Zeljka Korade

Lipid rafts, cholesterol, and the brain

Lipid rafts are specialized membrane microdomains that serve as organizing centers for assembly of signaling molecules, influence membrane fluidity and trafficking of membrane proteins, and regulate different cellular processes such as neurotransmission and receptor trafficking. In this article, we provide an overview of current methods for studying lipid rafts and models for how lipid rafts might form and function. Next, we propose a potential mechanism for regulating lipid rafts in the brain via local control of cholesterol biosynthesis by neurotrophins and their receptors. Finally, we discuss evidence that altered cholesterol metabolism and/or lipid rafts play a critical role in the pathophysiology of multiple CNS disorders, including Smith-Lemli-Opitz syndrome, Huntingtons, Alzheimers, and Niemann-Pick Type C diseases.

p75 Neurotrophin Receptor-mediated Apoptosis in Sympathetic Neurons Involves a Biphasic Activation of JNK and Up-regulation of Tumor Necrosis Factor-α-converting Enzyme/ADAM17

During the development of the sympathetic nervous system, the p75 neurotrophin receptor (p75NTR) has a dual function: promoting survival together with TrkA in response to NGF, but inducing cell death upon binding pro or mature brain-derived neurotrophic factor (BDNF). Apoptotic signaling through p75NTR requires activation of the stress kinase, JNK. However, the receptor also undergoes regulated proteolysis, first by a metalloprotease, and then by γ-secretase, in response to pro-apoptotic ligands and this is necessary for receptor mediated neuronal death (Kenchappa, R. S., Zampieri, N., Chao, M. V., Barker, P. A., Teng, H. K., Hempstead, B. L., and Carter, B. D. (2006) Neuron 50, 219–232). Hence, the relationship between JNK activation and receptor proteolysis remains to be defined. Here, we report that JNK3 activation is necessary for p75NTR cleavage; however, following release of the intracellular domain, there is a secondary activation of JNK3 that is cleavage dependent. Receptor proteolysis and apoptosis were prevented in sympathetic neurons from jnk3−/− mice, while activation of JNK by ectopic expression of MEKK1 induced p75NTR cleavage and cell death. Proteolysis of the receptor was not detected until 6 h after BDNF treatment, suggesting that JNK3 promotes cleavage through a transcriptional mechanism. In support of this hypothesis, BDNF up-regulated tumor necrosis factor-α-converting enzyme (TACE)/ADAM17 mRNA and protein in wild-type, but not jnk3−/− sympathetic neurons. Down-regulation of TACE by RNA interference blocked BDNF-induced p75NTR cleavage and apoptosis, indicating that this metalloprotease is responsible for the initial processing of the receptor. Together, these results demonstrate that p75NTR-mediated activation of JNK3 is required for up-regulation of TACE, which promotes receptor proteolysis, leading to prolonged activation of JNK3 and subsequent apoptosis in sympathetic neurons.

Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a metabolic and developmental disorder caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol reductase (Dhcr7). This reductase catalyzes the last step in cholesterol biosynthesis, and levels of 7-dehydrocholesterol (7-DHC), the substrate for this enzyme, are elevated in SLOS patients as a result of this defect. Our group has previously shown that 7-DHC is extremely prone to free radical autoxidation, and we identified about a dozen different oxysterols formed from oxidation of 7-DHC. We report here that 7-DHC-derived oxysterols reduce cell viability in a dose- and time-dependent manner, some of the compounds showing activity at sub-micromolar concentrations. The reduction of cell survival is caused by a combination of reduced proliferation and induced differentiation of the Neuro2a cells. The complex 7-DHC oxysterol mixture added to control Neuro2a cells also triggers the gene expression changes that were previously identified in Dhcr7-deficient Neuro2a cells. Based on the identification of overlapping gene expression changes in Dhcr7-deficient and 7-DHC oxysterol-treated Neuro2a cells, we hypothesize that some of the pathophysiological findings in the mouse SLOS model and SLOS patients might be due to accumulated 7-DHC oxysterols.

Protein Kinase A-Induced Phosphorylation of the p65 Subunit of Nuclear Factor-κB Promotes Schwann Cell Differentiation into a Myelinating Phenotype

Axon–Schwann cell interactions are critical for myelin formation during peripheral nerve development and regeneration. Axonal contact promotes Schwann cell precursors to differentiate into a myelinating phenotype, and cAMP-elevating agents can mimic this; however, the mechanisms underlying this differentiation are poorly understood. We demonstrated previously that the transcription factor nuclear factor-κB (NF-κB) is required for myelin formation by Schwann cells (Nickols et al., 2003), although how it is activated during this process remained to be determined. Here, we report that culturing Schwann cells with sensory neurons results in the activation of cAMP-dependent protein kinase (PKA), and this kinase phosphorylates the p65 subunit of NF-κB at S276. The phosphorylation was also induced in cultured Schwann cells by treatment with forskolin, dibutyryl-cAMP, or by overexpression of a catalytic subunit of PKA, and this increased the transcriptional activity of NF-κB. In developing perinatal rat sciatic nerve, the kinetics of p65 phosphorylation at S276 paralleled that of PKA and NF-κB activation. To elucidate the role of p65 phosphorylation in myelin formation, we overexpressed an S276A mutant of p65 in cultured Schwann cells, which blocked PKA-mediated transcriptional activation of NF-κB. When the Schwann cells expressing the mutant were cocultured with sensory neurons, there was a 45% reduction in the number of myelinated fibers relative to controls, demonstrating a requirement for p65 phosphorylation by PKA during myelin formation.

Late-migrating neuroepithelial cells from the spinal cord differentiate into sensory ganglion cells and melanocytes

During embryonic development, neural crest cells give rise to many structures in peripheral tissues. Other neural tube cells are thought to contribute only to structures within the CNS. In contrast to this idea, we report a second wave of migration of cells away from the spinal cord occurring after the emigration of crest cells is complete. Neuroepithelial cells from spinal cords in E5 chicken embryos migrate into the periphery and differentiate into neurons and satellite cells within sensory ganglia and into melanocytes in skin and feathers. These results show that some cell types previously considered to be the descendants exclusively of neural crest cells are also derived from neuroepithelial cells in the spinal cord.

An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.

The level of 7-dehydrocholesterol (7-DHC) is elevated in tissues and fluids of Smith-Lemli-Opitz syndrome (SLOS) patients due to defective 7-DHC reductase. Although over a dozen oxysterols have been identified from 7-DHC free radical oxidation in solution, oxysterol profiles in SLOS cells and tissues have never been studied. We report here the identification and complete characterization of a novel oxysterol, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), as a biomarker for 7-DHC oxidation in fibroblasts from SLOS patients and brain tissue from a SLOS mouse model. Deuterated (d7)-standards of 7-DHC and DHCEO were synthesized from d7-cholesterol. The presence of DHCEO in SLOS samples was supported by chemical derivatization in the presence of d7-DHCEO standard followed by HPLC-MS or GC-MS analysis. Quantification of cholesterol, 7-DHC, and DHCEO was carried out by isotope dilution MS with the d7-standards. The level of DHCEO was high and correlated well with the level of 7-DHC in all samples examined (R = 0.9851). Based on our in vitro studies in two different cell lines, the mechanism of formation of DHCEO that involves 5α,6α-epoxycholest-7-en-3β-ol, a primary free radical oxidation product of 7-DHC, and 7-cholesten-3β,5α,6β-triol is proposed. In a preliminary test, a pyrimidinol antioxidant was found to effectively suppress the formation of DHCEO in SLOS fibroblasts.

Presenilin-1-Dependent Transcriptome Changes

Familial forms of Alzheimers disease (FADs) are caused by the expression of mutant presenilin 1 (PS1) or presenilin 2. Using DNA microarrays, we explored the brain transcription profiles of mice with conditional knock-out of PS1 (cKO PS1) in the forebrain. In parallel, we performed a transcription profiling of the hippocampus and frontal cortex of the FAD-linked ΔE9 mutant transgenic (TG) mice and matched controls [TG mice expressing wild-type human PS1 (hPS1)]. When the TG and cKO datasets were cross-compared, the majority of the 30 common expression alterations were in opposite direction, suggesting that the FAD-linked PS1 variant produces transcriptome changes primarily by gain of aberrant function. Our microarray studies also revealed an unanticipated inverse correlation of transcript levels between the brains of mice that coexpress ΔE9 hPS1+ amyloid precursor protein (APP)695 Swe and ΔE9 hPS1 single transgenic mice. The opposite directionality of these changes in transcript levels must be a function of APP and/or APP derivatives.

Lipid biomarkers of oxidative stress in a genetic mouse model of Smith-Lemli-Opitz syndrome

Abstract7-Dehydrocholesterol (7-DHC) accumulates in tissues and fluids of patients with Smith-Lemli-Opitz syndrome (SLOS), which is caused by mutations in the gene encoding 3β-hydroxysterol-Δ7-reductase (DHCR7). We recently reported that 7-DHC is the most reactive lipid molecule toward free radical oxidation (lipid peroxidation) and 14 oxysterols have been identified as products of oxidation of 7-DHC in solution. As the high oxidizability of 7-DHC may lead to systemic oxidative stress in SLOS patients, we report here lipid biomarkers of oxidative stress in a Dhcr7-KO mouse model of SLOS, including oxysterols, isoprostanes (IsoPs), and neuroprostanes (NeuroPs) that are formed from the oxidation of 7-DHC, arachidonic acid and docosahexaenoic acid, respectively. In addition to a previously described oxysterol, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), we provide evidence for the chemical structures of three new oxysterols in the brain and/or liver tissue of Dhcr7-KO mice, two of which were quantified. We find that levels of IsoPs and NeuroPs are also elevated in brain and/or liver tissues of Dhcr7-KO mice relative to matching WT mice. While IsoPs and NeuroPs have been established as a reliable measurement of lipid peroxidation and oxidative stress in vivo, we show that in this genetic SLOS mouse model, 7-DHC-derived oxysterols are present at much higher levels than IsoPs and NeuroPs and thus are better markers of lipid oxidation and related oxidative stress.

DHCEO accumulation is a critical mediator of pathophysiology in a Smith-Lemli-Opitz syndrome model

Smith-Lemli-Opitz syndrome (SLOS) is an inborn error of metabolism caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase (DHCR7), the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol (7-DHC) in the brain tissue and blood of the SLOS patients. The objective of this study was to determine the consequences of the accumulation of an immediate cholesterol precursor, 7-DHC and its oxysterol metabolite, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), in the brain tissue of Dhcr7-KO mouse, a model for SLOS. We found that cholesterol, 7-DHC and DHCEO show region-specific distribution, suggesting that the midbrain and the cortex are the primary sites of vulnerability. We also report that neurons are ten fold more susceptible to a 7-DHC-derived oxysterol mixture than glial cells, and that DHCEO accelerates differentiation and arborization of cortical neurons. The overall results suggest that 7-DHC oxidative metabolites are critical contributors to altered neural development in SLOS. The future studies will test if antioxidant supplementation will ameliorate some of the clinical symptoms associated with this devastating disease.

Expression and p75 neurotrophin receptor dependence of cholesterol synthetic enzymes in adult mouse brain

Normal brain function depends critically on cholesterol. Although cholesterol is synthesized locally in the adult brain, the precise anatomical localization of cholesterogenic enzymes is not known. Here we show that 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAred) and 7-dehydrocholesterol reductase (7dhcred), the first and last enzymes, respectively, in the cholesterol biosynthesis pathway, are co-expressed in neurons throughout adult murine brain. Co-localization is most prominent in cortical, hippocampal, and cholinergic neurons. Since adult hippocampal and cholinergic neurons express p75 neurotrophin receptors (p75NTR) we hypothesized that p75NTR regulates expression of cholesterogenic enzymes. Treatment of Neuro2a neuroblastoma cells or primary cerebellar cultures with siRNA downregulates p75NTR and decreases the expression level of HMG-CoAred and 7dhcred. Native neuroblastoma cell lines with differential expression of p75NTR differentially express 7dhcred; 7dhcred expression correlates with p75NTR expression. This suggests that, in p75NTR-expressing cells, p75NTR regulates cholesterol synthesis through regulation of HMG-CoAred and 7dhcred expression. The unexpected localization of cholesterogenic enzymes in adult neurons suggests that at least some adult neurons retain the ability to synthesize cholesterol.