Folding Free-Energy Landscape of α-Synuclein (35-97) Via Replica Exchange Molecular Dynamics.

The misfolding and aggregation of α-synuclein (α-syn) in Lewy bodies are implicated in the pathogenesis of various neurodegenerative disorders, such as Parkinson's disease and dementia. The formation of α-syn fibrils is a complex process, involving various intermediates and oligomeric forms. These intermediates establish at an early stage of aggregation and subsequently lead to fibrillation. Determining which conformations are accessible to monomeric α-syn and especially, as shown in a recent work, to the central amino acids from residue 35 to residue 97 (63 residues) is thus crucial to understand the formation of these oligomers. Here, we carry out extensive replica exchange molecular dynamics (total time-18 µs) with an all-atom model and explicit solvent to characterize the free-energy landscape of human α-syn (residue 35 to residue 97). The simulation results lead us to identify two free-energy basins. Clustering analysis for the deepest free-energy minimum reveals a compact structure, with a secondary structure predominantly α-helix, while the shallower minimum corresponds to an elongated conformation, also predominantly α-helix. Furthermore, at physiological temperature, we find that conformational rearrangements happen via helix breaks due to the presence of glycine. We also show that the most likely conformations are characterized by the α-helix structure rather than the ß-hairpin structure (for residue 38 to residue 53), in contrast with prior simulation studies using coarse-grained models or an implicit solvent. For higher temperatures, we observe a shift in secondary structure with a decrease in the population of α-helix in favor of random coils, ß-bend, and ß-turns.

Palmitate-induced C/EBP homologous protein activation leads to NF-κB-mediated increase in BACE1 activity and amyloid beta genesis.

The etiology of Alzheimer's disease (AD) is egregiously comprehended, but epidemiological studies have posited that diets rich in the saturated fatty acid palmitic acid (palmitate) are a significant risk factor. The production and accumulation of amyloid beta peptide (Aß) is considered the core pathological molecular event in the pathogenesis of AD. The rate-limiting step in Aß genesis from amyloid-ß precursor protein (AßPP) is catalyzed by the enzyme ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), the expression and enzymatic activity of which is significantly up-regulated in the AD brain. In this study, we determined the molecular mechanisms that potentially underlie the palmitate-induced up-regulation in BACE1 expression and augmented Aß production. We demonstrate that a palmitate-enriched diet and exogenous palmitate treatment evoke an increase in BACE1 expression and activity leading to enhanced Aß genesis in the mouse brain and SH-SY5Y-APPSwe cells, respectively, through the activation of the transcription factor NF-κB. Chromatin immunoprecipitation (ChIP) assays and luciferase reporter assays revealed that palmitate enhances BACE1 expression by increasing the binding of NF-κB in the BACE1 promoter followed by an enhancement in the transactivation of the BACE1 promoter. Elucidation and delineation of upstream molecular events unveiled a critical role of the endoplasmic reticulum stress-associated transcription factor, C/EBP homologous protein (CHOP) in the palmitate-induced NF-κB activation, as CHOP knock-down cells and Chop-/- mice do not exhibit the same degree of NF-κB activation in response to the palmitate challenge. Our study delineates a novel CHOP-NF-κB signaling pathway that mediates palmitate-induced up-regulation of BACE1 expression and Aß genesis.

27-Hydroxycholesterol increases α-synuclein protein levels through proteasomal inhibition in human dopaminergic neurons.

BACKGROUND: Accumulation of the α-synuclein (α-syn) protein is a hallmark of a group of brain disorders collectively known as synucleinopathies. The mechanisms responsible for α-syn accumulation are not well understood. Several studies suggest a link between synucleinopathies and the cholesterol metabolite 27-hydroxycholesterol (27-OHC). 27-OHC is the major cholesterol metabolite in the blood that crosses the blood brain barrier, and its levels can increase following hypercholesterolemia, aging, and oxidative stress, which are all factors for increased synucleinopathy risk. In this study, we determined the extent to which 27-OHC regulates α-syn levels in human dopaminergic neurons, the cell type in which α-syn accumulates in PD, a major synucleinopathy disorder. RESULTS: Our results show that 27-OHC significantly increases the protein levels, not the mRNA expression of α-syn. The effects of 27-OHC appear to be independent of an action through liver X receptors (LXR), its cognate receptors, as the LXR agonist, GW3965, or the LXR antagonist ECHS did not affect α-syn protein or mRNA levels. Furthermore, our data strongly suggest that the 27-OHC-induced increase in α-syn protein levels emanates from inhibition of the proteasomal degradation of this protein and a decrease in the heat shock protein 70 (HSP70). CONCLUSIONS: Identifying 27-OHC as a factor that can increase α-syn levels and the inhibition of the proteasomal function and reduction in HSP70 levels as potential cellular mechanisms involved in regulation of α-syn. This may help in targeting the correct degradation of α-syn as a potential avenue to preclude α-syn accumulation.

The cholesterol metabolite 27-hydroxycholesterol stimulates cell proliferation via ERß in prostate cancer cells.

BACKGROUND: For every six men, one will be diagnosed with prostate cancer (PCa) in their lifetime. Estrogen receptors (ERs) are known to play a role in prostate carcinogenesis. However, it is unclear whether the estrogenic effects are mediated by estrogen receptor α (ERα) or estrogen receptor ß (ERß). Although it is speculated that ERα is associated with harmful effects on PCa, the role of ERß in PCa is still ill-defined. The cholesterol oxidized metabolite 27-hydroxycholesterol (27-OHC) has been found to bind to ERs and act as a selective ER modulator (SERM). Increased 27-OHC levels are found in individuals with hypercholesterolemia, a condition that is suggested to be a risk factor for PCa. METHODS: In the present study, we determined the extent to which 27-OHC causes deleterious effects in the non-tumorigenic RWPE-1, the low tumorigenic LNCaP, and the highly tumorigenic PC3 prostate cancer cells. We conducted cell metabolic activity and proliferation assays using MTS and CyQUANT dyes, protein expression analyses via immunoblots and gene expression analyses via RT-PCR. Additionally, immunocytochemistry and invasion assays were performed to analyze intracellular protein distribution and quantify transepithelial cell motility. RESULTS: We found that incubation of LNCaP and PC3 cells with 27-OHC significantly increased cell proliferation. We also demonstrate that the ER inhibitor ICI 182,780 (fulvestrant) significantly reduced 27-OH-induced cell proliferation, indicating the involvement of ERs in proliferation. Interestingly, ERß levels, and to a lesser extent ERα, were significantly increased following incubation of PCa cells with 27-OHC. Furthermore, in the presence of the ERß specific inhibitor, PHTPP, 27-OHC-induced proliferation is attenuated. CONCLUSIONS: Altogether, our results show for the first time that 27-OHC, through ER activation, triggers deleterious effect in prostate cancer cell lines. We propose that dysregulated levels of 27-OHC may trigger or exacerbate prostate cancer via acting on ERß.

Leptin attenuates BACE1 expression and amyloid-ß genesis via the activation of SIRT1 signaling pathway.

The aspartyl protease ß-site AßPP-cleaving enzyme 1 (BACE1) catalyzes the rate-limiting step in Aß production, a peptide at the nexus of neurodegenerative cascades in Alzheimer Disease (AD). The adipocytokine leptin has been demonstrated to reduce Aß production and decrease BACE1 activity and expression levels. However, the signaling cascades involved in the leptin-induced mitigation in Aß levels and BACE1 expression levels have not been elucidated. We have demonstrated that the transcription factor nuclear factor - kappa B (NF-κB) positively regulates BACE1 transcription. NF-κB activity is tightly regulated by the mammalian sirtuin SIRT1. Multiple studies have cogently evinced that leptin activates the metabolic master regulator SIRT1. In this study, we determined the extent to which SIRT1 expression and activity regulate the leptin-induced attenuation in BACE1 expression and Aß levels in cultured human neuroblastoma SH-SY5Y cells. This study also elucidated and delineated the signal transduction pathways involved in the leptin induced mitigation in BACE1 expression. Our results demonstrate for the first time that leptin attenuates the activation and transcriptional activity of NF-κB by reducing the acetylation of the p65 subunit in a SIRT1-dependent manner. Furthermore, our data shows that leptin reduces the NF-κB-mediated transcription of BACE1 and consequently reduces Amyloid-ß genesis. Our study provides a valuable insight and a novel mechanism by which leptin reduces BACE1 expression and Amyloid-ß production and may help design potential therapeutic interventions.

Epigenetics of inflammation, maternal infection, and nutrition.

Studies have demonstrated that epigenetic changes such as DNA methylation, histone modification, and chromatin remodeling are linked to an increased inflammatory response as well as increased risk of chronic disease development. A few studies have begun to investigate whether dietary nutrients play a beneficial role by modifying or reversing epigenetically induced inflammation. Results of these studies show that nutrients modify epigenetic pathways. However, little is known about how nutrients modulate inflammation by regulating immune cell function and/or immune cell differentiation via epigenetic pathways. This overview will provide information about the current understanding of the role of nutrients in the epigenetic control mechanisms of immune function.

The cholesterol metabolite 27-hydroxycholesterol regulates p53 activity and increases cell proliferation via MDM2 in breast cancer cells.

Estrogen is synthesized from cholesterol and high cholesterol levels are suggested to be associated with increased risk of estrogen receptor(ER)-positive breast cancer. The cholesterol metabolite 27-hydroxycholesterol (27-OHC) was recently identified as a selective estrogen receptor modulator (SERM) and may therefore impact breast cancer progression. However, the mechanisms by which 27-OHC may contribute to breast cancer are not all known. We determined the extent to which 27-OHC regulates cell proliferation in MCF7 ER-positive breast cancer cell line involving the tumor suppressor protein p53. We found that treatment of MCF7 cells with 27-OHC resulted reduced p53 transcriptional activity. Conversely, treatment of the ER-negative MDA-MB 231 cells with 27-OHC induced no significant change in p53 activity. Exposure of MCF7 cells to 27-OHC was also associated with increased protein levels of the E3 ubiquitin protein ligase MDM2 and decreased levels of p53. Moreover, 27-OHC also enhanced physical interaction between p53 and MDM2. Furthermore, 27-OHC-induced proliferation was attenuated using either the p53 activator Tenovin-1 or the MDM2 inhibitor Nutlin-3 and Mdm2 siRNA. Taken together, our results indicate that 27-OHC may contribute to ER-positive breast cancer progression by disrupting constitutive p53 signaling in an MDM2-dependent manner.

Cholesterol-enriched diet disrupts the blood-testis barrier in rabbits.

About 15% of heterosexual couples in the USA suffer from infertility issues; male infertility accounts for ∼50% of all infertility cases and roughly 50% of male infertility is idiopathic. Increased levels of plasma cholesterol affect spermatogenesis and male fertility negatively, but by unclear mechanisms. Clearly, spermatogenesis occurs in immune-privileged seminiferous tubules that are protected by the blood-testis barrier (BTB), and BTB disruption results in sperm damage and male infertility. Accordingly, using rabbits fed a 2% cholesterol-enriched diet for 2, 4, and 6 wk to raise levels of plasma cholesterol, we tested the hypothesis that elevated levels of plasma cholesterol disrupt the BTB functionally and biochemically. The cholesterol-enriched diet increased lipid deposition dramatically and time-dependently in the seminiferous tubules and disrupted the BTB as evidenced by increased IgG staining within the seminiferous tubules. Total protein levels of the tight-junction proteins ZO-1 and occludin were increased in the seminiferous tubules of rabbits fed the cholesterol-enriched diet, and the distribution patterns of tight-junction proteins were markedly affected, including an increased accumulation of tight-junction proteins in endosomes. Disruption of the integrity of the BTB due to increased plasma levels of cholesterol might play a role in male infertility.

Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis.

The development of glycomics increasingly requires the detection and quantification of large numbers of glycans, which is only partially achieved by current glycomics approaches. Taking advantage of selected reaction monitoring to enhance both sensitivity and selectivity, we report here a strategy termed targeted glycomics that enables highly sensitive and consistent identification and quantification of diverse glycans across multiple samples at the same time. In this proof-of-principle study, we validated the method by analyzing global N-glycans expressed in different systems: single proteins, cancer cells, and serum samples. A dynamic range of three orders of magnitude was obtained for the detection of all five glycans released from ribonuclease B. The limit of detection of 80 attomole for Man(9)GlcNAc(2) demonstrated the excellent sensitivity of the method. The capability of the strategy to identify diverse glycans was demonstrated by identification and detection of 162 different glycans and isomers from pancreatic cancer cells. The sensitivity of the method was illustrated further by the ability to detect eight glycans from 250 cancer cells and five glycans released from 100 cancer cells. In serum obtained from rabbits fed control diet or diet enriched with 2% cholesterol, differences to 42 glycans were accurately measured and this indicates that this strategy might find use in studies of biomarker discovery and validation.

Increased EID1 nuclear translocation impairs synaptic plasticity and memory function associated with pathogenesis of Alzheimer's disease.

Though loss of function in CBP/p300, a family of CREB-binding proteins, has been causally associated with a variety of human neurological disorders, such as Rubinstein-Taybi syndrome, Huntington's disease and drug addiction, the role of EP300 interacting inhibitor of differentiation 1 (EID1), a CBP/p300 inhibitory protein, in modulating neurological functions remains completely unknown. Through the examination of EID1 expression and cellular distribution, we discovered that there is a significant increase of EID1 nuclear translocation in the cortical neurons of Alzheimer's disease (AD) patient brains compared to that of control brains. To study the potential effects of EID1 on neurological functions associated with learning and memory, we generated a transgenic mouse model with a neuron-specific expression of human EID1 gene in the brain. Overexpression of EID1 led to an increase in its nuclear localization in neurons mimicking that seen in human AD brains. The transgenic mice had a disrupted neurofilament organization and increase of astrogliosis in the cortex and hippocampus. Furthermore, we demonstrated that overexpression of EID1 reduced hippocampal long-term potentiation and impaired spatial learning and memory function in the transgenic mice. Our results indicated that the negative effects of extra nuclear EID1 in transgenic mouse brains are likely due to its inhibitory function on CBP/p300 mediated histone and p53 acetylation, thus affecting the expression of downstream genes involved in the maintenance of neuronal structure and function. Together, our data raise the possibility that alteration of EID1 expression, particularly the increase of EID1 nuclear localization that inhibits CBP/p300 activity in neuronal cells, may play an important role in AD pathogenesis.

The oxysterol 27-hydroxycholesterol regulates α-synuclein and tyrosine hydroxylase expression levels in human neuroblastoma cells through modulation of liver X receptors and estrogen receptors--relevance to Parkinson's disease.

Loss of dopaminergic neurons and α-synuclein accumulation are the two major pathological hallmarks of Parkinson's disease. Currently, the mechanisms governing depletion of dopamine content and α-synuclein accumulation are not well understood. We showed that the oxysterol 27-hydroxycholesterol (27-OHC) reduces the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and increases α-synuclein levels in SH-SY5Y cells. However, the cellular mechanisms involved in 27-OHC effects were not elucidated. In this study, we demonstrate that 27-OHC regulates TH and α-synuclein expression levels through the estrogen receptors (ER) and liver X receptors (LXR). We specifically show that inhibition of ERß mediates 27-OHC-induced decrease in TH expression, an effect reversed by the ER agonist estradiol. We also show that 27-OHC and the LXR agonist GW3965 increase α-synuclein while the LXR antagonist 5α-6α-epoxycholesterol-3-sulfate significantly attenuated the 27-OHC-induced increase in α-synuclein expression. We further demonstrate that LXRß positively regulates α-synuclein expression and 27-OHC increases LXRß-mediated α-synuclein transcription. Our results demonstrate the involvement of two distinct pathways that are involved in the 27-OHC regulation of TH and α-synuclein levels. Concomitant activation of ERß and inhibition of LXRß prevent 27-OHC effects and may therefore reduce the progression of Parkinson's disease by precluding TH reduction and α-synuclein accumulation.

Cholesterol-enriched diet causes age-related macular degeneration-like pathology in rabbit retina.

BACKGROUND: Alzheimer's disease (AD) and age-related macular degeneration (AMD) share several pathological hallmarks including ß-amyloid (Aß) accumulation, oxidative stress, and apoptotic cell death. The causes of AD and AMD are likely multi-factorial with several factors such as diet, environment, and genetic susceptibility participating in the pathogenesis of these diseases. Epidemiological studies correlated high plasma cholesterol levels with high incidence of AD, and feeding rabbits with a diet rich in cholesterol has been shown to induce AD-like pathology in rabbit brain. High intake of cholesterol and saturated fat were also long been suspected to increase the risk for AMD. However, the extent to which cholesterol-enriched diet may also cause AMD-like features in rabbit retinas is not well known. METHODS: Male New Zealand white rabbits were fed normal chow or a 2% cholesterol-enriched diet for 12 weeks. At necropsy, animals were perfused with Dulbecco's phosphate-buffered saline and the eyes were promptly removed. One eye of each animal was used for immunohistochemistry and retina dissected from the other eye was used for Western blot, ELISA assays, spectrophotometry and mass spectrometry analyses. RESULTS: Increased levels of Aß, decreased levels of the anti-apoptotic protein Bcl-2, increased levels of the pro-apoptotic Bax and gadd153 proteins, emergence of TUNEL-positive cells, and increased generation of reactive oxygen species were found in retinas from cholesterol-fed compared to normal chow-fed rabbits. Additionally, astrogliosis, drusen-like debris and cholesterol accumulations in retinas from cholesterol-fed rabbits were observed. As several lines of evidence suggest that oxidized cholesterol metabolites (oxysterols) may be the link by which cholesterol contributes to the pathogenesis of AMD, we determined levels of oxysterols and found a dramatic increase in levels of oxysterols in retinas from cholesterol-fed rabbits. CONCLUSIONS: Our results suggest that cholesterol-enriched diets cause retinal degeneration that is relevant to AMD. Furthermore, our data suggests high cholesterol levels and subsequent increase in the cholesterol metabolites as potential culprits to AMD.

Alpha-Synuclein-induced DNA Methylation and Gene Expression in Microglia.

Synucleinopathy disorders are characterized by aggregates of α-synuclein (α-syn), which engage microglia to elicit a neuroinflammatory response. Here, we determined the gene expression and DNA methylation changes in microglia induced by aggregate α-syn. Transgenic murine Thy-1 promoter (mThy1)-Asyn mice overexpressing human α-syn are a model of synucleinopathy. Microglia from 3 and 13-month-old mice were used to isolate nucleic acids for methylated DNA and RNA-sequencing. α-Syn-regulated changes in gene expression and genomic methylation were determined and examined for functional enrichment followed by network analysis to further elucidate possible connections within the data. Microglial DNA isolated from our 3-month cohort had 5315 differentially methylated gene (DMG) changes, while RNA levels demonstrated a change in 119 differentially expressed genes (DEGs) between mThy1-Asyn mice and wild-type littermate controls. The 3-month DEGs and DMGs were highly associated with adhesion and migration signaling, suggesting a phenotypic transition from resting to active microglia. We observed 3742 DMGs and 3766 DEGs in 13-month mThy1-Asyn mice. These genes were often related to adhesion, migration, cell cycle, cellular metabolism, and immune response. Network analysis also showed increased cell mobility and inflammatory functions at 3 months, shifting to cell cycle, immune response, and metabolism changes at 13 months. We observed significant α-syn-induced methylation and gene expression changes in microglia. Our data suggest that α-syn overexpression initiates microglial activation leading to neuroinflammation and cellular metabolic stresses, which is associated with disease progression.

ß-Amyloid regulates leptin expression and tau phosphorylation through the mTORC1 signaling pathway.

High levels of the adipocytokine leptin are associated with reduced risk of Alzheimer's disease. Leptin treatment also reduces ß-amyloid (Aß) levels in in vivo and in vitro models of Alzheimer's disease. Aß and leptin interact with the Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Akt/mTORC1 activation reduces tau phosphorylation through the inhibition of the downstream enzyme GSK-3ß. mTORC1 also regulates translation of many proteins including leptin. While Aß has been shown to inactivate Akt, inhibit mTORC1, and facilitate the phosphorylation of tau, leptin activates both Akt and mTORC1 and reduces tau phosphorylation. However, the extent to which Aß may modulate leptin expression and increase tau phosphorylation involving Akt/mTORC1 has not been determined. In this study, we show that incubation of organotypic slices from rabbit hippocampus with Aß down-regulates leptin expression, inhibits Akt, activates GSK-3ß, increases tau phosphorylation, and inactivates mTORC1. Leptin treatment reverses Aß effects by alleviating Akt inhibition, preventing GSK-3ß activation, reducing tau phosphorylation, and activating mTORC1. On the other hand, Rapamycin, an allosteric inhibitor of mTORC1, down-regulates leptin expression, increases tau phosphorylation, and does not affect Akt and GSK-3ß. Our results demonstrate for the first time that Aß regulates leptin expression and tau phosphorylation through mTORC1.

The oxysterol 27-hydroxycholesterol increases ß-amyloid and oxidative stress in retinal pigment epithelial cells.

BACKGROUND: Alzheimer's disease (AD) and age-related macular degeneration (AMD) share several pathological features including ß-amyloid (Aß) peptide accumulation, oxidative damage, and cell death. The causes of AD and AMD are not known but several studies suggest disturbances in cholesterol metabolism as a culprit of these diseases. We have recently shown that the cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC) causes AD-like pathology in human neuroblastoma SH-SY5Y cells and in organotypic hippocampal slices. However, the extent to which and the mechanisms by which 27-OHC may also cause pathological hallmarks related to AMD are ill-defined. In this study, the effects of 27-OHC on AMD-related pathology were determined in ARPE-19 cells. These cells have structural and functional properties relevant to retinal pigmented epithelial cells, a target in the course of AMD. METHODS: ARPE-19 cells were treated with 0, 10 or 25 µM 27-OHC for 24 hours. Levels of Aß peptide, mitochondrial and endoplasmic reticulum (ER) stress markers, Ca2+ homeostasis, glutathione depletion, reactive oxygen species (ROS) generation, inflammation and cell death were assessed using ELISA, Western blot, immunocytochemistry, and specific assays. RESULTS: 27-OHC dose-dependently increased Aß peptide production, increased levels of ER stress specific markers caspase 12 and gadd153 (also called CHOP), reduced mitochondrial membrane potential, triggered Ca2+ dyshomeostasis, increased levels of the nuclear factor κB (NFκB) and heme-oxygenase 1 (HO-1), two proteins activated by oxidative stress. Additionally, 27-OHC caused glutathione depletion, ROS generation, inflammation and apoptotic-mediated cell death. CONCLUSIONS: The cholesterol metabolite 27-OHC is toxic to RPE cells. The deleterious effects of this oxysterol ranged from Aß accumulation to oxidative cell damage. Our results suggest that high levels of 27-OHC may represent a common pathogenic factor for both AMD and AD.

Broad DNA repair responses in neural injury are associated with activation of the IL-6 pathway in cholesterol-fed rabbits.

The importance of DNA repair in the pathogenic mechanism of Alzheimer's Disease (AD) is still poorly understood. Here, we report that a broad range of responses by DNA repair proteins plays a critical role in the regulation of inflammatory response in rabbits fed with cholesterol-rich diet, a model system for AD. We found accumulation of oxodG DNA adduct in the brain of rabbits fed with cholesterol-enriched diets compared to control diets, which subsequently induced a broad range of DNA repair protein activities. Also, the hippocampus was identified as the primary site of oxidative DNA damage and elevated OGG1 activity. In addition, a physical interaction between XPB and OGG1 may account for a potential mechanism involving these DNA repair responses. DNA repair proteins also impact activation of various signaling cascades, including Src in response to cholesterol oxidation. Furthermore, OGG1 deficient mice showed no IL-6 activation as seen in wt mice but a drastic increase of TNF-alpha, a pro-inflammatory cytokine. Thus, OGG1 may be associated with cytokine production induced by high cholesterol levels, impacting neurodegeneration. Together, our studies suggest that critical DNA repair proteins are associated with development of AD, and may serve as potential targets for the treatment of AD.

Palmitate-Induced SREBP1 Expression and Activation Underlies the Increased BACE 1 Activity and Amyloid Beta Genesis.

Numerous cross-sectional and longitudinal studies have implicated saturated fat-enriched diets in the etio-pathogenesis of Alzheimer's disease (AD). Emerging evidence shows that saturated fat-enriched diets, such as palmitate-enriched diets, increase amyloid-beta (Aß) production, the histopathological hallmark of AD. However, the molecular mechanisms that underlie the deleterious effects of palmitate-enriched diets in the augmentation of Aß genesis are yet to be characterized. Sterol response element binding protein 1 (SREBP1) is a transcription factor that is modulated by saturated fatty acids, such as palmitate, and consequently regulates the expression of genes that code for proteins involved in almost all facets of lipid metabolism. Herein, we determined the role of changes in SREBP1 expression and transcriptional activity in the palmitate-induced effects on Aß genesis and BACE1 expression, the enzyme that catalyzes the rate-limiting step in Aß biosynthesis. We demonstrate that palmitate-induced SREBP1 activation directly regulates BACE1 expression at the transcriptional level in the mouse hippocampus and mouse Neuro-2a (N2a) neuroblastoma cells. Chromatin immunoprecipitation (ChIP) studies show that palmitate increases the binding of SREBP1 to the Bace1 promoter region in the mouse hippocampus and mouse N2a neuroblastoma cells. Ectopic expression of the dominant negative SREBP1 mutant and knocking-down SREBP1 expression significantly reduced the palmitate-induced increase in BACE1 expression and subsequent Aß genesis in mouse N2a neuroblastoma cells. Our study unveils SREBP1 activation as a novel molecular player in the palmitate-induced upregulation of BACE1 expression and subsequent Aß genesis.

A Diet Enriched in Palmitate and Deficient in Linoleate Exacerbates Oxidative Stress and Amyloid-ß Burden in the Hippocampus of 3xTg-AD Mouse Model of Alzheimer's Disease.

Epidemiological studies have suggested a positive correlation between saturated fat intake and the risk for developing Alzheimer's disease (AD). While diets-enriched in the saturated free fatty acid (sFFA) palmitate has been shown to induce cognitive dysfunction and AD-like pathology, polyunsaturated fatty acids (PUFA) such as linoleate have been suggested to protect against AD in mouse models. However, the underlying cellular and molecular mechanisms that mediate the deleterious effects of palmitate or the protective effects of linoleate remain to be characterized. We fed 9-month-old cohorts of triple transgenic AD mice (3xTg-AD) and their-matched controls with a palmitate-enriched/linoleate-deficient diet for three months and determined the impact of the diet on oxidative stress, Bace1 promoter transactivation status, and amyloid-ß (Aß) burden. The palmitate-enriched/linoleate-deficient diet causes a profound increase in oxidative stress burden characterized by significant oxidative damage to lipids, proteins, and nucleic acids concomitant with deficits in the endogenous antioxidant defense capacity in the hippocampi of 3xTg-AD mice. These effects were also associated with increased NF-κB transcriptional activity resulting in NF-κB-mediated transactivation of the Bace1 promoter that culminated in higher BACE1 expression and activity, and Aß production. Our study unveils a novel mechanism by which a diet enriched in the sFFA palmitate and deficient in the PUFA linoleate exacerbates AD-like pathology involving signaling cross-talk between oxidative stress and NF-κB activation as a critical underlying factor in upregulating BACE1 activity and increasing Aß burden.

Differential effects of 24-hydroxycholesterol and 27-hydroxycholesterol on tyrosine hydroxylase and alpha-synuclein in human neuroblastoma SH-SY5Y cells.

Evidence suggests that environmental and dietary factors may contribute to the pathogenesis of Parkinson's disease (PD). High dietary intake of cholesterol is such a factor that has been shown to increase or decrease the risk of PD. However, because circulating cholesterol does not cross the blood-brain barrier, the mechanisms linking dietary cholesterol to the pathogenesis of PD remain to be understood. In contrast to cholesterol, the oxidized cholesterol metabolites (oxysterols), 24S-hydroxycholesterol (24-OHC) and 27-hydroxycholesterol (27-OHC), can cross the blood-brain barrier and may place the brain at risk of degeneration. In this study, we incubated the human neuroblastoma SH-SY5Y cells for 24 h with 24-OHC, 27-OHC, or a mixture of 24-OHC plus 27-OHC, and have determined effects on tyrosine hydroxylase (the rate-limiting enzyme in dopamine synthesis) levels, alpha-synuclein levels, and apoptosis. We demonstrate that while 24-OHC increases the levels of tyrosine hydroxylase, 27-OHC increases levels of alpha-synuclein, and induces apoptosis. Our findings show for the first time that oxysterols trigger changes in levels of proteins that are associated with the pathogenesis of PD. As steady state levels of 24-OHC and 27-OHC are tightly regulated in the brain, disturbances in these levels may contribute to the pathogenesis of PD.