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Alzheimer's disease (AD) is characterized by extracellular amyloid-β (Aβ) plaques and intracellular tau inclusions. However, the exact mechanistic link between these two AD lesions remains enigmatic. Through injection of human AD-brain-derived pathological tau (AD-tau) into Aβ plaque-bearing mouse models that do not overexpress tau, we recapitulated the formation of three major types of AD-relevant tau pathologies: tau aggregates in dystrophic neurites surrounding Aβ plaques (NP tau), AD-like neurofibrillary tangles (NFTs) and neuropil threads (NTs). These distinct tau pathologies have different temporal onsets and functional consequences on neural activity and behavior. Notably, we found that Aβ plaques created a unique environment that facilitated the rapid amplification of proteopathic AD-tau seeds into large tau aggregates, initially appearing as NP tau, which was followed by the formation and spread of NFTs and NTs, likely through secondary seeding events. Our study provides insights into a new multistep mechanism underlying Aβ plaque-associated tau pathogenesis.

The data reported in the Technical Comments by Fitz et al., Price et al., Tesseur et al., and Veeraraghavalu et al. replicate and validate our central conclusion that bexarotene stimulates the clearance of soluble β-amyloid peptides and results in the reversal of behavioral deficits in mouse models of Alzheimer's disease (AD). The basis of the inability to reproduce the drug-stimulated microglial-mediated reduction in plaque burden is unexplained. However, we concluded that plaque burden is functionally unrelated to improved cognition and memory elicited by bexarotene.

A hallmark pathological feature of the Alzheimer's disease (AD) brain is the presence of senile plaques, which comprise amyloid β (Aβ) peptides that are derived from the amyloid precursor protein (APP). The plaque-containing AD brain is thought to be under oxidative stress, as evidenced by increased lipid oxidation products that include isoprostane-F2αIII (iPF2αIII). IPF2αIII can bind to and activate the thromboxane A2-prostanoid (TP) receptor, and TP receptor activation causes increased Aβ production through enhancement of APP mRNA stability. Moreover, TP receptor antagonists have been shown to block iPF2αIII-induced increases of Aβ secretion. Thus, the TP receptor may be a potential drug target for AD therapy. However, here we show that existing TP receptor antagonists have poor blood-brain barrier (BBB) permeability, likely due to the presence of a carboxylic acid moiety that is believed to be important for receptor interaction, but which may hamper passive diffusion across the BBB. We now report selected analogues of a known tetrahydronaphthalene TP receptor antagonist, wherein the carboxylic acid moiety has been replaced by heterocyclic bioisosteres. These heterocyclic analogues retained relatively high affinity for the mouse and human TP receptors, and, unlike the parent carboxylic acid compound, several examples freely diffused across the BBB into the brain upon administration to mice. These results reveal that brain-penetrant tetrahydronaphthalene TP receptor antagonists can be developed by substituting the carboxylic acid moiety with a suitable nonacidic bioisostere. Compounds of this type hold promise as potential lead structures to develop drug candidates for the treatment of AD.

Alzheimer's disease (AD) is associated with impaired clearance of β-amyloid (Aβ) from the brain, a process normally facilitated by apolipoprotein E (apoE). ApoE expression is transcriptionally induced through the action of the nuclear receptors peroxisome proliferator-activated receptor gamma and liver X receptors in coordination with retinoid X receptors (RXRs). Oral administration of the RXR agonist bexarotene to a mouse model of AD resulted in enhanced clearance of soluble Aβ within hours in an apoE-dependent manner. Aβ plaque area was reduced more than 50% within just 72 hours. Furthermore, bexarotene stimulated the rapid reversal of cognitive, social, and olfactory deficits and improved neural circuit function. Thus, RXR activation stimulates physiological Aβ clearance mechanisms, resulting in the rapid reversal of a broad range of Aβ-induced deficits.

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine that is elevated in Alzheimer's disease (AD) brains. Because TNFalpha is released from cell membranes by the TNFalpha-converting enzyme (TACE), inhibition of TACE has the potential to mitigate TNFalpha effects in AD brain. TACE also cleaves amyloid precursor protein (APP) and generates sAPPalpha, precluding the formation of potentially harmful amyloid beta (Abeta) peptides by beta-site APP cleaving enzymes (BACE). Hence, the anti-inflammatory benefits of TACE inhibition might be offset by an increase in Abeta. We have examined the effects of the highly selective TACE inhibitor, BMS-561392, on APP processing in vitro and in vivo. In Chinese hamster ovary cells expressing APP, BMS-561392 significantly reduced secretion of sAPPalpha without a corresponding increase in Abeta production. Conversely, a BACE inhibitor decreased sAPPbeta and Abeta peptides with no change in the secretion of sAPPalpha. These data indicate an absence of TACE and BACE competition for the APP substrate. Despite this, we observed competition for APP when TACE activity was enhanced via phorbol ester treatment or if APP was modified such that it was retained within the trans-Golgi network (TGN). These results suggest that BACE and TACE share a common TGN localization, but under normal conditions do not compete for APP. To confirm this finding in vivo, BMS-561392 was infused into the brains of Tg2576 and wild-type mice. Although decreased brain sAPPalpha levels were observed, steady-state Abeta levels were not significantly changed. Accordingly, it is possible that TACE inhibitors could reduce TNFalpha levels without increasing Abeta levels within the AD brain.

Activated microglia surrounding amyloid beta-containing senile plaques synthesize interleukin-1, an inflammatory cytokine that has been postulated to contribute to Alzheimer's disease pathology. Studies have demonstrated that amyloid beta treatment causes increased cytokine release in microglia and related cell cultures. The present work evaluates the specificity of this cellular response by comparing the effects of amyloid beta to that of amylin, another amyloidotic peptide. Both lipopolysaccharide-treated THP-1 monocytes and mouse microglia showed significant increases in mature interleukin-1beta release 48 h following amyloid beta or human amylin treatment, whereas nonfibrillar rat amylin had no effect on interleukin-1beta production by THP-1 cells. Lipopolysaccharide-stimulated THP-1 cells treated with amyloid beta or amylin also showed increased release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6, as well as the chemokines interleukin-8 and macrophage inflammatory protein-1alpha and -1beta. THP-1 cells incubated with fibrillar amyloid beta or amylin in the absence of lipopolysaccharide also showed significant increases of both interleukin-1beta and tumor necrosis factor-alpha mRNA. Furthermore, treatment of THP-1 cells with amyloid fibrils resulted in an elevated expression of the immediate-early genes c-fos and junB. These studies provide further evidence that fibrillar amyloid peptides can induce signal transduction pathways that initiate an inflammatory response that is likely to contribute to Alzheimer's disease pathology.

Our laboratory has routinely used the methodologies described here to characterize the effects of fibrillar A beta and amylin on cytokine synthesis and secretion by LPS-differentiated THP-1 cells. Because LPS-treated THP-1 cells resemble macrophage and microglia, this assay system represents an in vitro model of the potential interactions between A beta-containing senile plaques and microglia in the AD brain. As such, these methodologies should prove useful in the identification of compounds that inhibit this A beta-induced inflammatory response.

Various data suggest that Alzheimer's disease results from the accumulation of amyloid beta (A beta) peptide fibrils and the consequent formation of senile plaques in the cognitive regions of the brain. One approach to lowering senile plaque burden in Alzheimer's disease brain is to identify compounds that will increase the degradation of existing amyloid fibrils. Previous studies have shown that proteoglycans and serum amyloid P (SAP), molecules that localize to senile plaques, bind to A beta fibrils and protect the amyloid peptide from proteolytic breakdown. Therefore, molecules that prevent the binding of SAP and/or proteoglycans to fibrillar A beta might increase plaque degradation and prove useful in the treatment of Alzheimer's disease. The nature of SAP and proteoglycan binding to A beta is defined further in the present study. SAP binds to both fibrillar and nonfibrillar forms of A beta. However, only the former is rendered resistant to proteolysis after SAP association. It is interesting that both SAP and proteoglycan binding to A beta fibrils can be inhibited by glycosaminoglycans and Congo red. Unexpectedly, Congo red protects fibrillar A beta from breakdown, suggesting that this compound and other structurally related molecules are unlikely to be suitable for use in the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a devastating neurological disorder characterized by loss of cognitive skills and progressive dementia. The pathological hallmark of AD is the presence of numerous senile plaques throughout the hippocampus and cerebral cortex associated with degenerating axons, neurofibrillary tangles, and gliosis. The core of the senile plaque primarily is composed of the 39-43 amino acid beta-amyloid peptide (Abeta), which forms fibrils of beta-pleated sheets. Although considerable genetic evidence implicates Abeta in the pathogenesis of AD, a direct causal link remains to be established. Senile plaques are foci of local inflammatory processes, as evidenced by the presence of numerous activated microglia and acute phase proteins. Abeta has been shown to elicit inflammatory responses in microglia; however, the intracellular events mediating these effects are largely unknown. We report that exposure of microglia and THP1 monocytes to fibrillar Abeta led to time- and dose-dependent increases in protein tyrosine phosphorylation of a population of proteins similar to that elicited by classical immune stimuli such as immune complexes. The tyrosine kinases Lyn, Syk, and FAK were activated on exposure of microglia and THP1 monocytes to Abeta, resulting in the tyrosine kinase-dependent generation of superoxide radicals. The present data support a role for oxidative damage in the pathogenesis of AD, provide an important mechanistic link between Abeta and the generation of reactive oxygen intermediates, and identify molecular targets for therapeutic intervention in AD.

Previous reports have demonstrated that IL-1 is elevated in the Alzheimer's disease brain. We propose that beta-amyloid (A beta) in senile plaques triggers microglial interleukin-1(IL-1) release. Since microglia and monocytes have similar lineage and functions, the human monocyte cell line, THP-1, was used to determine whether A beta peptides can stimulate release of IL-1 beta. THP-1 cells were grown in culture with LPS and incubated with various A beta peptides (0.5-10 microM). IL-1 released into the medium was measured using either an IL-1 beta ELISA or an IL-1 bioassay. Treatment of activated THP-1 cells with A beta 25-35, fibrillar A beta 1-40, or A beta 1-42 significantly elevated IL-1 beta release. A beta 25-35 with a scrambled sequence or non-fibrillar A beta 1-40 did not significantly change IL-1 beta release from activated THP-1 cells. The A beta 25-35- and fibrillar A beta 1-40 induced IL-1 beta release was dose-dependent. IL-1 released following treatment with A beta 25-35 and measured using an IL-1 bioassay gave similar results. The present report provides evidence that A beta is capable of elevating release of functional IL-1 beta, a potent pro-inflammatory cytokine, from macrophages/microglia and provides support that a chronic local inflammatory response is an ongoing phenomenon within and surrounding senile plaques.

Because the mechanisms of A beta degradation in normal and Alzheimer's disease brain are poorly understood, we have examined whether various cortical cells are capable of processing this peptide. Rat microglia and astrocytes, as well as the human THP-1 monocyte cell line, degraded A beta 1-42 added to culture medium. In contrast, neither rat cortical neurons or meningeal fibroblasts effectively catabolized this peptide. When A beta fibrils were immobilized as plaque-like deposits on culture dishes, both microglia and THP-1 cells removed the peptide. Astrocytes were incapable of processing the A beta deposits, but these cells released glycosaminoglycase-sensitive molecules that inhibited the subsequent removal of A beta by microglia. This implied that astrocyte-derived proteoglycans associated with the amyloid peptide and slowed its degradation. The addition of purified proteoglycan to A beta that was in medium or focally deposited also resulted in significant inhibition of peptide removal by microglia. These data suggest that A beta can be catabolized by microglia and proteoglycans which co-localize with senile plaques may slow the degradation of A beta within these pathologic bodies.

Senile plaques of Alzheimer's disease brain contain, in addition to beta amyloid peptide (A beta), multiple proteoglycans. Systemic amyloidotic deposits also routinely contain proteoglycan, suggesting that these glycoconjugates are generally involved in amyloid plaque formation and/or persistence. We demonstrate that heparan sulfate proteoglycan (HSPG) and chondroitin sulfate proteoglycan (CSPG) inhibit the proteolytic degradation of fibrillar, but not non-fibrillar, A beta at physiological pH. In accordance with the proteolysis studies, high affinity binding of proteoglycans to fibrillar A beta(1-40) and A beta(1-42) is observed from pH 4 to 9, whereas appreciable binding of HSPG or CSPG to non-fibrillar peptide is only seen at pH < 6. This differing pH dependence of binding suggests that a lysine residue is involved in proteoglycan association with fibrillar A beta, whereas a protonated histidine appears to be needed for binding of the glycoconjugates to non-fibrillar peptide. Scatchard analysis of fibrillar A beta association with proteoglycans indicates a single affinity interaction, and the binding of both HSPG and CSPG to fibrillar A beta is completely inhibited by free glycosaminoglycan chains. This implies that these sulfated carbohydrate moieties are primarily responsible for proteoglycan.A beta interaction. The ability of proteoglycans to bind fibrillar A beta and inhibit its proteolytic degradation suggests a possible mechanism of senile plaque accumulation and persistence in Alzheimer's disease.

The senile plaques found within the cerebral cortex and hippocampus of the Alzheimer disease brain contain beta-amyloid peptide (A beta) fibrils that are associated with a variety of macromolecular species, including dermatan sulfate proteoglycan and heparan sulfate proteoglycan. The latter has been shown recently to bind tightly to both amyloid precursor protein and A beta, and this binding has been attributed largely to the interaction of the core protein of heparan sulfate proteoglycan with A beta and its precursor. Here we have examined the ability of synthetic A beta s to bind to and interact with the glycosaminoglycan moieties of proteoglycans. A beta(1-28) associates with heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate. The interaction of these sulfated polysaccharides with the amyloid peptide results in the formation of large aggregates that are readily sedimented by centrifugation. The ability of both A beta(1-28) and A beta(1-40) to bind glycosaminoglycans is pH-dependent, with increasing interaction as the pH values fall below neutrality and very little binding at pH 8.0. The pH profile of heparin-induced aggregation of A beta(1-28) has a midpoint pH of approximately 6.5, suggesting that one or more histidine residues must be protonated for binding to occur. Analysis of the A beta sequence reveals a consensus heparin-binding domain at residues 12-17, and this motif contains histidines at positions 13 and 14 that may be involved in the interaction with glycosaminoglycans.(ABSTRACT TRUNCATED AT 250 WORDS)