APP-overexpressing Mice Research Articles

Neuroprotective Effects of Asparagus officinalis Stem Extract in Transgenic Mice Overexpressing Amyloid Precursor Protein.

To mimic Alzheimer's disease, transgenic mice overexpressing the amyloid precursor protein (APP) were used in this study. We hypothesize that the neuroprotective effects of ETAS®50, a standardized extract of Asparagus officinalis stem produced by Amino Up Co., Ltd. (Sapporo, Japan), are linked to the inhibition of the apoptosis cascade through an enhancement of the stress-response proteins: heat shock proteins (HSPs). APP-overexpressing mice (double-transgenic APP and PS1 mouse strains with a 129s6 background), ages 6-8 weeks old, and weighing 20-24 grams were successfully bred in our laboratory. The animals were divided into 5 groups. APP-overexpressing mice and wild-type (WT) mice were pretreated with ETAS®50 powder (50% elemental ETAS and 50% destrin) at 200 mg/kg and 1000 mg/kg body weight. Saline, the vehicle for ETAS®50, was administered in APP-overexpressing mice and WT mice. ETAS®50 and saline were administered by gavage daily for 1 month. Cognitive assessments, using the Morris Water Maze, demonstrated that memory was recovered following ETAS®50 treatment as compared to nontreated APP mice. At euthanization, the brain was removed and HSPs, amyloid β, tau proteins, and caspase-3 were evaluated through immunofluorescence staining with the appropriate antibodies. Our data indicate that APP mice have cognitive impairment along with elevated amyloid β, tau proteins, and caspase-3. ETAS®50 restored cognitive function in these transgenic mice, increased both HSP70 and HSP27, and attenuated pathogenic level of amyloid β, tau proteins, and caspsase-3 leading to neuroprotection. Our results were confirmed with a significant increase in HSP70 gene expression in the hippocampus.

Elucidating the relationship between TGF‐β signaling and Alzheimer’s disease

AbstractBackgroundThe TGF‐β signaling pathway represents a complex family and network of ligands and receptors. A body of evidence suggests that in Alzheimer’s Disease this signaling network may be perturbed and could contribute to disease development and progression. Other studies have shown a role for TGF‐β signaling in both Aβ clearance and deposition, as well as, in neuroprotection and neurotoxicity. It remains to be determined precisely the contributions of TGF‐β signaling to mechanisms underlying Alzheimer’s disease and other dementias and whether targeting TGF‐β may lead to suitable novel therapies.MethodsTo interrogate the contributions of TGF‐β signaling to Aβ and tau pathology in AD we developed several recombinant adeno‐associated virus (rAAV) constructs to express TGF‐β family ligands and decoy receptors. APP overexpressing (TgCRND8) mice were injected with rAAVs at postnatal day 0 or after 3 months of aging to examine contributions of the TGF‐β family prior to and during Aβ deposition. We also examined how TGF‐β family ligands and decoy receptors affect the development of tau pathology in tau brain slice culture (BSC) models.ResultsWe have successfully expressed TGF‐β family ligands and decoy receptors to determine their contributions to Aβ clearance and deposition in vivo in APP overexpressing mice, as well as, effects on tau pathology in ex vivo tau BSC models. Stimulating and blocking TGF‐β signaling cascades appears to have opposing effects on AD‐relevant pathology. Using this rAAV approach we can rapidly dissect how complex signaling cascades may contribute to the progression and development of human AD and how targeting these pathways may be therapeutically rewarding.ConclusionsThese findings suggest that interventions targeting TGF‐β signaling may be a novel therapeutic approach to AD. This data also enforces that pathways involved in immune function can have significant effects on the healthy and diseased brain and should be explored further in order to understand the development and progression of AD. Using a systems approach (NIH U01AG046139) we identified perturbed networks regulating innate immunity in the AD brain and have now validated how intervening with TGF‐β signaling may provide therapeutic benefit.

Plant sphingolipids promote extracellular vesicle release and alleviate amyloid-\u03b2 pathologies in a mouse model of Alzheimer\u2019s disease

The accumulation of amyloid-β protein (Aβ) in brain is linked to the early pathogenesis of Alzheimer’s disease (AD). We previously reported that neuron-derived exosomes promote Aβ clearance in the brains of amyloid precursor protein transgenic mice and that exosome production is modulated by ceramide metabolism. Here, we demonstrate that plant ceramides derived from Amorphophallus konjac, as well as animal-derived ceramides, enhanced production of extracellular vesicles (EVs) in neuronal cultures. Oral administration of plant glucosylceramide (GlcCer) to APP overexpressing mice markedly reduced Aβ levels and plaque burdens and improved cognition in a Y-maze learning task. Moreover, there were substantial increases in the neuronal marker NCAM-1, L1CAM, and Aβ in EVs isolated from serum and brain tissues of the GlcCer-treated AD model mice. Our data showing that plant ceramides prevent Aβ accumulation by promoting EVs-dependent Aβ clearance in vitro and in vivo provide evidence for a protective role of plant ceramides in AD. Plant ceramides might thus be used as functional food materials to ameliorate AD pathology.

Abstract 149: CD36 in Perivascular Macrophages Contributes to Neurovascular and Cognitive Dysfunction and Amyloid Angiopathy in Mice Overexpressing the Alzheimer Aβ Peptide

Amyloid-β (Aβ) exerts deleterious effects on the cerebral microcirculation, which may play a role in Alzheimer’s disease and mixed dementias. In mouse models of amyloid precursor protein (APP) overexpression, the vascular effects of Aβ require the innate immunity receptor CD36 in perivascular macrophages (PVM), immune cells located in the Virchow-Robin space surrounding penetrating cerebral vessels. PVM, in turn, induce vascular oxidative stress and dysfunction via NADPH oxidase (Park et al., Circ Res 2017). However, whether CD36 in PVM is involved in the cognitive impairment induced by Aβ remains unknown. We transplanted CD36 -/- bone marrow (BM) into irradiated male APP-overexpressing mice (Tg2576) (age 12 months) to repopulate their perivascular space with CD36 -/- PVM. Three months later, cerebral blood flow (CBF) was measured by laser-Doppler flowmetry in the somatosensory cortex of anesthetized mice with a cranial window (n=5/group). Cognition was also tested (n=10/group). In Tg2576 mice transplanted with WT BM (Wt→Tg), CBF responses induced by whisker stimulation (WS) or neocortical superfusion of the endothelium-dependent vasodilator acetylcholine (ACh) were attenuated compared to WT mice receiving WT BM (Wt→Wt) (WS, -35%; ACh, -36%; p<0.05). CD36 -/- BM transplantation in Tg2576 mice (CD36 -/- →Tg) prevented the cerebrovascular dysfunction (CBF increases: WS: Wt→Tg, 9±1% vs. CD36 -/- →Tg, 21±2%; ACh: Wt→Tg, 9±1% vs CD36 -/- →Tg, 17±2%; p<0.05), and reduced cerebrovascular amyloid deposition (thioflavin-S-based vascular amyloid load: -58%; p<0.05 from Wt→Tg). Wt→Tg mice spent more time finding the escape hole at the Barnes maze test, indicating impairment in spatial memory (Wt→Wt, 965±166 sec; Wt→Tg, 1807±166 sec; p<0.05), and showed impaired nest building ability (nesting score: Wt→Wt, 4.6±0.2; Wt→Tg, 1.8±0.2; p<0.05). These cognitive deficits were ameliorated in CD36 -/- →Tg mice (Barnes maze: 1217±136 sec; nesting score: 2.7±0.3; p<0.05 from Wt→Tg). We conclude that CD36 in PVM is critically involved in the cerebrovascular and cognitive dysfunctions induced by Aβ, and in cerebral amyloid angiopathy. CD36 in PVM may be a new therapeutic target to counteract the detrimental neurovascular and cognitive effects of amyloid pathology.

Decreased Plasma Aβ in Hyperlipidemic APPSL Transgenic Mice Is Associated with BBB Dysfunction.

Besides the continued focus on Aβ and Tau in Alzheimer's disease (AD), it is increasingly evident that other pathologic characteristics, such as vascular alterations or inflammation, are associated with AD. Whether these changes are an initial cause for the onset of AD or occur as a result of the disease in late stages is still under debate. In the present study, the impact of the high-fat diet (HFD) induced vascular risk factor hyperlipidemia on Aβ levels and clearance as well as cerebral vasculature and blood-brain barrier (BBB) integrity was examined in mice. For this purpose, human APP transgenic (APPSL) and wildtype (WT) mice were fed a HFD for 12 weeks. Plasma and tissues were subsequently investigated for Aβ distribution and concentrations of several vascular markers. Decreased plasma Aβ together with increased levels of insoluble Aβ and amyloid plaques in the brains of HFD fed APPSL mice point toward impaired Aβ clearance due to HFD. Additionally, HFD induced manifold alterations in the cerebral vasculature and BBB integrity exclusively in human APP overexpressing mice but not in wildtype mice. Therefore, HFD appears to enhance Aβ dependent vascular/BBB dysfunction in combination with an increased proportion of cerebral to plasma Aβ in APPSL mice.

Tc1 mouse model of trisomy-21 dissociates properties of short- and long-term recognition memory

The present study examined memory function in Tc1 mice, a transchromosomic model of Down syndrome (DS). Tc1 mice demonstrated an unusual delay-dependent deficit in recognition memory. More specifically, Tc1 mice showed intact immediate (30sec), impaired short-term (10-min) and intact long-term (24-h) memory for objects. A similar pattern was observed for olfactory stimuli, confirming the generality of the pattern across sensory modalities. The specificity of the behavioural deficits in Tc1 mice was confirmed using APP overexpressing mice that showed the opposite pattern of object memory deficits. In contrast to object memory, Tc1 mice showed no deficit in either immediate or long-term memory for object-in-place information. Similarly, Tc1 mice showed no deficit in short-term memory for object-location information. The latter result indicates that Tc1 mice were able to detect and react to spatial novelty at the same delay interval that was sensitive to an object novelty recognition impairment. These results demonstrate (1) that novelty detection per se and (2) the encoding of visuo-spatial information was not disrupted in adult Tc1 mice. The authors conclude that the task specific nature of the short-term recognition memory deficit suggests that the trisomy of genes on human chromosome 21 in Tc1 mice impacts on (perirhinal) cortical systems supporting short-term object and olfactory recognition memory.

Differential proteomic and behavioral effects of long-term voluntary exercise in wild-type and APP-overexpressing transgenics

Physical exercise may provide protection against the cognitive decline and neuropathology associated with Alzheimer's disease, although the mechanisms are not clear. In the present study, APP/PSEN1 double-transgenic and wild-type mice were allowed unlimited voluntary exercise for 7months. Consistent with previous reports, wheel-running improved cognition in the double-transgenic mice. Interestingly, the average daily distance run was strongly correlated with spatial memory in the water maze in wild-type mice (r2=.959), but uncorrelated in transgenics (r2=.013). Proteomics analysis showed that sedentary transgenic mice differed significantly from sedentary wild-types with respect to proteins involved in synaptic transmission, cytoskeletal regulation, and neurogenesis. When given an opportunity to exercise, the transgenics' deficiencies in cytoskeletal regulation and neurogenesis largely normalized, but abnormal synaptic proteins did not change. In contrast, exercise enhanced proteins associated with cytoskeletal regulation, oxidative phosphorylation, and synaptic transmission in wild-type mice. Soluble and insoluble Aβ40 and Aβ42 levels were significantly decreased in both cortex and hippocampus of active transgenics, suggesting that this may have played a role in the cognitive improvement in APP/PSEN1 mice. β-secretase was significantly reduced in active APP/PSEN1 mice compared to sedentary controls, suggesting a mechanism for reduced Aβ. Taken together, these data illustrate that exercise improves memory in wild-type and APP-overexpressing mice in fundamentally different ways.

Parkinson's disease iron deposition caused by nitric oxide-induced loss of β-amyloid precursor protein.

Elevation of both neuronal iron and nitric oxide (NO) in the substantia nigra are associated with Parkinson's disease (PD) pathogenesis. We reported previously that the Alzheimer-associated β-amyloid precursor protein (APP) facilitates neuronal iron export. Here we report markedly decreased APP expression in dopaminergic neurons of human PD nigra and that APP(-/-) mice develop iron-dependent nigral cell loss. Conversely, APP-overexpressing mice are protected in the MPTP PD model. NO suppresses APP translation in mouse MPTP models, explaining how elevated NO causes iron-dependent neurodegeneration in PD.

Pathology of SSLOW, a transmissible and fatal synthetic prion protein disorder, and comparison with naturally occurring classical transmissible spongiform encephalopathies

Naturally occurring transmissible spongiform encephalopathies (TSEs) accumulate disease-specific forms of prion protein on cell membranes in association with pathognomonic lesions. We wished to determine whether synthetic prion protein disorders recapitulated these and other subcellular TSE-specific changes. SSLOW is a TSE initiated with refolded synthetic prion protein. Five terminally sick hamsters previously intracerebrally inoculated with third passage SSLOW were examined using light and immunogold electron microscopy. SSLOW-affected hamsters showed widespread abnormal prion protein (PrP(SSLOW) ) and amyloid plaques. PrP(SSLOW) accumulated on plasma lemmas of neurites and glia without pathological changes. PrP(SSLOW) also colocalized with increased coated vesicles and pits, coated spiral membrane invaginations and membrane microfolding. PrP(SSLOW) was additionally observed in lysosomes of microglial cells but not of neurones or astrocytes. PrP(SSLOW) is propagated by cell membrane conversion of normal PrP and lethal disease may be linked to the progressive growth of amyloid plaques. Cell membrane changes present in SSLOW are indistinguishable from those of naturally occurring TSEs. However, some lesions found in SSLOW are absent in natural animal TSEs and vice versa. SSLOW may not entirely recapitulate neuropathological features previously described for natural disease. End-stage neuropathology in SSLOW, particularly the nature and distribution of amyloid plaques may be significantly influenced by the early redistribution of seeds within the inoculum and its recirculation following interstitial, perivascular and other drainage pathways. The way in which seeds are distributed and aggregate into plaques in SSLOW has significant overlap with murine APP overexpressing mice challenged with Aβ.

Actions of β-Amyloid Protein on Human Neurons Are Expressed through the Amylin Receptor

Disruption of neurotoxic effects of amyloid β protein (Aβ) is one of the major, but as yet elusive, goals in the treatment of Alzheimer's disease (AD). The amylin receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aβ in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an amylin receptor antagonist, blocks electrophysiological effects of Aβ. Pharmacological blockade of the amylin receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aβ-induced caspase-dependent and caspase-independent apoptotic cell death. In transgenic mice (TgCRND8) that overexpress amyloid precursor protein, amylin receptor is up-regulated in specific brain regions that also demonstrate an elevated amyloid burden. The expression of Aβ actions through the amylin receptor in human neurons and temporospatial interrelationship of Aβ and the amylin receptor in an in vivo model of AD together provide a persuasive rationale for this receptor as a novel therapeutic target in the treatment of AD.

Interaction of Reelin with Amyloid Precursor Protein Promotes Neurite Outgrowth

The processing of amyloid precursor protein (APP) to Abeta is an important event in the pathogenesis of Alzheimer's disease, but the physiological function of APP is not well understood. Our previous work has shown that APP processing and Abeta production are regulated by the extracellular matrix protein Reelin. In the present study, we examined whether Reelin interacts with APP, and the functional consequences of that interaction in vitro. Using coimmunoprecipitation, we found that Reelin interacted with APP through the central domain of Reelin (repeats 3-6) and the E1 extracellular domain of APP. Reelin increased cell surface levels of APP and decreased endocytosis of APP in hippocampal neurons in vitro. In vivo, Reelin levels were increased in brains of APP knock-out mice and decreased in APP-overexpressing mice. RNA interference knockdown of APP decreased neurite outgrowth in vitro and prevented Reelin from increasing neurite outgrowth. Knock-out of APP or Reelin decreased dendritic arborization in cortical neurons in vivo, and APP overexpression increased dendritic arborization. APP and Reelin have previously been shown to promote neurite outgrowth through interactions with integrins. We confirmed that APP interacted with alpha3beta1 integrin, and alpha3beta1 integrin altered APP trafficking and processing. Addition of an alpha3beta1 integrin antibody prevented APP and Reelin-induced neurite outgrowth. These findings demonstrate that Reelin interacts with APP, potentially having important effects on neurite development.

Hyperhomocysteinemia Increases β-Amyloid by Enhancing Expression of γ-Secretase and Phosphorylation of Amyloid Precursor Protein in Rat Brain

Hyperhomocysteinemia and beta-amyloid (Abeta) overproduction are critical etiological and pathological factors in Alzheimer disease, respectively; however, the intrinsic link between them is still missing. Here, we found that Abeta levels increased and amyloid precursor protein (APP) levels simultaneously decreased in hyperhomocysteinemic rats after a 2-week induction by vena caudalis injection of homocysteine. Concurrently, both the mRNA and protein levels of presenilin-1, a component of gamma-secretase, were elevated, whereas the expression levels of beta-secretase and presenilin-2 were not altered. We also observed that levels of phosphorylated APP at threonine-668, a crucial site facilitating the amyloidogenic cleavage of APP, increased in rats with hyperhomocysteinemia, although the phosphorylation per se did not increase the binding capacity of pT668-APP to the secretases. The enhanced phosphorylation of APP in these rats was not relevant to either c-Jun N-terminal kinase or cyclin-dependent kinase-5. A prominent spatial memory deficit was detected in rats with hyperhomocysteinemia. Simultaneous supplementation of folate and vitamin-B12 attenuated the hyperhomocysteinemia-induced abnormal processing of APP and improved memory. Our data revealed that hyperhomocysteinemia could increase Abeta production through the enhanced expression of gamma-secretase and APP phosphorylation, causing memory deficits that could be rescued by folate and vitamin-B12 treatment in these rats. It is suggested that hyperhomocysteinemia may serve as an upstream factor for increased Abeta production as seen in patients with Alzheimer disease.

A transgenic rat that develops Alzheimer's disease-like amyloid pathology, deficits in synaptic plasticity and cognitive impairment

In the last decade, multiple lines of transgenic APP overexpressing mice have been created that recapitulate certain aspects of Alzheimer's disease (AD). However, none of the previously reported transgenic APP overexpressing rat models developed AD-like β-amyloid (Aβ) deposits, or age-related learning and memory deficits. In the present study, we have characterized a transgenic rat model overexpressing transgenes with three, familial AD mutations (two in APP and one in PS1) that were developed by Flood et al. [Flood, D.G., et al., Aβ deposition in a transgenic rat model of Alzheimer's disease. Society for Neuroscience 2003, Washington, DC, 2003]. From the age of 9 months, these rats develop Aβ deposits in both diffuse and compact forms, with the latter being closely associated with activated microglia and reactive astrocytes. Impaired long-term potentiation (LTP) was revealed by electrophysiological recordings performed on hippocampal slices from rats at 7 months of age, which is 2 months before the appearance of amyloid plaques. The deficit in LTP was accompanied by impaired spatial learning and memory in the Morris water maze, which became more pronounced in transgenic rats of 13 months of age. For Tg rats of both ages, there was a trend for cognitive impairment to correlate with total Aβ42 levels in the hippocampus. The rat model therefore recapitulates AD-like amyloid pathology and cognitive impairment. The advantage of the rat model over the available mouse models is that rats provide better opportunities for advanced studies, such as serial CSF sampling, electrophysiology, neuroimaging, cell-based transplant manipulations, and complex behavioral testing.

Increased Neurogenesis in Young Transgenic Mice Overexpressing Human APPSw,Ind

APP overexpressing mice have been widely used in the study of Alzheimer's disease (AD), focusing mainly at older ages, with higher accumulation of amyloid-beta peptide (Abeta). A decrease in hippocampal adult neurogenesis has been described in these models and proposed to be a consequence of Abeta accumulation. Only one study demonstrates increased neurogenesis in the hippocampus of APP-overexpressing J20 mice, and suggests it is a compensatory effect due to a subtle Abeta-induced damage. We have previously reported that a specific aggregation of Abeta has neurogenic potential on neural stem cells (NSC) in vitro. In order to clarify the contradicting data reported in vivo, we investigated NSC proliferation and neuronal differentiation in the hippocampi of J20 mice at a broader range of ages. Using immunohistochemistry, we show increased proliferation and neuronal differentiation in the hippocampi of 3 month-old J20 mice that reverted when animals became older. The increase in neurogenesis correlated with detectable levels of oligomeric Abeta, measured by ELISA and western blot. We suggest that oligomeric Abeta directly induces neurogenesis in vivo as has been demonstrated in vitro. Understanding the mechanisms underlying these changes could lead to treatments to control the neuronal differentiation of endogenous precursors through the progress of AD.

Impaired spatial memory in APP-overexpressing mice on a homocysteinemia-inducing diet

Consumption of a diet that significantly elevates homocysteine (homocysteinemia) induces cell death in the CA3 hippocampal subfield in amyloid precursor protein (APP) over-expressing transgenic mice but not in wild-type controls. We assessed behavioral and other neuropathological effects of a homocysteinemia-inducing diet in aged APP-overexpressing mice. Starting at 16–18 months of age, mice were fed either a treatment diet lacking folate, choline, and methionine, and supplemented with homocysteine, or a control diet containing normal amounts of folate, choline and methionine but no homocysteine. After 5 months on the experimental diets, performance on a delayed non-matching-to-position working-memory task was unimpaired. In contrast, spatial reference memory in the water maze was impaired in transgenic mice on the treatment diet. Transgenic mice had higher homocysteine levels than wild-type mice even when fed the control diet, suggesting an effect of genotype on homocysteine metabolism. Methyl-donor deficiency did not alter amyloid deposition in the transgenic mice. These results suggest that disrupted homocysteine metabolism may induce Aβ-associated memory impairments and neurodegeneration in APP overexpressing mice.

Green Tea Epigallocatechin-3-Gallate (EGCG) Modulates Amyloid Precursor Protein Cleavage and Reduces Cerebral Amyloidosis in Alzheimer Transgenic Mice

Alzheimer's disease (AD) is a progressive neurodegenerative disorder pathologically characterized by deposition of beta-amyloid (Abeta) peptides as senile plaques in the brain. Recent studies suggest that green tea flavonoids may be used for the prevention and treatment of a variety of neurodegenerative diseases. Here, we report that (-)-epigallocatechin-3-gallate (EGCG), the main polyphenolic constituent of green tea, reduces Abeta generation in both murine neuron-like cells (N2a) transfected with the human "Swedish" mutant amyloid precursor protein (APP) and in primary neurons derived from Swedish mutant APP-overexpressing mice (Tg APPsw line 2576). In concert with these observations, we find that EGCG markedly promotes cleavage of the alpha-C-terminal fragment of APP and elevates the N-terminal APP cleavage product, soluble APP-alpha. These cleavage events are associated with elevated alpha-secretase activity and enhanced hydrolysis of tumor necrosis factor alpha-converting enzyme, a primary candidate alpha-secretase. As a validation of these findings in vivo, we treated Tg APPsw transgenic mice overproducing Abeta with EGCG and found decreased Abeta levels and plaques associated with promotion of the nonamyloidogenic alpha-secretase proteolytic pathway. These data raise the possibility that EGCG dietary supplementation may provide effective prophylaxis for AD.

Apolipoprotein E and Alzheimer's disease. A role in amyloid catabolism.

It has been shown over the past few years that apolipoprotein E (apoE) plays a central role in the brain response to injury and neurodegeneration in mammalian species. The coordinated expression of apoE and its different receptors, the so-called LDL receptor family, appears to regulate the transport of cholesterol and phospholipids during the early and middle phases of the reinnervation in the adult mammalian brain. As neurons undergo dendritic remodelling and synaptogenesis using cholesterol internalization through the apoE/LDL receptor pathway, they progressively shut down 3,3-hydroxymethylglutaryl-Coenzyme A (HMG CoA) reductase activity, the rate-limiting enzyme in the synthesis of cholesterol. These results suggest that cholesterol delivery and synthesis in the brain are tightly regulated through an apoE-dependent mechanism. The discovery that the apolipoprotein e4 allele is strongly linked to both sporadic and familial late-onset Alzheimer's disease (AD) has raised the possibility that a dysfunction of lipid transport could explain the poor compensatory synaptogenesis reported by several independent research groups in the brain of AD subjects. Recently, it has been shown that alterations of cholesterol homeostasis in the brain by exogenous administration of dietary cholesterol, or through inhibition of cholesterol synthesis, markedly affect beta amyloid production (1-40 and 1-42) and deposition and significantly impair amyloid precursor protein (APP) metabolism. In vivo, it has been shown that breeding of APP-overexpressing mice with apoE knockout mice completely abolishes amyloid plaque deposition in the brain of hybrid animals, without affecting beta amyloid steady state levels. Conversely, introduction of the human apoE3 and apoE4 genes in APP-overexpressing mice drastically reduced beta amyloid deposition in the brain of hybrid mice, confirming the proposed biological role of apoE in the clearance of extracellular beta amyloid. These results indicate that lipid homeostasis is controlled in large part by the apoE lipoprotein transport system in the extracellular space, whereas alterations in intracellular lipid homeostasis markedly affect APP processing, beta amyloid production and plaque formation in vivo. The convergence of the so-called amyloid cascade hypothesis (Hardy et al., 1992) and of the apoE/lipid recycling cascade model (Poirier, 1994) is consistent with the notion that alterations in lipid homeostasis could serve as the common denominator for apoE and beta amyloid dysfunctions in Alzheimer's disease. It is also interesting to note that lipid homeostasis is also a central feature of one of the most important neurotransmitter systems in the brain: the cholinergic system. This system is unique in the CNS since it relies heavily on lipid bioavailability to locally synthesize acetylcholine. It is thus quite tempting to propose that two of the most common neuropathologic landmarks of AD--namely, cholinergic dysfunction and amyloid deposition--may in fact depend on the integrity of local lipid homeostatic processes, which in turn are strongly dependent upon proper lipid delivery by the apoE transport system.

CD45 Opposes β-Amyloid Peptide-Induced Microglial Activation via Inhibition of p44/42 Mitogen-Activated Protein Kinase

Reactive microglia have been suggested to play a role in the Alzheimer's disease (AD) process, and previous studies have shown that expression of CD45, a membrane-bound protein-tyrosine phosphatase (PTP), is elevated in microglia in AD brain compared with controls. To investigate the possible role of CD45 in microglial responsiveness to beta-amyloid (Abeta) peptides, we first co-treated primary cultured microglia with a tyrosine phosphatase inhibitor [potassium bisperoxo (1,10-phenanthroline) oxovanadate (phen), 5 micrometer] and freshly solubilized Abeta peptides (1000 nm). Data show synergistic induction of microglial activation as evidenced by tumor necrosis factor alpha (TNF-alpha) production and nitric oxide (NO) release, both of which we show to be dependent on activation of p44/42 mitogen-activated protein kinase (MAPK). Furthermore, co-treatment with phen and Abeta peptides results in microglia-induced neuronal cell injury. Stimulation of microglial CD45 by anti-CD45 antibody markedly inhibits these effects via inhibition of p44/42 MAPK, suggesting that CD45 is a negative regulator of microglial activation. Accordingly, primary cultured microglia from CD45-deficient mice demonstrate hyper-responsiveness to Abeta, as evidenced by TNF-alpha release, NO production, and neuronal injury after stimulation with Abeta peptides. As a validation of these findings in vivo, brains from a transgenic mouse model of AD [transgenic Swedish APP-overexpressing (Tg APP(sw)) mice] deficient for CD45 demonstrate markedly increased production of TNF-alpha compared with Tg APP(sw) mice. Taken together, these results suggest that therapeutic agents that stimulate the CD45 PTP signaling pathway may be effective in suppressing microglial activation associated with AD.

Measurement of cholinergic receptors in APP-overexpressing transgenic mice

Measurement of cholinergic receptors in APP-overexpressing transgenic mice