Faculty Bibliography

AIM/OBJECTIVE:After the continued failure of β-secretase (BACE1) inhibitor clinical trials in prodromal as well as mild-to-moderate Alzheimer's disease (AD), they are shifting to further earlier or asymptomatic stages. The aim of this study is to explore a cognitive paradigm that allows us to more sensitively detect beneficial effects of BACE1 inhibitors in presymptomatic AD. METHODS:GRL-8234 (33.4 mg/kg, ip), a small-molecule BACE1 inhibitor, was administered once daily for 28 days to the 5XAFD transgenic mouse model of AD. The contextual fear conditioning was used to evaluate the effects of GRL-8234 on memory deficits in 5XFAD mice at different ages. RESULTS:Chronic administration of GRL-8234 to 5XFAD mice rescued their contextual memory deficits, when tested 1 day after training at 6-8 months but not at 12 months of age. Importantly, 4-month-old 5XFAD mice retain the ability to form contextual memory equivalent to wild-type controls, demonstrating that the standard method of 1-day memory assessment is not suitable for evaluating BACE1 inhibitor efficacy in ameliorating cognitive declines during earlier disease stages. Despite normal contextual memory formation, young 5XFAD mice showed faster forgetting when a longer delay (28 days) intervened between training and memory testing. Notably, GRL-8234 administered to 4-month-old 5XFAD mice during the 28-day delay reversed accelerated long-term forgetting almost completely back to wild-type control levels. CONCLUSION/CONCLUSIONS:The results provide experimental evidence that accelerated long-term forgetting represents more sensitive memory testing that can help evaluate BACE1 inhibitor therapy in presymptomatic AD populations.

Cell signaling in response to an array of diverse stress stimuli converges on the phosphorylation of eukaryotic initiation factor-2alpha (eIF2alpha). In the brain, eIF2alpha is a hub for controlling learning and memory function and for maintaining neuronal integrity in health and disease. Among four eIF2alpha kinases, PERK is emerging as a key regulator for memory impairments and neurodegeneration in Alzheimer's disease (AD). Genetic and pharmacological manipulations of PERK-eIF2alpha signaling have revealed that the overactivation of this pathway is not a mere consequence of the neurodegenerative process but play critical roles in AD pathogenesis and the occurrence of memory deficits. This review provides an overview of recent progress in animal model studies, which demonstrate that dysregulated PERK accounts for memory deficits and neurodegeneration not only as a detrimental mediator downstream of beta-amyloidosis and tauopathy but also as an important regulator upstream of both pathogenic mechanisms in AD. A therapeutic perspective is also discussed, in which interventions targeting the PERK-eIF2alpha pathway are expected to provide multiple beneficial outcomes in AD, including enhanced mnemonic function, neuroprotection and disease modification.

2-hydroxypropyl-beta-cyclodextrin (CYCLO), a modifier of cholesterol efflux from cellular membrane and endo-lysosomal compartments, reduces lysosomal lipid accumulations and has therapeutic effects in animal models of Niemann-Pick disease type C and several other neurodegenerative states. Here, we investigated CYCLO effects on autophagy in wild-type mice and TgCRND8 mice - an Alzheimer's Disease (AD) model exhibiting beta-amyloidosis, neuronal autophagy deficits leading to protein and lipid accumulation within greatly enlarged autolysosomes. A 14-day intracerebroventricular administration of CYCLO to 8 month old TgCRND8 mice that exhibit moderately advanced neuropathology markedly diminished the sizes of enlarged autolysosomes and lowered their content of GM2 ganglioside and Abeta-immunoreactivity without detectably altering amyloid precursor protein processing or extracellular Abeta/beta-amyloid burden. We identified two major actions of CYCLO on autophagy underlying amelioration of lysosomal pathology. First, CYCLO stimulated lysosomal proteolytic activity by increasing cathepsin D activity, levels of cathepsins B and D and two proteins known to interact with cathepsin D, NPC1 and ABCA1. Second, CYCLO impeded autophagosome-lysosome fusion as evidenced by accumulation of LC3, SQSTM1/p62, and ubiquitinated substrates in an expanded population of autophagosomes in the absence of greater autophagy induction. By slowing substrate delivery to lysosomes, autophagosome maturational delay, as further confirmed by our in vitro studies, may relieve lysosomal stress due to accumulated substrates. These findings provide in vivo evidence for lysosomal enhancing properties of CYCLO, but caution that prolonged interference with cellular membrane fusion/autophagosome maturation could have unfavorable consequences, which might require careful optimization of dosage and dosing schedules.

Accumulating evidence points to the amyloid-beta (Abeta) peptide as the culprit in the pathogenesis of Alzheimer's disease (AD). beta-Site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) is a protease that is responsible for initiating Abeta production. Although precise mechanisms that trigger Abeta accumulation remain unclear, BACE1 inhibition undoubtedly represents an important intervention that may prevent and/or cure AD. Remarkably, animal model studies with knockouts, virus-delivered small interfering RNAs, immunization and bioavailable small-molecule agents that specifically inhibit BACE1 activity strongly support the idea for the therapeutic BACE1 inhibition. Meanwhile, a growing number of BACE1 substrates besides APP uncover new physiological roles of this protease, raising some concern regarding the safety of BACE1 inhibition. Here, I review recent progress in preclinical studies that have evaluated the efficacies and potential limitations of genetic/pharmacological inhibition of BACE1, with special focus on AD-associated phenotypes including synaptic dysfunction, neuron loss and memory deficits in animal models.

Memantine, a noncompetitive NMDA receptor antagonist with neuroprotective properties, has been used for the treatment of Alzheimer's disease (AD). Administration of memantine to various transgenic AD mice has been reported to improve cognitive deficits, very often completely back to normal wild-type control levels. However, such great benefits of memantine in preclinical studies do not translate into clinical results of this drug, showing only marginal and transient efficacy in moderate to severe AD. To further address in vivo efficacy, we compared the effects of memantine at different disease stages in 5XFAD mice, one of the rapid-onset and most aggressive amyloid models. Specifically, we administered memantine once daily for 30days to 5XFAD mice, which showed moderate (6-7months of age) and robust (12-15months) beta-amyloid (Abeta) accumulation. Treatments with memantine (10mg/kg, i.p.) reversed memory impairments in the younger 5XFAD mice, as tested by the contextual fear conditioning and spontaneous alternation Y-maze paradigms. Memantine had no effects on soluble Abeta oligomer or total Abeta42 levels in 5XFAD mouse brains. In contrast, subchronic treatments with memantine showed no behavioral benefits in the older 5XFAD group, which exhibited more profound memory deficits concomitant with highly increased concentrations of Abeta as compared with those of the younger 5XFAD group. Since subchronic memantine at the higher dose (30mg/kg) impaired memory performances in wild-type controls, we further tested acute administration of 50mg/kg memantine, which was reported to enhance hippocampal adult neurogenesis and memory function. However, this treatment also failed to rescue memory deficits in 12-15-month-old 5XFAD mice. Collectively, our results demonstrate that cognitive benefits of memantine independent of Abeta reductions were no longer observed in the 5XFAD Alzheimer mouse model during advanced stages, which may be reflective of the limited efficacy of memantine in clinical settings.

beta-site APP-cleaving enzyme 1 (BACE1) initiates the generation of amyloid-beta (Abeta), thus representing a prime therapeutic target for Alzheimer's disease (AD). Previous work including ours has used BACE1 haploinsufficiency (BACE1+/-; i.e., 50% reduction) as a therapeutic relevant model to evaluate the efficacy of partial beta-secretase inhibition. However, it is unclear whether the extent of Abeta reductions in amyloid precursor protein (APP) transgenic mice with BACE1+/- gene ablation may vary with sex or disease progression. Here, we compared the impacts of BACE1 haploinsufficiency on Abeta concentrations and APP processing in 5XFAD Alzheimer mice (1) between males and females and (2) between different stages with moderate and robust Abeta accumulation. First, male and female 5XFAD mice at 6-7months of age showed equivalent levels of Abeta, BACE1, full-length APP and its metabolites. BACE1 haploinsufficiency significantly lowered soluble Abeta oligomers, total Abeta42 levels and plaque burden in 5XFAD mouse brains irrespective of sex. Furthermore, there was no sex difference in reductions of beta-cleavage products of APP (C99 and sAPPbeta) found in BACE1+/-.5XFAD mice relative to BACE1+/+.5XFAD controls. Meanwhile, APP and sAPPalpha levels in BACE1+/-.5XFAD mice were higher than those of 5XFAD controls regardless of sex. Based on these observations, we next combined male and female data to examine the effects of BACE1 haploinsufficiency in 5XFAD mice at 12-14months of age, as compared with those in 6-7-month-old 5XFAD mice. Oligomeric Abeta and C99 levels were dramatically elevated in older 5XFAD mice. Although the beta-metabolites of APP were significantly reduced by BACE1 haploinsufficiency in both age groups, high levels of these toxic amyloidogenic fragments remained in 12-14-month-old BACE1+/-.5XFAD mice. The present findings are consistent with our previous behavioral data showing that BACE1 haploinsufficiency rescues memory deficits in 5XFAD mice irrespective of sex but only in the younger age group.

Accumulating evidence shows that brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) significantly decrease early in Alzheimer's disease (AD). However, it remains unclear whether BDNF/TrkB reductions may be mechanistically involved in the pathogenesis of AD. To address this question, we generated 5XFAD transgenic mice with heterozygous TrkB knockout (TrkB(+/-).5XFAD), and tested the effects of TrkB reduction on AD-like features in this mouse model during an incipient stage that shows only modest amyloid-beta (Abeta) pathology and retains normal mnemonic function. TrkB(+/-) reduction exacerbated memory declines in 5XFAD mice at 4-5 months of age as assessed by the hippocampus-dependent spontaneous alternation Y-maze task, while the memory performance was not affected in TrkB(+/-) mice. Meanwhile, TrkB(+/-).5XFAD mice were normal in nest building, a widely used measure for social behavior, suggesting the memory-specific aggravation of AD-associated behavioral impairments. We found no difference between TrkB(+/-).5XFAD and 5XFAD control mice in cerebral plaque loads, Abeta concentrations including total Abeta42 and soluble oligomers and beta-amyloidogenic processing of amyloid precursor protein. Interestingly, reductions in hippocampal expression of AMPA/NMDA glutamate receptor subunits as well as impaired signaling pathways downstream to TrkB such as CREB (cAMP response element-binding protein) and Akt/GSK-3beta (glycogen synthase kinase-3beta) were observed in TrkB(+/-).5XFAD mice but not in 5XFAD mice. Among these signaling aberrations, only Akt/GSK-3beta dysfunction occurred in TrkB(+/-) mice, while others were synergistic consequences between TrkB reduction and subthreshold levels of Abeta in TrkB(+/-).5XFAD mice. Collectively, our results indicate that reduced TrkB does not affect beta-amyloidosis but exacerbates the manifestation of hippocampal mnemonic and signaling dysfunctions in early AD.

BACKGROUND: Accumulating evidence indicates that partial inhibition of beta-site APP-cleaving enzyme 1 (BACE1), which initiates amyloid-beta (Abeta) production, mitigates Alzheimer's disease (AD)-like pathologies and memory deficits in a battery of transgenic mouse models. However, our previous investigations suggest that therapeutic BACE1 suppression may be beneficial only if targeted on earlier stages of AD and encounter dramatic reductions in efficacy during disease progression. This study was designed to test the possibility that a combination approach, aimed at inhibiting BACE1 and boosting neprilysin (a major Abeta-degrading enzyme) activities, may be able to mechanistically overcome the limited efficacy of anti-Abeta therapy in advanced AD. RESULTS: After crossbreeding between BACE1 heterozygous knockout (BACE1(+/-)), neprilysin transgenic (NEP) and 5XFAD mice, we analyzed the resultant mice at 12 months of age when 5XFAD controls showed robust amyloid-beta (Abeta) accumulation and elevation of BACE1 expression (~2 folds). Although haploinsufficiency lowered BACE1 expression by ~50% in concordance with reduction in gene copy number, profound beta-amyloidosis, memory deficits and cholinergic neuron death were no longer rescued in BACE1(+/-) . 5XFAD mice concomitant with their persistently upregulated BACE1 (i.e., equivalent to wild-type control levels). Notably, neprilysin overexpression not only prevented Abeta accumulation but also suppressed the translation initiation factor eIF2alpha-associated elevation of BACE1 and lowered levels of the beta-secretase-cleaved C-terminal fragment of APP (C99) in NEP . 5XFAD mice. Interestingly, these markers for beta-amyloidogenesis in BACE1(+/-) . NEP . 5XFAD mice were further reduced to the levels reflecting a combination of single BACE1 allele ablation and the abolishment of translational BACE1 upregulation. However, since neprilysin overexpression was striking (~8-fold relative to wild-type controls), memory impairments, cholinergic neuronal loss and beta-amyloidosis were similarly prevented in NEP . 5XFAD and BACE1(+/-) . NEP . 5XFAD mice. CONCLUSIONS: Our findings indicate that robust overexpression of neprilysin is sufficient to ameliorate AD-like phenotypes in aged 5XFAD mice. We also found that Abeta-degrading effects of overexpressed neprilysin can block deleterious BACE1-elevating mechanisms that accelerate Abeta production, warranting further study to test whether interventions moderately activating neprilysin may be useful for boosting the limited efficacy of therapeutic BACE1 inhibition in treating AD with established Abeta pathology.

The beta-secretase enzyme BACE1, which initiates the cleavage of amyloid precursor protein (APP) into the amyloid-beta (Abeta) peptide, is a prime therapeutic target for Alzheimer's disease (AD). However, recent investigations using genetic animal models raise concern that therapeutic BACE1 inhibition may encounter the dramatic reduction of efficacy in ameliorating AD-like pathology and memory deficits during disease progression. Here, we compared the effects of the potent and selective small-molecule BACE1 inhibitor GRL-8234 in different pathological stages of AD mouse model. Specifically, we administered GRL-8234 (33.4 mg/kg, i.p.) once daily for 2 months to 5XFAD transgenic mice, which showed modest (4 months) and massive (10 months of age) Abeta plaque deposition at starting points. Chronic treatments with GRL-8234 reversed memory impairments, as tested by the spontaneous alternation Y-maze task, in the younger 5XFAD group concomitant with significant reductions in cerebral Abeta42 levels. In contrast, only marginal reductions of Abeta42 were observed in 12-month-old 5XFAD mice treated with GRL-8234 and their memory function remained impaired. We found that not only BACE1 but also full-length APP expression was significantly elevated with progressive Abeta accumulation in 5XFAD mice, while GRL-8234 failed to affect these detrimental mechanisms that further accelerate plaque growth in brains of older 5XFAD mice. Therefore, our results provide important insights into the mechanisms by which Abeta accumulation and related memory impairments become less responsive to rescue by BACE1 inhibition during the course of AD development.