Determining the safety of the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-Cembratriene-4,6-diol (4R): A translational study in nonhuman primates.

The tobacco cembranoid known as (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (4R) has been shown to offer neuroprotection against conditions such as brain ischemia, systemic inflammation, Parkinson's disease, and organophosphate toxicity in rodents. Previous safety studies conducted on male and female Sprague Dawley rats revealed no significant side effects following a single injection of 4R at varying concentrations (6, 24, or 98 mg/kg of body weight). This study aimed to assess the potential of 4R for clinical trials in neurotherapy in male nonhuman primates. Ten macaques (Macacca mulatta) were randomly separated into two groups of 5 and then intravenously injected with 4R or vehicle for 11 consecutive days at a dose of 1.4 mg/kg. Throughout the study, we monitored brain activity by electroencephalogram, somatosensory evoked potentials, and transcranial motor evoked potentials on days 0, 4, 8, and 12 and found no significant changes. The spontaneous behavior of the primates remained unaffected by the treatment. Minor hematological and blood composition variations were also detected in the experimental animals but lacked clinical significance. In conclusion, our results reinforce the notion that 4R is non-toxic in nonhuman primates under the conditions of this study.

In Vivo Evaluation of the Acute Systemic Toxicity of (1S,2E,4R,6R,7E,11E)-Cembratriene-4,6-diol (4R) in Sprague Dawley Rats.

The tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (4R) interacts with nicotinic acetylcholine receptors, which results in neuroprotection against organophosphate toxicity, brain ischemia, and Parkinson's disease. The present study is a continuation of our previous research in which we applied a single dose of 4R 1 h before or 24 h after exposure to diisopropylfluorophosphate (DFP) (analog of the nerve agent sarin). The 4R dose robustly decreased neuroinflammation and neuronal death at both timepoints. Here, we investigated the toxicity of a single dose of 4R in male and female Sprague Dawley (SD) rats after a subcutaneous (s.c.) injection of 6, 24, or 98 mg/kg. Body weight was not affected by 4R during the 7-day observation period. No histopathologic changes in the organs were attributed to 4R. Minor hematological and blood composition variations were detected on Day 3 in the mid- and the high-dose males, but these were resolved by Day 8. At the area of the s.c. injection site, alopecia and dry skin were detected in both the 4R-treated males and females and in the female controls.

Two Pathways for the Activity-Dependent Growth and Differentiation of Synaptic Boutons in Drosophila.

Synapse formation can be promoted by intense activity. At the Drosophila larval neuromuscular junction (NMJ), new synaptic boutons can grow acutely in response to patterned stimulation. We combined confocal imaging with electron microscopy and tomography to investigate the initial stages of growth and differentiation of new presynaptic boutons at the Drosophila NMJ. We found that the new boutons can form rapidly in intact larva in response to intense crawling activity, and we observed two different patterns of bouton formation and maturation. The first pathway involves the growth of filopodia followed by a formation of boutons that are initially devoid of synaptic vesicles (SVs) but filled with filamentous matrix. The second pathway involves rapid budding of synaptic boutons packed with SVs, and these more mature boutons are sometimes capable of exocytosis/endocytosis. We demonstrated that intense activity predominantly promotes the second pathway, i.e., budding of more mature boutons filled with SVs. We also showed that this pathway depends on synapsin (Syn), a neuronal protein which reversibly associates with SVs and mediates their clustering via a protein kinase A (PKA)-dependent mechanism. Finally, we took advantage of the temperature-sensitive mutant sei to demonstrate that seizure activity can promote very rapid budding of new boutons filled with SVs, and this process occurs at scale of minutes. Altogether, these results demonstrate that intense activity acutely and selectively promotes rapid budding of new relatively mature presynaptic boutons filled with SVs, and that this process is regulated via a PKA/Syn-dependent pathway.

Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission.

Many neurons influence their targets through co-release of neuropeptides and small-molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small-molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both stimulated emission depletion super-resolution and transmission electron microscopy. Ptp4E also controls the presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content.This article has an associated First Person interview with the first author of the paper.

FM1-43 Photoconversion and Electron Microscopy Analysis at the Drosophila Neuromuscular Junction.

We developed a protocol for photoconversion of endocytic marker FM1-43 followed by electron microscopy analysis of synaptic boutons at the Drosophila neuromuscular junction. This protocol allows detection of stained synaptic vesicle even when release rates are very low, such as during the spontaneous release mode. The preparations are loaded with the FM1-43 dye, pre-fixed, treated and illuminated to photoconvert the dye, and then processed for conventional electron microscopy. This procedure enables clear identification of stained synaptic vesicles at electron micrographs.

Complexin Mutants Reveal Partial Segregation between Recycling Pathways That Drive Evoked and Spontaneous Neurotransmission.

Synaptic vesicles fuse at morphological specializations in the presynaptic terminal termed active zones (AZs). Vesicle fusion can occur spontaneously or in response to an action potential. Following fusion, vesicles are retrieved and recycled within nerve terminals. It is still unclear whether vesicles that fuse spontaneously or following evoked release share similar recycling mechanisms. Genetic deletion of the SNARE-binding protein complexin dramatically increases spontaneous fusion, with the protein serving as the synaptic vesicle fusion clamp at Drosophila synapses. We examined synaptic vesicle recycling pathways at complexin null neuromuscular junctions, where spontaneous release is dramatically enhanced. We combined loading of the lipophilic dye FM1-43 with photoconversion, electron microscopy, and electrophysiology to monitor evoked and spontaneous recycling vesicle pools. We found that the total number of recycling vesicles was equal to those retrieved through spontaneous and evoked pools, suggesting that retrieval following fusion is partially segregated for spontaneous and evoked release. In addition, the kinetics of FM1-43 destaining and synaptic depression measured in the presence of the vesicle-refilling blocker bafilomycin indicated that spontaneous and evoked recycling pools partially intermix during the release process. Finally, FM1-43 photoconversion combined with electron microscopy analysis indicated that spontaneous recycling preferentially involves synaptic vesicles in the vicinity of AZs, whereas vesicles recycled following evoked release involve a larger intraterminal pool. Together, these results suggest that spontaneous and evoked vesicles use separable recycling pathways and then partially intermix during subsequent rounds of fusion. SIGNIFICANCE STATEMENT: Neurotransmitter release involves fusion of synaptic vesicles with the plasma membrane in response to an action potential, or spontaneously in the absence of stimulation. Upon fusion, vesicles are retrieved and recycled, and it is unclear whether recycling pathways for evoked and spontaneous vesicles are segregated after fusion. We addressed this question by taking advantage of preparations lacking the synaptic protein complexin, which have elevated spontaneous release that enables reliable tracking of the spontaneous recycling pool. Our results suggest that spontaneous and evoked recycling pathways are segregated during the retrieval process but can partially intermix during stimulation.

GRP78 rescues the ABCG5 ABCG8 sterol transporter in db/db mice.

OBJECTIVE: Mice lacking leptin (ob/ob) or its receptor (db/db) are obese, insulin resistant, and have reduced levels of biliary cholesterol due, in part, to reduced levels of hepatic G5G8. Chronic leptin replacement restores G5G8 abundance and increases biliary cholesterol concentrations, but the molecular mechanisms responsible for G5G8 regulation remain unclear. In the current study, we used a series of mouse models to address potential mechanisms for leptin-mediated regulation of G5G8. METHODS AND RESULTS: We acutely replaced leptin in ob/ob mice and deleted hepatic leptin receptors in lean mice. Neither manipulation altered G5G8 abundance or biliary cholesterol. Similarly, hepatic vagotomy had no effect on G5G8. Alternatively, G5G8 may be decreased in ob/ob and db/db mice due to ER dysfunction, the site of G5G8 complex assembly. Overexpression of the ER chaperone GRP78 using an adenoviral vector restores ER function and reduces steatosis in ob/ob mice. Therefore, we determined if AdGRP78 could rescue G5G8 in db/db mice. As in ob/ob mice, AdGRP78 reduced expression of lipogenic genes and plasma triglycerides in the db/db strain. Both G5 and G8 protein levels increased as did total biliary cholesterol, but in the absence of changes in G5 or G8 mRNAs. The increase in G5G8 was associated with increases in a number of proteins, including the ER lectin chaperone, calnexin, a key regulator of G5G8 complex assembly. CONCLUSIONS: Leptin signaling does not directly regulate G5G8 abundance. The loss of G5G8 in mice harboring defects in the leptin axis is likely associated with compromised ER function.

Acceleration of biliary cholesterol secretion restores glycemic control and alleviates hypertriglyceridemia in obese db/db mice.

OBJECTIVE: Recent studies support a role for cholesterol in the development of obesity and nonalcoholic fatty liver disease. Mice lacking the ABCG5 ABCG8 (G5G8) sterol transporter have reduced biliary cholesterol secretion and are more susceptible to steatosis, hepatic insulin resistance, and loss of glycemic control when challenged with a high-fat diet. We hypothesized that accelerating G5G8-mediated biliary cholesterol secretion would correct these phenotypes in obese mice. APPROACH AND RESULTS: Obese (db/db) male and their lean littermates were administered a cocktail of control adenovirus or adenoviral vectors encoding ABCG5 and ABCG8 (AdG5G8). Three days after viral administration, measures of lipid and glucose homeostasis were determined, and tissues were collected for biochemical analyses. AdG5G8 increased biliary cholesterol and fecal sterol elimination. Fasting glucose and triglycerides declined, and glucose tolerance improved in obese mice expressing G5G8 compared with mice receiving control adenovirus. These changes were associated with a reduction in phosphorylated eukaryotic initiation factor 2α and c-Jun N-terminal kinase in liver, suggesting alleviation of endoplasmic reticulum stress. Phosphorylated insulin receptor and protein kinase B were increased, indicating restored hepatic insulin signaling. However, there was no reduction in hepatic triglycerides after the 3-day treatment period. CONCLUSIONS: Accelerating biliary cholesterol secretion restores glycemic control and reduces plasma triglycerides in obese db/db mice.

The ABCG5 ABCG8 sterol transporter opposes the development of fatty liver disease and loss of glycemic control independently of phytosterol accumulation.

ABCG5 and ABCG8 form a complex (G5G8) that opposes the absorption of plant sterols but is also expressed in liver where it promotes the excretion of cholesterol into bile. Hepatic G5G8 is transcriptionally regulated by a number of factors implicated in the development of insulin resistance and nonalcoholic fatty liver disease. Therefore, we hypothesized that G5G8 may influence the development of diet-induced obesity phenotypes independently of its role in opposing phytosterol absorption. G5G8 knock-out (KO) mice and their wild type (WT) littermates were challenged with a plant sterol-free low fat or high fat (HF) diet. Weight gain and the rise in fasting glucose were accelerated in G5G8 KO mice following HF feeding. HF-fed G5G8 KO mice had increased liver weight, hepatic lipids, and plasma alanine aminotransferase compared with WT controls. Consistent with the development of nonalcoholic fatty liver disease, macrophage infiltration, the number of TUNEL-positive cells, and the expression of proinflammatory cytokines were also increased in G5G8 KO mice. Hepatic lipid accumulation was associated with increased peroxisome proliferator activated receptor γ, CD36, and fatty acid uptake. Phosphorylation of eukaryotic translation initiation factor 2α (eiF2α) and expression of activating transcription factor 4 and tribbles 3 were elevated in HF-fed G5G8 KO mice, a pathway that links the unfolded protein response to the development of insulin resistance through inhibition of protein kinase B (Akt) phosphorylation. Phosphorylation of Akt and insulin receptor was reduced, whereas serine phosphorylation of insulin receptor substrate 1 was elevated.

Phytosterols differentially influence ABC transporter expression, cholesterol efflux and inflammatory cytokine secretion in macrophage foam cells.

Phytosterol supplements lower low-density lipoprotein (LDL) cholesterol, but accumulate in vascular lesions of patients and limit the anti-atherosclerotic effects of LDL lowering in apolipoprotein E (Apo E)-deficient mice, suggesting that the cholesterol-lowering benefit of phytosterol supplementation may not be fully realized. Individual phytosterols have cell-type specific effects that may be either beneficial or deleterious with respect to atherosclerosis, but little is known concerning their effects on macrophage function. The effects of phytosterols on ABCA1 and ABCG1 abundance, cholesterol efflux and inflammatory cytokine secretion were determined in cultured macrophage foam cells. Among the commonly consumed phytosterols, stigmasterol increased expression of ABCA1 and ABCG1 and increased efflux of cholesterol to apolipoprotein (Apo) AI and high-density lipoprotein (HDL). Campesterol and sitosterol had no effect on ABCA1 or ABCG1 levels. Sitosterol had no effect on cholesterol efflux to Apo AI or HDL, whereas campesterol had a modest but significant reduction in cholesterol efflux to HDL in THP-1 macrophages. Whereas stigmasterol blunted aggregated LDL (agLDL) induced increases in tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1ß secretion, sitosterol exacerbated these effects. The presence of campesterol had no effect on agLDL-induced inflammatory cytokine secretion from THP-1 macrophages. In conclusion, the presence of stigmasterol in modified lipoproteins promoted cholesterol efflux and suppressed inflammatory cytokine secretion in response to lipid loading in macrophage foam cells. While campesterol was largely inert, the presence of sitosterol increased the proinflammatory cytokine secretion.

ABCG5/ABCG8-independent biliary cholesterol excretion in lactating rats.

Lactation is associated with increased expression of bile acid transporters and an increased size and hydrophobicity of the bile acid pool in rats. ATP-binding cassette (ABC) transporters multidrug resistance protein 2 (Mdr2), Abcb11 [bile salt export pump (Bsep)], and Abcg5/Abcg8 heterodimers are essential for the biliary secretion of phospholipids, bile acids, and cholesterol, respectively. We investigated the expression of these transporters and secretion of their substrates in female control and lactating Sprague Dawley rats and C57BL/6 mice. Expression of Abcg5/Abcg8 mRNA was decreased by 97 and 60% by midlactation in rats and mice, respectively; protein levels of Abcg8 were below detection limits in lactating rats. Mdr2 mRNA expression was decreased in lactating rats and mice by 47 and 59%, respectively. Despite these changes in transporter expression, basal concentrations of cholesterol and phospholipid in bile were unchanged in rats and mice, whereas increased Bsep mRNA expression in early lactation coincided with an increased basal biliary bile acid concentration in lactating mice. Following taurocholate infusion, coupling of phospholipid and taurocholate secretion in bile of lactating mice was significantly impaired relative to control mice, with no significant changes in maximal secretion of cholesterol or bile acids. In rats, taurocholate infusion revealed a significantly impaired coupling of cholesterol to taurocholate secretion in bile in lactating vs. control animals. These data reveal marked utilization of an Abcg5/Abcg8-independent mechanism for basal biliary cholesterol secretion in rats during lactation, but a dependence on Abcg5/g8 for maximal biliary cholesterol secretion.

ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat.

The ATP binding cassette transporter, ABCD2 (D2), is a peroxisomal protein whose mRNA has been detected in the adrenal, brain, liver, and fat. Although the role of this transporter in neural tissues has been studied, its function in adipose tissue remains unexplored. The level of immunoreactive D2 in epididymal fat is >50-fold of that found in brain or adrenal. D2 is highly enriched in adipocytes and is upregulated during adipogenesis but is not essential for adipocyte differentiation or lipid accumulation in day 13.5 mouse embryonic fibroblasts isolated from D2-deficient (D2(-/-)) mice. Although no differences were appreciated in differentiation percentage, total lipid accumulation was greater in D2(-/-) adipocytes compared with the wild type. These results were consistent with in vivo observations in which no significant differences in adiposity or adipocyte diameter between wild-type and D2(-/-) mice were observed. D2(-/-) adipose tissue showed an increase in the abundance of 20:1 and 22:1 fatty acids. When mice were challenged with a diet enriched in erucic acid (22:1), this lipid accumulated in the adipose tissue in a gene-dosage-dependent manner. In conclusion, D2 is a sterol regulatory element binding protein target gene that is highly abundant in fat and opposes the accumulation of dietary lipids generally absent from the triglyceride storage pool within adipose tissue.

The ABCG5 ABCG8 sterol transporter and phytosterols: implications for cardiometabolic disease.

PURPOSE OF REVIEW: This review summarizes recent developments in the activity, regulation, and physiology of the ABCG5 ABCG8 (G5G8) transporter and the use of its xenobiotic substrates, phytosterols, as cholesterol lowering agents in the treatment of cardiovascular disease. Recent progress has significant implications for the role of G5G8 and its substrates in complications associated with features of the metabolic syndrome. RECENT FINDINGS: Recent reports expand the clinical presentation of sitosterolemia to include platelet and adrenal dysfunction. The G5G8 sterol transporter is critical to hepatobiliary excretion of cholesterol under nonpathological conditions and has been linked to the cholesterol gallstone susceptibility. Finally, the cardiovascular benefits of cholesterol lowering through the use of phytosterol supplements were offset by vascular dysfunction, suggesting that alternative strategies to reduced cholesterol absorption offer greater benefit. SUMMARY: Insulin resistance elevates G5G8 and increases susceptibility to cholesterol gallstones. However, this transporter is critical for the exclusion of phytosterols from the absorptive pathways in the intestine. Challenging the limits of this protective mechanism through phytosterol supplementation diminishes the cardioprotective benefits of cholesterol lowering in mouse models of cardiovascular disease.

Defects in the leptin axis reduce abundance of the ABCG5-ABCG8 sterol transporter in liver.

ABGG5 (G5) and ABCG8 (G8) are ABC half-transporters that dimerize within the endoplasmic reticulum, traffic to the cell surface, and mediate cholesterol excretion into bile. Mice harboring defects in the leptin axis (db/db and ob/ob) have reduced biliary cholesterol concentrations. Rapid weight loss brought about by administration of leptin or dietary restriction increases biliary cholesterol excretion. We hypothesized that the reduction in biliary cholesterol in mice harboring defects in the leptin axis is associated with a reduction in G5G8 transporters and that levels of the transporter would increase with leptin administration and dietary restriction. We examined mRNA and protein levels for G5 and G8 in db/db and ob/ob mice. In both models G5 and G8 protein levels were reduced. In ob/ob mice, both leptin administration and dietary restriction increased G5 and G8 protein and biliary cholesterol concentrations. Finally, we examined the effects of tauroursodeoxycholate, which has been shown to increase biliary cholesterol excretion and function as a molecular chaperone. Tauroursodeoxycholate increased G5 and G8 protein and biliary cholesterol concentrations in both wild-type and db/db mice. Our results indicate that the mechanism for reduced biliary cholesterol excretion in db/db and ob/ob mice involves reductions in G5 and G8 protein levels and that this may occur at the level of G5G8 heterodimer assembly within the endoplasmic reticulum.