Zhi-Xing Yao

Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis, neuropathology and neurological disorders

The peripheral-type benzodiazepine receptor is a mitochondrial protein expressed at high levels in steroid synthesizing tissues, including the glial cells of the brain. Peripheral-type benzodiazepine receptor binds cholesterol with high affinity and is a key element of the cholesterol mitochondrial import machinery responsible for supplying the substrate cholesterol to the first steroidogenic enzyme, thus initiating and maintaining neurosteroid biosynthesis. Neurosteroid formation and metabolism of steroid intermediates are critical components of normal brain function. Peripheral-type benzodiazepine receptor also binds with high affinity various classes of compounds. Upon ligand activation peripheral-type benzodiazepine receptor-dependent cholesterol transport into mitochondria is accelerated leading in increased formation of neuroactive steroids. These steroids, such as allopregnanolone, have been shown to be involved in various neurological disorders, such as anxiety and mood disorders. Thus, peripheral-type benzodiazepine receptor drug ligand-induced neuroactive steroid formation offers a means to regulate brain dysfunction. Peripheral-type benzodiazepine receptor basal expression is upregulated in a number of neuropathologies, including gliomas and neurodegenerative disorders, as well as in various forms of brain injury and inflammation. In Alzheimers disease pathology neurosteroid biosynthesis is altered and a decrease in the intermediate 22R-hydroxycholesterol levels is observed. This steroid was found to exert neuroprotective properties against beta-amyloid neurotoxicity. Based on this observation, a stable spirostenol derivative showing to display neuroprotective properties was identified, suggesting that compounds developed based on critical intermediates of neurosteroid biosynthesis could offer novel means for neuroprotection. In conclusion, changes in peripheral-type benzodiazepine receptor and neurosteroid levels are part of the phenotype seen in neuropathology and neurological disorders and offer potential targets for new therapies.

The Ginkgo biloba extract EGb 761 rescues the PC12 neuronal cells from β-amyloid-induced cell death by inhibiting the formation of β-amyloid-derived diffusible neurotoxic ligands

Abstract β Amyloid (Aβ) treatment induced free radical production and increased glucose uptake, apoptosis and cell death in PC12 nerve cells. Addition of the standardized extract of Ginkgo biloba leaves, EGb 761 together with the Aβ protein prevented, in a dose-dependent manner, the Aβ-induced free radical production, increased glucose uptake, apoptosis and cell death. However, pretreatment of the cells with EGb 761 did not rescue the cells from the Aβ-induced toxicity although it prevented the Aβ-induced reactive oxygen species generation. Moreover, the terpene and flavonoid-free EGb 761 extract, HE 208, although inhibited the Aβ-induced increased glucose uptake, it failed to protect the cells from apoptosis and cytotoxicity induced by Aβ. In conclusion, these results indicate that the terpenoid and flavonoid constituents of EGb 761, acting probably in combination with components present in HE 208, are responsible for rescuing the neuronal cells from Aβ-induced apoptosis and cell death; their mechanism of action being distinct of their antioxidant properties. Because pre- and post-treatment with EGb 761 did not protect the cells from Aβ-induced neurotoxicity, we examined whether EGb 761 interacts directly with Aβ. Indeed, in vitro reconstitution studies demonstrated that EGb 761 inhibits, in a dose-dependent manner, the formation of β-amyloid-derived diffusible neurotoxic soluble ligands (ADDLs), suggested to be involved in the pathogenesis of Alzheimer’s disease.

Effect of Peroxisome Proliferators on Leydig Cell Peripheral-Type Benzodiazepine Receptor Gene Expression, Hormone-Stimulated Cholesterol Transport, and Steroidogenesis: Role of the Peroxisome Proliferator-Activator Receptor α

In this study, we hypothesized that many of the reported effects of phthalate esters and other peroxisome proliferators (PPs) in the testis are mediated by members of the PPactivated receptor (PPAR) family of transcription factors through alterations in proteins involved in steroidogenesis. Exposure of Leydig cells to PPs prevented cholesterol transport into the mitochondria after hormonal stimulation and inhibited steroid synthesis, without altering total cell protein synthesis or mitochondrial and DNA integrity. PPs also reduced the levels of the cholesterol-binding protein peripheraltype benzodiazepine receptor (PBR) because of a direct transcriptional inhibition of PBR gene expression in MA-10 Leydig cells. MA-10 cells contain mRNAs for PPAR and PPAR/, but not for PPAR. In vivo treatment of mice with PPs resulted in the reduction of both testis PBR mRNA and circulating testosterone levels, in agreement with the proposed role of PBR in steroidogenesis. By contrast, liver PBR mRNA levels were increased, in agreement with the proposed role of PBR in cell growth/tumor formation in nonsteroidogenic tissues. However, PPs did not inhibit testosterone production and testis PBR expression in PPAR-null mice. These results suggest that the antiandrogenic effect of PPs is mediated by a PPARdependent inhibition of Leydig cell PBR gene expression. (Endocrinology 143: 2571–2583, 2002)

Function of β-amyloid in cholesterol transport: a lead to neurotoxicity

Amyloid β‐peptide (Aβ), Aβ precursor protein (APP), apolipoprotein E (apoE), and elevated cholesterol levels have been linked to Alzheimers disease (AD) pathology. High cholesterol levels increase APP and apoE expression in human NT2 neuron progenitor cells. A cholesterol‐ rich environment also induces processing of APP, leading to the formation of Aβ and Aβ peptide fragments. Using a novel method, we determined that 1) cholesterol binds to Aβ at a‐secretase cleavage site; 2) Aβ17–40 rather than Aβ1–40 prevents cholesterol from binding to apoE; 3) Aβ1–40 inhibits cholesterol from binding to low‐density lipoprotein (LDL), leading to decrease cholesterol influx and intracellular cholesterol levels; 4) the binding of cholesterol to apoE or LDL was abolished completely in presence of Aβ1–42. Increased extracellular free cholesterol levels are toxic to neurons; this toxicity is prevented by specific lipoproteins, such as high‐ density lipoproteins, which maintain their ability to bind cholesterol in the presence of Aβ. We propose that one of the physiological functions of Aβ and APP is to control cholesterol transport. AD is associated with increased Aβ production. High cholesterol levels also lead to overproduction of Aβ. Aβ blocks cholesterol trafficking and changes cholesterol homeostasis leading to neurodegeneration and the onset and/or progression of AD pathology.

Free radicals and lipid peroxidation do not mediate β-amyloid-induced neuronal cell death

Abstract “β Amyloid (Aβ)-induced free radical-mediated neurotoxicity” is a leading hypothesis as a cause of Alzheimers disease (AD). Aβ increased free radical production and lipid peroxidation in PC12 nerve cells, leading to increased 4-hydroxy-2-nonenal (HNE) production and modification of specific mitochondrial target proteins, apoptosis and cell death. Pretreatment of the cells with isolated ginkgolides, the anti-oxidant component of Ginkgo biloba leaves, or vitamin E, prevented the Aβ-induced increase of reactive oxygen species (ROS). Ginkgolides, but not vitamin E, inhibited the Aβ-induced HNE modification of mitochondrial proteins. However, treatment with these anti-oxidants did not rescue the cells from Aβ-induced apoptosis and cell death. These results indicate that free radicals and lipid peroxidation may not mediate Aβ-induced neurotoxicity.

Control of hypercholesterolemia and atherosclerosis using the cholesterol recognition/interaction amino acid sequence of the translocator protein TSPO

The translocator protein (18-kDa) TSPO is an ubiquitous high affinity cholesterol-binding protein reported to be present in the endothelial and smooth muscle cells of the blood vessels; its expression dramatically increased in macrophages found in atherosclerotic plaques. A domain in the carboxy-terminus of TSPO was identified and characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide could be used as an hypocholesterolemic, with potential anti-atherogenic properties, agent. We report herein the therapeutic benefit that resulted for the administration of the VLNYYVWR human CRAC sequence to guinea pigs fed with a high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. CRAC treatment (3 and 30mg/kg once daily for 6 weeks) resulted in reduced circulating cholesterol levels in guinea pigs fed with 2% high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. In high cholesterol fed guinea pigs, CRAC treatment administered once daily induced an increase in circulating HDL, decreased total, free and LDL cholesterol, and removed atheroma deposits in the aorta in a dose-dependent manner. The treatment also prevented the high cholesterol diet-induced increase in serum creatine kinase, total and isoforms, markers of neurological, cardiac and muscular damage. No toxicity was observed. Taken together these results support a role of TSPO in lipid homeostasis and atherosclerosis and indicate that CRAC may constitute a novel and safe treatment of hypercholesterolemia and atherosclerosis.

The Ginkgo biloba extract EGB 761 rescues the PC12 neuronal cells from β-amyloid-induced cell death by inhibiting the formation of β-amyloid-derived diffusible neurotoxic ligands

beta Amyloid (Abeta) treatment induced free radical production and increased glucose uptake, apoptosis and cell death in PC12 nerve cells. Addition of the standardized extract of Ginkgo biloba leaves, EGb 761 together with the Abeta protein prevented, in a dose-dependent manner, the Abeta-induced free radical production, increased glucose uptake, apoptosis and cell death. However, pretreatment of the cells with EGb 761 did not rescue the cells from the Abeta-induced toxicity although it prevented the Abeta-induced reactive oxygen species generation. Moreover, the terpene and flavonoid-free EGb 761 extract, HE 208, although inhibited the Abeta-induced increased glucose uptake, it failed to protect the cells from apoptosis and cytotoxicity induced by Abeta. In conclusion, these results indicate that the terpenoid and flavonoid constituents of EGb 761, acting probably in combination with components present in HE 208, are responsible for rescuing the neuronal cells from Abeta-induced apoptosis and cell death; their mechanism of action being distinct of their antioxidant properties. Because pre- and post-treatment with EGb 761 did not protect the cells from Abeta-induced neurotoxicity, we examined whether EGb 761 interacts directly with Abeta. Indeed, in vitro reconstitution studies demonstrated that EGb 761 inhibits, in a dose-dependent manner, the formation of beta-amyloid-derived diffusible neurotoxic soluble ligands (ADDLs), suggested to be involved in the pathogenesis of Alzheimers disease.