Examination of Longitudinal Alterations in Alzheimer's Disease-Related Neurogenesis in an APP/PS1 Transgenic Mouse Model, and the Effects of P33, a Putative Neuroprotective Agent Thereon.

Neurogenesis plays a crucial role in cognitive processes. During aging and in Alzheimer's disease (AD), altered neurogenesis and neuroinflammation are evident both in C57BL/6J, APPSwe/PS1dE9 (Tg) mice and humans. AD pathology may slow down upon drug treatment, for example, in a previous study of our group P33, a putative neuroprotective agent was found to exert advantageous effects on the elevated levels of APP, Aß, and neuroinflammation. In the present study, we aimed to examine longitudinal alterations in neurogenesis, neuroinflammation and AD pathology in a transgenic (Tg) mouse model, and assessed the putative beneficial effects of long-term P33 treatment on AD-specific neurological alterations. Hippocampal cell proliferation and differentiation were significantly reduced between 8 and 12 months of age. Regarding neuroinflammation, significantly elevated astrogliosis and microglial activation were observed in 6- to 7-month-old Tg animals. The amounts of the molecules involved in the amyloidogenic pathway were altered from 4 months of age in Tg animals. P33-treatment led to significantly increased neurogenesis in 9-month-old animals. Our data support the hypothesis that altered neurogenesis may be a consequence of AD pathology. Based on our findings in the transgenic animal model, early pharmacological treatment before the manifestation of AD symptoms might ameliorate neurological decline.

Neuroinflammatory processes are augmented in mice overexpressing human heat-shock protein B1 following ethanol-induced brain injury.

BACKGROUND: Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury. METHODS: In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry. RESULTS: Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions. CONCLUSIONS: Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.

Synaptic mitochondrial dysfunction and septin accumulation are linked to complement-mediated synapse loss in an Alzheimer's disease animal model.

Synaptic functional disturbances with concomitant synapse loss represent central pathological hallmarks of Alzheimer's disease. Excessive accumulation of cytotoxic amyloid oligomers is widely recognized as a key event that underlies neurodegeneration. Certain complement components are crucial instruments of widespread synapse loss because they can tag synapses with functional impairments leading to their engulfment by microglia. However, an exact understanding of the affected synaptic functions that predispose to complement-mediated synapse elimination is lacking. Therefore, we conducted systematic proteomic examinations on synaptosomes prepared from an amyloidogenic mouse model of Alzheimer's disease (APP/PS1). Synaptic fractions were separated according to the presence of the C1q-tag using fluorescence-activated synaptosome sorting and subjected to proteomic comparisons. The results raised the decline of mitochondrial functions in the C1q-tagged synapses of APP/PS1 mice based on enrichment analyses, which was verified using flow cytometry. Additionally, proteomics results revealed extensive alterations in the level of septin protein family members, which are known to dynamically form highly organized pre- and postsynaptic supramolecular structures, thereby affecting synaptic transmission. High-resolution microscopy investigations demonstrated that synapses with considerable amounts of septin-3 and septin-5 show increased accumulation of C1q in APP/PS1 mice compared to the wild-type ones. Moreover, a strong positive correlation was apparent between synaptic septin-3 levels and C1q deposition as revealed via flow cytometry and confocal microscopy examinations. In sum, our results imply that deterioration of synaptic mitochondrial functions and alterations in the organization of synaptic septins are associated with complement-dependent synapse loss in Alzheimer's disease.

Male and Female Animals Respond Differently to High-Fat Diet and Regular Exercise Training in a Mouse Model of Hyperlipidemia.

Inappropriate nutrition and a sedentary lifestyle can lead to obesity, one of the most common risk factors for several chronic diseases. Although regular physical exercise is an efficient approach to improve cardiometabolic health, the exact cellular processes are still not fully understood. We aimed to analyze the morphological, gene expression, and lipidomic patterns in the liver and adipose tissues in response to regular exercise. Healthy (wild type on a normal diet) and hyperlipidemic, high-fat diet-fed (HFD-fed) apolipoprotein B-100 (APOB-100)-overexpressing mice were trained by treadmill running for 7 months. The serum concentrations of triglyceride and tumor necrosis factor α (TNFα), as well as the level of lipid accumulation in the liver, were significantly higher in HFD-fed APOB-100 males compared to females. However, regular exercise almost completely abolished lipid accumulation in the liver of hyperlipidemic animals. The expression level of the thermogenesis marker, uncoupling protein-1 (Ucp1), was significantly higher in the subcutaneous white adipose tissue of healthy females, as well as in the brown adipose tissue of HFD-fed APOB-100 females, compared to males. Lipidomic analyses revealed that hyperlipidemia essentially remodeled the lipidome of brown adipose tissue, affecting both the membrane and storage lipid fractions, which was partially restored by exercise in both sexes. Our results revealed more severe metabolic disturbances in HFD-fed APOB-100 males compared to females. However, exercise efficiently reduced the body weight, serum triglyceride levels, expression of pro-inflammatory factors, and hepatic lipid accumulation in our model.

Effects of the Pentapeptide P33 on Memory and Synaptic Plasticity in APP/PS1 Transgenic Mice: A Novel Mechanism Presenting the Protein Fe65 as a Target.

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) leads to the formation of fragments, among which the intracellular domain of APP (AICD) was also identified to be a causative of early pathological events. AICD-counteracting proteins, such as Fe65, may serve as alternative therapeutic targets of Alzheimer's disease (AD). The detection of elevated levels of Fe65 in the brains of both human patients and APP transgenic mice may further strengthen the hypothesis that influencing the interaction between Fe65 and APP may have a beneficial effect on the course of AD. Based on a PXP motif, proven to bind to the WW domain of Fe65, a new pentapeptide was designed and tested. The impedimental effect of P33 on the production of beta amyloid (Aß) (soluble fraction and aggregated plaques) and on the typical features of the AD pathology (decreased dendritic spine density, synaptic markers, elevated inflammatory reactions) was also demonstrated. Significant enhancements of both learning ability and memory function were observed in a Morris water maze paradigm. The results led us to formulate the theory that P33 acts by altering the conformation of Fe65 via binding to its WW domain, consequently hindering any interactions between Fe65 and key members involved in APP processing.

Heat Shock Proteins and Autophagy Pathways in Neuroprotection: from Molecular Bases to Pharmacological Interventions.

Neurodegenerative diseases (NDDs) such as Alzheimer's disease, Parkinson's disease and Huntington's disease (HD), amyotrophic lateral sclerosis, and prion diseases are all characterized by the accumulation of protein aggregates (amyloids) into inclusions and/or plaques. The ubiquitous presence of amyloids in NDDs suggests the involvement of disturbed protein homeostasis (proteostasis) in the underlying pathomechanisms. This review summarizes specific mechanisms that maintain proteostasis, including molecular chaperons, the ubiquitin-proteasome system (UPS), endoplasmic reticulum associated degradation (ERAD), and different autophagic pathways (chaperon mediated-, micro-, and macro-autophagy). The role of heat shock proteins (Hsps) in cellular quality control and degradation of pathogenic proteins is reviewed. Finally, putative therapeutic strategies for efficient removal of cytotoxic proteins from neurons and design of new therapeutic targets against the progression of NDDs are discussed.

Brain cortical cholesterol metabolism is highly affected by human APP overexpression in mice.

Processing of the amyloid precursor protein (APP) and amyloid beta (Aß) has been for decades in the center of Alzheimer's disease (AD) research. Beside many other variables, lipids, especially cholesterol and its derivatives, are discussed to contribute to AD pathogenesis. Several studies show that cholesterol affects APP metabolism. Also the converse mechanism, the direct influence of Aß on cholesterol metabolism, has been described. To further investigate this crosstalk between cholesterol- and APP metabolism, a high-fat feeding study was conducted with animals overexpressing human APPSL and/or human ApoB-100. The impact of diet and genotype on cerebral cholesterol metabolism and content as well as spatial learning and memory was examined. While behavioral performance was not influenced by this high fat diet (HFD), reduction of cortical free cholesterol levels and mRNA expression patterns under normal diet and HFD conditions in human APPSL overexpressing mice argue for an important role of APP in cerebral lipid metabolism. From our results we conclude that increased APP metabolism in ApoBxAPP and APPSL mice induces mechanisms to reduce free cholesterol levels.

Nonlocality and conflicting interest games.

Nonlocality enables two parties to win specific games with probabilities strictly higher than allowed by any classical theory. Nevertheless, all known such examples consider games where the two parties have a common interest, since they jointly win or lose the game. The main question we ask here is whether the nonlocal feature of quantum mechanics can offer an advantage in a scenario where the two parties have conflicting interests. We answer this in the affirmative by presenting a simple conflicting interest game, where quantum strategies outperform classical ones. Moreover, we show that our game has a fair quantum equilibrium with higher payoffs for both players than in any fair classical equilibrium. Finally, we play the game using a commercial entangled photon source and demonstrate experimentally the quantum advantage.

Hepatic mitochondrial and ER stress induced by defective PPARα signaling in the pathogenesis of hepatic steatosis.

Emerging evidence demonstrates a close interplay between disturbances in mitochondrial function and ER homeostasis in the development of the metabolic syndrome. The present investigation sought to advance our understanding of the communication between mitochondrial dysfunction and ER stress in the onset of hepatic steatosis in male rodents with defective peroxisome proliferator-activated receptor-α (PPARα) signaling. Genetic depletion of PPARα or perturbation of PPARα signaling by high-fructose diet compromised the functional activity of metabolic enzymes involved in mitochondrial fatty acid ß-oxidation and induced hepatic mitochondrial stress in rats and mice. Inhibition of PPARα activity further enhanced the expression of apolipoprotein B (apoB) mRNA and protein, which was associated with reduced mRNA expression of the sarco/endoplasmic reticulum calcium ATPase (SERCA), the induction of hepatic ER stress, and hepatic steatosis. Restoration of PPARα activity recovered the metabolic function of the mitochondria and ER, alleviated systemic hypertriglyceridemia, and improved hepatic steatosis. These findings unveil novel roles for PPARα in mediating stress signals between hepatic subcellular stress-responding machinery and in the onset of hepatic steatosis under conditions of metabolic stress.

Role of interleukin-6 and interleukin-10 in morphological and functional changes of the blood-brain barrier in hypertriglyceridemia.

BACKGROUND: Hypertriglyceridemia is closely linked to atherosclerosis related inflammatory processes and blood-brain barrier (BBB) dysfunction. Using apolipoprotein B-100 (APOB-100) transgenic mice, an animal model of chronic hypertriglyceridemia, we analyzed BBB function and morphology in vitro and ex vivo. Our objective was to determine which BBB characteristics are produced mainly by interleukin (IL)-6, an atherosclerosis promoting cytokine, and whether these actions can be antagonized by IL-10, an anti-inflammatory cytokine. METHODS: Brain endothelial and glial cell cultures and brain microvessels were isolated from wild type (WT) and APOB-100 transgenic mice and were treated with IL-6, IL-10 and their combination. First, IL-6 and IL-10 production was measured in WT and APOB-100 microvessels using qPCR. Then functional parameters of endothelial cell cultures were analyzed and immunocytochemistry for key BBB proteins was performed. RESULTS: IL-6 mRNA levels were higher in brain microvessels than in brain parenchyma of APOB-100 transgenic mice. Transendothelial electric resistance and P-glycoprotein activity were lower, and paracellular permeability was higher in cultured APOB-100 brain endothelial cells. These features were sensitive to both IL-6 and IL-10 treatments. A decreased P-glycoprotein immunostaining was measured in transgenic endothelial cells under control conditions and in WT cells after treating them with IL-6. This effect was antagonized by IL-10. Changes in immunostaining for tight junction proteins were observed after IL-6 exposure, which were in part antagonized by IL-10. In glial cell cultures an increase in aquaporin-4 immunolabeling in the transgenic group and an increase in microglia cell density in WT glia cultures was detected after IL-6 treatment, which was antagonized by IL-10. In isolated brain microvessels a decrease in P-glycoprotein immunolabeled area fraction was measured in APOB-100 microvessels under control conditions and in WT microvessels after every cytokine treatment. ZO-1 immunolabeling showed characteristics similar to that of P-glycoprotein. No change was seen in claudin-5 and occludin immunoreactive area fractions in microvessels. A decrease in aquaporin-4 immunoreactivity was measured in WT microvessels treated by IL-6, which was antagonized by IL-10. CONCLUSION: IL-6 produced in microvessels contributes to BBB impairment observed in the APOB-100 mice. We showed that IL-10 partly antagonizes the effects of IL-6 at the BBB.

Exercise training worsens cardiac performance in males but does not change ejection fraction and improves hypertrophy in females in a mouse model of metabolic syndrome.

BACKGROUND: Metabolic syndrome (MetS) refers to a cluster of co-existing cardio-metabolic risk factors, including visceral obesity, dyslipidemia, hyperglycemia with insulin resistance, and hypertension. As there is a close link between MetS and cardiovascular diseases, we aimed to investigate the sex-based differences in MetS-associated heart failure (HF) and cardiovascular response to regular exercise training (ET). METHODS: High-fat diet-fed male and female APOB-100 transgenic (HFD/APOB-100, 3 months) mice were used as MetS models, and age- and sex-matched C57BL/6 wild-type mice on standard diet served as healthy controls (SD/WT). Both the SD/WT and HFD/APOB-100 mice were divided into sedentary and ET groups, the latter running on a treadmill (0.9 km/h) for 45 min 5 times per week for 7 months. At month 9, transthoracic echocardiography was performed to monitor cardiac function and morphology. At the termination of the experiment at month 10, blood was collected for serum low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-cholesterol measurements and homeostatic assessment model for insulin resistance (HOMA-IR) calculation. Cardiomyocyte hypertrophy and fibrosis were assessed by histology. Left ventricular expressions of selected genes associated with metabolism, inflammation, and stress response were investigated by qPCR. RESULTS: Both HFD/APOB-100 males and females developed obesity and hypercholesterolemia; however, only males showed insulin resistance. ET did not change these metabolic parameters. HFD/APOB-100 males showed echocardiographic signs of mild HF with dilated ventricles and thinner walls, whereas females presented the beginning of left ventricular hypertrophy. In response to ET, SD/WT males developed increased left ventricular volumes, whereas females responded with physiologic hypertrophy. Exercise-trained HFD/APOB-100 males presented worsening HF with reduced ejection fraction; however, ET did not change the ejection fraction and reversed the echocardiographic signs of left ventricular hypertrophy in HFD/APOB-100 females. The left ventricular expression of the leptin receptor was higher in females than males in the SD/WT groups. Left ventricular expression levels of stress response-related genes were higher in the exercise-trained HFD/APOB-100 males and exercise-trained SD/WT females than exercise-trained SD/WT males. CONCLUSIONS: HFD/APOB-100 mice showed sex-specific cardiovascular responses to MetS and ET; however, left ventricular gene expressions were similar between the groups except for leptin receptor and several stress response-related genes.

Interplay between glycogen synthase kinase-3ß and tau in the cerebellum of Hsp27 transgenic mouse.

The association between heat shock protein 27 (Hsp27) and hyperphosphorylated tau has gained attention for more than a decade, but it has never been explored in vivo. In the present study, we found that tau phosphorylated at S396/404 (PHF-1) and S262 sites was significantly increased in the cerebellum of Hsp27 transgenic mice, which was concomitant with increased glycogen synthase kinase-3ß (GSK3ß) phosphorylated at Y216 and decreased GSK3ß phosphorylated at S9. Neither 70-kDa ribosomal protein S6 kinase (p70S6K; total p70S6K, p70S6K at T389, and p70S6K at T421/S424) nor protein phosphatase PP2A (total PP2A, PP2A at Y307, methylated or demethylated PP2A) was changed. This suggests that the increased tau phosphorylation at S396/404 and S262 sites may be induced by Hsp27 through enhancement of GSK3ß activity in the mouse cerebellum.

Age-Related Inflammatory Balance Shift, Nasal Barrier Function, and Cerebro-Morphological Status in Healthy and Diseased Rodents.

Increased blood-brain barrier (BBB) permeability and extensive neuronal changes have been described earlier in both healthy and pathological aging like apolipoprotein B-100 (APOB-100) and amyloid precursor protein (APP)-presenilin-1 (PSEN1) transgenic mouse models. APOB-100 hypertriglyceridemic model is a useful tool to study the link between cerebrovascular pathology and neurodegeneration, while APP-PSEN1 humanized mouse is a model of Alzheimer's disease. The aim of the current study was to characterize the inflammatory changes in the brain with healthy aging and in neurodegeneration. Also, the cerebro-morphological and cognitive alterations have been investigated. The nose-to-brain delivery of a P-glycoprotein substrate model drug (quinidine) was monitored in the disease models and compared with the age-matched controls. Our results revealed an inflammatory balance shift in both the healthy aged and neurodegenerative models. In normal aging monocyte chemoattractant protein-1, stem cell factor and Rantes were highly upregulated indicating a stimulated leukocyte status. In APOB-100 mice, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB), and interleukin-17A (IL-17A) were induced (vascular reaction), while in APP-PSEN1 mice resistin, IL-17A and GM-CSF were mostly upregulated. The nasal drug absorption was similar in the brain and blood indicating the molecular bypass of the BBB. The learning and memory tests showed no difference in the cognitive performance of healthy aged and young animals. Based on these results, it can be concluded that various markers of chronic inflammation are present in healthy aged and diseased animals. In APOB-100 mice, a cerebro-ventricular dilation can also be observed. For development of proper anti-aging and neuroprotective compounds, further studies focusing on the above inflammatory targets are suggested.

Q134R: Small chemical compound with NFAT inhibitory properties improves behavioral performance and synapse function in mouse models of amyloid pathology.

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging-related Alzheimer's disease (AD)-like pathology. However, concerns over adverse effects have slowed the transition of common CN-inhibiting drugs to the clinic for the treatment of AD and AD-related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN-mediated dephosphorylation of a non-NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild-type mice (3-4 months old) infused with oligomeric Aß peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild-type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aß plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders.

Biglycan protects cardiomyocytes against hypoxia/reoxygenation injury: role of nitric oxide.

Biglycan, a proteoglycan component of extracellular matrix, has been suspected to contribute to the development of atherosclerosis, but overexpression of biglycan in transgenic mice has been shown to induce cardioprotective genes including nitric oxide (NO) synthases in the heart. Therefore, here we hypothesized if exogenous administration of biglycan exerts cytoprotection. Primary cardiomyocytes from neonatal rats were subjected to 150 min hypoxia and 2 h reoxygenation. Mortality of cardiomyocytes was dose-dependently attenuated by pretreatment with 1-100 nM biglycan. Biglycan enhanced eNOS mRNA and protein, and significantly increased NO content of cardiomyocytes. The NO synthase inhibitor l-nitro-arginine-methyl-ester significantly attenuated the cytoprotective effect of biglycan. This is the first demonstration that biglycan leads to cytoprotection against hypoxia/reoxygenation injury, and that this phenomenon is partially mediated by an NO-dependent mechanism.

Apolipoprotein B100 acts as a molecular link between lipid-induced endoplasmic reticulum stress and hepatic insulin resistance.

UNLABELLED: Accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) results in ER stress and lipid overload-induced ER stress has been implicated in the development of insulin resistance. Here, evidence is provided for a molecular link between hepatic apolipoprotein B100 (apoB100), induction of ER stress, and attenuated insulin signaling. First, in vivo upregulation of hepatic apoB100 by a lipogenic diet was found to be closely associated with ER stress and attenuated insulin signaling in the liver. Direct in vivo overexpression of human apoB100 in a mouse transgenic model further supported the link between excessive apoB100 expression and hepatic ER stress. Human apoB100 transgenic mice exhibited hypertriglyceridemia and hyperglycemia. In vitro, accumulation of cellular apoB100 by free fatty acid (oleate) stimulation or constant expression of wild-type or N-glycosylation mutant apoB50 in hepatic cells induced ER stress. This led to perturbed activation of glycogen synthase kinase 3 and glycogen synthase by way of the activation of c-Jun N-terminal kinase and suppression of insulin signaling cascade, suggesting that dysregulation of apoB was sufficient to disturb ER homeostasis and induce hepatic insulin resistance. Small interfering (si)RNA-mediated attenuation of elevated apoB level in the apoB50-expressing cells rescued cells from lipid-induced ER stress and reversed insulin insensitivity. CONCLUSION: These findings implicate apoB100 as a molecular link between lipid-induced ER stress and hepatic insulin resistance.

Biologia futura: animal testing in drug development-the past, the present and the future.

Animal experiments have served to improve our knowledge on diseases and treatment approaches since ancient times. Today, animal experiments are widely used in medical, biomedical and veterinary research, and are essential means of drug development and preclinical testing, including toxicology and safety studies. Recently, great efforts have been made to replace animal experiments with in vitro organoid culture methods and in silico predictions, in agreement with the 3R strategy to "reduce, refine and replace" animals in experimental testing, as outlined by the European Commission. Here we present a mini-review on the development of animal testing, as well as on alternative in vitro and in silico methods, that may at least partly replace animal experiments in the near future.

Cerebrovascular Changes and Neurodegeneration Related to Hyperlipidemia: Characteristics of the Human ApoB-100 Transgenic Mice.

Serum lipid levels are closely related to the structure and function of blood vessels. Chronic hyperlipidemia may lead to damage in both the cardio- and the cerebrovascular systems. Vascular dysfunctions, including impairments of the blood-brain barrier, are known to be associated with neurodegenerative diseases. A growing number of evidence suggests that cardiovascular risk factors, such as hyperlipidemia, may increase the likelihood of developing dementia. Due to differences in lipoprotein metabolism, wild-type mice are protected against dietinduced hypercholesterolemia, and their serum lipid profile is different from that observed in humans. Therefore, several transgenic mouse models have been established to study the role of different apolipoproteins and their receptors in lipid metabolism, as well as the complications related to pathological lipoprotein levels. This minireview focused on a transgenic mouse model overexpressing an apolipoprotein, the human ApoB-100. We discussed literature data and current advancements on the understanding of ApoB-100 induced cardio- and cerebrovascular lesions in order to demonstrate the involvement of this type of apolipoprotein in a wide range of pathologies, and a link between hyperlipidemia and neurodegeneration.

Bruch's membrane changes in transgenic mice overexpressing the human biglycan and apolipoprotein b-100 genes.

Age-Related Macular Degeneration (AMD) is characterized by the accumulation of lipid- and protein-rich deposits in Bruch's Membrane (BrM). A consequent decrease in hydraulic conductivity and impairment of transport through BrM may play a central role in the pathogenesis of AMD. The mechanism of deposit formation in AMD had been suggested to show similarities to the formation of atherosclerotic plaques in which the interactions of extracellular matrix proteoglycans with apolipoprotein-B 100 (apoB-100) play an important role. A prime candidate for this interaction is the small leucin-rich proteoglycan biglycan. The aim of our study was to test the effect of the simultaneous overexpression of human apoB-100 and biglycan genes in combination with a high-cholesterol diet on BrM morphology in transgenic mice. Six-weeks-old homozygous apoB-100 or biglycan, hemizygous apoB-100/biglycan transgenic and wild-type C57Bl/6 mice were fed either a standard chow or a diet supplemented with 2% cholesterol for 17 weeks. Animals were sacrificed, serum lipid levels were measured and eyes were processed for transmission electron microscopy (TEM) according to standard protocol. Morphometric analysis of digitally acquired TEM images of BrM showed that in apoB-100 and double transgenic animals fed a high-cholesterol diet, the BrM thickness was significantly increased compared to wild-type animals. Both groups had electron-lucent profiles in clusters, scattered throughout the collagenous layers of BrM, and focal nodules of an amorphous material of intermediate electron-density between the plasma and basement membranes of the retinal pigment epithelium (RPE). BrM thickness in these two groups correlated well with elevated cholesterol levels. Unexpectedly, animals overexpressing biglycan alone showed a marked, diet-independent increase in BrM thickness associated with a layer of a basement membrane-like material in outer BrM. The effects of biglycan overexpression are intriguing and further investigations are needed to elucidate the underlying mechanisms.

Noladin ether, a putative endocannabinoid, inhibits mu-opioid receptor activation via CB2 cannabinoid receptors.

We examined the occurrence of possible changes in mRNA expression and the functional activity of opioid receptors after acute in vivo and in vitro treatment with the putative endogenous cannabinoid noladin ether. While noladin ether (NE) demonstrates agonist activity at CB1 cannabinoid receptors, recent data indicate that NE acts as a full agonist at CB2 cannabinoid receptors too. Considering the functional interactions between opioids and cannabinoids, it is of interest to examine whether NE affects the opioid system. To that end, we studied the influence of NE on mu-opioid receptor (MOR) mRNA expression and MOR mediated G-protein signaling. We used real-time PCR and [35S]GTPgammaS binding assays to examine the changes of MOR mRNA levels and the capability of the mu-opioid agonist peptide ([D-Ala2,(NMe)Phe4,Gly5-ol]enkephalin (DAMGO) in activating regulatory G-proteins via MORs in forebrain membrane fractions of wild-type (w.t., CB1+/+) and CB1 receptor deficient transgenic mice (knockout, CB1-/-). We found, that the expression of MOR mRNAs significantly decreased both in CB1+/+ and CB1-/- forebrain after a single injection of NE at 1 mg/kg when compared to control. Consequently, MOR-mediated signaling is attenuated after acute in vivo treatment with NE in both CB1+/+ and CB1-/- mice. Inhibition on MOR mediated activation is observed after in vitro NE administration as well. Radioligand binding competition studies showed that the noticed effect of NE on MOR signaling is not mediated through MORs. Both in vivo and in vitro attenuations of NE can be antagonized by the CB2 selective antagonist SR144528. Taken together, our data suggest that the NE caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors.