The Effects Of 27-Hydroxycholesterol And Palmitic Acid On Parkinson\u27s Disease-Like Pathology

Synucleinopathies is the overarching term used to describe a group of neurodegenerative disorders characterized by aggregates of α-synuclein (α-syn) protein in the cytoplasm of neurons, nerve fibers, and glial cells. The cause(s) for synucleinopathies are likely multi-factorial with genetic predisposition and environmental factors participating in the pathogenesis of the diseases. Dietary factors including dyslipidemia of cholesterol, its metabolites, and fatty acids have shown conflicting results as risk factors in recent years. Our overarching hypothesis is that dietary factors including 27-Hydroxycholesterol (27-OHC) and Palmitic Acid (PA) can affect key proteins involved in synucleinopathies. Findings regarding risk related specifically to dietary cholesterol have indicated either an increased risk, decreased risk, or no association. We believe the reason for the conflicting association between cholesterol and synucleinopathies lies in the metabolites of cholesterol and not cholesterol per se. Supporting our hypothesis many studies have shown increased levels of many different oxysterols, including 27-OHC, within the brains of synucleinopathy patients. However, the extent to which increased 27-OHC levels in the brain causes α-syn deposition and promotes synucleinopathies is yet to be determined. Therefore, in this dissertation we explore the effects of 27-OHC on the accumulation of α-syn and investigate the mechanisms of such involvement. We demonstrate that 27-OHC induces an increase in α-syn levels in human dopaminergic neurons. The mechanism involved in the α-syn increase does not appear to involve LXRs as we did not observe any significant changes in α-syn mRNA with 27-OHC or LXR agonist and antagonistic treatments. To the best of our knowledge, our results are the first to show that 27-OHC increases α-syn in dopaminergic neurons and that this increase may emanate from inhibition of the proteasomal function. Also, 27-OHC decreases levels of HSP70 protein which is involved in protein folding, and protein degradation through the Ubiquitin-Proteasomal System (UPS). The extent to which a decrease in HSP70 protein levels leads to decreased protein folding and degradation through specific pathways needs to be further elucidated. All-together, our results potentially suggest that restoring proteasomal function and HSP70 protein levels may attenuate the 27-OHC-induced increase in α-syn protein levels in vitro and reduce α-syn accumulation that can increase the risk for synucleinopathies. Additionally, to date, studies focused on the contributions of dietary fat intake to the risk of PD type synucleinopathy have yielded inconsistent results. Epidemiological studies of dietary fat intake and PD have found positive associations, no association, and even protective effects. As implied by all the conflicting studies, the jury is still out on the role(s) of FAs in PD-type synucleinopathy risk. Palmitic acid (PA) (16:0) is the most abundant saturated fatty acid in the body and the most abundant fatty acid in our diet. It has been shown to increase ER stress proinflammatory cytokine expression in astrocytes and microglia, and activation of TLRs via NFKβ but its role in pathological hallmark formation of PD-type synucleinopathy remains unknown. Throughout this dissertation we aim to examine the role(s) of PA on various hallmarks of PD-type synucleinopathy pathology in various animal and cellular models. We demonstrate that a PA-enriched diet induces an increase in α-syn and TH protein and mRNA expression in both B6D2 and m-Thy1 mice. We also show that the PA-enriched diet does not affect biogenic amine content in control B6D2 mice but significantly changes dopamine and serotonin levels in m-Thy1 mice relative to control-fed mice. Our results demonstrate that a diet enriched in PA increases the levels of Tyrosine Hydroxylase (TH), and serotonin, an effect that can provide beneficial effects in a variety of conditions. Additionally, we demonstrate that PA treatment in mouse dopaminergic neurons decreases α-syn protein and mRNA expression as well as it decreases TH protein content. Our study is the first to show that within MPTP-injected C57BL/6 mice a PA-enriched diet preserves motor function, decreases α-syn accumulation, increases TH protein, and increases dopaminergic neuronal survival. Altogether, our results suggest that a diet enriched in PA is protective against MPTP-induced Parkinsonism. Future studies are needed to elucidate the mechanisms by which a PA-enriched diet modulates these proteins. Establishing the effects of a smaller percentage of PA in the diet may reveal beneficial effects of this saturated free fatty acid in neurodegenerative conditions including PD and other synucleinopathies

Molecular interplay between leptin, insulin-like growth factor-1, and β-amyloid in organotypic slices from rabbit hippocampus

<p>Abstract</p> <p>Background</p> <p>Evidence shows that the insulin-like growth factor-1 (IGF-1) and leptin reduce β-amyloid (Aβ) production and tau phosphorylation, two major hallmarks of Alzheimer's disease (AD). IGF-1 expression involves the JAK/STAT pathway and the expression of leptin is regulated by the mammalian target of rapamycin complex 1 (mTORC1). We have previously shown that Aβ reduces leptin by inhibiting the mTORC1 pathway and Aβ was also suggested to inhibit the JAK/STAT pathway, potentially attenuating IGF-1 expression. As IGF-1 can activate mTORC1 and leptin can modulate JAK/STAT pathway, we determined the extent to which IGF-1 and leptin can upregulate the expression of one another and protect against Aβ-induced downregulation.</p> <p>Results</p> <p>We demonstrate that incubation of organotypic slices from adult rabbit hippocampus with Aβ42 downregulates IGF-1 expression by inhibiting JAK2/STAT5 pathway. Leptin treatment reverses these Aβ42 effects on IGF-1 and treatment with the STAT5 inhibitor completely abrogated the leptin-induced increase in IGF-1. Furthermore, EMSA and ChIP analyses revealed that leptin increases the STAT5 binding to the IGF-1 promoter. We also show that IGF-1 increases the expression of leptin and reverses the Aβ42-induced attenuation in leptin expression via the activation of mTORC1 signaling as the mTORC1 inhibitor rapamycin completely precluded the IGF-1-induced increase in leptin expression.</p> <p>Conclusion</p> <p>Our results demonstrate for the first time that Aβ42 downregulates IGF-1 expression and that leptin and IGF-1 rescue one another from downregulation by Aβ42. Our study provides a valuable insight into the leptin/IGF-1/Aβ interplay that may be relevant to the pathophysiology of AD.</p

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

Abstract 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

Palmitic Acid-Enriched Diet Increases α-Synuclein and Tyrosine Hydroxylase Expression Levels in the Mouse Brain

Background: Accumulation of the α-synuclein (α-syn) protein and depletion of dopaminergic neurons in the substantia nigra are hallmarks of Parkinson’s disease (PD). Currently, α-syn is under scrutiny as a potential pathogenic factor that may contribute to dopaminergic neuronal death in PD. However, there is a significant gap in our knowledge on what causes α-syn to accumulate and dopaminergic neurons to die. It is now strongly suggested that the nature of our dietary intake influences both epigenetic changes and disease-related genes and may thus potentially increase or reduce our risk of developing PD.Objective: In this study, we determined the extent to which a 3 month diet enriched in the saturated free fatty acid palmitate (PA) influences levels of α-syn and tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis in mice brains.Methods: We fed the m-Thy1-αSyn (m-Thy1) mouse model for PD and its matched control, the B6D2F1/J (B6D2) mouse a PA-enriched diet or a normal diet for 3 months. Levels of α-syn, tyrosine hydroxylase, and the biogenic amines dopamine and dopamine metabolites, serotonin and noradrenaline were determined.Results: We found that the PA-enriched diet induces an increase in α-syn and TH protein and mRNA expression levels in m-Thy1 transgenic mice. We also show that, while it didn’t affect levels of biogenic amine content in the B6D2 mice, the PA-enriched diet significantly reduces dopamine metabolites and increases the level of serotonin in m-Thy1 mice.Conclusion: Altogether, our results demonstrate that a diet rich in the saturated fatty acid palmitate can modulate levels of α-syn, TH, dopamine, and serotonin which all are proteins and neurochemicals that play key roles in increasing or reducing the risk for many neurodegenerative diseases including PD

Silencing GADD153/CHOP gene expression protects against Alzheimer's disease-like pathology induced by 27-hydroxycholesterol in rabbit hippocampus.

Endoplasmic reticulum (ER) stress is suggested to play a key role in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). Sustained ER stress leads to activation of the growth arrest and leucine zipper transcription factor, DNA damage inducible gene 153 (gadd153; also called CHOP). Activated gadd153 can generate oxidative damage and reactive oxygen species (ROS), increase β-amyloid (Aβ) levels, disturb iron homeostasis and induce inflammation as well as cell death, which are all pathological hallmarks of AD. Epidemiological and laboratory studies suggest that cholesterol dyshomeostasis contributes to the pathogenesis of AD. We have previously shown that the cholesterol oxidized metabolite 27-hydroxycholesterol (27-OHC) triggers AD-like pathology in organotypic slices. However, the extent to which gadd153 mediates 27-OHC effects has not been determined. We silenced gadd153 gene with siRNA and determined the effects of 27-OHC on AD hallmarks in organotypic slices from adult rabbit hippocampus. siRNA to gadd153 reduced 27-OHC-induced Aβ production by mechanisms involving reduction in levels of β-amyloid precursor protein (APP) and β-secretase (BACE1), the enzyme that initiates cleavage of APP to yield Aβ peptides. Additionally, 27-OHC-induced tau phosphorylation, ROS generation, TNF-α activation, and iron and apoptosis-regulatory protein levels alteration were also markedly reduced by siRNA to gadd153. These data suggest that ER stress-mediated gadd153 activation plays a central role in the triggering of AD pathological hallmarks that result from incubation of hippocampal slices with 27-OHC. Our results add important insights into cellular mechanisms that underlie the potential contribution of cholesterol metabolism in AD pathology, and suggest that preventing gadd153 activation protects against AD related to cholesterol oxidized products

Method for organotypic tissue culture in the aged animal

Organotypic slicing of brain tissue from young rodents has been used as a powerful model system for biomedical research [1–3]. Organotypic slicing complements cell culture and in vivo studies in multiple facets. This system can be useful for investigating manipulation of cellular signaling pathways without the hindrance of the blood-brain barrier while sacrificing fewer animals in the process. It also allows for preserved cellular connectivity and local intact circuitry which is a drawback of isolated cell cultures. Studies on age-related diseases have mainly used embryonic or early postnatal organotypic slice tissue. Excluding synaptic plasticity studies that are usually carried-out over a few hours and use adult mice or rats, a handful of studies performed on adult animals have had success for survival of slices [4,5]. Here we describe a method for culturing organotypic slices with high viability from hippocampus of aged mice and rabbits. • Our method permits slices from mice as old as 16 months and rabbits as old as years of age to survive ex vivo up to 8 weeks [6–9]. Such a slice system may be relevant to investigating age-related brain diseases