The oDGal Mouse: A Novel, Physiologically Relevant Rodent Model of Sporadic Alzheimer's Disease.

Sporadic Alzheimer's disease (sAD) represents a serious and growing worldwide economic and healthcare burden. Almost 95% of current AD patients are associated with sAD as opposed to patients presenting with well-characterized genetic mutations that lead to AD predisposition, i.e., familial AD (fAD). Presently, the use of transgenic (Tg) animals overexpressing human versions of these causative fAD genes represents the dominant research model for AD therapeutic development. As significant differences in etiology exist between sAD and fAD, it is perhaps more appropriate to develop novel, more sAD-reminiscent experimental models that would expedite the discovery of effective therapies for the majority of AD patients. Here we present the oDGal mouse model, a novel model of sAD that displays a range of AD-like pathologies as well as multiple cognitive deficits reminiscent of AD symptomology. Hippocampal cognitive impairment and pathology were delayed with N-acetyl-cysteine (NaC) treatment, which strongly suggests that reactive oxygen species (ROS) are the drivers of downstream pathologies such as elevated amyloid beta and hyperphosphorylated tau. These features demonstrate a desired pathophenotype that distinguishes our model from current transgenic rodent AD models. A preclinical model that presents a phenotype of non-genetic AD-like pathologies and cognitive deficits would benefit the sAD field, particularly when translating therapeutics from the preclinical to the clinical phase.

Aß reduction in BACE1 heterozygous null 5XFAD mice is associated with transgenic APP level.

BACKGROUND: The ß-secretase, BACE1, cleaves APP to initiate generation of the ß-amyloid peptide, Aß, that comprises amyloid plaques in Alzheimer's disease (AD). Reducing BACE1 activity is an attractive therapeutic approach to AD, but complete inhibition of BACE1 could have mechanism-based side-effects as BACE1-/- mice show deficits in axon guidance, myelination, memory, and other neurological processes. Since BACE1+/- mice appear normal there is interest in determining whether 50% reduction in BACE1 is potentially effective in preventing or treating AD. APP transgenic mice heterozygous for BACE1 have decreased Aß but the extent of reduction varies greatly from study to study. Here we assess the effects of 50% BACE1 reduction on the widely used 5XFAD mouse model of AD. RESULTS: 50% BACE1 reduction reduces Aß42, plaques, and BACE1-cleaved APP fragments in female, but not in male, 5XFAD/BACE1+/- mice. 5XFAD/BACE1+/+ females have higher levels of Aß42 and steady-state transgenic APP than males, likely caused by an estrogen response element in the transgene Thy-1 promoter. We hypothesize that higher transgenic APP level in female 5XFAD mice causes BACE1 to no longer be in excess over APP so that 50% BACE1 reduction has a significant Aß42 lowering effect. In contrast, the lower APP level in 5XFAD males allows BACE1 to be in excess over APP even at 50% BACE1 reduction, preventing lowering of Aß42 in 5XFAD/BACE1+/- males. We also developed and validated a dot blot assay with an Aß42-selective antibody as an accurate and cost-effective alternative to ELISA for measuring cerebral Aß42 levels. CONCLUSIONS: 50% BACE1 reduction lowers Aß42 in female 5XFAD mice only, potentially because BACE1 is not in excess over APP in 5XFAD females with higher transgene expression, while BACE1 is in excess over APP in 5XFAD males with lower transgene expression. Our results suggest that greater than 50% BACE1 inhibition might be necessary to significantly lower Aß, given that BACE1 is likely to be in excess over APP in the human brain. Additionally, in experiments using the 5XFAD mouse model, or other Thy-1 promoter transgenic mice, equal numbers of male and female mice should be used, in order to avoid artifactual gender-related differences.

Linking vascular disorders and Alzheimer's disease: potential involvement of BACE1.

The etiology of Alzheimer's disease (AD) remains unknown. However, specific risk factors have been identified, and aging is the strongest AD risk factor. The majority of cardiovascular events occur in older people and a close relationship between vascular disorders and AD exists. Amyloid plaques, composed of the beta amyloid peptide (Abeta), are hallmark lesions in AD and evidence indicates that Abeta plays a central role in AD pathophysiology. The BACE1 enzyme is essential for Abeta generation, and BACE1 levels are elevated in AD brain. The cause(s) of this BACE1 elevation remains undetermined. Here we review the potential contribution of vascular disease to AD pathogenesis. We examine the putative vasoactive properties of Abeta and how the cellular changes associated with vascular disease may elevate BACE1 levels. Despite increasing evidence, the exact role(s) vascular disorders play in AD remains to be determined. However, given that vascular diseases can be addressed by lifestyle and pharmacologic interventions, the potential benefits of these therapies in delaying the clinical appearance and progression of AD may warrant investigation.

Phosphorylation of the translation initiation factor eIF2alpha increases BACE1 levels and promotes amyloidogenesis.

beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for beta-amyloid (Abeta) production, is elevated in Alzheimer's disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2alpha (eIF2alpha-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2alpha-P phosphatase PP1c, directly increases BACE1 and elevates Abeta production in primary neurons. Preventing eIF2alpha phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant-negative eIF2alpha kinase PERK, or PERK inhibitor P58(IPK) blocks the energy-deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2alpha-P, BACE1, Abeta, and amyloid plaques. Importantly, eIF2alpha-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2alpha-P, and amyloid load are correlated in humans with AD. These results strongly suggest that eIF2alpha phosphorylation increases BACE1 levels and causes Abeta overproduction, which could be an early, initiating molecular mechanism in sporadic AD.

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology.

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.

BACE1 structure and function in health and Alzheimer's disease.

Amyloid plaques, hallmark neuropathological lesions in Alzheimer's disease (AD) brain, are composed of the beta-amyloid peptide (Abeta). Much evidence suggests that Abeta is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. Given the strong correlation between Abeta and AD, therapeutic strategies to lower cerebral Abeta levels should prove beneficial for AD treatment. Abeta is derived from amyloid precursor protein (APP) via cleavage by two proteases, beta- and gamma-secretase. The beta-secretase has been identified as a novel aspartic protease named BACE1 (beta-site APP Cleaving Enzyme 1) that initiates Abeta formation. Importantly, BACE1 appears to be dysregulated in AD. As the rate-limiting enzyme in Abeta generation, BACE1, in principle, is an excellent therapeutic target for strategies to reduce the production of Abeta in AD. While BACE1 knockout (BACE1-/-) mice have been instrumental in validating BACE1 as the authentic beta-secretase in vivo, data indicates that complete abolishment of BACE1 may be associated with specific behavioral and physiological alterations. Recently a number of non-APP BACE1 substrates have been identified. It is plausible that failure to process certain BACE1 substrates may underlie some of the reported abnormalities in the BACE1-/- mice. Here we review the basic biology of BACE1, focusing on the regulation, structure and function of this enzyme. We pay special attention to the putative function of BACE1 during normal conditions and discuss in detail the relationship that exists between key risk factors for AD and the pathogenic alterations in BACE1 that are observed in the diseased state.

The Alzheimer's disease beta-secretase enzyme, BACE1.

The pathogenesis of Alzheimer's disease is highly complex. While several pathologies characterize this disease, amyloid plaques, composed of the beta-amyloid peptide are hallmark neuropathological lesions in Alzheimer's disease brain. Indeed, a wealth of evidence suggests that beta-amyloid is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. The BACE1 enzyme is essential for the generation of beta-amyloid. BACE1 knockout mice do not produce beta-amyloid and are free from Alzheimer's associated pathologies including neuronal loss and certain memory deficits. The fact that BACE1 initiates the formation of beta-amyloid, and the observation that BACE1 levels are elevated in this disease provide direct and compelling reasons to develop therapies directed at BACE1 inhibition thus reducing beta-amyloid and its associated toxicities. However, new data indicates that complete abolishment of BACE1 may be associated with specific behavioral and physiological alterations. Recently a number of non-APP BACE1 substrates have been identified. It is plausible that failure to process certain BACE1 substrates may underlie some of the reported abnormalities in the BACE1-deficient mice. Here we review BACE1 biology, covering aspects ranging from the initial identification and characterization of this enzyme to recent data detailing the apparent dysregulation of BACE1 in Alzheimer's disease. We pay special attention to the putative function of BACE1 during healthy conditions and discuss in detail the relationship that exists between key risk factors for AD, such as vascular disease (and downstream cellular consequences), and the pathogenic alterations in BACE1 that are observed in the diseased state.

Amyloid precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein receptor LRP1.

Mutations in the amyloid precursor protein (APP) cause early-onset Alzheimer's disease (AD), but the only genetic risk factor for late-onset AD is the varepsilon4 allele of apolipoprotein E (apoE), a major cholesterol carrier. Using Cre-lox conditional knockout mice, we demonstrate that lipoprotein receptor LRP1 expression regulates apoE and cholesterol levels within the CNS. We also found that deletion of APP and its homolog APLP2, or components of the gamma-secretase complex, significantly enhanced the expression and function of LRP1, which was reversed by forced expression of the APP intracellular domain (AICD). We further show that AICD, together with Fe65 and Tip60, interacts with the LRP1 promoter and suppresses its transcription. Together, our findings support that the gamma-secretase cleavage of APP plays a central role in regulating apoE and cholesterol metabolism in the CNS via LRP1 and establish a biological linkage between APP and apoE, the two major genetic determinants of AD.

BACE1 gene deletion prevents neuron loss and memory deficits in 5XFAD APP/PS1 transgenic mice.

Evidence suggests that beta-amyloid (Abeta) peptide triggers a pathogenic cascade leading to neuronal loss in Alzheimer's disease (AD). However, the causal link between Abeta and neuron death in vivo remains unclear since most animal models fail to recapitulate the dramatic cell loss observed in AD. We have recently developed transgenic mice that overexpress human APP and PS1 with five familial AD mutations (5XFAD mice) and exhibit robust neuron death. Here, we demonstrate that genetic deletion of the beta-secretase (BACE1) not only abrogates Abeta generation and blocks amyloid deposition but also prevents neuron loss found in the cerebral cortex and subiculum, brain regions manifesting the most severe amyloidosis in 5XFAD mice. Importantly, BACE1 gene deletion also rescues memory deficits in 5XFAD mice. Our findings provide strong evidence that Abeta ultimately is responsible for neuron death in AD and validate the therapeutic potential of BACE1-inhibiting approaches for the treatment of AD.

Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation.

Mutations in the genes for amyloid precursor protein (APP) and presenilins (PS1, PS2) increase production of beta-amyloid 42 (Abeta42) and cause familial Alzheimer's disease (FAD). Transgenic mice that express FAD mutant APP and PS1 overproduce Abeta42 and exhibit amyloid plaque pathology similar to that found in AD, but most transgenic models develop plaques slowly. To accelerate plaque development and investigate the effects of very high cerebral Abeta42 levels, we generated APP/PS1 double transgenic mice that coexpress five FAD mutations (5XFAD mice) and additively increase Abeta42 production. 5XFAD mice generate Abeta42 almost exclusively and rapidly accumulate massive cerebral Abeta42 levels. Amyloid deposition (and gliosis) begins at 2 months and reaches a very large burden, especially in subiculum and deep cortical layers. Intraneuronal Abeta42 accumulates in 5XFAD brain starting at 1.5 months of age (before plaques form), is aggregated (as determined by thioflavin S staining), and occurs within neuron soma and neurites. Some amyloid deposits originate within morphologically abnormal neuron soma that contain intraneuronal Abeta. Synaptic markers synaptophysin, syntaxin, and postsynaptic density-95 decrease with age in 5XFAD brain, and large pyramidal neurons in cortical layer 5 and subiculum are lost. In addition, levels of the activation subunit of cyclin-dependent kinase 5, p25, are elevated significantly at 9 months in 5XFAD brain, although an upward trend is observed by 3 months of age, before significant neurodegeneration or neuron loss. Finally, 5XFAD mice have impaired memory in the Y-maze. Thus, 5XFAD mice rapidly recapitulate major features of AD amyloid pathology and may be useful models of intraneuronal Abeta42-induced neurodegeneration and amyloid plaque formation.

Statins cause intracellular accumulation of amyloid precursor protein, beta-secretase-cleaved fragments, and amyloid beta-peptide via an isoprenoid-dependent mechanism.

The use of statins, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that block the synthesis of mevalonate (and downstream products such as cholesterol and nonsterol isoprenoids), as a therapy for Alzheimer disease is currently the subject of intense debate. It has been reported that statins reduce the risk of developing the disorder, and a link between cholesterol and Alzheimer disease pathophysiology has been proposed. Moreover, experimental studies focusing on the cholesterol-dependent effects of statins have demonstrated a close association between cellular cholesterol levels and amyloid production. However, evidence suggests that statins are pleiotropic, and the potential cholesterol-independent effects of statins on amyloid precursor protein (APP) metabolism and amyloid beta-peptide (A beta) genesis are unknown. In this study, we developed a novel in vitro system that enabled the discrete analysis of cholesterol-dependent and -independent (i.e. isoprenoid-dependent) statin effects on APP cleavage and A beta formation. Given the recent interest in the role that intracellular A beta may play in Alzheimer disease, we analyzed statin effects on both secreted and cell-associated A beta. As reported previously, low cellular cholesterol levels favored the alpha-secretase pathway and decreased A beta secretion presumably within the endocytic pathway. In contrast, low isoprenoid levels resulted in the accumulation of APP, amyloidogenic fragments, and A beta likely within biosynthetic compartments. Importantly, low cholesterol and low isoprenoid levels appeared to have completely independent effects on APP metabolism and A beta formation. Although the implications of these effects for Alzheimer disease pathophysiology have yet to be investigated, to our knowledge, these results provide the first evidence that isoprenylation is involved in determining levels of intracellular A beta.