Valerio Leoni

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.

Progressive dysfunction of the cholesterol biosynthesis pathway in the R6/2 mouse model of Huntington's disease

We have recently reported significantly reduced levels of the mRNA of genes critical for the cholesterol biosynthesis pathway in the brains of mice and patients with Huntingtons disease (HD), which are indicative of a biological dysfunction. We here show that the brains of R6/2 transgenic mice have progressively decreasing levels of the cholesterol precursors, lathosterol and lanosterol, and declining 3-hydroxy-3-methylglutaryl coenzyme A reductase activity starting from pre-symptomatic stages. We also show that, despite the progressive reduction of brain cholesterol biosynthesis, steady-state levels of total cholesterol remain constant, thus suggesting that compensatory mechanisms are in operation. These in vivo findings indicate a consistent and progressive reduction in the activity of the cholesterol biosynthesis pathway in HD brain. The defect occurs early in these mice and generates lower levels of newly synthesized cholesterol and its intermediates, which may affect different aspects of the disease.

Metabolic consequences of mitochondrial coenzyme A deficiency in patients with PANK2 mutations.

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare, inborn error of metabolism characterized by iron accumulation in the basal ganglia and by the presence of dystonia, dysarthria, and retinal degeneration. Mutations in pantothenate kinase 2 (PANK2), the rate-limiting enzyme in mitochondrial coenzyme A biosynthesis, represent the most common genetic cause of this disorder. How mutations in this core metabolic enzyme give rise to such a broad clinical spectrum of pathology remains a mystery. To systematically explore its pathogenesis, we performed global metabolic profiling on plasma from a cohort of 14 genetically defined patients and 18 controls. Notably, lactate is elevated in PKAN patients, suggesting dysfunctional mitochondrial metabolism. As predicted, but never previously reported, pantothenate levels are higher in patients with premature stop mutations in PANK2. Global metabolic profiling and follow-up studies in patient-derived fibroblasts also reveal defects in bile acid conjugation and lipid metabolism, pathways that require coenzyme A. These findings raise a novel therapeutic hypothesis, namely, that dietary fats and bile acid supplements may hold potential as disease-modifying interventions. Our study illustrates the value of metabolic profiling as a tool for systematically exploring the biochemical basis of inherited metabolic diseases.

Are the CSF levels of 24S-hydroxycholesterol a sensitive biomarker for mild cognitive impairment?

There is a need for effective biomarkers showing whether or not a patient with mild cognitive impairment (MCI) will progress to Alzheimers disease (AD) with dementia. At the present three cerebrospinal fluid (CSF) biomarkers are in general use: total tau, phospho-tau and beta-Amyloid. These markers are regarded to have high capacity to differentiate early AD from normal ageing. We have analysed CSF levels of a new marker for neuronal degeneration, 24S-hydroxycholesterol (24OHC) in patients with MCI. For reasons of comparison, we also analysed these levels in patients with AD. There was a significant correlation between CSF levels of 24OHC and total tau (as well as phospho-tau) in both groups of patients. Fifty percent of the patients contemplated for MCI were found to have elevated levels of 24OHC (using a 95th upper percentile set cut-off). All the MCI patients with normal levels of 24OHC had normal levels of the other markers. In patients with AD, the percentages of those with increased levels of 24OHC, tau and phospho tau were similar (55-67%). In this pilot study, we discuss the possibility that 24OHC may be a sensitive test for MCI.

Plasma levels of 24S-hydroxycholesterol reflect brain volumes in patients without objective cognitive impairment but not in those with Alzheimer's disease.

OBJECTIVES Cholesterol has been linked to Alzheimers disease (AD) and plasma 24S-hydroxycholesterol (24OHC) has been suggested as a surrogate marker for brain cholesterol metabolism. This study investigates the relation of 24OHC as well as markers of extracerebral cholesterol homeostasis (lanosterol, lathosterol, cholesterol, LDL-C, HDL-C and 27-hydroxycholesterol) with brain volumes in memory clinic patients. METHODS 96 patients (33 with subjective cognitive impairment--SCI; 36 with mild cognitive impairment--MCI; 27 with AD) referred to the Memory Clinic at Karolinska University Hospital, Sweden. Plasma assessments were done by isotope dilution-mass spectrometry. MRI measurements were done using custom-made software BMAP (imaging laboratory, Karolinska Institutet), running on HERMES platform. RESULTS Ratios of 24-hydroxycholesterol, 27-hydroxycholesterol, lanosterol and lathosterol to cholesterol (R_24OHC, R_27OHC, R_lanosterol and R_lathosterol) were significantly lower in patients with AD. In the whole population, after controlling for age, sex, APOE genotype and statins, R_24OHC was positively related to gray matter (GM) fraction. However, when groups were considered separately, the relation to GM volume, GM and parenchymal fractions was significant in the SCI group only (p<0.05). There was a significant positive association between cholesterol and white matter (WM) volume, WM and parenchymal fractions in patients with AD. CONCLUSIONS Plasma R_24OHC was lower in patients with AD, but R_24OHC was significantly related to brain volumes in the control group only. One reason may be the previously demonstrated abnormal expression of cholesterol 24S-hydroxylase in astrocytes in AD, which may limit the usefulness of this plasma marker in this specific disease. The findings on cholesterol agree with previous reports of decreasing plasma cholesterol levels in AD patients, suggesting a CNS-mediated effect on extracerebral cholesterol homeostasis.

Levels of ApoE in cerebrospinal fluid are correlated with Tau and 24S-hydroxycholesterol in patients with cognitive disorders.

Evidence was recently presented from in vitro studies that 24S-hydroxycholesterol acts as a signalling molecule inducing apoE-mediated cholesterol efflux from astrocytoma cells, and that there is a direct effect of the oxysterol on apoE transcription, protein synthesis and secretion. Consistent with this mechanism, a significant correlation is demonstrated here between levels of apoE and 24S-hydroxycholesterol in cerebrospinal fluid from patients with Alzheimers disease and patients with mild cognitive impairment. Such a correlation was not found in control patients. There was no correlation between levels of apoE and cholesterol in cerebrospinal fluid from controls. The results are consistent with a close coupling between release of 24S-hydroxycholesterol and apoE secretion under conditions with neuronal degeneration. The levels of apoE in cerebrospinal fluid were also correlated to the levels of Tau and the possibility is discussed that the level of apoE in cerebrospinal fluid may be used as a marker of neurodegeneration.

The impairment of cholesterol metabolism in Huntington disease.

Huntington disease (HD), an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG trinucleotide repeat in the Huntingtin (HTT) gene, is characterized by extensive neurodegeneration of striatum and cortex and severe diffuse atrophy at MRI. The expression of genes involved in the cholesterol biosynthetic pathway and the amount of cholesterol, lanosterol, lathosterol and 24S-hydroxycholesterol were reduced in murine models of HD. In case of HD-patients, the decrease of plasma 24OHC follows disease progression proportionally to motor and neuropsychiatric dysfunction and MRI brain atrophy, together with lanosterol and lathosterol (markers of cholesterol synthesis), and 27-hydroxycholesterol. A significant reduction of total plasma cholesterol was observed only in advanced stages. It is likely that mutant HTT decreases the maturation of SREBP and the up-regulation LXR and LXR-targeted genes (SREBP, ABCG1 and ABCG4, HMGCoA reductase, ApoE) resulting into a lower synthesis and transport of cholesterol from astrocytes to neurons via ApoE. In primary oligodendrocytes, mutant HTT inhibited the regulatory effect of PGC1α on cholesterol metabolism and on the expression of MBP. HTT seems to play a regulatory role in lipid metabolism. The impairment of the cholesterol metabolism was found to be proportional to the CAG repeat length and to the load of mutant HTT. A dysregulation on PGC1α and mitochondria dysfunction may be involved in an overall reduction of acetyl-CoA and ATP synthesis, contributing to the cerebral and whole body cholesterol impairment. This article is part of a Special Issue entitled Brain Lipids.

Links between ApoE, brain cholesterol metabolism, tau and amyloid β-peptide in patients with cognitive impairment

Brain neurons remove the excess of cholesterol via conversion into the more polar 24OHC [(24S)-hydroxycholesterol]. 24OHC acts as a signalling molecule inducing ApoE (apolipoprotein E)-mediated cholesterol efflux from astrocytes, by a direct effect on ApoE transcription, protein synthesis and secretion. In CSF (cerebrospinal fluid) collected form from patients with cognitive impairment (Alzheimers disease and patients with mild cognitive impairment) the levels of ApoE, tau, p-tau (hyperphosphorylated tau) were significantly increased, together with 24OHC, compared with controls. We also found that the levels of tau and p-tau were significantly correlated with ApoE and 24OHC in the same samples. Such a correlation was not found in control patients. Increased levels of cholesterol in membranes and impairment in brain cholesterol metabolism were found to be involved both in APP (amyloid precursor protein) processing and amyloid beta-peptide deposition and, recently, in tau pathology. The CSF tau levels are considered to be related to the neurodegenerative process in Alzheimers disease. During neurodegeneration, the cholesterol accumulated in neurons is converted into 24OHC. The release of 24OHC from neurons induces ApoE secretion by astrocytes, and both are related to the intensity of the neurodegenerative process and neuronal injury. ApoE can also be involved in the scavenging of tau from neurons. The direct correlations between ApoE, 24OHC and tau suggest that cholesterol metabolism may be involved in generation of both tau and amyloid beta-peptide and that the ApoE is released by astrocytes in order to counteract this ongoing process.

High levels of 15-oxygenated steroids in circulation of patients with multiple sclerosis: fact or fiction?

15-Oxygenated cholesterol species such as 5α-cholest-8(14)ene-3β,15α-diol (15HC) and 3β-hydroxy-5α-cholest-8(14)-en-15-one (15KC) are commercially available synthetic products unlikely to occur in biological systems. Surprisingly, Farez et al. recently reported that these two steroids occur in human circulation at levels considerably higher than those of any other endogenous oxysterol [Farez, M. et al. 2009. Toll-like receptor 2 and poly(ADP-ribose) polymerase 1 promote central nervous system neuroinflammation in progressive EAE. Nat. Immunol. 10: 958–964]. The levels were reported to be increased in patients with multiple sclerosis in a progressive phase and the authors suggested that this could be utilized diagnostically. Based on extensive in vitro experiments exposing cells to the same high levels of 15HC as found in vivo (1000 ng/ml) the authors concluded that 15HC may be an important pathogenetic factor in multiple sclerosis. Using combined gas chromatography-mass spectrometry we fail to detect significant plasma levels of 15HC either in healthy controls or in patients with multiple sclerosis (levels < 2 ng/ml). If 15KC is present in these plasma samples, the concentration of it must be <10 ng/ml. Our failure to detect significant levels of the above steroids could not be due to loss during hydrolysis and work-up because recovery of the added two oxysterols was close to 100%. Autoxidation of lipoprotein-bound cholesterol resulted in extensive conversion of cholesterol into 7-oxygenated but not 15-oxygenated sterols. We conclude that if present there are trace amounts only of the above 15-oxygenated steroids in human circulation and that the role of such oxysterols as pathogenetic factors and biomarkers must be reconsidered.

Cholesterol‐loaded nanoparticles ameliorate synaptic and cognitive function in Huntington's disease mice

Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntingtons disease (HD), suggesting that locally synthesized, newly formed cholesterol is less available to neurons. This may be detrimental for neuronal function, especially given that locally synthesized cholesterol is implicated in synapse integrity and remodeling. Here, we used biodegradable and biocompatible polymeric nanoparticles (NPs) modified with glycopeptides (g7) and loaded with cholesterol (g7‐NPs‐Chol), which per se is not blood–brain barrier (BBB) permeable, to obtain high‐rate cholesterol delivery into the brain after intraperitoneal injection in HD mice. We report that g7‐NPs, in contrast to unmodified NPs, efficiently crossed the BBB and localized in glial and neuronal cells in different brain regions. We also found that repeated systemic delivery of g7‐NPs‐Chol rescued synaptic and cognitive dysfunction and partially improved global activity in HD mice. These results demonstrate that cholesterol supplementation to the HD brain reverses functional alterations associated with HD and highlight the potential of this new drug‐administration route to the diseased brain.