Aβ Deposition In Brain Research Articles

Interacting with γSecretase for Treating Alzheimer's Disease: From Inhibition to Modulation

Drugs currently used for the treatment of Alzheimer's disease (AD) produce limited clinical benefits, and there is no disease-modifying therapy yet available. Compounds that inhibit or modulate γ-secretase, the pivotal enzyme that generates β-amyloid (Aβ), are potential therapeutics for AD. This article briefly reviews the profile of γ-secretase inhibitors and modulators that have reached the clinic. Studies in both transgenic and non-transgenic animal models of AD have indicated that γ-secretase inhibitors, administered by the oral route, are able to lower brain Aβ concentrations. However, scanty data are available on the effects of these compounds on brain Aβ deposition after prolonged administration. γ-Secretase inhibitors may cause abnormalities in the gastrointestinal tract, thymus, spleen, skin, and decrease in lymphocytes and alterations in hair color in experimental animals and in man, effects believed to be associated with the inhibition of the cleavage of Notch, a transmembrane receptor involved in regulating cell-fate decisions. Unfortunately, two large Phase III clinical trials of semagacestat in mild-to-moderate AD patients were prematurely interrupted because of the observation of a detrimental cognitive and functional effect of the drug. These detrimental effects were mainly ascribed to the inhibition of the processing of an unknown substrate of γ-secretase. It has been also hypothesized that the detrimental cognitive effects observed after semagacestat administration are due to the accumulation of the neurotoxic precursor of Aβ (the carboxy-terminal fragment of amyloid precursor protein, APP, or CTFβ) resulting from the block of the γ-secretase cleavage activity on APP. Some non-steroidal anti-inflammatory drugs and other small organic molecules have been found to modulate γ-secretase shifting its cleavage activity from longer to shorter Aβ species without affecting Notch cleavage. However, two large Phase III studies in mild AD patients with tarenflurbil, a putative γ-secretase modulator, were also completely negative. The failure of tarenflurbil was ascribed to low potency and brain penetration. New more selective γ-secretase inhibitors and more potent, more brain penetrant γ-secretase modulators are being developed with the hope of overcoming the previous setbacks. Further understanding of the reasons of the failures of these γ-secretase-based drugs in AD may be important for the future research on effective treatments for this devastating disease.

γ-secretase inhibitors for treating Alzheimer’s disease: rationale and clinical data

There are no drugs available for slowing down the rate of deterioration of patients with Alzheimer’s disease (AD). With the aim of altering the natural history of the disease, the pharmaceutical industry has designed and developed several compounds inhibiting γ-secretase, the enzymatic complex generating β-amyloid (Aβ) peptides (Aβ1–40 and Aβ1–42), believed to be involved in the pathophysiological cascade of AD, from amyloid precursor protein (APP). This article briefly reviews the profile of γ-secretase inhibitors that have reached the clinic. Studies in both transgenic and nontransgenic animal models of AD have indicated that γ-secretase inhibitors, administered by the oral route, are able to lower brain Aβ concentrations. However, few data are available on the effects of these compounds on brain Aβ deposition after prolonged administration. γ-secretase inhibitors may cause abnormalities in the gastrointestinal tract, thymus, spleen, skin, and decrease in lymphocytes and alterations in hair color in ex...

Novel Anti-Inflammatory Compound SEN1176 Alleviates Behavioral Deficits Induced Following Bilateral Intrahippocampal Injection of Aggregated Amyloid-β1-42

Behavioral effects of a novel anti-inflammatory SEN1176 were investigated. This pyrrolo[3,2-e][1,2,4]triazolo[1,5-a]pyrimidine suppresses amyloid-β (Aβ)1-42-induced macrophage production of nitric oxide, TNF-α, IL-1β, and IL-6 in a dose-dependent fashion, an activity profile consistent with SEN1176 being a neuroinflammation inhibitor. Using male Sprague-Dawley rats, SEN1176 was examined relative to detrimental behavioral effects induced following bilateral intrahippocampal (IH) injections of aggregated Aβ1-42. The rats were trained to respond under an alternating-lever cyclic-ratio (ALCR) schedule of food reinforcement, enabling measurement of parameters of operant performance that reflect aspects of learning and memory. Under the ALCR schedule, orally administered SEN1176 at 5, 20, or 30 mg/kg was effective in reducing the behavioral deficit caused by bilateral IH aggregated Aβ1-42 injections in a dose-related manner over a 90-day treatment period. SEN1176 at 20 and 30 mg/kg significantly reduced lever switching errors and, at doses of 5, 10, and 30 mg/kg, significantly reduced incorrect lever perseverations, indicating a reduction of the behavioral deficit induced as a result of inflammation following IH Aβ1-42 injections. When treatment with SEN1176 was instigated 30 days after IH Aβ1-42 injections, it resulted in progressive protection, and withdrawal of SEN1176 treatment 60 days after IH Aβ1-42 injections revealed partial retention of the protective effect. SEN1176 also significantly reduced numbers of activated astrocytes adjacent to the aggregated Aβ1-42 injection sites. These results indicate the potential of SEN1176 for alleviating chronic neuroinflammatory processes related to brain Aβ deposition that affect learning and memory in Alzheimer's disease.

γ-Secretase Inhibitors and Modulators for the Treatment of Alzheimer's Disease: Disappointments and Hopes

According to the β-amyloid (Aβ) hypothesis, compounds that inhibit or modulate γ secretase, the pivotal enzyme that generates Aβ, are potential therapeutics for Alzheimer's disease (AD). Studies in both transgenic and non-transgenic animal models of AD have indicated that γ-secretase inhibitors, administered by the oral route, are able to lower brain Aβ concentrations. However, scanty data are available on the effects of these compounds on brain Aβ deposition after chronic administration. Behavioral studies are also scarce with only one study indicating positive cognitive effects of a peptidomimetic compound acutely administered (DAPT). γ-Secretase inhibitors may cause abnormalities in the gastrointestinal tract, thymus, spleen and skin in experimental animals and in man. These toxic effects are likely due to inhibition of Notch cleavage, a transmembrane receptor involved in regulating cell-fate decisions. Some non-steroidal anti-inflammatory drugs (NSAIDs) and other small organic molecules have been found to modulate γ secretase shifting its cleavage activity from longer to shorter β-amyloid species without affecting Notch cleavage. Long-term histopathological and behavioral animal studies are available with these NSAIDs (mainly ibuprofen) but it is unclear if the observed in vivo effects on Aβ brain pathology and learning depend on their activity on γ-secretase or on other biological targets. The most studied γ-secretase inhibitor, semagacestat (LY-450139), was shown to dose-dependently decrease the generation of Aβ in the cerebrospinal fluid of healthy humans. Unfortunately, two large Phase 3 clinical trials of semagacestat in mild-to-moderate AD patients were prematurely interrupted because of the observation of detrimental effects on cognition and functionality in patients receiving the drug compared to those receiving placebo. These detrimental effects were mainly ascribed to the inhibition of Notch processing and to the accumulation of the neurotoxic precursor of Aβ (the carboxy-terminal fragment of APP, or CTFβ) resulting from the block of the γ-secretase cleavage activity on APP. Two large Phase 3 studies in mild AD patients with tarenflurbil (R flurbiprofen), a putative γ-secretase modulator, were also completely negative. The failure of tarenflurbil was ascribed to low potency and brain penetration. New Notch-sparing γ-secretase inhibitors and more potent, more brain penetrant γ-secretase modulators are being developed with the hope of overcoming the previous setbacks.

Perindopril, a centrally active angiotensin‐converting enzyme inhibitor, prevents cognitive impairment in mouse models of Alzheimer's disease

The purpose of this work was to test whether brain-penetrating angiotensin-converting enzyme (ACE) inhibitors (e.g., perindopril), as opposed to non-brain-penetrating ACE inhibitors (e.g., enalapril and imidapril), may reduce the cognitive decline and brain injury in Alzheimer's disease (AD). We first compared the effect of perindopril, enalapril, and imidapril on cognitive impairment and brain injury in a mouse model of AD induced by intracerebroventricular (i.c.v.) injection of amyloid-β (Aβ)₁₋₄₀. Perindopril, with significant inhibition of hippocampal ACE, significantly prevented cognitive impairment in this AD mouse model. This beneficial effect was attributed to the suppression of microglia/astrocyte activation and the attenuation of oxidative stress caused by iNOS induction and extracellular superoxide dismutase down-regulation. In contrast, neither enalapril nor imidapril prevented cognitive impairment and brain injury in this AD mouse. We next examined the protective effects of perindopril on cognitive impairment in PS2APP-transgenic mice overexpressing Aβ in the brain. Perindopril, without affecting brain Aβ deposition, significantly suppressed the increase in hippocampal ACE activity and improved cognition in PS2APP-transgenic mice, being associated with the suppression of hippocampal astrocyte activation and attenuation of superoxide. Our data demonstrated that the brain-penetrating ACE inhibitor perindopril, as compared to non-brain-penetrating ACE inhibitors, protected against cognitive impairment and brain injury in experimental AD models.

F-18]FDDNP microPET imaging correlates with brain Aβ burden in a transgenic rat model of Alzheimer disease: Effects of aging, in vivo blockade, and anti-Aβ antibody treatment

In vivo detection of Alzheimer's disease (AD) neuropathology in living patients using positron emission tomography (PET) in conjunction with high affinity molecular imaging probes for β-amyloid (Aβ) and tau has the potential to assist with early diagnosis, evaluation of disease progression, and assessment of therapeutic interventions. Animal models of AD are valuable for exploring the in vivo binding of these probes, particularly their selectivity for specific neuropathologies, but prior PET experiments in transgenic mice have yielded conflicting results. In this work, we utilized microPET imaging in a transgenic rat model of brain Aβ deposition to assess [F-18]FDDNP binding profiles in relation to age-associated accumulation of neuropathology. Cross-sectional and longitudinal imaging demonstrated that [F-18]FDDNP binding in the hippocampus and frontal cortex progressively increases from 9 to 18months of age and parallels age-associated Aβ accumulation. Specificity of in vivo [F-18]FDDNP binding was assessed by naproxen pretreatment, which reversibly blocked [F-18]FDDNP binding to Aβ aggregrates. Both [F-18]FDDNP microPET imaging and neuropathological analyses revealed decreased Aβ burden after intracranial anti-Aβ antibody administration. The combination of this non-invasive imaging method and robust animal model of brain Aβ accumulation allows for future longitudinal in vivo assessments of potential therapeutics for AD that target Aβ production, aggregation, and/or clearance. These results corroborate previous analyses of [F-18]FDDNP PET imaging in clinical populations.

Peripheral reduction of β-amyloid is sufficient to reduce brain β-amyloid: Implications for Alzheimer's disease

Three loci that modify β-amyloid (Aβ) accumulation and deposition in the brains of a mouse model of Alzheimer's disease have been previously described. One encompasses the Psen2 gene encoding presenilin 2, a component of the γ-secretase activity responsible for generating Aβ by proteolysis. We show that the activity of mouse Psen2, as measured by levels of mRNA accumulation, unexpectedly is heritable in the liver but not the brain, suggesting liver as the origin of brain Aβ deposits. Administration of STI571, a cancer therapeutic that does not cross the blood-brain barrier, reduced accumulation of Aβ in both the blood and the brain, confirming brain Aβ's peripheral origin and suggesting that STI571 and related compounds might have therapeutic/prophylactic value in human Alzheimer's disease. The genes Cib1 and Zfhx1b reside within the other modifier loci and also exhibit heritable expression in the liver, suggesting that they too contribute to Aβ accumulation.

Lithium Improves Hippocampal Neurogenesis, Neuropathology and Cognitive Functions in APP Mutant Mice

BackgroundAlzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive deterioration of cognitive functions, extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles within neocortex and hippocampus. Adult hippocampal neurogenesis plays an important role in learning and memory processes and its abnormal regulation might account for cognitive impairments associated with AD.Methodology/Principal FindingsThe double transgenic (Tg) CRND8 mice (overexpressing the Swedish and Indiana mutations in the human amyloid precursor protein), aged 2 and 6 months, were used to examine in vivo the effects of 5 weeks lithium treatment. BrdU labelling showed a decreased neurogenesis in the subgranular zone of Tg mice compared to non-Tg mice. The decrease of hippocampal neurogenesis was accompanied by behavioural deficits and worsened with age and pathology severity. The differentiation into neurons and maturation of the proliferating cells were also markedly impaired in the Tg mice. Lithium treatment to 2-month-old Tg mice significantly stimulated the proliferation and neuron fate specification of newborn cells and fully counteracted the transgene-induced impairments of cognitive functions. The drug, by the inhibition of GSK-3β and subsequent activation of Wnt/ß-catenin signalling promoted hippocampal neurogenesis. Finally, the data show that the lithium's ability to stimulate neurogenesis and cognitive functions was lost in the aged Tg mice, thus indicating that the lithium-induced facilitation of neurogenesis and cognitive functions declines as brain Aβ deposition and pathology increases.ConclusionsLithium, when given on time, stimulates neurogenesis and counteracts AD-like pathology.

Silymarin Attenuated the Amyloid β Plaque Burden and Improved Behavioral Abnormalities in an Alzheimer’s Disease Mouse Model

Alzheimer's disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.

β-Amyloid peptide increases levels of iron content and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease

Recent studies indicate that the deposition of β-amyloid peptide (Aβ) is related to the pathogenesis of Alzheimer disease (AD); however, the underlying mechanism is still not clear. The abnormal interactions of Aβ with metal ions such as iron are implicated in the process of Aβ deposition and oxidative stress in AD brains. In this study, we observed that Aβ increased the levels of iron content and oxidative stress in SH-SY5Y cells overexpressing the Swedish mutant form of human β-amyloid precursor protein (APPsw) and in Caenorhabditis elegans Aβ-expressing strain CL2006. Intracellular iron and calcium levels and reactive oxygen species and nitric oxide generation significantly increased in APPsw cells compared to control cells. The activity of superoxide dismutase and the antioxidant levels of APPsw cells were significantly lower than those of control cells. Moreover, iron treatment decreased cell viability and mitochondrial membrane potential and aggravated oxidative stress damage as well as the release of Aβ1–40 from the APPsw cells. The iron homeostasis disruption in APPsw cells is very probably associated with elevated expression of the iron transporter divalent metal transporter 1, but not transferrin receptor. Furthermore, the C. elegans with Aβ-expression had increased iron accumulation. In aggregate, these results demonstrate that Aβ accumulation in neuronal cells correlated with neuronal iron homeostasis disruption and probably contributed to the pathogenesis of AD.

Role of neuroinflammation in hypertension-induced brain amyloid pathology

Hypertension and sporadic Alzheimer's disease (AD) have been associated but clear pathophysiological links have not yet been demonstrated. Hypertension and AD share inflammation as a pathophysiological trait. Thus, we explored if modulating neuroinflammation could influence hypertension-induced β-amyloid (Aβ) deposition. Possible interactions among hypertension, inflammation and Aβ-deposition were studied in hypertensive mice with transverse aortic coarctation (TAC). Given that brain Aβ deposits are detectable as early as 4 weeks after TAC, brain pathology was analyzed in 3-week TAC mice, before Aβ deposition, and at a later time (8-week TAC mice). Microglial activation and interleukin (IL)-1β upregulation were already found in 3-week TAC mice. At a later time, along with evident Aβ deposition, microglia was still activated. Finally, immune system stimulation (LPS) or inhibition (ibuprofen), strategies described to positively or negatively modulate neuroinflammation, differently affected Aβ deposition. We demonstrate that hypertension per se triggers neuroinflammation before Aβ deposition. The finding that only immune system activation, but not its inhibition, strongly reduced amyloid burden suggests that stimulating inflammation in the appropriate time window may represent a promising strategy to limit vascular-triggered AD-pathology.

Oxidative modification to LDL receptor-related protein 1 in hippocampus from subjects with Alzheimer disease: Implications for Aβ accumulation in AD brain

Alzheimer disease (AD) is a neurodegenerative disorder characterized histopathologically by the presence of senile plaques (SPs), neurofibrillary tangles, and synapse loss. The main component of SPs is amyloid-β peptide (Aβ), which has been associated with increased oxidative stress, leading to oxidative modification of proteins and consequently to neurotoxicity and neurodegeneration. Low-density lipoprotein receptor-related protein 1 (LRP1) is the primary moiety responsible for the efflux of Aβ from the brain to the blood across the blood–brain barrier. Impaired brain-to-blood transport of Aβ by LRP1 has been hypothesized to contribute to increased levels of Aβ in AD brain. The cause of LRP1 dysfunction is unknown, but we have hypothesized that Aβ oxidizes LRP1, thus damaging its own transporter. Consistent with this notion, we report in this study a significant increase in the levels of the lipid peroxidation product 4-hydroxy-2-nonenal bound to transmembrane LRP1 in AD hippocampus. In contrast, the levels of LRP1-resident 3-nitrotyrosine did not show a significant increase in AD hippocampus compared to age-matched controls. Based on this study, we propose that Aβ impairs its own efflux from the brain by oxidation of its transporter LRP1, leading to increased Aβ deposition in brain, thereby contributing to subsequent cognitive impairment in AD.

An aminopeptidase from Streptomyces sp. KK565 degrades beta amyloid monomers, oligomers and fibrils

The accumulation of beta amyloid (Aβ) has been a primary target for Alzheimer disease therapeutic strategies. Previously, we discovered an activity from Streptomyces sp. KK565 growth media that inhibits Aβ aggregation. The active component was an aminopeptidase and named Streptomyces sp. KK565 aminopeptidase (SKAP). SKAP cleaved N-terminal amino-acids of Aβ 1–42 monomer, inhibited formation of fibrils and protected Aβ 1–42-induced neurotoxicity. Over-expression of a human homolog of SKAP, glutamate carboxypeptidase II (hGCPII) in Aβ-oversynthesizing cells dramatically reduced the Aβ levels. These findings suggest a possible role of M28 family peptidases in preventing Aβ deposits in mammalian brain. Structured summary MINT- 7992796: SKAP (uniprotkb: Q306T3) physically interacts (MI: 0915) with Abeta (uniprotkb: P05067) by protease assay (MI: 0435) MINT- 7992752, MINT- 7992778: SKAP (uniprotkb: Q306T3) binds (MI: 0407) to Abeta (uniprotkb: P05067) by protease assay (MI: 0435)

Severe In Vivo Hyper-Homocysteinemia is not Associated with Elevation of Amyloid-β Peptides in the Tg2576 Mice

Since hyper-homocysteinemia (HHcy) was recognized as a risk factor for Alzheimer's disease (AD), many studies tried to induce HHcy in animal models to investigate its effect on amyloid-beta protein precursor (AbetaPP) metabolism. Previous reports found that HHcy induced in AD transgenic mouse models, by either feedina a methionine-enriched diet or vitamin Bs deficient diet, is associated with elevation of amyloid-beta (Abeta) levels. However, there is no data available on the effect of dietary intervention which combines both excessive methionine and low levels of vitamin Bs on amyloidogenesis in any of these models. In the current study, we investigated the effect of a combination diet, which was both enriched in methionine and deficient in folate, vitamin B6 and B12, in an AD mouse model, the Tg2576. We found that 7 months treatment of this diet induced severe HHcy in these mice with plasma homocysteine level higher than 150 microM. However, no difference was detected in brain Abeta levels or deposition between the diet-treated and control group. As shown by western blot, severe HHcy did not alter the steady state levels of proteins involved in AbetaPP metabolism, either. These results demonstrate that this combination diet-induced severe HHcy does not influence amyloidogenesis in vivo.

Intramuscular delivery of a single chain antibody gene prevents brain Aβ deposition and cognitive impairment in a mouse model of Alzheimer’s disease

Anti-beta-amyloid (Aβ) immunotherapy is effective in removing brain Aβ, but has shown to be associated with detrimental effects. We have demonstrated that Adeno-associated virus (AAV)-mediated delivery of an anti-Aβ single chain antibody (scFv) gene was effective in clearing brain Aβ without eliciting any inflammatory side effects in old APP Swe/PS1dE9 transgenic mice. In the present study, we tested the efficacy and safety of intramuscular delivery of the scFv gene in preventing brain Aβ deposition. The scFv gene was intramuscularly delivered to APP Swe/PS1dE9 transgenic mice at 3 months of age, prior to Aβ deposition in the brain. Six months later, we found that the transgenes were expressed in a stable form at the delivered sites, with a small amount of ectopic expression in the liver and olfactory bulb. Brain Aβ plaque formation, Aβ accumulation, AD-type pathologies and cognitive impairment were significantly attenuated in scFv-treated APP Swe/PS1dE9 transgenic mice relative to EGFP-treated mice. Intramuscular delivery of scFv gene was well tolerated by the animals, did not cause inflammation or microhemorrhage at the gene expression site and in the brain, and did not induce neutralizing antibodies in the animals. These findings suggest that peripheral application of scFv is effective and safe in preventing the development of Alzheimer’s disease (AD), and would be a promising non-inflammatory immunological modality for prevention and treatment of AD.

Human intravenous immunoglobulin provides protection against Aβ toxicity by multiple mechanisms in a mouse model of Alzheimer's disease

BackgroundPurified intravenous immunoglobulin (IVIG) obtained from the plasma of healthy humans is indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. IVIG contains naturally occurring auto-antibodies, including antibodies (Abs) against β-amyloid (Aβ) peptides accumulating in the brains of Alzheimer's disease (AD) patients. IVIG has been shown to alleviate AD pathology when studied with mildly affected AD patients. Although its mechanisms-of-action have been broadly studied, it remains unresolved how IVIG affects the removal of natively formed brain Aβ deposits by primary astrocytes and microglia, two major cell types involved in the neuroinflammatory responses.MethodsWe first determined the effect of IVIG on Aβ toxicity in primary neuronal cell culture. The mechanisms-of-action of IVIG in reduction of Aβ burden was analyzed with ex vivo assay. We studied whether IVIG solubilizes natively formed Aβ deposits from brain sections of APP/PS1 mice or promotes Aβ removal by primary glial cells. We determined the role of lysosomal degradation pathway and Aβ Abs in the IVIG-promoted reduction of Aβ. Finally, we studied the penetration of IVIG into the brain parenchyma and interaction with brain deposits of human Aβ in a mouse model of AD in vivo.ResultsIVIG was protective against Aβ toxicity in a primary mouse hippocampal neuron culture. IVIG modestly inhibited the fibrillization of synthetic Aβ1-42 but did not solubilize natively formed brain Aβ deposits ex vivo. IVIG enhanced microglia-mediated Aβ clearance ex vivo, with a mechanism linked to Aβ Abs and lysosomal degradation. The IVIG-enhanced Aβ clearance appears specific for microglia since IVIG did not affect Aβ clearance by astrocytes. The cellular mechanisms of Aβ clearance we observed have potential relevance in vivo since after peripheral administration IVIG penetrated to mouse brain tissue reaching highest concentrations in the hippocampus and bound selectively to Aβ deposits in co-localization with microglia.ConclusionsOur results demonstrate that IVIG promotes recognition and removal of natively formed brain Aβ deposits by primary microglia involving natural Aβ Abs in IVIG. These findings may have therapeutic relevance in vivo as IVIG penetrates through the blood-brain barrier and specifically binds to Aβ deposits in brain parenchyma.

Anti-Amyloid Effects of Small Molecule Aβ-Binding Agents in PS1/APP Mice

AIMS: One promising approach for treatment of Alzheimer's disease (AD) is use of anti-amyloid therapies, based on the hypothesis that increases in amyloid-beta (Aβ) deposits in brain are a major cause of AD. Several groups have focused on Aβ immunotherapy with some success. Small molecules derivatives of Congo red have been shown to inhibit Aβ aggregation and protect against Aβ neurotoxicity in vitro. The agents described here are all small molecule Aβ-binding agents (SMAβBA's) derivatives of Congo red. MAIN METHODS: Here, we have explored the anti-amyloid properties of these SMAβBA's in mice doubly transgenic for human prensenilin-1 (PS1) and APP gene mutations that cause early-onset AD. Mice were treated with either methoxy-X04, X:EE:B34 and X:034-3-OMe1. After treatment, brains were examined for Aβ-deposition, using histochemistry, and soluble and insoluble Aβ levels were determined using ELISA. KEY FINDINGS: A range of anti-amyloid activity was observed with these three compounds. PS1/APP mice treated with methoxy-X04 and X:EE:B34 showed decrease in total Aβ load, a decrease in Aβ fibril load, and a decrease in average plaque size. Treatment with methoxy-X04 also resulted in a decrease in insoluble Aβ levels. The structurally similar compound, X:034:3-OMe1, showed no significant effect on any of these measures. The effectiveness of the SMAβBA's may be related to a combination of binding affinity for Aβ and entry into brain, but other factors appear to apply as well. SIGNIFICANCE: These data suggest that SMAβBA's may significantly decrease amyloid burden in brain during the pathogenesis of AD and could be useful therapeutics alone, or in combination with immunotherapy.

Divalent metal transporter 1 is involved in amyloid precursor protein processing and Aβ generation

The amyloid-beta precursor protein (APP) and its pathogenic byproduct beta-amyloid peptide (Abeta) play central roles in the pathogenesis of Alzheimer's disease (AD). Reduction in levels of the potentially toxic Abeta is one of the most important therapeutic goals in AD. Recent studies have shown that bivalent metals such as iron, copper, and zinc are involved in APP expression, Abeta deposition, and senile plaque formation in the AD brain. However, the underlying mechanisms involved in abnormal homeostasis of bivalent metals in AD brain remain unclear. In the present study, we found that two isoforms of the divalent metal transporter 1 (DMT1), DMT1-IRE, and DMT1-nonIRE, were colocalized with Abeta in the plaques of postmortem AD brain. Using the APP/PS1 transgenic mouse model, we found that the levels of both DMT1-IRE and DMT1-nonIRE were significantly increased in the cortex and hippocampus compared with wild type-control. We further verified the proposed mechanisms by which DMT1 might be involved in APP processing and Abeta secretion by using the SH-SY5Y cell line stably overexpressing human APP Swedish mutation (APPsw) as a cell model. We found that overexpression of APPsw resulted in increased expression levels of both DMT1-IRE and DMT1-nonIRE in SH-SY5Y cells. Interestingly, silencing of endogenous DMT1 by RNA interference, which reduced bivalent ion influx, led to reductions of APP expression and Abeta secretion. These findings suggest both that DMT1 plays a critical role in ion-mediated neuropathogenesis in AD and that pharmacological blockage of DMT1 may provide novel therapeutic strategies against AD.

HumanAPOEIsoform-Dependent Effects on Brain β-Amyloid Levels in PDAPP Transgenic Mice

To investigate the role of human apolipoprotein E (apoE) on Abeta deposition in vivo, we crossed PDAPP mice lacking mouse Apoe to targeted replacement mice expressing human apoE (PDAPP/TRE2, PDAPP/TRE3, or PDAPP/TRE4). We then measured the levels of apoE protein and Abeta peptides in plasma, CSF, and brain homogenates in these mice at different ages. We also quantified the amount of brain Abeta and amyloid burden in 18-month-old mice. In young PDAPP/TRE4 mice that were analyzed at an age before brain Abeta deposition, we observed a significant decrease in the levels of apoE in CSF and brain when compared with age-matched mice expressing either human E2 or E3. The brain levels of Abeta42 in PDAPP/TRE4 mice were substantially elevated even at this very early time point. In older PDAPP/TRE4 mice, the levels of insoluble apoE protein increased in parallel to the dramatic rise in brain Abeta burden, and the majority of apoE was associated with Abeta. In TRE4 only mice, we also observed a significant decrease in the level of apoE in brain homogenates. Since the relative level of apoE mRNA was equivalent in PDAPP/TRE and TRE only mice, it appears that post-translational mechanisms influence the levels of apoE protein in brain (E4 < E3 << E2), resulting in early and dramatic apoE isoform-dependent effects on brain Abeta levels (E4 >> E3 > E2) that increase with age. Therapeutic strategies aimed at increasing the soluble levels of apoE protein, regardless of isoform, may effectively prevent and (or) treat Alzheimer's disease.

Macrophage-Mediated Degradation of β-Amyloid via an Apolipoprotein E Isoform-Dependent Mechanism

Recent studies suggest that bone marrow-derived macrophages can effectively reduce beta-amyloid (Abeta) deposition in brain. To further elucidate the mechanisms by which macrophages degrade Abeta, we cultured murine macrophages on top of Abeta plaque-bearing brain sections from transgenic mice expressing PDAPP [human amyloid precursor protein (APP) with the APP(717V>F) mutation driven by the platelet-derived growth factor promoter]. Using this ex vivo assay, we found that macrophages from wild-type mice very efficiently degrade both soluble and insoluble Abeta in a time-dependent manner and markedly eliminate thioflavine-S positive amyloid deposits. Because macrophages express and secrete apolipoprotein E (apoE), we compared the efficiency of Abeta degradation by macrophages prepared from apoE-deficient mice or mice expressing human apoE2, apoE3, or apoE4. Macrophages expressing apoE2 were more efficient at degrading Abeta than apoE3-expressing, apoE4-expressing, or apoE-deficient macrophages. Moreover, macrophage-induced degradation of Abeta was effectively blocked by an anti-apoE antibody and receptor-associated protein, an antagonist of the low-density lipoprotein (LDL) receptor family, suggesting involvement of LDL receptors. Measurement of matrix metalloproteinase-9 (MMP-9) activity in the media from human apoE-expressing macrophages cocultured with Abeta-containing brain sections revealed greater levels of MMP-9 activity in apoE2-expressing than in either apoE3- or apoE4-expressing macrophages. Differences in MMP-9 activity appear to contribute to the isoform-specific differences in Abeta degradation by macrophages. These apoE isoform-dependent effects of macrophages on Abeta degradation suggest a novel "peripheral" mechanism for Abeta clearance from brain that may also, in part, explain the isoform-dependent effects of apoE in determining the genetic risk for Alzheimer's disease.