. Fatty acid synthase expression is also induced but the hepatic lipogenesis measured in vivo is not significantly different between WT and Tg mice. In addition, expression of carnitine palmitoyl transferase 1, the rate-limiting enzyme of beta-oxidation, is slightly upregulated. Finally, we show that overexpressing H-apoD in HepG2 cells in presence of arachidonic acid (AA), the main apoD ligand, increases the transcriptional activity of PPARγ. Supporting the role of apoD in AA transport, we observed enrichment in hepatic AA and a decrease in plasmatic AA concentration. Taken together, our results demonstrate that the hepatic steatosis observed in apoD Tg mice is a consequence of increased PPARγ transcriptional activity by AA leading to increased fatty acid uptake by the liver.

Apolipoprotein D Internalization Is a Basigin-dependent Mechanism.

Apolipoprotein D (apoD), a member of the lipocalin family, is a 29-kDa secreted glycoprotein that binds and transports small lipophilic molecules. Expressed in several tissues, apoD is up-regulated under different stress stimuli and in a variety of pathologies. Numerous studies have revealed that overexpression of apoD led to neuroprotection in various mouse models of acute stress and neurodegeneration. This multifunctional protein is internalized in several cells types, but the specific internalization mechanism remains unknown. In this study, we demonstrate that the internalization of apoD involves a specific cell surface receptor in 293T cells, identified as the transmembrane glycoprotein basigin (BSG, CD147); more particularly, its low glycosylated form. Our results show that internalized apoD colocalizes with BSG into vesicular compartments. Down-regulation of BSG disrupted the internalization of apoD in cells. In contrast, overexpression of basigin in SH-5YSY cells, which poorly express BSG, restored the uptake of apoD. Cyclophilin A, a known ligand of BSG, competitively reduced apoD internalization, confirming that BSG is a key player in the apoD internalization process. In summary, our results demonstrate that basigin is very likely the apoD receptor and provide additional clues on the mechanisms involved in apoD-mediated functions, including neuroprotection.

Apolipoprotein D modulates amyloid pathology in APP/PS1 Alzheimer's disease mice.

Apolipoprotein D (apoD) is expressed in the brain and levels are increased in affected brain regions in Alzheimer's disease (AD). The role that apoD may play in regulating AD pathology has not been addressed. Here, we crossed both apoD-null mice and Thy-1 human apoD transgenic mice with APP-PS1 amyloidogenic AD mice. Loss of apoD resulted in a nearly 2-fold increase in hippocampal amyloid plaque load, as assessed by immunohistochemical staining. Conversely, transgenic expression of neuronal apoD reduced hippocampal plaque load by approximately 35%. This latter finding was associated with a 60% decrease in amyloid ß 1-40 peptide levels, and a 34% decrease in insoluble amyloid ß 1-42 peptide. Assessment of ß-site amyloid precursor protein cleaving enzyme-1 (BACE1) levels and proteolytic products of amyloid precursor protein and neuregulin-1 point toward a possible association of altered BACE1 activity in association with altered apoD levels. In conclusion, the current studies provide clear evidence that apoD regulates amyloid plaque pathology in a mouse model of AD.