Doris Dea

Cholesterol synthesis and lipoprotein reuptake during synaptic remodelling in hippocampus in adult rats

Apolipoprotein E is synthesized and secreted by astrocytes in the hippocampus following lesions of the entorhinal cortex. It was proposed that apolipoprotein E, by analogy to its role in cholesterol transport in circulation, could be involved in the salvage and reutilization of non-esterified cholesterol released during terminal breakdown. The salvaged cholesterol could then be transported to neurons by apolipoprotein E-complexes and taken up via the apolipoprotein E/apolipoprotein B (low-density lipoprotein) receptor. To test this hypothesis, we have examined low-density lipoprotein receptor binding in brain sections of rats undergoing hippocampal reinnervation. The number of neuronal cells labelled by fluorescent Dil-low-density lipoprotein as well as the density of [125I]low-density lipoprotein binding sites in the dentate gyrus were found to increase in parallel with the extent of cholinergic reinnervation occurring in the deafferented hippocampus. In contrast, hippocampal cholesterol synthesis fell by more than 60% at eight days post-lesion, but eventually returned to control levels at 30 days post-lesion. The transient loss of cholesterol synthesis coincided with a peak in hippocampal apolipoprotein E expression. A concomitant accumulation of sudanophilic lipids (cholesterol esters and phospholipids) was detected in the outer molecular layer of the dentate gyrus and in the hilar region. The present findings suggest that non-esterified cholesterol released during terminal breakdown is esterified, transported via the apolipoprotein E transport system to neurons undergoing reinnervation, and take-up through the low-density lipoprotein receptor pathway where it is presumably used as a precursor molecule for the synthesis of new synapses and terminals.

Increased caveolin-1 expression in Alzheimer’s disease brain

Increasing evidence suggests that cholesterol plays a central role in the pathophysiology of Alzheimers disease (AD). Caveolin is a cholesterol-binding membrane protein involved in cellular cholesterol transport. We investigated the changes in the protein amount of hippocampal caveolin of autopsy-confirmed AD and aged-matched control subjects. Our results demonstrate that caveolin protein levels in the hippocampus and caveolin mRNA in the frontal cortex are up-regulated in AD by approximately two-fold, compared to control brains. These results suggest a relationship between caveolin-1 expression levels and a dysregulation of cholesterol homeostasis at the plasma membrane of brain cells. In support of this hypothesis, a significant increase in caveolin protein levels has also been observed in hippocampal tissue from ApoE-deficient (knockout) and aged wild-type mice; two situations associated with modifications of transbilayer distribution of cholesterol in brain synaptic plasma membranes. These results indicate that caveolin over-expression is linked to alterations of cholesterol distribution in the plasma membrane of brain cells and are consistent with the notion of a deterioration of cholesterol homeostasis in AD.

β-Amyloid Peptides Increase the Binding and Internalization of Apolipoprotein E to Hippocampal Neurons

Abstract: The frequency of the ε4 allele of apolipoprotein E(apoE) is increased in late‐onset and sporadic forms of Alzheimers disease (AD). ApoE also binds to β‐amyloid (Aβ) and both proteins are found in AD plaques. To further investigate the potential interaction of apoE and Aβ in the pathogenesis of AD, we have determined the binding, internalization, and degradation of human apoE isoforms in the presence and absence of Aβ peptides to rat primary hippocampal neurons. We demonstrate that the lipophilic Aβ peptides, in particular Aβ1–42, Aβ1–40, and Aβ25–35, increase significantly apoE‐liposome binding to hippocampal neurons. For each Aβ peptide, the increase was significantly greater for the apoE4 isoform than for the apoE3 isoform. The most effective of the Aβ peptides to increase apoE binding, Aβ25–35, was further shown to increase significantly the internalization of both apoE3‐ and apoE4‐liposomes, without affecting apoE degradation. Conversely, Aβ1–40 uptake by hippocampal neurons was shown to be increased in the presence of apoE‐liposomes, more so in the presence of the apoE4 than the apoE3 isoform. These results provide evidence that Aβ peptides interact directly with apoE lipoproteins, which may then be transported together into neuronal cells through apoE receptors.

Apolipoprotein E isoform-specific reduction of extracellular amyloid in neuronal cultures

Both apolipoprotein E (apoE) and amyloid peptides are associated with Alzheimers disease (AD). Using primary hippocampal neurons, we demonstrate that apoE is capable of reducing potentially toxic extracellular amyloid peptides, likely through a receptor mediated mechanism. We hypothesize that isoform-specific differences in apoE-mediated amyloid clearance and intracellular accumulation may be responsible, at least in part, for the increased number of amyloid plaques observed in apoE epsilon4 allele AD individuals.

The cholesterol-lowering drug probucol increases apolipoprotein E production in the hippocampus of aged rats: implications for Alzheimer's disease.

Several recent epidemiological studies have proposed that cholesterol-lowering drug Statin may provide protection against Alzheimers disease (AD). Probucol is a non-Statin cholesterol-lowering drug and a potent inducer of apolipoprotein E (apoE) production in peripheral circulation. A recent clinical study using Probucol in elderly AD subjects revealed a concomitant stabilisation of cognitive symptoms and significant increases in apoE levels in the cerebral spinal fluid in these patients. To gain insight into the mechanisms underlying these effects, we treated a cohort of aged male rats (26-month-old) with oral dose of Probucol for 30 days. Specifically, we examined the effects of Probucol on apoE production and its receptors (low density lipoprotein receptor [LDLr] and low density lipoprotein receptor-related protein [LRP]), astroglial marker of cell damage (glial fibrillary acidic protein [GFAP]), markers of neuronal synaptic plasticity and integrity (synaptosomal associated protein of 25 kDa [SNAP-25] and synaptophysin) as well as cholesterol biosynthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase [HMGCoAr]) in the hippocampus. We report that Probucol induces the production of apoE and one of its main receptors, LRP, increases HMGCoAr (rate-limiting enzyme in cholesterol synthesis), substantially attenuates age-related increases in glial activation, and induces production of synaptic marker SNAP-25, a molecule commonly associated with synaptogenesis and dendritic remodeling. These findings suggest that Probucol could promote neural and synaptic plasticity to counteract the synaptic deterioration associated with brain aging through an apoE/LRP-mediated system. Consistent with the beneficial effects of other cholesterol-lowering drugs such as the Statin, Probucol could also offers additional benefits based on apoE neurobiology.

Apolipoprotein E and Low‐Density Lipoprotein Binding and Internalization in Primary Cultures of Rat Astrocytes: Isoform‐Specific Alterations

Abstract: Apolipoprotein (apo) E is likely involved in redistributing cholesterol and phospholipids during compensatory synaptogenesis in the injured CNS. Three common isoforms of apoE exist in human (E2, E3, and E4). The apoE4 allele frequency is markedly increased in both late‐onset sporadic and familial Alzheimers disease (AD). ApoE concentration in the brain of AD subjects follows a gradient: ApoE levels decrease as a function of E2 > E3 ≫ E4. It has been proposed that the poor reinnervation capacity reported in AD may be caused by impairment of the apoE/low‐density lipoprotein (LDL) receptor activity. To understand further the role of this particular axis in lipid homeostasis in the CNS, we have characterized binding, internalization, and degradation of human 125I‐LDL to primary cultures of rat astrocytes. Specific binding was saturable, with a KD of 1.8 nM and a Bmax of 0.14 pmol/mg of proteins. Excess unlabeled human LDL or very LDL (VLDL) displaced 70% of total binding. Studies at 37°C confirmed that astrocytes bind, internalize, and degrade 125I‐LDL by a specific, saturable mechanism. Reconstituted apoE (E2, E3, and E4)‐liposomes were labeled with 125I and incubated with primary cultures of rat astrocytes and hippocampal neurons to examine specific binding. Human LDL and VLDL displaced binding and internalization of all apoE isoforms similarly in both astrocytes and neurons. 125I‐ApoE2 binding was significantly lower than that of the other 125I‐apoE isoforms in both cell types. 125I‐ApoE4 binding was similar to that of 125I‐apoE3 in both astrocytes and neurons. On the other hand, 125I‐apoE3 binding was significantly higher in neurons than in astrocytes. These isoform‐specific alterations in apoE‐lipoprotein pathway could explain some of the differences reported in the pathophysiology of AD subjects carrying different apoE alleles.

A polymorphism in lipoprotein lipase affects the severity of Alzheimer's disease pathophysiology

Emerging evidences indicate a role for lipoprotein lipase (LPL) in degenerative states. Genetic variations in the LPL gene were previously associated to lipid imbalance and coronary artery disease (CAD) risk and severity, a condition that shares pathological features with common Alzheimers disease (AD). To evaluate whether these genetic variations associate with the risk and pathophysiology of common AD, autopsy‐confirmed patients (242 controls, 153 AD) were genotyped for a PvuII single nucleotide polymorphism (SNP; rs285; referred to as the P+ allele) of LPL. Brain LPL mRNA levels, cholesterol levels, amyloid concentration, senile plaques and neurofibrillary tangles density counts were measured and contrasted with specific LPL genotypes. When adjusted for age and sex, homozygosity for the P+ allele resulted in an odds ratio of 2.3 for the risk of developing AD. More importantly, we report that the presence of the P+ allele of LPL significantly affects its mRNA expression level (n = 51; P = 0.026), brain tissue cholesterol levels (n = 55; P = 0.0013), neurofibrillary tangles (n = 52; P = 0.025) and senile plaque (n = 52; P = 0.022) densities. These results indicate that a common polymorphism in the lipoprotein lipase gene modulates the risk level for sporadic AD in the eastern Canadian population but more importantly, indirectly modulates the pathophysiology of the brain in autopsy‐confirmed cases.

Involvement of paraoxonase 1 genetic variants in Alzheimer’s disease neuropathology

Evidence suggests that the genes involved in brain lipid homeostasis are of particular relevance for Alzheimer’s disease (AD) etiology. Among these genes, that encoding paraoxonase 1 (PON1) has gained newfound interest from a public health perspective, as recent studies have suggested that PON1 L55M and Q192R genetic variants might affect individual susceptibility to environmental events, such as exposure to cholinesterase inhibitors. Cholinesterase inhibitor therapy being the treatment of choice for patients with mild to moderate AD, we sought to answer two main questions: (i) are these genetic variants associated with increased AD risk, earlier age of onset/death, or shorter AD duration; and (ii) do they affect the neuropathological hallmarks of AD? This genetic study used a large cohort of clinical and autopsy‐confirmed AD cases and age‐matched, cognitively intact controls from the Douglas Hospital Brain Bank, Quebec, Canada (n = 1066). The evidence presented here suggests multiple gender‐specific effects of PON1 polymorphisms on AD etiopathology. The L55M Met allele exerts an AD risk‐enhancing effect only in men (P < 0.001), whereas both men and women carrying the M55M/Q192Q genotype exhibit increased survival (2.5 years, P < 0.05) and later age of onset (1.5 years, P < 0.05). These genetic variants are also individually and significantly associated, sometimes in opposite directions for both genders, with β‐amyloid levels (P < 0.001), senile plaque accumulation (P < 0.001) and choline acetyltransferase activity (P < 0.05) in, respectively, two of two, five of six, and three of six brain areas. These results suggest an involvement of the PON1 gene in AD etiopathology and responses to treatment.

Superoxide Dismutase Expression in Parkinson's Disease

Although the biochemical basis for the extrapyramidal symptoms of idiopathic Parkinson’s disease (IPD), namely the brain nigrostriatal dopamine deficiency has been well defined (for review, see reference l), very little is known about the etiological factor(s) responsible for the neuronal loss in the substantia nigra (SN). One of the most popular research hypothesis deals with the possible involvement of free radicals’ species as a cause for nigral cell death in 1PD.I It assumes that the loss of nigral cells is a direct consequence of excessive oxidative stress due to defective antioxydant protection, leading to excessive lipid peroxidation. Indicators of oxidative stress such as glutathione content of the SN was shown to be lowered in patients with Parkinson’s disease,2J whereas lipid peroxide formation was found to be increased in the SN of IPD.4 The cerebrospinal fluid lipid hydroperoxide content remains unaltered in 1PD.S The increased lipid peroxidation in IPD was recently correlated with a significant increase of iron level^^*^ and a reduction of ferritin concentration’ in the SN of Parkinsonian patients. These two compounds are known as catalists of lipid peroxidation. The key antioxidant enzyme responsible for the scavenging of the toxic superoxide radicals, the superoxide dismutase (SOD), was found to be increased in the striatum and SN of IPD patients.”l0 The increased SOD activity in IPD has to be interpreted with caution since high SOD activity should lead to chronic synthesis of hydrogen peroxide:

Normalization of gene expression using SYBR green qPCR: a case for paraoxonase 1 and 2 in Alzheimer's disease brains.

Validating the expression stability of reference genes is crucial for reliable normalization of real-time quantitative PCR (qPCR) data, but relatively few studies have investigated this issue in brain human tissues. The present study thus aimed at identifying in human post-mortem brain tissues a set of suitable endogenous reference genes (ERG) for the expression analysis of potential candidate genes associated with Alzheimers disease (AD). The mRNA levels of ten common ERGs (ACTB, GAPDH, GPS1, GUSB, M-RIP, PGK1, POL2RF, PPIA, UBE2D2, and YES1) were determined in the frontal cortex of autopsy-confirmed AD and non-demented control cases (n=20) using SYBR Green technology. Then, these levels were ranked according to their expression stability using three software applications: geNorm, NormFinder and BestKeeper. Whereas PPIA and UBE2D2 were among the ERGs with the most reliable expression, ACTB was the worst. Subsequently, using PPIA and UBE2D2 as ERGs for normalization, the mRNA levels of paraoxonase 1 (PON1) and paraoxonase 2 (PON2) were quantified in the frontal cortex of AD and control cases (n=80) and analyzed using the REST 2009 program. Our results indicate that both paraoxonases are expressed in the human frontal cortex and that PON2 but not PON1 mRNA levels are up-regulated in AD relative to non-demented controls. However, re-analysis of the results by ANCOVA indicated that the significance of the difference between AD and control groups depended upon the ERG used for normalization. The use of a computational method allowing the inclusion of possible confounding factors is thus recommended for the analysis of data.