Peripheral nerve regeneration and cholesterol reutilization are normal in the low-density lipoprotein receptor knockout mouse

Following peripheral nerve injury, cholesterol from degenerating myelin is retained locally within macrophages and subsequently reutilized by Schwann cells for synthesis of new myelin during nerve regeneration. Substantial evidence indicates this conservation and reutilization of cholesterol is accomplished via lipoprotein-mediated intercellular transport, although the identities of the lipoproteins and their receptors are unresolved. Because Schwann cells in regenerating nerve are reported to express the low-density lipoprotein (LDL) receptor (LDLR), we used the LDLR knockout mouse to examine the potential role of this receptor in cholesterol reutilization. Sciatic nerves were crushed in knockout and wild-type mice and examined 3 days to 10 weeks later. Morphometric analyses and measures of mRNA levels for myelin protein P(0), indicate that axon regeneration and myelination proceed normally in the LDLR knockout mouse. We therefore measured hydroxy-methylglutaryl-coenzyme A (HMG-CoA) reductase activity and mRNA levels to determine whether Schwann cells compensated for the absence of the LDLR by upregulating cholesterol synthesis. Unexpectedly, these measures remained at the same downregulated levels found in regenerating nerves of wild-type animals. The apparently normal nerve regeneration, coupled with the lack of any compensatory upregulation of cholesterol synthesis in the LDLR knockout mice, indicates that other lipoprotein receptors must be primarily involved in cholesterol uptake by Schwann cells.     

The role of pleiotropic effects of statins in dementia

High serum cholesterol is associated with ischemic heart disease. Recent reports also indicate that cholesterol modulates amyloid beta-peptide interactions in the brain. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme involved in cholesterol synthesis. Statin treatment significantly reduces the levels of low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL). In the past decade, cardiovascular mortality and morbidity has been reduced by the use of statins. However, evidence from in vivo and in vitro research has indicated that statins may confer multiple effects because of the inhibition of the production of intermediates in the mevalonate pathway. The aim of this review was to discuss the biological effects of statins on regulation of processes involved in the pathogenesis of dementia.     

Hypertriglyceridemia and lower LDL cholesterol concentration in relation to apolipoprotein E phenotypes in myotonic dystrophy

Plasma lipid, lipoprotein levels and apolipoprotein apo E phenotypes were determined in 70 patients with myotonic dystrophy (MyD) and 81 controls. Marked differences were noticed in the apo E phenotype frequencies between the two groups. Plasma triglycerides and VLDL cholesterol were higher in MyD than controls, but only the latter was related to differences in the apo E phenotypes between two groups. Accordingly, the ratio of VLDL cholesterol/plasma triglycerides was increased significantly in MyD, suggesting accumulation of intermediary density particles due to lower affinity of E2 containing lipoproteins for lipoprotein cell receptors. The LDL cholesterol concentration was lower in MyD than controls and was related to differences in the apo E phenotype frequencies between the two groups. These results indicate increased removal of LDL particles in the apo E2 phenotypes, perhaps due to upregulation of LDL (B, E) receptor activity. Plasma cholesterol and HDL cholesterol concentrations were similar in both groups. Another feature of the study was lower levels of plasma cholesterol, triglycerides, VLDL and LDL cholesterol in the homozygous E4:E4 phenotype. These results suggest increased clearance rate of both VLDL and LDL particles and support the concept that apo E4-containing lipoproteins have higher in vivo affinity for ape E and/or B, E receptors.     

Regulation of sterol synthesis and of 3-hydroxy-3-methylglutaryl coenzyme a reductase by lipoproteins in glial cells in primary culture

Although plasma lipoproteins have been demonstrated to have a major role in regulating cholesterol biosynthesis in extraneural cells, no data concerning such regulation are available for developing brain, when cholesterol synthesis is especially active. Glial primary cultures derived from neonatal rat brain and by morphological and biochemical criteria essentially exclusively composed of astrocytes were utilized to examine such regulation. When the primary cultures, which had been maintained in 10% fetal calf serum, were placed in 10% lipoprotein-poor serum on day 7 of culture, an induction of sterol synthesis (1.6–2.2-fold) and of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-specific activity (1.5-2-fold) resulted after 24 hr. Addition of low-density lipopro-tein (LDL) to the 10% lipoprotein-poor serum prevented the induction of both sterol synthesis and HMG-CoA reductase. However, addition of high-den-sity lipoprotein (HDL) to the 10% lipoprotein-poor serum caused a 1.5–2-fold further induction of sterol synthesis relative to that in cultures containing 10% lipoprotein-poor serum alone. In contrast to the glial primary cultures, cultures of C-6 glioma cells responded to replacement of 10% fetal calf serum with 10% lipoprotein-poor serum with much more marked increases of sterol synthesis and HMG-CoA reductase. Although, as with the primary cultures, addition of LDL to the C-6 glioma cell cultures prevented the increases in sterol synthesis and reductase activity, addition of HDL had no effect. Thus, these results indicate that in developing glial cells in primary culture, cholesterol synthesis and HMG-CoA reductase are capable of responsiveness to both LDL and HDL. Moreover, at least in terms of regulation of sterol synthesis, C-6 glioma cells and developing glia in primary culture clearly are not identical.     

Differential effects of lovastatin treatment on brain cholesterol levels in normal and apoE-deficient mice

Growing evidence indicates that membrane cholesterol is involved in the development of Alzheimer's disease. Therefore, the availability of pharmacological strategies to modify brain cholesterol is of increasing importance. Accordingly, we investigated the effects of the HMG-CoA reductase inhibitor lovastatin on brain cholesterol levels in vivo. Brain cholesterol was significantly decreased by lovastatin treatment (100 mg/kg/day) in 1- and 12-month-old C57BL/6J mice. Reduced brain cholesterol was associated with decreased pyrene-excimer fluorescence, indicating altered membrane function. Lovastatin had no effect on brain cholesterol ApoE-/- mice. Peripheral cholesterol levels were not affected by lovastatin in all three groups of mice. We demonstrate for the first time that lovastatin represents a valid pharmacological tool to significantly modulate brain cholesterol levels.     

3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA in Alzheimer and control brain

Statins are widely used pharmaceutical agents which lower plasma cholesterol by inhibiting the rate controlling enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase. One epidemiological study suggests that statin therapy may provide protection against Alzheimer disease (AD). The aim of the present study was to determine the relative expression of HMG-CoA reductase mRNAs in various areas of brain as well as in peripheral organs and to compare values in AD and control cases. High levels of the mRNA were found in all areas of brain but no obvious differences were found between AD and controls. We conclude that brain has a robust capacity to synthesize cholesterol which appears to be unaffected by AD pathology.     

Simvastatin induces cell death in a mouse cerebellar slice culture (CSC) model of developmental myelination

Statins (inhibitors of HMG-CoA reductase) have shown promise in treating multiple sclerosis (MS). However, their effect on oligodendrocyte remyelination of demyelinated axons has not been clarified. Since developmental myelination shares many features with the remyelination process, we investigated the effect of lipophilic simvastatin on developmental myelination in organotypic cerebellar slice cultures (CSC). In this study, we first characterized developmental myelination in CSC from postnatal day (P)5 and P10 mice that express enhanced green fluorescence protein (eGFP) in oligodendrocyte-lineage cells. We then examined the effect of simvastatin on three developmental myelination stages: early myelination (P5 CSC, 2DIV), late myelination (P10 CSC, 2DIV) and full myelination (P10 CSC, 10DIV). We found that treatment with simvastatin (0.1 μM) for 6 days decreased the survival of Purkinje cells and oligodendrocytes drastically during the early myelination stage, while moderately during the late and full myelination stages. Oligodendrocytes are more resistant than Purkinje cells. The toxic effect of simvastatin could be rescued by the product of HMG-CoA reductase mevalonate but not low-density lipoprotein (LDL). Additionally, this toxic effect is independent of isoprenylation since farnesyl pyrophosphate (Fpp) but not geranylgeranyl pyrophosphate (GGpp) provided partial rescue. Our findings therefore suggest that inhibition of cholesterol synthesis is detrimental to neuronal tissue.     

Cholesterol depletion with physiological concentrations of a statin decreases the formation of the Alzheimer amyloid Abeta peptide

Epidemiological studies have demonstrated that hypercholsterolemia is a significant risk factor for Alzheimer's disease (AD). The mechanism by which increased cholesterol may contribute to AD is unknown. However, as the generation and accumulation of the amyloid Abeta peptide in the brain appears to be significant for the initiation and progression of AD, it is possible that cholesterol levels can regulate Abeta formation and/or clearance. To test the effects of altering cholesterol on Abeta formation, we incubated cells in the presence of lipid depleted serum, with or without the active metabolite of the HMG-CoA reductase inhibitor lovastatin. After confirming that cholesterol was depleted in the cells, we then measured the fraction of Abeta formed from its precursor betaPP under each condition. We observed that cholesterol depletion led to a profound decrease in the levels of Abeta released from the cells. This effect of lovastatin acid was observed at concentrations of 0.05-5 &mgr;M, ranges where this compound is effective at inhibiting HMG-CoA reductase, thereby inhibiting cholesterol synthesis. In contrast, the release of an additional AbetaPP fragment, AbetaPPs, was only modestly reduced by cholesterol treatment. In further studies, we determined that the decreased release of Abeta was not due to its accumulation in the cell, but rather due to decreased formation of Abeta. Finally, we were able to exclude decreased maturation (glycosylation and sulfation) of newly synthesized AbetaPP as a cause for the effects of lovastatin acid on betaPP processing and Abeta formation. Our results demonstrate that reducing cellular cholesterol by the use of an HMG-CoA reductase inhibitor regulates Abeta formation. This effect may involve alterations in the trafficking of AbetaPP and/or alterations in the activity of the proteases that cleave AbetaPP. The results suggest a mechanism by which hypercholesterolemia may increase risk for AD and indicate that reduction in cholesterol may delay the onset and/or slow the progression of AD.     

In vivo regulation of mu-opioid receptor density and gene expression in CXBK and outbred Swiss Webster mice

Chronic in vivo treatment with the opioid agonist etorphine downregulates mu-opioid receptor density, produces tolerance, and regulates gene expression in the mouse. After cessation of treatment, there is an increase in mu-opioid receptor mRNA level associated with the recovery of mu-opioid receptors. However, the effect of etorphine on the regulation of mRNA during treatment is currently not known. In this study, etorphine-induced changes in mu-opioid receptor density, mRNA, and opioid analgesic potency were determined in two mouse strains that differ in basal mu-opioid receptor density in brain. CXBK mice (mu-opioid receptor deficient) and outbred Swiss Webster mice were implanted s.c. with placebo pellets (controls) or etorphine minipumps (250 microg/kg/day) for 1-7 days and mu-opioid receptor density or mRNA levels in whole brain were assessed or mice were tested for etorphine analgesia following 7 days of treatment. In control CXBK mice, mu-receptor density was approximately 40% less than that for the Swiss Webster, although mRNA abundance was similar in both strains. Etorphine's potency was 4-fold greater in control Swiss Webster compared to CXBK mice. Etorphine treatment decreased ( approximately 25-40%) mu-receptor density similarly in both strains throughout treatment. The magnitude of analgesic tolerance to etorphine was 8-fold in both mouse strains. Etorphine produced a biphasic effect on receptor mRNA in both strains with levels decreased (25%) by 3 days and increased (30-40%) at 7 days. mRNA levels remained elevated (55%) 16 h following the end of the 7 day etorphine treatment. Taken together, these data suggest that in vivo etorphine treatment that produces mu-opioid receptor downregulation and tolerance, can regulate mu-opioid receptor mRNA abundance. Receptor downregulation may initially induce decreases in mRNA levels since downregulation preceded a decrease in gene expression. Prolonged (>3 days) receptor downregulation may be responsible for increasing message levels and may be important in recovery of receptors following treatment. In addition, the magnitude of changes in receptor density, mRNA, and tolerance were similar in both CXBK and Swiss Webster mice, indicating that the mechanisms required for receptor regulation and its functional consequences are independent of basal mu-opioid receptor density.     

Combined treatment with LDL-apheresis, chenodeoxycholic acid and HMG-CoA reductase inhibitor for cerebrotendinous xanthomatosis

The effects of combined treatment with low-density lipoprotein (LDL)-apheresis, chenodeoxycholic acid (CDCA) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor were studied in 2 patients with cerebrotendinous xanthomatosis. Patient 1 was initially treated with LDL-apheresis alone: serum cholestanol levels decreased by 50% after each apheresis, but returned to their initial levels within 2 weeks. After an addition of CDCA administration, the serum cholestanol levels steadily decreased, resulting in slight improvement of neurological symptoms. Patient 2 received a combined treatment with LDL-apheresis, CDCA and HMG-CoA reductase inhibitor. This combination showed less LDL-apheresis-dependent fluctuation and more rapid decrease of serum cholestanol levels than those in Patient 1, resulting in improvement and stabilization of the symptoms. Our results suggest that LDL-apheresis in combination with CDCA and HMG-CoA reductase inhibitor may have beneficial effects and can be one of the treatment options.     

Differential ontogenetic patterns of levocabastine-sensitive neurotensin NT2 receptors and of NT1 receptors in the rat brain revealed by in situ hybridization

The postnatal ontogeny of the levocabastine-sensitive neurotensin receptor (NT2) mRNA was studied by in situ hybridization in the rat brain and compared with the distribution of the levocabastine-insensitive NT1 receptor. NT2 receptor mRNA was absent at birth from all brain structures except the ependymal cell layer lining the ventricles. The development of NT2 receptor mRNA followed three ontogenetic patterns. The first pattern, involving the majority of the cerebral gray matter, was characterized by a continuous increase from postnatal day 5 (P5) to P30. The second one, involving regions rich in myelinated fibers such as the corpus callosum and lacunosum moleculare layer of the hippocampus, exhibited a pronounced increase between P5 and P10, peaked at P15 and was followed by a plateau or a slight decrease. The third pattern was observed in the ependymal cell layer lining the olfactory and lateral ventricles, where the high labeling already present at birth continued to increase during development. These different developmental patterns could reflect the variety of cells expressing NT2 receptor mRNA, including neurons, protoplasmic astrocytes in gray matter, fibrous astrocytes present in myelinated fibers tracts, and ependymal cells. In contrast, NT1 receptor mRNA, which seems to be associated only with neurons, was highly and transiently expressed during the perinatal period in the cerebral cortex, hippocampus and striatal neuroepithelium. Other regions, notably the ventral tegmental area and substantia nigra compacta, exhibited a gradual increase in NT1 receptor signal, reaching adult levels by P21. Both the differential localization and ontogenetic profiles of NT1 and NT2 receptor mRNAs suggest different involvement of these two receptors in brain functions and development.     

Relationship between DRD4 polymorphism and lipid metabolism: what is the role of novelty seeking?

OBJECTIVE: This study examined the association of the dopamine receptor D4 (DRD4) gene polymorphism with the temperament dimension of novelty seeking (NS) on cardiovascular heart disease risk factors [the levels of high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides]. METHOD: From the ongoing population-based study of 'Cardiovascular Risk in Young Finns', 125 participants were DRD4 genotyped in 1997 and responded to the NS scale of the Temperament and Character Inventory in 2001. Their cholesterol and triglyceride concentrations were assessed in 2001. RESULTS: Having a 2- or 5-allele DRD4 polymorphism was related to high HDL cholesterol levels in men, but to low HDL cholesterol levels in women. NS was related to triglycerides in men and to LDL in women, but this was mediated by behavioral, age, and weight factors, and NS was not the underlying factor for the association between the polymorphism and lipids. CONCLUSION: Our preliminary findings suggest that there is a link between the dopaminergic receptor gene DRD4 and lipid metabolism, but this link is dependent on gender.     

Brain cholesterol, statins and Alzheimer's Disease

Growing evidence suggests that cellular cholesterol homeostasis is causally involved in different steps leading to pathological events in the brain of Alzheimer's Disease (AD) patients. It was previously demonstrated that the processing of the amyloid beta-peptide precursor protein (APP) is modulated by pronounced alterations in cellular cholesterol levels using statins or cholesterol extracting agents. However, a cholesterol-rich diet was found to enhance amyloid beta-peptide (Abeta) burden in the brain of transgenic mice without clearly affecting total brain cholesterol levels. Recent retrospective epidemiological studies have reported that the use of statins potentially suppresses the development of AD. Although some HMG-CoA reductase inhibitors seem to influence the central cholesterol pool in vivo, the above epidemiological findings are probably not linked to statin-induced changes in brain membrane cholesterol levels per se since not all statins active in preventing AD enter the central nervous system (CNS). Recently, we reported that different statins, regardless of their brain availability, induce alterations in cellular cholesterol distribution in the brain. Such pleiotropic, cholesterol-synthesis independent statin effects might be indirect and are possibly mediated at the blood-brain barrier (BBB) via nitric oxide (NO) or apolipoprotein E (ApoE).     

Regional expression of 5-HT1B receptor mRNA in the human brain

The regional mRNA expression pattern of 5-HT(1B) receptors has been extensively characterized in the rodent and guinea pig brain, but a detailed mapping of the 5-HT(1B) receptor mRNA expression in the human brain has not previously been performed. In the present study, the mRNA expression of 5-HT(1B) receptors was analyzed using in situ hybridization histochemistry and whole hemisphere sections of the human postmortem brain. The mRNA expression was compared with the autoradiographic distribution of 5-HT(1B) receptors. High levels of mRNA expression were found in the striatum, cortex, lateral geniculate nucleus, and raphe nuclei. The expression was higher in ventral than in dorsal striatal regions and was absent from the substantia nigra and pallidum, where high levels of 5-HT(1B) receptors were found. A layer-specific expression pattern was observed in cortical regions. The results extend previous knowledge about the localization of the 5-HT(1B) receptor in the human brain. This study provides evidence of a mismatch of the regional expression of 5-HT(1B) receptor mRNA and the 5-HT(1B) receptor distribution in human brain, similar to what has been demonstrated in other species. This is in line with the localization of this receptor subtype in nerve terminals. The results give support to species differences in the cortical mRNA expression pattern of this receptor subtype.