Steve Meaney

Oxysterols and neurodegenerative diseases.

In contrast to their parent molecule cholesterol, two of its side-chain oxidized metabolites are able to cross the blood-brain barrier. There is a concentration-driven flux of 24S-hydroxycholesterol (24S-OHC) from the brain into the circulation, which is of major importance for elimination of excess cholesterol from the brain. The opposite flux of 27-hydroxycholesterol (27-OHC) from the circulation into the brain may regulate a number of key enzymes within the brain. In vitro experiments suggest that the balance between the levels of these two molecules may be of importance for the generation of beta-amyloid peptides. In primary cultures of rat hippocampal cells 27-OHC is able to suppress expression of the activity regulated cytoskeleton-associated protein (Arc), a protein important in memory consolidation which is reduced in patients with Alzheimers disease (AD). In the present work we explore the possibility that the flux of 27-OHC from the circulation into the brain represents the missing link between AD and hypercholesterolemia, and discuss the possibility that modification of this flux may be a therapeutic strategy. Lastly, we discuss the use of oxysterols as diagnostic markers in neurodegenerative disease.

Oxysterols and Alzheimer's disease.

There is a clear link between cholesterol turnover and neurodegenerative diseases and hypercholesterolemia is an established risk factor for Alzheimers disease (AD). The failure to demonstrate a transfer of cholesterol from the circulation into the brain in humans and experimental animals makes it difficult to explain the link between hypercholesterolemia and AD. In contrast to cholesterol itself, side‐chain oxidized cholesterol metabolites such as 24S‐hydroxycholesterol and 27‐hydroxycholesterol are able to pass the blood–brain barrier (BBB). Formation of 24S‐hydroxycholesterol is the quantitatively most important mechanism for elimination of cholesterol from the brain and we recently demonstrated a significant net uptake of 27‐hydroxycholesterol by the brain from the circulation. We have also shown that patients with AD have increased brain levels of 27‐hydroxycholesterol, which may affect the production of β‐amyloid in the brain. The levels of 27‐hydroxycholesterol in the circulation are correlated with the levels of cholesterol and the possibility must be considered that the flux of 27‐hydroxycholesterol into the brain is the missing link between hypercholesterolemia and Alzheimers disease. Current knowledge about the role of the two oxysterols for cholesterol homeostasis in the brain as well as their diagnostic potential are reviewed.

Genetic connections between neurological disorders and cholesterol metabolism

Cholesterol is an essential component of both the peripheral and central nervous systems of mammals. Over the last decade, evidence has accumulated that disturbances in cholesterol metabolism are associated with the development of various neurological conditions. In addition to genetically defined defects in cholesterol synthesis, which will be covered in another review in this Thematic Series, defects in cholesterol metabolism (cerebrotendinous xanthomatosis) and intracellular transport (Niemann Pick Syndrome) lead to neurological disease. A subform of hereditary spastic paresis (type SPG5) and Huntingtons disease are neurological diseases with mutations in genes that are of importance for cholesterol metabolism. Neurodegeneration is generally associated with disturbances in cholesterol metabolism, and presence of the E4 isoform of the cholesterol transporter apolipoprotein E as well as hypercholesterolemia are important risk factors for development of Alzheimers disease. In the present review, we discuss the links between genetic disturbances in cholesterol metabolism and the above neurological disorders.

Enhanced production of 24S-hydroxycholesterol is not sufficient to drive liver X receptor target genes in vivo

Abstract.u2002 Shafaati M, Olin M, Båvner A, Pettersson H, Rozell B, Meaney S, Parini P, Björkhem I (Karolinska University Hospital Huddinge, Huddinge, Sweden; Dublin Institute of Technology, Dublin, Ireland). Enhanced production of 24S‐hydroxycholesterol is not sufficient to drive liver X receptor target genes in vivo. J Intern Med 2011; 270: 377–387.

Oxysterols and Parkinson's disease: Evidence that levels of 24S-hydroxycholesterol in cerebrospinal fluid correlates with the duration of the disease

Oxysterols are important for cholesterol homeostasis in the brain and may be affected in neurodegenerative diseases. The levels of the brain-derived oxysterol 24S-hydroxycholesterol (24S-OH) have been reported to be markedly reduced in the circulation of patients with Parkinsons disease (PD) (Lee et al., Antioxid. Redox Signal. 11 (2009) 407-420). The finding is surprising in view of the fact that other neurodegenerative diseases are associated with relatively modest effects on the circulating levels of 24S-OH. We determined the plasma and cerebrospinal fluid (CSF) levels of 24S-OH and 27-hydroxycholesterol (27-OH) in patients with PD with different disease duration using a highly accurate method based on isotope dilution-mass spectrometry. All the patients had plasma levels of the different oxysterols within the normal range. When analyzing CSF, 10% of the PD patients were found to have levels of 24S-OH above the cut-off level and interestingly there was a significant correlation between levels of 24S-OH in CSF and duration of the disease (r=0.40, P<0.05). The CSF level of 27-OH was found to be above the cut-off level in 10% of the patients, indicating a defect blood-brain barrier function. There was no correlation between levels of 27-OH in CSF and duration of the disease. These data indicates that oxysterol levels in CSF may be of value to follow disease progression.

Transcriptional regulation of cholesterol 24-hydroxylase by histone deacetylase inhibitors

The mechanistic basis for the tissue specific expression of cholesterol elimination pathways is poorly understood. To gain additional insight into this phenomenon we considered it of interest to investigate if epigenetic mechanisms are involved in the regulation of the brain-specific enzyme cholesterol 24-hydroxylase (CYP46A1), a key regulator of brain cholesterol elimination. We demonstrated a marked time-dependent derepression of the expression of CYP46A1, in response to treatment with the potent histone deacetylase (HDAC) inhibitor Trichostatin A. The pattern of expression of the genes in the genomic region surrounding CYP46A1 was found to be diametrically opposite in brain and liver. Intraperitoneal injection of HDAC inhibitors in mice led to a significant derepression of hepatic Cyp46a1 mRNA expression and tissue specific changes in Hmgcr and Cyp39a1 mRNA expression. These results are discussed in the context of the phenomenology of tissue specific cholesterol balance.

Protein Replacement Therapy Partially Corrects the Cholesterol-Storage Phenotype in a Mouse Model of Niemann-Pick Type C2 Disease

Niemann-Pick type C2 (NPC2) disease is a fatal autosomal recessive neurovisceral degenerative disorder characterized by late endosomal-lysosomal sequestration of low-density lipoprotein derived cholesterol. The breach in intracellular cholesterol homeostasis is caused by deficiency of functional NPC2, a soluble sterol binding protein targeted to the lysosomes by binding the mannose-6-phosphate receptor. As currently there is no effective treatment for the disorder, we have investigated the efficacy of NPC2 replacement therapy in a murine gene-trap model of NPC2-disease generated on the 129P2/OlaHsd genetic background. NPC2 was purified from bovine milk and its functional competence assured in NPC2-deficient fibroblasts using the specific cholesterol fluorescent probe filipin. For evaluation of phenotype correction in vivo, three-week-old NPC2 −/− mice received two weekly intravenous injections of 5 mg/kg NPC2 until trial termination 66 days later. Whereas the saline treated NPC2 −/− mice exhibited massive visceral cholesterol storage as compared to their wild-type littermates, administration of NPC2 caused a marked reduction in cholesterol build up. The histological findings, indicating an amelioration of the disease pathology in liver, spleen, and lungs, corroborated the biochemical results. Little or no difference in the overall cholesterol levels was observed in the kidneys, blood, cerebral cortex and hippocampus when comparing NPC2 −/− and wild type mice. However, cerebellum cholesterol was increased about two fold in NPC2 −/− mice compared with wild-type littermates. Weight gain performance was slightly improved as a result of the NPC2 treatment but significant motor coordination deficits were still observed. Accordingly, ultrastructural cerebellar abnormalities were detected in both saline treated and NPC2 treated NPC2 −/− animals 87 days post partum. Our data indicate that protein replacement may be a beneficial therapeutic approach in the treatment of the visceral manifestations in NPC2 disease and further suggest that neurodegeneration is not secondary to visceral dysfunction.

Unique patient with cerebrotendinous xanthomatosis. Evidence for presence of a defect in a gene that is not identical to sterol 27-hydroxylase

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder believed to be exclusively caused by mutations in the CYP27A1 gene coding for the enzyme sterol 27‐hydroxylase. Common findings in CTX are tendon xanthomas, cataracts and progressive neurological dysfunction. Here, we characterize an adult female patient with tendon xanthomas and classic biochemical findings of CTX (i.e. high levels of bile alcohols and cholestanol and extremely low levels of 27‐hydroxycholesterol in plasma). Additionally, sterol 27‐hydroxylase activity in cultured monocyte‐derived macrophages from this patient was <5% of normal. Sequencing the CYP27A1 gene uncovered that the patient is heterozygous for two previously undescribed base substitutions in exon 8, C478A and C479A, which are expected to affect the haeme‐binding domain of the enzyme. When expressed in HEK293 cells, the corresponding protein had only 8% of normal enzymatic activity. No other mutation was found in the open reading frame of the CYP27A1 gene, intron–exon boundaries or in the 5′‐untranslated region up to 5000u2003bp distal to the translational start site. Sequencing mRNA isolated from leucocytes from the patient revealed a 1u2003:u20031 ratio of mutated and nonmutated species, with total mRNA levels that were not significantly different from the controls. It is concluded that the patient is heterozygous for two mutations affecting one allele of the CYP27A1 gene and with at least one additional yet undefined gene that is of critical importance for the activity of sterol 27‐hydroxylase.

Is C-26 hydroxylation an evolutionarily conserved steroid inactivation mechanism?

Sterols are essential components of virtually all higher eukaryotic organisms, though the exact identity of the dominating sterol varies between species, from the C‐27 of cholesterol in vertebrates to the C‐28 and C‐29 sterols of plants and invertebrates. In addition to their role as structural components of cell membranes these sterols are also converted into a variety of biologically active hormones. This conversion generally involves modifications of the basic structure of the sterol by dealkylation, hydroxylation and/or isomerization. Recent studies have demonstrated that irreversible inactivation of both plant and insect hormones is achieved by a specific C‐26 hydroxylation. The concept of sterol deactivation by 26‐hydroxylation appears to be an example of an evolutionarily conserved mechanism that has persisted despite the widely varying requirements for sterols in the species where it has been detected. Meaney, S. Is C‐26 hydroxylation an evolutionarily conserved steroid inactivation mechanism? FASEB J. 19, 1220–1224 (2005)

Epigenetic Regulation of Cholesterol Homeostasis

Although best known as a risk factor for cardiovascular disease, cholesterol is a vital component of all mammalian cells. In addition to key structural roles, cholesterol is a vital biochemical precursor for numerous biologically important compounds including oxysterols and bile acids, as well as acting as an activator of critical morphogenic systems (e.g., the Hedgehog system). A variety of sophisticated regulatory mechanisms interact to coordinate the overall level of cholesterol in cells, tissues and the entire organism. Accumulating evidence indicates that in additional to the more “traditional” regulatory schemes, cholesterol homeostasis is also under the control of epigenetic mechanisms such as histone acetylation and DNA methylation. The available evidence supporting a role for these mechanisms in the control of cholesterol synthesis, elimination, transport and storage are the focus of this review.