DNA hypomethylation of inflammation-associated genes in adipose tissue of female mice after multigenerational high fat diet feeding.

OBJECTIVE: Maternal obesity significantly increases the susceptibility of offspring to develop obesity and chronic diseases in adulthood. The offspring of obese mothers are shown to prefer high fat diet (HFD) due to their altered neural circuitry, creating a 'feed-forward cycle' across generations. We hypothesized that the 'feed-forward cycle' caused by multigenerational HFD feeding would have exacerbated effects in adipose tissue of the offspring. METHODS: Three generations (F0, F1 and F2) of HFD (60% Kcal fat)-fed and corresponding normal chow (NC)-fed C57BL/6 mice were generated. Body weight (BW) and food intake were monitored weekly. Parametrial adipose tissue (pAT) weight and endocrine parameters were measured in 9-month-old female offspring. Gene expression microarray, quantitative RT-PCR and bisulfite sequencing were performed using pAT. RESULTS: BW and pAT weight increased in female mice across generations under continuous HFD stress, with the most severe phenotype found in the F2 generation. Genes involved in inflammatory response showed increased expression across generations in the pAT, accompanied by increased macrophage infiltration. The promoters of Toll-like receptor 1 (Tlr1), Tlr2 and linker for activation of T cells (Lat) were hypomethylated in the HF groups compared with the NC group, with additional hypomethylation on some specific CpG sites in the F2 generation. CONCLUSIONS: A feed-forward cycle exists in female mice after continuous HFD stress as demonstrated by increased adiposity and progressive inflammation in adipose tissue across generations. DNA hypomethylation over generations lead to epigenetically altered expression of Tlr1, Tlr2 and Lat, which may contribute to the inflammation in adipose tissue. Our study provides a potential mechanism for enhanced inflammation in adipose tissue under multigenerational HFD-fed stress.

Cost-effectiveness of MRI in managing suspected scaphoid fractures.

In a cost-effectiveness study, we compared a treatment algorithm using repeated radiological examination with an algorithm using subacute MRI in patients with clinical signs of scaphoid fracture but normal initial radiography. Twenty-seven patients were included in both groups, and MRI reduced the immobilisation time from 20 days (range, 6-54) to 4 days (range, 1-19) and sick leave from 27 days (1-92) to 11 days (0-28). Use of MRI increased hospital costs by 151 euro (P<0.05), but reduced non-hospital costs by 2869 euro (P<0.05), making MRI cost-effective in the treatment of suspected scaphoid fractures.

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions.

Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27 - 30 detectable by the 3F4 antibody against human PrP109 - 112. We recently identified a new PK-resistant PrP species, designated PrP*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97 - 108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP*20 but also a small amount of 3F4-detected PK-resistant PrP27 - 30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP*20 to the occurrence of the 3F4-detected PrP27 - 30 was observed. Our study suggests that an increase in the level of PrP*20 characterizes the early stages of prion diseases.

Circulating cholesterol levels, apolipoprotein E genotype and dementia severity influence the benefit of atorvastatin treatment in Alzheimer's disease: results of the Alzheimer's Disease Cholesterol-Lowering Treatment (ADCLT) trial.

CONTEXT: Recent evidence suggests that treatment of mild-to-moderate Alzheimer's disease (AD) with atorvastatin provides significant benefit on the Alzheimer Disease Assessment Scale-Cognitive (ADAS-cog) after 6 months. OBJECTIVE: To determine if benefit on ADAS-cog performance produced by atorvastatin is influenced by severity of cognitive impairment, circulating cholesterol levels, or apolipoprotein E genotype. DESIGN: A double-blind, placebo-controlled, randomized (1:1) trial with a 1-year exposure to atorvastatin calcium or placebo. SETTING: A single-site study at the clinical research center of the Sun Health Research Institute. PARTICIPANTS: Ninety-eight individuals with mild-to-moderate AD (MMSE score of 12-28) provided informed consent, and 67 were randomized. Stable dose use of cholinesterase inhibitors, estrogen and vitamin E was allowed, as was the use of many other medications in the treatment of co-morbidities. Participants using cholesterol-lowering medications or being treated for major depression or a psychiatric condition were excluded. INTERVENTION: Once daily atorvastatin calcium (80 mg; two 40 mg tablets) or placebo. MAIN OUTCOME MEASURES: A primary outcome measure was change ADAS-cog sub-scale score. Secondary outcome measures included scores on the MMSE, and circulating cholesterol levels. The Apolipoprotein E genotype was established for each participant. RESULTS: A significant positive effect on ADAS-cog performance occurred after 6 months of atorvastatin therapy compared with placebo. This positive effect was more prominent among individuals entering the trial with, (i) higher MMSE scores, (ii) cholesterol levels above 200 mg/dl or (iii) if they harbored an apolipoprotein-E-4 allele compared with participants not responding to atorvastatin treatment. Individuals in the placebo group tended to experience more pronounced deterioration if their cholesterol levels exceeded 200 mg/dl or they harbored an apolipoprotein-E-4 allele. CONCLUSION: Atorvastatin therapy may be of benefit in the treatment of mild-to-moderately affected AD patients, but the level of benefit produced may be predicated on earlier treatment, an individual's apolipoprotein E genotype or whether the patient exhibits elevated cholesterol levels.

Pathobiology of familial hypercholesterolemic atherosclerosis.

Many factors play a role in the development of atherosclerotic lesions. One of the leading risk factors for development of atherosclerosis is familial hypercholesterolemia (FH). FH is a genetic disease characterized by a deficiency, and/or mutation, of receptors for low density lipoprotein (LDL) on the plasmalemma of endothelial cells (EC), a high level of low density lipoprotein in the plasma, and early, spontaneous development of atherosclerosis and skin xanthoma. In this review we describe Watanabe heritable hyperlipidemic (WHHL) rabbits, which represent such an animal model for human FH. This strain of the rabbits is characterized by a genetic deficiency or mutation of functional LDL receptors and develops severe atherosclerosis, which is pathologically similar to familial homozygous hyperlipidemic patients. The most completely characterized animal model is the Watanabe rabbit, a model of homozygous and heterozygous type IIa hypercholesterolemia related to an LDL receptor deficiency. Additional manipulation such as aortic injury in this rabbit model induces the development of atherosclerotic lesions that are structurally similar to those found in humans. Thus, this model of hypercholesterolemia fulfils the above criteria set, i.e. it is able to provide new insights for a better understanding of the pathogenesis of atherosclerosis and for testing new treatment strategies.

Creutzfeldt-Jakob disease associated with a deletion of two repeats in the prion protein gene.

A two-octapeptide repeat deletion of the prion protein gene has been recently observed in a patient with a 2-year history of dementia and a clinical diagnosis of possible Creutzfeldt-Jakob disease (CJD). The authors report a similar deletion in a patient with a definitive diagnosis of CJD. Since the two-repeat deletion has not been observed in large, population-based studies, the two cases suggest that this deletion is a new pathogenic mutation associated with CJD.

Novel differences between two human prion strains revealed by two-dimensional gel electrophoresis.

The phenotype of human sporadic prion diseases is affected by patient genotype at codon 129 of the prion protein (PrP) gene, the site of a common methionine/valine polymorphism, and by the type of the scrapie PrP (PrP(Sc)), which likely reflects the prion strain. However, two distinct disease phenotypes, identified as sporadic Creutzfeldt-Jakob disease (M/M2 sCJD) and sporadic fatal insomnia (sFI), share methionine homozygosity at codon 129 and PrP(Sc) type 2. One-dimensional gel electrophoresis and immunoblotting reveal no difference between the M/M2 sCJD and sFI species of PrP(Sc) in gel mobility and glycoform ratio. In contrast, the two-dimensional immunoblot demonstrates that in M/M2 sCJD the full-length PrP(Sc) form is overrepresented and carries glycans that are different from those present in the PrP(Sc) of sFI. Because the altered glycans are detectable only in the PrP(Sc) and not in the normal or cellular PrP (PrP(C)), they are likely to result from preferential conversion to PrP(Sc) of rare PrP(C) glycoforms. This is the first evidence that a qualitative difference in glycans contributes to prion diversity.

Oxidative damage is the earliest event in Alzheimer disease.

Recently, we demonstrated a significant increase of an oxidized nucleoside derived from RNA, 8-hydroxyguanosine (8OHG), and an oxidized amino acid, nitrotyrosine in vulnerable neurons of patients with Alzheimer disease (AD). To determine whether oxidative damage is an early- or end-stage event in the process of neurodegeneration in AD, we investigated the relationship between neuronal 8OHG and nitrotyrosine and histological and clinical variables, i.e. amyloid-beta (A beta) plaques and neurofibrillary tangles (NFT), as well as duration of dementia and apolipoprotein E (ApoE) genotype. Our findings show that oxidative damage is quantitatively greatest early in the disease and reduces with disease progression. Surprisingly, we found that increases in A beta deposition are associated with decreased oxidative damage. These relationships are more significant in ApoE epsilon4 carriers. Moreover, neurons with NFT show a 40%-56% decrease in relative 8OHG levels compared with neurons free of NFT. Our observations indicate that increased oxidative damage is an early event in AD that decreases with disease progression and lesion formation. These findings suggest that AD is associated with compensatory changes that reduce damage from reactive oxygen.

Mitochondrial abnormalities in Alzheimer's disease.

The finding that oxidative damage, including that to nucleic acids, in Alzheimer's disease is primarily limited to the cytoplasm of susceptible neuronal populations suggests that mitochondrial abnormalities might be part of the spectrum of chronic oxidative stress of Alzheimer's disease. In this study, we used in situ hybridization to mitochondrial DNA (mtDNA), immunocytochemistry of cytochrome oxidase, and morphometry of electron micrographs of biopsy specimens to determine whether there are mitochondrial abnormalities in Alzheimer's disease and their relationship to oxidative damage marked by 8-hydroxyguanosine and nitrotyrosine. We found that the same neurons showing increased oxidative damage in Alzheimer's disease have a striking and significant increase in mtDNA and cytochrome oxidase. Surprisingly, much of the mtDNA and cytochrome oxidase is found in the neuronal cytoplasm and in the case of mtDNA, the vacuoles associated with lipofuscin. Morphometric analysis showed that mitochondria are significantly reduced in Alzheimer's disease. The relationship shown here between the site and extent of mitochondrial abnormalities and oxidative damage suggests an intimate and early association between these features in Alzheimer's disease.

Induction of HO-1 and NOS in doppel-expressing mice devoid of PrP: implications for doppel function.

Ectopic expression of the doppel (Dpl) protein, a homologue of the prion protein (PrP), was recently associated with cerebellar Purkinje cell degeneration observed in two aging prion protein knock-out (Prnp(0/0)) mouse lines. We investigated the possible role of Dpl in oxidative metabolism. Two Prnp(0/0) mouse lines of similar genetic background were studied. One line expresses Dpl in the brain and displays Dpl-associated cerebellar abnormalities. The other has no elevated expression of Dpl and no cerebellar abnormalities. We observed a correlation between Dpl expression and the induction of both heme oxygenase 1 (HO-1) and nitric oxide synthase systems (nNOS and iNOS). These responses are suggestive of increased oxidative stress in the brains of the Dpl-expressing Prnp(0/0) mice. No induction was observed with Hsp-60, indicating a specific response by the HO/NOS system. We proposed that Dpl expression exacerbates oxidative damage that is antagonistic to the protective function of wild-type PrP.

Differential expression of cellular prion protein in mouse brain as detected with multiple anti-PrP monoclonal antibodies.

The normal cellular prion protein (PrP(C)) plays an essential role in the development of prion diseases. Indirect evidence has suggested that different PrP(C) glycoforms may be expressed in different brain regions and perform distinct functions. However, due to a lack of monoclonal antibodies (Mabs) that are specific for mouse PrP(C), the expression of PrP(C) in the mouse brain has not been studied in great detail. We used Mabs specific for either the N-terminus or the C-terminus of the mouse PrP(C) to study its expression in the mouse brain by immunoblotting and immunohistochemistry. Immunoblotting studies demonstrated that the expression of PrP(C) differed quantitatively as well as qualitatively in different regions of the brain. The anti-C-terminus Mabs reacted with all three molecular weight bands of PrP(C); the anti-N-terminus Mabs only reacted with the 39-42 kDa PrP(C). The results from immunohistochemical staining revealed the spatial distribution of PrP(C) in the mouse brain, which were consistent with that from immunoblotting. Although expression of PrP(C) has been reported to be required for long-term survival of Purkinje cells, we were unable to detect PrP(C) in the Purkinje cell layer in the cerebellum with multiple anti-PrP Mabs. Our findings suggest that PrP(C) variants, i.e. various glycoforms and truncated forms, might be specifically expressed in different regions of mouse brain and might have different functions.

Normal cellular prion protein is preferentially expressed on subpopulations of murine hemopoietic cells.

We studied the expression of normal cellular prion protein (PrP(C)) in mouse lymphoid tissues with newly developed mAbs to PrP(C). Most of the mature T and B cells in the peripheral lymphoid organs do not express PrP(C). In contrast, most thymocytes are PrP(C+). In the bone marrow, erythroid cells and maturing granulocytes are PrP(C+). Approximately 50% of the cells in the region of small lymphocytes and progenitor cells also express PrP(C). Most of these PrP(C+) cells are CD43(+), but B220(-), surface IgM(-) (sIgM(-)), and IL-7R(-), a phenotype that belongs to cells not yet committed to the B cell lineage. Another small group of the PrP(C+) cell are B220(+), and some of these are also sIgM(+). The majority of the B220(+) cells, however, are PrP(C-). Therefore, PrP(C) is preferentially expressed in early bone marrow progenitor cells and subsets of maturing B cells. Supporting this interpretation is our observation that stimulation of bone marrow cells in vitro with PMA results in a decrease in the number of PrP(C+)B220(-) cells with a corresponding increase of sIgM(+)B220(high) mature B cells. This result suggests that the PrP(C+)B220(-) cells are potential progenitors. Furthermore, in the bone marrow of Rag-1(-/-) mice, there are an increased number of PrP(C+)B220(-) cells, and most of the developmentally arrested pro-B cells in these mice are PrP(C+). Collectively, these results suggest that PrP(C) is expressed preferentially in immature T cells in the thymus and early progenitor cells in the bone marrow, and the expression of PrP(C) is regulated during hemopoietic differentiation.

The expression and potential function of cellular prion protein in human lymphocytes.

We examined expression of the normal cellular prion protein (PrP(C)) in human peripheral blood mononuclear cells (PBMC) and in transfected neuroblastoma cells with a panel of six monoclonal antibodies (Mabs). While all six of the Mabs reacted strongly with the neuroblastoma cells, only four of the Mabs reacted with PrP(C) expressed by human PBMC. PrP(C) is expressed at high levels in human T cells, B cells, monocytes, and dendritic cells, but not in red blood cells. Immunoblotting studies revealed that the PrP(C) glycoforms and the composition of the N-linked glycans on PrP(C) in human PBMC are different from those of the brain or the neuroblastoma cells. In human PBMC and the neuroblastoma cell lines the N-terminal portion of the PrP(C) is hypersensitive to proteolytic digestion, suggesting that the N-terminus of the PrP(C) on the surface of a living cell lacks secondary structure. We found that the level of PrP(C) expressed on the surface of human T lymphocytes was up-regulated as a consequence of cellular activation. Accordingly, memory T cells express more PrP(C) than naïve T cells. In addition, the proliferation of human T lymphocytes stimulated with an anti-CD3 Mab was inhibited by anti-PrP(C) Mabs. Collectively, these results suggest that PrP(C) can participate in signal transduction in human T lymphocytes.

Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein.

Although minor abnormalities have been reported in prion protein (PrP) knock-out (Prnp-/-) mice, the normal physiological function of PrP, the causative agent implicated in transmissible spongiform encephalopathies (TSE), remains unresolved. Since there are increasing correlations between oxidative stress and amyloidoses, we decided to investigate whether PrP plays a role in oxidative modulation. We found higher levels of oxidative damage to proteins and lipids in the brain lysates of Prnp-/- as compared to wild-type (WT) mice of the same genetic background. These two indicators, protein oxidation and lipid peroxidation, are hallmarks of cellular oxidative damage. Elevated levels of ubiquitin-protein conjugates were also observed in Prnp-/- mice, a probable consequence of cellular attempts to remove the damaged proteins as indicated by increased proteasome activity. Taken together, these findings are indicative of a role for PrP in oxidative homeostasis in vivo.

Expression and structural characterization of the recombinant human doppel protein.

The doppel protein (Dpl) is a newly recognized prion protein (PrP)-like molecule encoded by a novel gene locus, prnd, located on the same chromosome as the PrP gene. To study the structural features of Dpl, we have expressed recombinant human Dpl corresponding to the putative mature protein domain (residues 24-152) in Escherichia coli. The primary structure of the recombinant Dpl 24-152 was characterized using gel electrophoresis, N-terminal Edman sequencing, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization mass spectrometry. Dpl 24-152 was shown to contain two disulfide bonds (Cys94-Cys145 and Cys108-Cys140). The secondary structure of Dpl was analyzed using far-UV circular dichroism spectroscopy. Dpl 24-152 was found to be an alpha-helical protein having a high helical content (40%). Dpl 24-152 exhibited characteristics of a thermodynamically stable protein that undergoes reversible and cooperative thermal denaturation. In addition, Dpl was found to be soluble and sensitive to proteinase K digestion. Therefore, Dpl 24-152 possesses biochemical properties similar to those of recombinant PrP. This study provides knowledge about the molecular features of human Dpl that will be useful in further investigation into its normal function and the role it may play in neurodegenerative diseases.

Prion disease: A loss of antioxidant function?

Prion disease, a neurodegenerative disorder, is widely believed to arise when a cellular prion protein (PrP(C)) undergoes conformational changes to a pathogenic isoform (PrP(Sc)). Recent data have shown PrP(C) to be copper binding and that it acquires antioxidant activity as a result. This enzymatic property is dependent mainly on copper binding to the octarepeats region. In normal human brain and human prion disease, there is a population of brain-derived PrP that has been truncated at the N-terminal which encompassed the octarepeats region. Increasing evidences have suggested imbalances of metal-catalyzed reactions to be the common denominator for several neurodegenerative diseases. Therefore, we propose that one of the causative factors for prion disease could be due to the imbalances in metal-catalyzed reactions resulting in an alteration of the antioxidant function. These result in an increase level of oxidative stress and, as such, trigger the neurodegenerative cascade.

Effect of the E200K mutation on prion protein metabolism. Comparative study of a cell model and human brain.

The hallmark of prion diseases is the cerebral accumulation of a conformationally altered isoform (PrP(Sc)) of a normal cellular protein, the prion protein (PrP(C)). In the inherited form, mutations in the prion protein gene are thought to cause the disease by altering the metabolism of the mutant PrP (PrP(M)) engendering its conversion into PrP(Sc). We used a cell model to study biosynthesis and processing of PrP(M) carrying the glutamic acid to lysine substitution at residue 200 (E200K), which is linked to the most common inherited human prion disease. PrP(M) contained an aberrant glycan at residue 197 and generated an increased quantity of truncated fragments. In addition, PrP(M) showed impaired transport of the unglycosylated isoform to the cell surface. Similar changes were found in the PrP isolated from brains of patients affected by the E200K variant of Creutzfeldt-Jakob disease. Although the cellular PrP(M) displayed some characteristics of PrP(Sc), the PrP(Sc) found in the E200K brains was quantitatively and qualitatively different. We propose that the E200K mutation cause the same metabolic changes of PrP(M) in the cell model and in the brain. However, in the brain, PrP(M) undergoes additional modifications, by an age-dependent mechanism that leads to the formation of PrP(Sc) and the development of the disease.

Altered cell-matrix associated ADAM proteins in Alzheimer disease.

Alterations in cell-matrix 'contact' are often related to a disruption of cell cycle regulation and, as such, occur variously in neoplasia. Given the recent findings showing cell cycle alterations in Alzheimer disease, we undertook a study of ADAM-1 and 2 (A Disintegrin And Metalloprotease), developmentally-regulated, integrin-binding, membrane-bound metalloproteases. Our results show that whereas ADAM-1 and 2 are found in susceptible hippocampal neurons in Alzheimer disease, these proteins were not generally increased in similar neuronal populations in younger or age-matched controls except in association with age-related neurofibrillary alterations. This increase in both ADAM-1 and 2 in cases of Alzheimer disease was verified by immunoblot analysis (P < 0.05). An ADAM-induced loss of matrix integration would effectively "reset" the mitotic clock and thereby stimulate re-entry into the cell cycle in neurons in Alzheimer disease. Furthermore, given the importance of integrins in maintaining short-term memory, alterations in ADAM proteins or their proteolytic activity could also play a proximal role in the clinico-pathological manifestations of Alzheimer disease.