Laura Mateos

Activity‐Regulated Cytoskeleton‐Associated Protein in Rodent Brain is Down‐Regulated by High Fat Diet in vivo and by 27‐Hydroxycholesterol in vitro

Growing evidence strongly suggests that high fat diet (HFD) has an important role in some neurodegenerative disorders, including Alzheimers disease (AD). To identify new cellular pathways linking hypercholesterolemia and neurodegeneration, we analyzed the effects of HFD on gene expression in mouse brain. Using cDNA microarrays and real time RT‐PCR, we found that HFD has a mild, but significant effect on the expression of several genes. The altered genes include molecules linked to AD pathology and others of potential interest for neurodegeneration. We further investigated the effect of HFD on the activity‐regulated cytoskeleton‐associated protein (Arc). Expression of Arc was decreased in cerebral cortex and hippocampus of HFD‐fed animals. From the known regulatory mechanisms of Arc expression, HFD reduced N‐methyl‐D‐aspartate receptor (NMDAR) activity, as seen by decreases in tyrosine phosphorylation of NMDAR2A and levels of NMDAR1. Additionally, we demonstrated that 27‐hydroxycholesterol, a cholesterol metabolite that enters the brain from the blood, decreases Arc levels as well as NMDAR and Src kinase activities in rat primary hippocampal neurons. Finally, we showed that Arc levels are decreased in the cortex of AD brains. We propose that one of the mechanisms, by which hypercholesterolemia contributes to neurodegenerative diseases, could be through Arc down‐regulation caused by 27‐hydroxycholesterol.

Impaired long term memory consolidation in transgenic mice overexpressing the human soluble form of IL-1ra in the brain

Interleukin-1 (IL-1) is expressed following LTP induction and is required for long-term memory consolidation. We demonstrate that the long-term, but not short-term memory is impaired in a transgenic mouse strain overexpressing the human soluble interleukin-1 receptor antagonist (hsIL-1ra) in the brain. Overexpression of IL-1ra was found to reduce the basal as well as the novelty-induced upregulation of activity-regulated cytoskeleton-associated protein (Arc) in the dentate gyrus and in the retrosplenial cortex. Together with the finding that blocking IL-1 receptors interferes with the BDNF-ERK1/2 pathway, our data suggest an essential role played by physiological levels of IL-1 in long-term memory consolidation.

Cholinergic hypofunction impairs memory acquisition possibly through hippocampal Arc and BDNF downregulation

Recent evidence suggests that activity‐regulated cytoskeleton associated protein (Arc) and brain‐derived neurotrophic factor (BDNF) are key players in the cellular mechanisms that trigger synaptic changes and memory consolidation. Cholinergic deafferentiation of hippocampus has been largely shown to induce memory impairments in different behavioral tasks. However, the mechanisms underlying cholinergic‐induced memory formation remain unclear. The role of hippocampal cholinergic denervation on synaptic consolidation and further acquisition of spatial memory was hereby examined by analyzing Arc and BDNF in standard environment and after behavioral training in Morris water maze (MWM). In standard environment, a cholinergic hypofunction induced by the toxin 192IgG‐saporin led to significant decreases in Arc protein and mRNA as well as in BDNF. Lesioned rats subjected to MWM showed a worse acquisition performance that was reversed after galantamine treatment. Recovery of memory acquisition was accompanied by normalization of Arc and BDNF levels in hippocampus. Stimulation of muscarinic, but not nicotinic receptors, in hippocampal primary neurons caused a rapid induction of Arc production. These data suggest that cholinergic denervation of hippocampus leads to deficits in muscarinic‐dependent induction of Arc and a subsequent impairment of spatial memory acquisition.

Upregulation of Brain Renin Angiotensin System by 27-Hydroxycholesterol in Alzheimer's Disease

In spite of the fact that cholesterol does not pass the blood-brain barrier, hypercholesterolemia has been linked to increase Alzheimers disease (AD) risk. Hypertension is another risk factor and angiotensin converting enzyme (ACE) activity is known to be increased in AD. Furthermore, a lower incidence of AD has been reported in patients taking anti-hypertensive drugs. Here we show that the levels of angiotensinogen (AGT) and ACE are increased in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment and AD. Moreover, we show ACE activity in the CSF to be positively correlated with both plasma and CSF levels of 27-hydroxycholesterol (27-OH), an oxysterol known to pass through the BBB and taken up from the circulation by the brain. In addition, treatment of rat primary neurons, astrocytes, and human neuroblastoma cells with 27-OH resulted in increased production of AGT. Our results demonstrate that upregulation of renin-angiotensin system (RAS) in AD brains occurs not only at the enzymatic level (ACE) but also at the substrate level (AGT). The possibility that 27-OH is part of a mechanism linking hypercholesterolemia with increased brain RAS activity and increased AD risk is discussed.

Side Chain-oxidized Oxysterols Regulate the Brain Renin-Angiotensin System through a Liver X Receptor-dependent Mechanism

Disturbances in cholesterol metabolism have been associated with hypertension and neurodegenerative disorders. Because cholesterol metabolism in the brain is efficiently separated from plasma cholesterol by the blood-brain barrier (BBB), it is an unsolved paradox how high blood cholesterol can cause an effect in the brain. Here, we discuss the possibility that cholesterol metabolites permeable to the BBB might account for these effects. We show that 27-hydroxycholesterol (27-OH) and 24S-hydroxycholesterol (24S-OH) up-regulate the renin-angiotensin system (RAS) in the brain. Brains of mice on a cholesterol-enriched diet showed up-regulated angiotensin converting enzyme (ACE), angiotensinogen (AGT), and increased JAK/STAT activity. These effects were confirmed in in vitro studies with primary neurons and astrocytes exposed to 27-OH or 24S-OH, and were partially mediated by liver X receptors. In contrast, brain RAS activity was decreased in Cyp27a1-deficient mice, a model exhibiting reduced 27-OH production from cholesterol. Moreover, in humans, normocholesterolemic patients with elevated 27-OH levels, due to a CYP7B1 mutation, had markers of activated RAS in their cerebrospinal fluid. Our results demonstrate that side chain-oxidized oxysterols are modulators of brain RAS. Considering that levels of cholesterol and 27-OH correlate in the circulation and 27-OH can pass the BBB into the brain, we suggest that this cholesterol metabolite could be a link between high plasma cholesterol levels, hypertension, and neurodegeneration.

Influence of Residue 22 on the Folding, Aggregation Profile, and Toxicity of the Alzheimer's Amyloid β Peptide

Several biophysical techniques have been used to determine differences in the aggregation profile (i.e., the secondary structure, aggregation propensity, dynamics, and morphology of amyloid structures) and the effects on cell viability of three variants of the amyloid beta peptide involved in Alzheimers disease. We focused our study on the Glu22 residue, comparing the effects of freshly prepared samples and samples aged for at least 20 days. In the aged samples, a high propensity for aggregation and beta-sheet secondary structure appears when residue 22 is capable of establishing polar (Glu22 in wild-type) or hydrophobic (Val22 in E22V) interactions. The Arctic variant (E22G) presents a mixture of mostly disordered and alpha-helix structures (with low beta-sheet contribution). Analysis of transmission electron micrographs and atomic force microscopy images of the peptide variants after aging showed significant quantitative and qualitative differences in the morphology of the formed aggregates. The effect on human neuroblastoma cells of these Abeta(12-28) variants does not correlate with the amount of beta-sheet of the aggregates. In samples allowed to age, the native sequence was found to have an insignificant effect on cell viability, whereas the Arctic variant (E22G), the E22V variant, and the slightly-aggregating control (F19G-F20G) had more prominent effects.

Thioredoxin-80 is a product of alpha-secretase cleavage that inhibits amyloid-beta aggregation and is decreased in Alzheimer's disease brain

Thioredoxin‐1 (Trx1) is an endogenous dithiol reductant and antioxidant that was shown to be decreased in Alzheimers disease (AD) neurons. A truncated form of Trx1, thioredoxin 80 (Trx80), was reported to be secreted from monocytes having cytokine activity. Here, we show that Trx80 is present in human brain in an aggregated form. Trx80 localizes mainly to neurons and is dramatically decreased in AD brains. Trx80 levels in cerebrospinal fluid (CSF) correlate with those of the classical AD biomarkers amyloid‐β (Aβ) 1–42 and total tau. Moreover, Trx80 measurements in CSF discriminate between patients with stable mild cognitive impairment, prodomal AD and mild AD. We report that ADAM10 and 17, two α‐secretases processing the Aβ precursor protein, are responsible for Trx80 generation. In contrast to the periphery, Trx80 has no pro‐inflammatory effects in glia, either by itself or in combination with Aβ or apolipoprotein E. Instead, Trx80 inhibits Aβ(1–42) aggregation and protects against its toxicity. Thus, a reduction in Trx80 production would result in increased Aβ polymerization and enhanced neuronal vulnerability. Our data suggest that a deficit in Trx80 could participate in AD pathogenesis.

Plasma cholesterol and risk for late-onset Alzheimer's disease.

Evaluation of: Reitz C, Tang MX, Schupf N et al. Association of higher levels of high-density lipoprotein cholesterol in elderly individuals and lower risk of late-onset Alzheimer disease. Arch. Neurol. 67(12), 1491–1497 (2010). Alzheimer’s disease (AD), the most common cause of dementia, is a progressive neurodegenerative disorder affecting millions of people worldwide. AD has a multifactorial origin, resulting from an interaction between genetic susceptibility and environmental risk factors. Genetic, epidemiological, experimental and clinical data strongly suggest that the metabolism of cholesterol has an important role in AD pathogenesis. Several studies have demonstrated that high concentrations of serum cholesterol increase the risk of AD. Statins, drugs that reduce cholesterol levels, have been investigated as a possible treatment for AD. However, the literature is not exempt of contradictory results. In this article, we review a recent article by Reitz et al. demonstrating that higher levels of high-density lipoprotein cholesterol, total cholesterol and non-high-density lipoprotein cholesterol are associated with lower risk of AD. In addition, we discuss the current state of knowledge regarding the relationship between plasma cholesterol and AD, stressing the need for understanding the molecular mechanisms behind this association.

Estrogen protects against amyloid-β toxicity by estrogen receptor α-mediated inhibition of Daxx translocation.

Estrogen was shown to promote neuronal survival against several neurotoxic insults including β-amyloid (Aβ). The proposed mechanism includes the activation of the mitogen activated protein kinase/extracellular signal-regulated kinase (Mapk/Erk), phosphatidylinositol 3-kinase/Akt pathways and the upregulation of antiapoptotic proteins. On the other hand, Aβ neurotoxicity depends on the activation of apoptosis signal-regulating kinase 1 (Ask1), and both Ask1 activity and Aβ toxicity are inhibited by thioredoxin-1 (Trx1). Here, we explored the possibility that estrogen could protect cells against Aβ(1-42) toxicity by inhibiting the Ask1 cascade or by modulating Trx1. Cytosolic translocation of death-associated protein Daxx was used as indicator of Ask1 activity. Using human SH-SY5Y neuroblastoma cells, 17β-estradiol (E2) and specific agonists for estrogen receptor (ER) α or β we demonstrated that nM concentrations of E2 protected against Aβ(1-42) by a mechanism depending upon ERα stimulation, Akt activation and Ask1 inhibition. Moreover, this protection would occur independently of ERβ and the induction of Trx1 expression. Our results emphasize the importance of Ask1 cascade in Aβ toxicity, and of ERα and Ask1 as targets for developing new neuroprotective drugs.

Parkin‐mediated ubiquitination regulates phospholipase C‐γ1

Mutations in parkin cause autosomal recessive forms of Parkinson’s disease (PD), with an early age of onset and similar pathological phenotype to the idiopathic disease. Parkin has been identified as an E3 ubiquitin ligase that mediates different types of ubiquitination, which has made the search for substrates an intriguing possibility to identify pathological mechanisms linked to PD. In this study, we present PLCγ1 as a novel substrate for parkin. This association was found in non‐transfected human neuroblastoma SH‐SY5Y cells as well as in stable cell lines expressing parkin WT and familial mutants R42P and G328E. Analysis of cortical, striatal and nigral human brain homogenates revealed that the interaction between parkin and PLCγ1 is consistent throughout these regions, suggesting that the interaction is likely to have a physiological relevance for humans. Unlike many of the previously identified substrates, we could also show that the steady‐state levels of PLCγ1 is significantly higher in parkin KO mice and lower in parkin WT human neuroblastoma cells, suggesting that parkin ubiquitination of PLCγ1 is required for proteasomal degradation. In line with this idea, we show that the ability to ubiquitinate PLCγ1 in vitro differs significantly between WT and familial mutant parkin. In this study, we demonstrate that parkin interacts with PLCγ1, affecting PLCγ1 steady state protein levels in human and murine models with manipulated parkin function and expression levels. This finding could be of relevance for finding novel pathogenic mechanisms leading to PD.