Loss Of Membrane Cholesterol Research Articles

Glutamate affects cholesterol homeostasis within the brain via the up-regulation of CYP46A1 and ApoE

Chronic glutamate excitotoxicity has been thought to be involved in numerous neurodegenerative disorders. A small but significant loss of membrane cholesterol has been reported following a short stimulation of ionotropic glutamate receptors (iGluRs). We investigated the alteration of brain cholesterol following chronic glutamate treatment. The alteration of cholesterol levels was evaluated in the hippocampus from the adult rats that received the subcutaneous injection with monosodium l-glutamate at 1, 3, 5, and 7 days of age. The regulation of CYP46A1, LXRα, and ApoE levels were assayed following subtoxic glutamate treatment in SH-SY5Y cells as well as HT-22 cells lacking iGluRs. The ratio of 24S-hydroxycholesterol to cholesterol was elevated in the adult rats exposed to monosodium l-glutamate before the weaning age, compared to the control. The blockers of NMDA receptor (MK801) and mGluR5 (MPEP) attenuated the glutamate-induced loss of cholesterol and elevation of 24S-hydroxycholesterol level in SH-SY5Y cells. The induction of the mRNA levels of CYP46A1, LXRα, and ApoE by glutamate was observed in both SH-SY5Y cells and HT-22 cells; additionally, MK801 and MPEP attenuated the increases in these genes in SH-SY5Y cells. The increase in the binding of LXRα proteins with ApoE promoter following glutamate treatment was attenuated by MK801. The luciferase assay indicated the binding of CREB protein with CYP46A1 promoter, and the glutamate-induced CREB expression was inhibited by MK801. The results suggest that glutamate, the major excitatory neurotransmitter, may affect the metabolism and redistribution of cholesterol in the neuronal cells via its specific receptors during chronic exposure.

Aging Increases Hippocampal DUSP2 by a Membrane Cholesterol Loss-Mediated RTK/p38MAPK Activation Mechanism

Numerous studies suggest that the increased activity of p38MAPK plays an important role in the abnormal immune and inflammatory response observed in the course of neurodegenerative diseases such as Alzheimer's disease. On the other hand, high levels of p38MAPK are present in the brain during normal aging, suggesting the existence of mechanisms that keep the p38MAPK-regulated pro-inflammatory activity within physiological limits. In this study, we show that high p38MAPK activity in the hippocampus of old mice is in part due to the reduction in membrane cholesterol that constitutively occurs in the aging brain. Mechanistically, membrane cholesterol reduction increases p38MAPK activity through the stimulation of a subset of tyrosine kinase receptors (RTKs). In turn, activated p38MAPK increases the expression and activity of the phosphatase DUSP2, which is known to reduce the activity of different MAPKs, including p38MAPK. These results suggest that the loss of membrane cholesterol that constitutively occurs with age takes part in a negative-feedback loop that keeps p38MAPK activity levels within physiological range. Thus, conditions that increase p38MAPK activity such as cellular stressors or that inhibit DUSP2 will amplify inflammatory activity with its consequent deleterious functional changes.

The Loss of Membrane Cholesterol Caused by UVA Irradiation Affects Collagen Metabolism in Dermal Fibroblasts

The Loss of Membrane Cholesterol Caused by UVA Irradiation Affects Collagen Metabolism in Dermal Fibroblasts

Cholesterol loss during glutamate-mediated excitotoxicity

The deregulation of brain cholesterol metabolism is typical in acute neuronal injury (such as stroke, brain trauma and epileptic seizures) and chronic neurodegenerative diseases (Alzheimer's disease). Since both conditions are characterized by excessive stimulation of glutamate receptors, we have here investigated to which extent excitatory neurotransmission plays a role in brain cholesterol homeostasis. We show that a short (30 min) stimulation of glutamatergic neurotransmission induces a small but significant loss of membrane cholesterol, which is paralleled by release to the extracellular milieu of the metabolite 24S-hydroxycholesterol. Consistent with a cause-effect relationship, knockdown of the enzyme cholesterol 24-hydroxylase (CYP46A1) prevented glutamate-mediated cholesterol loss. Functionally, the loss of cholesterol modulates the magnitude of the depolarization-evoked calcium response. Mechanistically, glutamate-induced cholesterol loss requires high levels of intracellular Ca(2+), a functional stromal interaction molecule 2 (STIM2) and mobilization of CYP46A1 towards the plasma membrane. This study underscores the key role of excitatory neurotransmission in the control of membrane lipid composition, and consequently in neuronal membrane organization and function.

Sphingomyelin upregulation in mature neurons contributes to TrkB activity by Rac1 endocytosis

A developmentally regulated loss of membrane cholesterol was reported to be sufficient and necessary for activation of neurotrophic tyrosine kinase receptor type 2 (TrkB) in aged neurons in vitro. However, TrkB activity in low cholesterol neurons remains confined to detergent-resistant membrane fractions, indicating that additional lipidic changes occur with age. Analysis of neuronal lipids at different developmental stages revealed a sharp increase in sphingomyelin (SM) during neuronal maturation. Reduction of SM abrogated TrkB activation in mature neurons, whereas increasing SM in immature neurons triggered receptor activation. TrkB activity in high SM background was the consequence of enhanced phosphorylation in the detergent-resistant fractions and increased Rac1-mediated endocytosis. The current results reveal developmental upregulation of SM as an important mechanism for sustaining TrkB activity in the mature nervous system, in addition to the presence of brain-derived neurotrophic factor (BDNF).

Oxidative stress activates the pro-survival TrkA pathway through membrane cholesterol loss

Neuronal activity is a highly demanding energetic process, resulting in the gradual accumulation of reactive oxygen species (ROS). Despite comparatively weak anti-oxidant defence systems, neurons outlive the pressure of ROS by activating most robust anti-stress mechanisms. We recently showed that one such mechanism is the activation of the TrkB receptor pathway, in turn determined by the loss of membrane cholesterol. It is not known however what causes the loss of membrane cholesterol. We here show that in differentiated PC12 cells induction of ROS is paralleled by a moderate loss of membrane cholesterol and the activation of the pro-survival TrkA receptor. Pharmacological reduction of cholesterol in non-stressed cells triggers TrkA activation while cholesterol replenishment inhibits receptor activation induced by stress. Moreover, addition of a ROS inhibitor prevented cholesterol loss and receptor activation under stress. These results highlight cholesterol loss as a compensatory protective mechanism against acute stress.

Cyp46-mediated cholesterol loss promotes survival in stressed hippocampal neurons

Aged neurons constitute an outstanding example of survival robustness, outliving the accumulation of reactive oxygen species (ROS) derived from various physiological activities. Since during aging hippocampal neurons experience a progressive loss of membrane cholesterol and, by virtue of this, a gradual and sustained increase in the activity of the survival receptor tyrosine kinase TrkB, we have tested in this study if cholesterol loss is functionally associated to survival robustness during aging. We show that old neurons that did not undergo the cholesterol drop, upon knockdown of the cholesterol hydroxylating enzyme Cyp46, presented low TrkB activity and increased apoptotic levels. In further agreement, inducing cholesterol loss in young neurons led to the early appearance of TrkB activity. In vivo, Cyp46 knockdown led to the appearance of damaged hippocampal neurons in old mice exposed to exogenous stressful stimuli. Cholesterol loss seems therefore to contribute to neuronal survival in conditions of prominent stress, either acute or chronic. The relevance of this pathway in health and disease is discussed.

Loss of membrane cholesterol influences lysosomal permeability to potassium ions and protons

Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K + and H +, and the organelle stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-β-cyclodextrin (MβCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K +/H + exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MβCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K + and H +. Loss of this membrane sterol is critical for the organelle acidification and stability.

Differential effects of cholesterol and 7-dehydrocholesterol on ligand binding of solubilized hippocampal serotonin 1A receptors: Implications in SLOS

The serotonin 1A receptor is an important member of the G-protein coupled receptor family, and is involved in the generation and modulation of a variety of cognitive, behavioral, and developmental functions. Solubilization of the hippocampal serotonin 1A receptor by 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity. Replenishment of cholesterol to solubilized membranes restores the cholesterol content of the membrane and significantly enhances specific agonist binding activity. In order to test the stringency of the requirement of cholesterol in this process, we solubilized native hippocampal membranes followed by replenishment with 7-dehydrocholesterol (7-DHC). 7-DHC is an immediate biosynthetic precursor of cholesterol differing only in a double bond at the 7th position in its sterol ring. Our results show, for the first time, that replenishment of solubilized hippocampal membranes with 7-DHC does not restore ligand binding activity of the serotonin 1A receptor, in spite of recovery of the overall membrane order. This observation shows that the requirement for restoration of ligand binding activity is more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane sterols with this important neuronal receptor under pathogenic conditions such as the Smith–Lemli–Opitz syndrome.

Lipid homeostasis and apolipoprotein E in the development and progression of Alzheimer's disease

Extracellular amyloid plaques, intracellular neurofibrillary tangles, and loss of basal forebrain cholinergic neurons in the brains of Alzheimer's disease (AD) patients may be the end result of abnormalities in lipid metabolism and peroxidation that may be caused, or exacerbated, by beta-amyloid peptide (Abeta). Apolipoprotein E (apoE) is a major apolipoprotein in the brain, mediating the transport and clearance of lipids and Abeta. ApoE-dependent dendritic and synaptic regeneration may be less efficient with apoE4, and this may result in, or unmask, age-related neurodegenerative changes. The increased risk of AD associated with apoE4 may be modulated by diet, vascular risk factors, and genetic polymorphisms that affect the function of other transporter proteins and enzymes involved in brain lipid homeostasis. Diet and apoE lipoproteins influence membrane lipid raft composition and the properties of enzymes, transporter proteins, and receptors mediating Abeta production and degradation, tau phosphorylation, glutamate and glucose uptake, and neuronal signal transduction. The level and isoform of apoE may influence whether Abeta is likely to be metabolized or deposited. This review examines the current evidence for diet, lipid homeostasis, and apoE in the pathogenesis of AD. Effects on the cholinergic system and response to cholinesterase inhibitors by APOE allele carrier status are discussed briefly.

Role of cholesterol in ligand binding and G-protein coupling of serotonin 1A receptors solubilized from bovine hippocampus

The serotonin 1A (5-HT 1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We report here that solubilization of the hippocampal 5-HT 1A receptor by the zwitterionic detergent CHAPS is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity and extent of G-protein coupling. Importantly, replenishment of cholesterol to solubilized membranes using MβCD–cholesterol complex restores the cholesterol content of the membrane and significantly enhances the specific agonist binding activity and G-protein coupling. These novel results provide useful information on the role of cholesterol in solubilization of G-protein-coupled receptors, an important step for molecular characterization of these receptors.

Neuronal membrane cholesterol loss results in low plasmin activity yet higher beta-secretase cleavage of APP

Neuronal membrane cholesterol loss results in low plasmin activity yet higher beta-secretase cleavage of APP

Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation

Bovine seminal vesicles secrete a family of similar proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30-kDa (collectively called bovine seminal plasma (BSP) proteins). The biochemical properties of these proteins are well documented and considerable progress has been made concerning their biological role. At ejaculation these BSP proteins bind to the sperm surface. The binding sites on the sperm surface have been identified as choline phospholipids (specifically phosphatidylcholine (PC), phophatidylcholine plasmalogen (PC plasm) and sphingomyelin (SPM)) composed of sperm plasma membrane. Our previous studies have shown that the BSP proteins interact specifically with heparin and high-density lipoproteins (HDL), the capacitation factors in bovine. In addition, we have shown that the BSP proteins potentiate epididymal sperm capacitation induced by heparin and HDL. Recently, we showed that the BSP proteins stimulated cholesterol and phospholipid efflux from the sperm membrane. Furthermore, the lipid efflux from sperm is dependent on BSP protein concentration and duration of incubation. The loss of membrane cholesterol is an important step in the capacitation process. These results together indicate that BSP proteins play an important role in sperm membrane lipid modification events that occur during sperm capacitation.

Effect of bilirubin on toxicity induced by trifluoperazine, dibucaine and praziquantel to erythrocytes

Unconjugated bilirubin (UCB), like trifluoperazine (TFP), dibucaine (DBC) and praziquantel (PZQ), induces erythrocyte morphological changes, lysis and lipid exfoliation. In the present study we determined whether TFP, DBC and PZQ toxicity to erythrocytes was potentiated or reverted by UCB. Human erythrocytes were either treated or non-treated with 34.2 μmol/L UCB for 10 min prior to the incubation with toxic concentrations of TFP (0.12 mmol/L), DBC (1.5 mmol/L) or PZQ (3.0 mmol/L), for 1 h (37°C). Studies of toxic effects included morphological analysis of erythrocytes, evaluation of hemoglobin release and loss of membrane lipids. Although UCB has an echinocytogenic effect, its co-incubation with TFP or PZQ did not alter the stomatocytogenic effect of the drug but enhanced DBC-induced stomatocytosis. Cell fusion was a common feature in experiments with DBC. Injurious effect of DBC to erythrocytes was potentiated by UCB as manifested by a marked increase in hemolysis (171%, p<0.05), and in elution of membrane cholesterol (73%, p<0.01) and phospholipids (123%, p<0.01). In opposite, toxic events produced by TFP and PZQ to erythrocytes were not aggravated by UCB. Interestingly, UCB prevented the loss of membrane cholesterol by PZQ (−36%, p<0.01), as well as that of phospholipids by TFP (−28%, p<0.05). These findings indicate that UCB potentiates DBC injury to erythrocytes, while protects membrane lipid elution by PZQ and TFP. Therefore, the relation of the benefits and risks of the administration of DBC to jaundiced patients should be carefully considered.

On the in vitro vasoactivity of bile acids.

We compared the vasorelaxant action of nine different bile acids and correlated their vasorelaxant activity with their individual indices for hydrophobicity or lipophilicity. Vasorelaxant activity correlated with the relative lipid solubility of bile acids with lipophilic bile acids exhibiting the greatest vasorelaxant activity with modest to no vasorelaxant activity exhibited by hydrophilic bile acids. We also investigated whether bile acid-induced vasorelaxation is mediated by antagonism of a prototypal contractile receptor, the alpha(1)-adrenoceptor, by stimulation of a bile acid surface membrane receptor, by the release of endothelium-derived relaxant factors, by promoting the generation of reactive oxygen species and increasing the extent of lipid peroxidation, or by modifying membrane fluidity. Lipophilic bile acids induce vasorelaxation possibly by antagonizing alpha(1)-adrenoceptors, a phenomenon that manifests itself as a lowering of the affinity of vascular alpha(1)-adrenoceptors. Bile acid-induced vasorelaxation was not dependent upon stimulation of a bile acid surface membrane receptor or the release of endothelium-derived relaxant factors. Lipophilic bile acids can also increase the extent of lipid peroxidation with a subtle reduction in the fluidity of rat vascular smooth muscle membranes not associated with loss of membrane cholesterol or phospholipid. We have concluded that lipophilic bile acids are non-selective vasorelaxants whose mechanism of action is a multifaceted process involving antagonism of contractile surface membrane receptors possibly effected by an increased extent of lipid peroxidation and/or membrane fluidity but occurs independent of the release of endothelial-derived relaxant factors or stimulation of a surface membrane bile acid binding site.

ASSOCIATION OF UREAPLASMA UREALYTICUM WITH ABNORMAL REACTIVE OXYGEN SPECIES LEVELS AND ABSENCE OF LEUKOCYTOSPERMIA

Ureaplasma urealyticum is a commensal of the lower genitourinary tract of many sexually active adults. The organism is more common in partners of infertile than fertile marriages. We conducted a prospective study at our tertiary care center to confirm a possible association between U. urealyticum and abnormal sperm function parameters. A total of 50 consecutive male patients seeking general urology consultation for lower urinary tract symptoms characteristic of chronic prostatitis were evaluated. Urine and semen localization cultures were performed with additional semen cultures for U. urealyticum, Chlamydia trachomatis and Mycoplasma hominis. Specimens from 21 healthy men were used as controls. Specimens were analyzed by a computer assisted semen analyzer, and verified manually for concentration, percent motility and morphology. Leukocytospermia was measured by the Endtz test. Semen specimens were also analyzed for reactive oxygen species (ROS), acrosome reaction and mannose binding assay. Of the patients 17 had positive U. urealyticum cultures and the other cultures were negative. Patients with U. urealyticum had significantly higher ROS levels (log [ROS + 1] = 2.52 +/- 0.25) than those without U. urealyticum (1.49 +/- 0.20, p = 0.002) or controls (1.31 +/- 0.19, p = 0.002). Leukocytospermia was detected in only 1 of the 17 (6%) positive specimens and 4 (12%) negative specimens. Seminal ROS levels are elevated among patients with U. urealyticum. ROS induces lipid peroxidation, which reduces membrane fluidity and sperm fertilization capability, and may be the mechanism by which U. urealyticum impairs sperm function. Absence of leukocytospermia does not exclude U. urealyticum.

Head-specific mannose-ligand receptor expression in human spermatozoa is dependent on capacitation-associated membrane cholesterol loss.

D-Mannose binding lectins appear on the human sperm head following in-vitro incubation under capacitating conditions. Surface expression of lectin is dependent on reduction of the sterol content of sperm membranes. Mannose-specific lectin distribution over the head differs in acrosome-intact and acrosome-reacted spermatozoa. Sugar competition experiments show that mannose is the only saccharide competitive with D-mannosylated albumin binding and that free mannose induces an acrosome reaction in capacitated spermatozoa. A total of 183 men with normozoospermic semen parameters were screened for the ability of their spermatozoa to bind fluorescein isothiocyanate (FITC)-labelled mannosylated albumins, before and after incubation in vitro. The spermatozoa from 176 men 'responded' to incubation by exhibiting time-dependent increases in head-directed mannosylated albumin binding, accompanied by increases in the percentage of spermatozoa showing spontaneous acrosome loss. Motile spermatozoa from the remaining seven men failed to express mannose-lectin binding activity after 18 h of incubation and only low percentages of their spermatozoa showed spontaneous acrosome loss. These seven men were classified as 'non-responders'. The relative amounts of non-esterified cholesterol in the sperm membranes of the responder and non-responder males were analysed by gas-liquid chromatography. Responder spermatozoa showed decreases in free cholesterol content whereas non-responder spermatozoa exhibited either no decrease or increases in relative free cholesterol per cell. Fresh swim-up spermatozoa contain sub-plasma membrane stores of mannose lectins which are revealed by FITC-mannosylated albumin labelling before and after removal of the plasma membrane by vortexing. In contrast, the mannose-lectin binding activity of capacitated spermatozoa is entirely limited to the sperm surface. Western blots of proteins isolated from sperm plasma membranes after capacitation revealed two molecular species reactive with polyclonal antibodies against human macrophage mannose receptors. A model is proposed for the molecular mechanism whereby mannose lectins are transposed from sub-plasma membrane sites to an integral membrane position as a consequence of the loss of cholesterol from the sperm membrane.

Cholesterol efflux from bovine sperm: II. Effect of reducing sperm cholesterol on penetration of zona-free hamster and in vitro matured bovine ova

Several reports have indicated that sperm capacitation includes loss of membrane cholesterol (Chol) with a concomitant decrease in the Chol-to-phospholipid (PL) ratio. Methods were developed for quantifiable removal of bovine sperm Chol, which predisposed sperm to induction of the acrosome reaction upon addition of lysophosphatidylcholine (LPC). The objective of this study was to evaluate the effect of Chol removal from bovine sperm on penetration of zona-free hamster and intact bovine ova in vitro. Washed ejaculated bovine sperm were incubated (2 h, 39 degrees C) in a modified Tyrode's solution (TALP) containing 1) Chol-free liposomes (-Chol, 50 x 10(6) sperm and 600 nmol phospholipid/ml); 2) liposomes containing 30 mol% Chol (+Chol, 2 x 10(8) sperm and 300 nmol total lipid/ml); or 3) no liposomes (Control). We have previously shown that net Chol efflux from sperm is 31% of the total sperm Chol with -Chol liposomes and less than 1% with control media. Sperm were then washed twice and challenged with LPC bound to bovine serum albumin (BSA) using celite as a carrier. Treated sperm (25 x 10(6)) were incubated immediately with either zona-free hamster ova (HO) or in vitro matured bovine ova (BO) in 50-microliters droplets of TALP under medical fluid in an atmosphere of 5% CO2 in air (3 h, 39 degrees C). Ova were fixed in ethanol:acetic acid, stained with 1% orcein, and examined.(ABSTRACT TRUNCATED AT 250 WORDS)

The selective and conjoint loss of red cell lipids

The pattern of lipid loss from the membrane of red cells incubated in serum is influenced by the availability of glucose. Under homeostatic conditions with respect to glucose, cholesterol alone is lost. This results from esterification of free cholesterol in serum by the serum enzyme, lecithin:cholesterol acyltransferase, and is associated with a proportional decrease in membrane surface area, reflected by an increased osmotic fragility. This selective loss of membrane cholesterol also occurs in hereditary spherocytosis (HS) red cells, even after incubation for 65 hr in the presence of glucose. The loss of free cholesterol from red cells relative to its loss from serum, under these conditions, is greatest at higher hematocrits, similar to those found in the spleen. Although the selective loss of membrane cholesterol increases the spherodicity of normal red cells, it does not lead to a change in their rate of glucose consumption, and both the loss of cholesterol and the increase in osmotic fragility are reversible in vitro. Moreover, normal red cells made osmotically fragile by cholesterol depletion in vitro rapidly become osmotically normal and survive normally after their reinfusion in vivo.In contrast to this selective loss of membrane cholesterol, red cells incubated in the absence of glucose lose both cholesterol and phospholipid. This occurs more rapidly in HS than normal red cells and is followed by a disruption of cation gradients and then by hemolysis. Cholesterol and phospholipid lost under these conditions is not restored during subsequent incubations in vitro. Selective loss of membrane cholesterol is a physiologic event secondary to an altered state of serum lipids. It is reversible both in vitro and in vivo and neither influences cellular metabolism nor impairs viability. Conjoint loss of phospholipid and cholesterol, however, results from intrinsic injury to the red cell membrane which results from prolonged metabolic depletion.