Neuronal expression and subcellular localization of cholesterol 24-hydroxylase in the mouse brain

Cholesterol 24-hydroxylase is a cytochrome P450 (CYP46A1) that is selectively expressed in the brain and is responsible for the majority of cholesterol turnover in the central nervous system. Mice deficient in 24-hydroxylase exhibit impaired learning and defective hippocampal long-term potentiation, suggesting that the metabolism of cholesterol by this enzyme is required for learning and memory formation. To determine where in the neuron cholesterol turnover was taking place, monoclonal antibodies directed against 24-hydroxylase were generated by immunization of mice with recombinant protein and used to detect the enzyme in brain homogenates, cultured neurons, and histological sections. 24-Hydroxylase was localized to the endoplasmic reticulum and was distributed throughout the cell bodies and dendrites of multiple types of neurons; the enzyme was not detected in axon terminals or in the cells of 24-hydroxylase knockout mice. 24-Hydroxylase was highly expressed in pyramidal neurons of the hippocampus and cortex, in Purkinje cells of the cerebellum, and in hippocampal and cerebellar interneurons. Within the retina, 24-hydroxylase was detected in ganglion cells and some but not all cells of the inner nuclear layer. These findings reveal the microsomal localization of 24-hydroxylase and provide subcellular insight into cholesterol turnover in the brain.     

Cholesterol metabolism in the brain: importance of 24S-hydroxylation

During the last three to four decades there has been an increasing interest in the interaction of circulating and brain cholesterol. Recent in vivo and in vitro studies have furthered our knowledge of cholesterol metabolism in the central nervous system (CNS). As the CNS matures and cholesterol pools in the brain become constant, the rate of de novo synthesis of cholesterol in the brain markedly declines. Besides some excretion of apoE-bound cholesterol via the CSF, another quantitatively more important mechanism has been described – the conversion of cholesterol into 24S-hydroxycholesterol, that is, in contrast to cholesterol, able to traverse the blood–brain barrier (BBB). The enzyme (CYP46a1) mediating this conversion has been characterized at the molecular level and is mainly located in neurons. Like other oxysterols, 24S-hydroxycholesterol is efficiently converted into normal bile acids or excreted in bile in its sulfated and glucuronidated form. Within the last 10 years the interest in studying the mechanisms of this and other cholesterol transport systems has increased and the results from these in vivo and in vitro investigations are reviewed.     

24S-hydroxycholesterol in relation to disease manifestations of acute experimental autoimmune encephalomyelitis

Levels of the brain-specific cholesterol metabolite 24S-hydroxycholesterol are proposed as possible biomarkers for multiple sclerosis (MS). It is not yet clear for which aspect of the MS disease manifestations 24S-hydroxycholesterol is a reflection. We studied the relation of serum levels of 24S-hydroxycholesterol and other sterols to the disease characteristics of acute experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Serum was analyzed for cholesterol precursors, oxysterols, and plant sterols during the course of disease development. Significantly increased levels of the cholesterol metabolites 24S-hydroxycholesterol and 27-hydroxycholesterol were observed on day 9, before the onset of clinical signs. The serum levels of these oxysterols gradually increased up to 193% and 415%, respectively, at day 17, when clinical symptoms had recovered. Total cholesterol levels were slightly but significantly decreased on day 9 and day 17 in treated animals. Serum levels of cholesterol precursors and plant sterols decreased gradually from day 11 and day 14, respectively. Immunostaining of the 24S-hydroxycholesterol-forming enzyme Cyp46 was shown in macrophage infiltrates. In vitro experiments confirmed the presence of Cyp46 in macrophages and showed a decreased expression after LPS treatment. The data indicate that changes in serum oxysterols occur early in EAE and can be formed by macrophages. These early changes indicate an important role for oxysterols in the development of EAE.     

Distribution and regulation of estrogen-2-hydroxylase in the quail brain

The anatomical distribution and endocrine regulation of the estrogen-2-hydroxylase activity were investigated in the brain of adult male and female Japanese quail. Significant levels of enzymatic activity were detected in all brain regions that were studied, but the highest levels were observed in preoptic and hypothalamic brain nuclei that are known to contain high levels of aromatase activity. These data are consistent with previous results suggesting that the placental aromatase is also responsible for the estrogen-2-hydroxylase activity. However, there is a marked sex difference and a control by T of aromatase activity in the quail brain, and no such difference in 2-hydroxylase activity could generally be detected except in the VMN. Further studies will be needed to know whether the previously published conclusions concerning the human placenta also apply to the brain. The present data are consistent with the idea that estrogens formed locally in the brain by testosterone aromatization could affect reproduction by interfering with the catecholaminergic transmission after being metabolized into catechol-estrogens.     

Reserpine effect on the axonal transport of dopamine-β-hydroxylase and tyrosine hydroxylase in rat brain

Selective, unilateral lesions with 6-hydroxydopamine in the rostral forebrain of rats were used to interrupt ascending noradrenergic axons to produce an accumulation of dopamine-β-hydroxylase (D-β-H) and tyrosine hydroxylase (TH) activities proximal to the lesion. The difference between endogenous plus accumulated enzyme activity on the D-β-H-damaged side, and endogenous activity on the nondamaged side was used as an index of axonal transport. Both D-β-H and TH activities accumulated linearly for 48 h in the anterior hypothalamic sections proximal to such lesions. While D-β-H activity decreased toward baseline over the next 4 days, TH activity remained at the 48-h levels, suggesting a faster turnover time of D-β-H activity at the lesion site. Accumulation of both enzymes was totally blocked by ipsilateral 6-hydroxydopamine lesions in the ascending dorsal and ventral noradrenergic bundles, suggesting that dopaminergic fibers did not contribute significantly to the accumulation of TH activity. Reserpine induced D-β-H and TH production in noradrenergic neurons of the locus coeruleus was followed, after a delay of days, by increased levels in distally innervated regions. Single-dose reserpine (5 mg/kg, i.p.) administration produced transient and sporadic decreases in the axonal transport of D-β-H and TH during the first 5 days after injection, followed by a steady increase to 693% of control for D-β-H at 7 days and 270% at 8 days for TH. Hypothalamic enzyme activities were unchanged until the 5th day when D-β-H activities decreased and then, in association with increased axonal transport, D-β-H increased steadily to a peak of 160% at 6 days and TH values to 270% of control on the 8th postreserpine day. Axonal transport of D-β-H returned to baseline at 22 days and TH transport returned to baseline at 15 days and was increased by 40% at 22 days. These results suggest that although reserpine induces the formation of new enzyme in noradrenergic cell bodies in 1 to 3 days, the subsequent appearance of increased enzyme activity in terminal regions innervated by these neurons is delayed due to a delay in the onset of increased enzyme transport from the cell bodies.     

Oxygen dependence of dopamine-β-hydroxylase activity and lactate metabolism in synaptosomes from rat brain

Dopamine-β-hydroxylase activity can be assayed in vitro in suspensions of rat hypothalamic synaptosomes by following tritium release from [2-³H]dopamine in the presence of monoamine oxidase inhibitors. The activity is inhibited by diethyldithiocarbamate, fusaric acid, amphetamine, reserpine, and desipramine. It is not sensitive to addition of tyrosine at the concentration present in cerebrospinal fluid, to added fumarate or to a rat heart extract known to contain potent inhibitors of the isolated enzyme. The reaction in air follows simple saturation kinetics with respect to variation in dopamine concentration with aK_m of 0.075 μM. There is reversible inhibition of activity when it is assayed at low oxygen tension. The apparent oxygen affinity is such that the enzyme does not appear saturated with respect to oxygen even at arterial oxygen tensions. Other reactions assayed exhibit less sensitivity to hypoxia: production of lactic acid from glucose becomes stimulated at 10–12 mm Hg; and oxidation of lactic acid is not inhibited at oxygen tensions above 3–4 mm Hg. Comparison of these data with the distribution of oxygen tensions present in brain suggests a basis for classifying oxygen dependent neurochemical reactions.     

24S-hydroxycholesterol: a marker of brain cholesterol metabolism

The enzymatic conversion of CNS cholesterol to 24S-hydroxycholesterol, which readily crosses the blood-brain barrier, is the major pathway for brain cholesterol elimination and brain cholesterol homeostasis maintenance. The enzyme mediating this conversion has been characterized at the molecular level (CYP46) and is mainly located in neurons. Like other oxysterols, 24S-hydroxycholesterol is efficiently converted into normal bile acids or excreted in bile in its sulfated and glucuronidated form. Levels of 24S-hydroxycholesterol in the circulation decrease with age in infants and children. In adults, however, the levels appear to be stable. There is accumulating evidence pointing toward a potentially important link between cholesterol, beta-amyloid, and Alzheimer's disease. Concentrations of 24S-hydroxycholesterol in plasma and cerebrospinal fluid (CSF) are significantly higher in Alzheimer's disease and vascular demented patients at early stages of the disease compared to healthy subjects. Variations in genetic background, time of disease onset, and severity of dementia are potential sources of variance. Inhibitors of cholesterol biosynthesis, also termed statins, seem to have a reductive influence on the generation of the amyloid precursor protein, the neuronal secretion of beta-amyloid, and on de novo cholesterol synthesis. Recent epidemiological studies indicate that the prevalence of diagnosed AD and vascular dementia is reduced among people taking statins for a longer period of time. High-dose simvastatin treatment (80 mg/day) in patients with hypercholesterolemia leads to a significant decrease in brain-specific serum 24S-hydroxycholesterol concentrations and indicates a diminished cholesterol metabolism in the brain. CSF levels of cholesterol and lathosterol, a cholesterol precursor considered to be an indicator for cholesterol neogenesis, were significantly decreased in statin-treated subjects compared to non-treated normo- and hypercholesterolemic subjects. Also, CSF concentrations of 24S-hydroxycholesterol were significantly lower in statin-treated patients compared to normocholesterolemic subjects. Treatment with high-dose simvastatin in normocholesterolemic Alzheimer patients for 26 weeks at early stages of the disease results in a significant decrease in Abeta-levels in cerebrospinal fluid. This decrease correlates with the reduction of 24S-hydroxycholesterol.     

Distribution of the histamine H(2) receptor in monkey brain and its mRNA localization in monkey and human brain

The distribution of histamine H(2) receptor mRNA was determined by in situ hybridization histochemistry in human and monkey brain. In the case of monkey brain, we combined this technique with receptor ligand autoradiography to compare the distribution of mRNA and receptor binding sites. [(125)I]Iodoaminopotentidine ([(125)I]-APT), a reversible, high specific activity antagonist with high affinity and selectivity for the H(2) receptor, was used for receptor autoradiography. Radiolabeled oligonucleotides derived from the human mRNA sequence encoding this receptor were used as hybridization probes. The highest density of the H(2) receptor mRNA in human and monkey brain was found in caudate and putamen nuclei and external layers of cerebral cortex. Moderate levels were seen in the hippocampal formation and lower densities in the dentate nucleus of cerebellum. Areas such as globus pallidus, amygdaloid complex, cerebellar cortex, and substantia nigra were devoid of hybridization signal. The distribution of H(2) receptor mRNA in monkey brain is generally in good agreement with that of the corresponding binding sites: prominent in caudate, putamen, accumbens nuclei, and cortical areas. The hippocampus showed lower densities of receptors and low levels were detected in the globus pallidus pars lateralis. No binding sites were seen in amygdaloid complex and substantia nigra. The distribution of histaminergic innervation is in good correlation with the areas of high density for H(2) receptors: caudate, putamen, and external layers of cerebral cortex in monkey and human brain. The presence of mRNA in caudate and putamen nuclei, together with its absence from substantia nigra, suggests that the H(2) receptors found in the striatum are synthesized by intrinsic cells and not by nigral dopaminergic cells. These striatal H(2) receptors may be located on short circuit striatal interneurons or somatodendritically on striatal projection neurons which project to the globus pallidus pars lateralis. In conclusion, the present results, which constitute, to our knowledge, the first report of the regional distribution of mRNA encoding H(2) receptors detected by in situ hybridization, define the sites of synthesis of H(2) receptors and are the basis for future, more detailed studies that should result in a better understanding of H(2) receptor function.     

Plasma HDL cholesterol in epileptics with elevated triglyceride and cholesterol

Due to the metabolic changes induced, e.g. in the liver by anti-epileptic drugs, the significance of high serum triglyceride and cholesterol in epileptics was studied and the plasma HDL cholesterol level was compared in 190 epileptic patients with elevated or normal triglyceride and cholesterol, with the corresponding values in 43 healthy subjects. One-third of the epileptic patients showed elevated plasma HDL cholesterol levels. Female epileptics had higher plasma HDL cholesterol than the normolipidemic healthy subjects. Epileptics with elevated triglyceride or a combination of elevated triglyceride and cholesterol had a lower plasma HDL cholesterol level than normolipidemic patients with epilepsy. HDL cholesterol level in epileptics with high serum cholesterol did not diverge from the level in epileptics with normal serum triglyceride and cholesterol. The results show significant differences of plasma HDL cholesterol between epileptic patients and normolipidemic healthy controls on one hand, and between epileptics with altered and epileptics with normal serum lipid levels on the other. The findings suggest that the increase of plasma HDL cholesterol level in epileptics undergoing anticonvulsant treatment is influenced by endogenous triglyceride metabolism.     

Early nutritional changes modify the kinetics and phosphorylation capacity of tryptophan-5-hydroxylase

Gestational malnutrition induces an acceleration of the serotonin biosynthetic pathway in the developing brain with an increase in brain L-tryptophan (L-Trp), tryptophan-5-hydroxylase (TrpOH) activity and serotonin content. In the present work we report results on the possible mechanism of TrpOH activation. Kinetic experiments were done with different L-Trp concentrations in the rat brain at different ages. Also various phosphorylating conditions of the enzyme were tested in order to compare its activation in developmentally malnourished and normal brains. The results showed lower K_m values and no changes in the V_max in the malnourished as compared to controls. Interestingly, in the malnourished group, TrpOH showed an increased activity under the phosphorylating conditions employed. We propose that in the activation of brain TrpOH by developmental malnutrition, not only is an elevation of L-Trp involved, but also a change in the enzyme itself reflected in a higher affinity for L-Trp and in a greater response to phosphorylation. This allows us to propose the possibility that early chronic malnutrition induces structural changes in the enzymatic molecule.     

Normal plasma dopamine- β -hydroxylase in non-treated and treated Parkinson patients

Plasma dopamine- β -hydroxylase activity (DBH) was estimated in 50 drug-free Parkinson patients, 30 of whom had never been treated, in 26 patients treated chronically with L-Dopa, and in 44 controls. 22 of the drug-free patients were examined again after two weeks treatment with L-Dopa plus decarboxylase inhibitor. In disagreement with other reports, no differences were found among the groups and no change in the enzyme activity occurred after treatment, although urinary homovanillic acid increased 20 fold. Low DBH activities could not be attributed to the age of onset or duration of illness, main symptom, depression, autonomic disturbances, or duration of drug treatment. The only difference was found in patients with dementia associated to their illness, who showed low enzyme activities.     

Descending projections of the locus coeruleus and subcoeruleus/medial parabrachial nuclei in monkey: Axonal transport studies and dopamine-β-hydroxylase immunocytochemistry

Spinal projections originating in the dorsolateral pons in the ventral locus coeruleus and in the subcoeruleus and medial parabrachial nuclei were identified in monkeys (Macaco fascicularis) using the retrograde horseradish peroxidase tracing technique. Anterograde autoradiographic tracing studies were then carried out to determine the brain stem and spinal cord terminations of the neurons. Finally, results obtained with the axonal transport tracing methods were compared with the pattern of staining for noradrenergic cells and terminals revealed immunocytochemically with an antibody to dopamine-β-hydroxylase (DβH), the synthesizing enzyme for norepinephrine. The major findings of these studies are that two presumed noradrenergic cell groups of the dorsolateral pons, one corresponding to the nucleus locus coeruleus, the second to the subcoeruleus/medial parabrachial nuclei, give rise to descending project-ions. They differ significantly in their patterns of termination in the lower brain stem and spinal cord. Among the major terminations of the locus coeruleus pathway are projections to parasympathetic neurons of the dorsal motor nucleus of the vagus, the region of the nucleus ambiguus, and the sacral spinal cord. The terminations of the descending subcoeruleus/medial parabrachial pathway, in contrast, include project-ions to sympathetic preganglionic neurons of the intermediolateral cell column of the thoracic cord and heavier projections to somatic cranial nerve nuclei. Both pathways have additional widespread and bilateral terminations in various nuclei of the reticular formation, in the spinal dorsal horn (including the marginal zone), in the region around the central canal and in the ventral spinal gray matter. Since the origins and terminations of both these pathways correspond closely to the locations and patterns of terminations of noradrenaline-containing neurons, demonstrated here with DβH immunocytochemistry, norepinephrine (or epinephrine) is suggested to be the transmitter in both these descending systems.     

Identification of catecholamine cell bodies in the pons and pons-mesencephalon junction of the cat brain, using tyrosine hydroxylase and dopamine-β-hydroxylase immunohistochemistry

Using a double labeling immunohistochemical method with antibodies to tyrosine hydroxylase and dopamine-beta-hydroxylase, we reexamined the topographical distribution of dopaminergic (DA) and noradrenergic (NA) cells in the pons-mesencephalon junction of the cat. Besides DA cells of SN and its extensions, DA cells were also observed more caudally in nucleus raphe linearis intermedius and dorsalis, decussation of brachium conjunctivum, fasciculus longitudinalis medialis and periaqueductal gray. In nucleus locus coeruleus, cells exhibited variable levels of DBH, but dopaminergic cells were not evidenced in this structure.     

Cytochrome P4502D6 (debrisoquine 4-hydroxylase) and Parkinson's disease in Chinese and Caucasians

Four polymorphic sites (C/T188, C/T2938, G/C4268, G/A1934) in the cytochrome P4502D6 (debrisoquine 4-hydroxylase) gene were investigated for their association with sporadic Parkinson's disease (PD). Three mutant alleles (C/T188, C/T2938 and G/C4268) result in amino acid changes which could alter the substrate specificity or alter its ability to metabolize their substrates; the fourth (G/A1934) causes a loss of enzyme activity. The study was carried out in two ethnically homogenous populations: Chinese (123 PD patients, 124 controls); and Caucasian (95 PD patients, 62 controls). Haplotype status, which took into account amino acid changes at three polymorphic sites, was deduced from genotyping results in order to investigate whether substrate specificity was important rather than loss of enzyme activity. There was no gender difference in the distribution of the alleles in either race. There was, however, significant association among the three polymorphic sites (C/T188, C/T2938, G/C4268) in both ethnic groups. T/T188:C/C2938:C/C4268 is the most common genotype in the Chinese population, in contrast to C/C188:C/T2938:C/G4268 (followed by C/C188:C/C2938:G/G4268) in Caucasians. All 69 of the sub-group of Chinese patients tested were homozygous for the wild-type allele at the G/A1934 polymorphic site. Neither the CYP2D6 allele nor haplotype was associated with PD in either ethnic group.     

Distribution of tyrosine hydroxylase, dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase activities in coronal sections of the rat lower brainstem

The activities of the three major catecholamines synthesizing enzymes were determined on supernatants from microcubes dissected out from 500 μm thick coronal sections of the rat lower brainstem. The microcubes exhibiting significant or high enzymatic activities represented only one-third of the microcubes analyzed and were generally localized either in the dorso-medial or in the ventro-lateral region of the lower brainstem. In the ventro-lateral region, referred to as the A₁-C₁ region, the rostro-caudal distribution of the enzymatic activities exhibited a small difference between the maximal TH and DBH activities and the maximum PNMT activity located 500 μm more rostrally. Conversely, in the dorso-medoal region, referred to as the A₂-C₂ region, there was a much larger rostro-caudal difference (about 1500 μm) between the peaks of TH and DBH activities and the more rostral peak of PNMT activity. Such a distribution suggests a separation between the adrenaline (A) and the noradrenaline (NA) containing neurons within the dorso-medial region. According to the results of this study, a preferential microdissection of the A versus NA neurons seems possible in the A₂–C₂ region of the rat medulla oblongata.     

Polymorphisms of the cholesterol 24-hydroxylase (CYP46A1) gene and the risk of Alzheimer's disease in a Chinese population

BACKGROUND: An increasing number of studies have suggested a link between cholesterol metabolism and Alzheimer's disease (AD), which may be mediated by its effect on amyloid processing. Intracranial cholesterol is primarily eliminated into the bloodstream through conversion into 24-hydroxycholesterol by the enzyme cholesterol 24-hydroxylase (encoded by the CYP46A1 gene). CYP46A1 is an essential gene modulating cholesterol metabolism in the brain. METHOD: To investigate whether polymorphisms in the CYP46A1 gene modulate the risk of AD, we studied four common polymorphisms (IVS1-192, IVS2-150, IVS3-128 and IVS4-122) in 182 Chinese AD patients and 179 age-matched healthy Chinese subjects. RESULTS AND CONCLUSION: We found that the IVS3-128 polymorphism was associated with the risk of AD (p < 0.05). Subjects homozygous for the C alleles were protected from AD with an adjusted odds ratio (OR) of 1.53 [95% confidence interval (95% CI) 0.98-2.37, p = 0.047]. However, another minor allele, IVS1-192 C, was more prevalent in the AD group and was associated with an increased risk. Haplotype analysis revealed that two of the eight common haplotypes formed by the four polymorphisms were rarely found in the AD group, suggesting a protective effect of these two haplotypes (GTCA and CCTA). The results supported the involvement of the CYP46A1 gene and cholesterol metabolism in the pathogenesis of AD.     

Inhibition of LH and prolactin release in the cycling rat following inhibition of dopamine-ß-hydroxylase

The effect of fusaric acid, an inhibitor of dopamine-ß-hydroxylase (DBH), on luteinizing hormone (LH) and prolactin levels during the estrous cycle was determined.Fusaric acid was found to cause a selective dose- and time-dependent inhibition of DBH activity in the medial basal hypothalamus without altering tyrosine hydroxylase activity. When DBH was inhibited during the afternoon of diestrus, the proestrous surges of both LH and, to a lesser extent, prolactin were inhibited. These results suggest that noradrenergic neuronal activity in the mediobasal hypothalamus is required during afternoon preceding proestrus in order for the LH and prolactin surges to occur. It is possible that the rise in serum estrogen during late diestrus 2 is blocked by fusaric acid treatment. This estrogen increase is necessary for LH and prolactin surges to occur during proestrus. When DBH was inhibited during the afternoon of proestrus, the LH and prolactin surges were completely eliminated. This indicates that noradrenergic neuronal activity in the mediobasal hypothalamus during the afternoon of proestrus is important for both the LH and prolactin surges to occur.