Cerebral microvascular amyloid beta protein deposition induces vascular degeneration and neuroinflammation in transgenic mice expressing human vasculotropic mutant amyloid beta precursor protein

Cerebral vascular amyloid beta-protein (Abeta) deposition, also known as cerebral amyloid angiopathy, is a common pathological feature of Alzheimer's disease. Additionally, several familial forms of cerebral amyloid angiopathy exist including the Dutch (E22Q) and Iowa (D23N) mutations of Abeta. Increasing evidence has associated cerebral microvascular amyloid deposition with neuroinflammation and dementia in these disorders. We recently established a transgenic mouse model (Tg-SwDI) that expresses human vasculotropic Dutch/Iowa mutant amyloid beta-protein precursor in brain. Tg-SwDI mice were shown to develop early-onset deposition of Abeta exhibiting high association with cerebral microvessels. Here we present quantitative temporal analysis showing robust and progressive accumulation of cerebral microvascular fibrillar Abeta accompanied by decreased cerebral vascular densities, the presence of apoptotic cerebral vascular cells, and cerebral vascular cell loss in Tg-SwDI mice. Abundant neuroinflammatory reactive astrocytes and activated microglia strongly associated with the cerebral microvascular fibrillar Abeta deposits. In addition, Tg-SwDI mouse brain exhibited elevated levels of the inflammatory cytokines interleukin-1beta and -6. Together, these studies identify the Tg-SwDI mouse as a unique model to investigate selective accumulation of cerebral microvascular amyloid and the associated neuroinflammation.     

Metallothionein-I and -III expression in animal models of Alzheimer disease

Previous studies have described altered expression of metallothioneins (MTs) in neurodegenerative diseases like multiple sclerosis (MS), Down syndrome, and Alzheimer's disease (AD). In order to gain insight into the possible role of MTs in neurodegenerative processes and especially in human diseases, the use of animal models is a valuable tool. Several transgenic mouse models of AD amyloid deposits are currently available. These models express human beta-amyloid precursor protein (AbetaPP) carrying different mutations that subsequently result in a varied pattern of beta-amyloid (Abeta) deposition within the brain. We have evaluated the expression of MT-I and MT-III mRNA by in situ hybridization in three different transgenic mice models of AD: Tg2576 (carrying AbetaPP harboring the Swedish K670N/M671L mutations), TgCRND8 (Swedish and the Indiana V717F mutations), and Tg-SwDI (Swedish and Dutch/Iowa E693Q/D694N mutations). MT-I mRNA levels were induced in all transgenic lines studied, although the pattern of induction differed between the models. In the Tg2576 mice MT-I was weakly upregulated in cells surrounding Congo Red-positive plaques in the cortex and hippocampus. A more potent induction of MT-I was observed in the cortex and hippocampus of the TgCRND8 mice, likely reflecting their higher amyloid plaques content. MT-I upregulation was also more significant in Tg-SwDI mice, especially in the subiculum and hippocampus CA1 area. Immunofluorescence stainings demonstrate that astrocytes and microglia/macrophages surrounding the plaques express MT-I&II. In general, MT-I regulation follows a similar but less potent response than glial fibrillary acidic protein (GFAP) expression. In contrast to MT-I, MT-III mRNA expression was not significantly altered in any of the models examined suggesting that the various MT isoforms may have different roles in these experimental systems, and perhaps also in human AD.     

Early-onset subicular microvascular amyloid and neuroinflammation correlate with behavioral deficits in vasculotropic mutant amyloid beta-protein precursor transgenic mice

Cerebral microvascular amyloid beta protein (Abeta) deposition and associated neuroinflammation are increasingly recognized as an important component leading to cognitive impairment in Alzheimer's disease and related cerebral amyloid angiopathy (CAA) disorders. Transgenic mice expressing the vasculotropic Dutch/Iowa (E693Q/D694N) mutant human Abeta precursor protein in brain (Tg-SwDI) accumulate abundant cerebral microvascular fibrillar amyloid deposits exhibiting robust neuroinflammation. In the present study, we sought to determine if the unique amyloid pathology of Tg-SwDI mice was associated with deficits in behavioral performance. Behavioral performance tests that assessed a variety of psychological functions, including overall activity, motor ability, balance and strength, anxiety, impulsivity, and learning were conducted on homozygous Tg-SwDI mice and similarly aged wild-type C57Bl/6 mice. Our results indicate that Tg-SwDI mice were impaired in the performance of the Barnes maze learning and memory task at 3, 9, and 12 months of age. While more widespread cerebral microvascular Abeta pathology was evident in older animals, the evaluation of the Abeta pathology in the 3 months old transgenic animals revealed specific accumulation of microvascular amyloid and markedly elevated numbers of reactive astrocytes and activated microglia restricted to the subiculum. These findings indicate that early-onset accumulation of subicular microvascular amyloid and accompanying neuroinflammation correlates with impaired performance in the learning and memory task in Tg-SwDI mice.     

Expression of macrophage colony-stimulating factor receptor is increased in the AbetaPP(V717F) transgenic mouse model of Alzheimer's disease

Inflammation is an important neuropathological change in Alzheimer's disease (AD). However, the pathophysiological factors that initiate and maintain the inflammatory response in AD are unknown. We examined AbetaPP(V717F) transgenic mice, which show numerous brain amyloid-beta (Abeta) deposits, for expression of the macrophage colony-stimulating factor (M-CSF) and its receptor (M-CSFR). M-CSF is increased in the brain in AD and dramatically augments the effects of Abeta on cultured microglia. AbetaPP(V717F) animals 12 months of age showed large numbers of microglia strongly labeled with an M-CSFR antibody near Abeta deposits. M-CSFR mRNA and protein levels were also increased in brain homogenates from AbetaPP(V717F) animals. Dystrophic neurites and astroglia showed no M-CSFR labeling in the transgenic animals. A M-CSF antibody decorated neuritic structures near hippocampal Abeta deposits in transgenic animals. M-CSF mRNA was also increased in AbetaPP(V717F) animals in comparison with wild-type controls. Simultaneous overexpression of M-CSFR and its ligand in AbetaPP(V717F) animals could result in augmentation of Abeta-induced activation of microglia. Because chronic activation of microglia is thought to result in neuronal injury, the M-CSF system may be a potential target for therapeutic intervention in AD.     

Amyloid-beta peptide remnants in AN-1792-immunized Alzheimer's disease patients: a biochemical analysis

Experiments with amyloid-beta (Abeta)-42-immunized transgenic mouse models of Alzheimer's disease have revealed amyloid plaque disruption and apparent cognitive function recovery. Neuropathological examination of patients vaccinated against purified Abeta-42 (AN-1792) has demonstrated that senile plaque disruption occurred in immunized humans as well. Here, we examined tissue histology and quantified and biochemically characterized the remnant amyloid peptides in the gray and white matter and leptomeningeal/cortical vessels of two AN-1792-vaccinated patients, one of whom developed meningoencephalitis. Compact core and diffuse amyloid deposits in both vaccinated individuals were focally absent in some regions. Although parenchymal amyloid was focally disaggregated, vascular deposits were relatively preserved or even increased. Immunoassay revealed that total soluble amyloid levels were sharply elevated in vaccinated patient gray and white matter compared with Alzheimer's disease cases. Our experiments suggest that although immunization disrupted amyloid deposits, vascular capture prevented large-scale egress of Abeta peptides. Trapped, solubilized amyloid peptides may ultimately have cascading toxic effects on cerebrovascular, gray and white matter tissues. Anti-amyloid immunization may be most effective not as therapeutic or mitigating measures but as a prophylactic measure when Abeta deposition is still minimal. This may allow Abeta mobilization under conditions in which drainage and degradation of these toxic peptides is efficient.     

A dynamic relationship between intracellular and extracellular pools of Abeta

The accumulation of the amyloid-beta peptide (Abeta) in the brain is considered to have a primary role in Alzheimer's disease (AD). In addition to the extracellular accumulation of Abeta in the parenchyma and cerebrovasculature, emerging evidence indicates that intraneuronal Abeta also plays a pathophysiological role in AD. It is unclear, however, if the intracellular and extracellular pools of Abeta are unrelated or connected. In these studies, we sought to establish a relationship between these two pools of Abeta. We identified an inverse relationship between intracellular and extracellular Abeta in the 3xTg-AD transgenic model of AD. Using an immunotherapy approach, we further found that extracellular Abeta was cleared before intracellular Abeta. After the antibody dissipated, however, the reappearance of extracellular plaques was preceded by the accumulation of intraneuronal Abeta. Taken together, these results provide strong experimental evidence that intraneuronal Abeta may serve as a source for some of the extracellular amyloid deposits.     

Antibodies from a DNA peptide vaccination decrease the brain amyloid burden in a mouse model of Alzheimer’s disease

The neuropathology of Alzheimer's disease(AD) is characterized by the accumulation of amyloid peptide Abeta in the brain derived from proteolytic cleavage of the amyloid precursor protein (APP). Vaccination of mice with plasmid DNA coding for the human Abeta42 peptide together with low doses of preaggregated peptide induced antibodies with detectable titers after only 2 weeks. One serum was directed against the four aminoterminal amino acids DAEF and differs from previously described ones. Both immune sera and monoclonal antibodies solubilized preformed aggregates of Abeta42 in vitro and recognized amyloid plaques in brain sections of mice transgenic for human APP. Passive immunization of transgenic AD mice caused a significant and rapid reduction in brain amyloid plaques within 24 h. The combined DNA peptide vaccine may prove useful for active immunization with few inoculations and low peptide dose which may prevent the recently described inflammatory reactions inpatients. The monoclonal antibodies are applicable for passive immunization studies and may lead to a therapy of AD.     

Primate-like amyloid-beta sequence but no cerebral amyloidosis in aged tree shrews.

A central pathological feature of Alzheimer's disease is the profuse deposition of amyloid-beta protein (Abeta) in the brain parenchyma and vessel walls. Abeta also forms deposits in the brains of a variety of mammals, including all aged non-human primates studied to date. The sequence of Abeta in these animals is identical to that in humans. No Abeta deposits have been found in the brains of wild-type rats and mice, suggesting that the three amino acid differences between their Abeta and that of amyloid-bearing mammals impedes the fibrillogenicity of Abeta. Analysis of the primary sequence of the beta-amyloid precursor protein in tree shrews revealed a 98% similarity and 97% identity with the human protein. Furthermore, the predicted amino acid sequence of Abeta in tree shrews is identical to that in humans. However, immunohistochemical analysis failed to reveal beta-amyloid deposits in the neural parenchyma or vasculature of eight aged (7-8 years) tree shrews (Tupaia belangeri). The lack of correlation between the Abeta sequence and amyloid formation suggests that other factors contribute to cerebral amyloid deposition in aged animals.     

Elevated abeta42 in skeletal muscle of Alzheimer disease patients suggests peripheral alterations of AbetaPP metabolism

The levels of amyloid-beta40 (Abeta40) and Abeta42 peptides were quantified in temporalis muscles and brain of neuropathologically diagnosed Alzheimer disease (AD) and of nondemented individuals. This was achieved by using a novel analytical approach consisting of a combination of fast-performance liquid chromatographic (FPLC) size exclusion chromatography developed under denaturing conditions and europium immunoassay on the 4.0- to 4.5-kd fractions. In the temporalis muscles of the AD and nondemented control groups, the average values for Abeta42 were 15.7 ng/g and 10.2 ng/g (P = 0.010), and for Abeta40 they were 37.8 ng/g and 29.8 ng/g (P = 0.067), respectively. Multiple regression analyses of the AD and control combined populations indicated that 1) muscle Abeta40 and muscle Abeta42 levels were correlated with each other (P < 0.001), 2) muscle Abeta40 levels were positively correlated with age (P = 0. 036), and 3) muscle Abeta42 levels were positively correlated with Braak stage (P = 0.042). Other forms of the Abeta peptide were discovered by mass spectrometry, revealing the presence of Abeta starting at residues 1, 6, 7, 9, 10, and 11 and ending at residues 40, 42, 44, 45, and 46. It is possible that in AD the skeletal muscle may contribute to the elevated plasma pool of Abeta and thus indirectly to the amyloid deposits of the brain parenchyma and cerebral blood vessels. The increased levels of Abeta in the temporalis muscles of AD patients suggest that alterations in AbetaPP and Abeta metabolism may be manifested in peripheral tissues.     

Pathophysiology of dementias and implications for therapy

Dementia is characterized clinically by progressive cognitive decline, often with impairment of memory. The pathology of dementias is either focal as with infarcts in Vascular Dementia or diffuse as typified by Alzheimer's disease. In many cases of Alzheimer's disease there is a mixture of focal infarcts and diffuse changes. Diffuse pathology in dementias comprises mainly intracellular and extracellular protein deposits. Intracellular inclusions are of tau protein (Alzheimer's disease; and some frontotemporal dementias), alpha-synuclein (Dementia with Lewy bodies) and huntingtin (Huntington's disease). Soluble and insoluble peptides also accumulate in the extracellular spaces of brain parenchyma in dementias with diffuse pathology, mainly amyloid-beta (Abeta) in parenchymal plaques and in artery walls as cerebral amyloid angiopathy (Alzheimer's disease and Dementia with Lewy bodies). Insoluble prion protein (PrP) is deposited in brain parenchyma in Creutzfeldt-Jakob disease and other insoluble amyloid peptides accumulate in brain and vessel walls infamilial dementias. The pattern of extracellular deposits in brain and artery walls suggests that there is a failure of elimination of peptides, such as Abeta along perivascular interstitial fluid drainage pathways ("lymphatics") from the aged brain and in Alzheimer's disease. Such failure may be due to reduced pulsations as arteries stiffen with age and cerebrovascular disease. Immunization against Abeta removes insoluble deposits of Abeta from brain parenchyma and may allow improved clearance of soluble Abeta. Reducing cerebrovascular disease and facilitating elimination of Abeta along perivascular drainage routes may offer long-term preventative measuresfor both Vascular Dementia and for Alzheimer's disease.     

Deficient cerebral clearance of vasculotropic mutant Dutch/Iowa Double A beta in human A betaPP transgenic mice

Cerebral amyloid angiopathy (CAA) is a prominent pathological feature of Alzheimer's disease and related familial CAA disorders. However, the mechanisms that account for the cerebral vascular accumulation of amyloid beta-peptide (A beta) have not been defined. Recently, we reported novel transgenic mice (Tg-SwDI) expressing neuronally derived Swedish/Dutch/Iowa vasculotropic mutant human A beta precursor (A betaPP) that develop early-onset and robust accumulation of fibrillar cerebral microvascular A beta. Deficient clearance of Dutch/Iowa mutant A beta from brain across the capillary blood-brain barrier into the circulation may contribute to its potent cerebral accumulation. To further evaluate this theory, we generated a new transgenic mouse (Tg-Sw) that is nearly identical to Tg-SwDI, except lacking the Dutch/Iowa A beta mutations. Tg-Sw and Tg-SwDI mice expressed comparable levels of human A betaPP in brain and not in peripheral tissues. However, Tg-SwDI mice strongly accumulated Dutch/Iowa mutant A beta in brain, particularly in the cerebral microvasculature, whereas Tg-Sw mice exhibited no accumulations of wild-type A beta. Conversely, Tg-SwDI mice had no detectable Dutch/Iowa mutant A beta in plasma whereas Tg-Sw mice exhibited consistent levels of human wild-type A beta in plasma. Together, these findings suggest that while wild-type A beta is readily transported out of brain into plasma, Dutch/Iowa mutant A beta is deficient in this clearance process, likely contributing to its robust accumulation in the cerebral vasculature.     

Lessons from the AN 1792 Alzheimer vaccine: lest we forget

Recent clinical and neuropathological data show that the AN 1792 vaccine enhanced the production of Abeta antibodies in the sera of Alzheimer's disease (AD) patients, but it appears to have been ineffective at stimulating the removal of Abeta deposits from the brain or at slowing the rate of cognitive decline. The 19 cases of meningoencephalitis were not linked in an obvious way to serum antibody titre, but they may have been linked to infiltration of the brain by antibodies and/or T-cells. Brain imaging indicated that oedema associated with the neuroinflammation did not reflect the typical distribution of neuritic plaques in AD. These outcomes were not anticipated by experiments on transgenic mice because compared to humans, these mice have less genetic variability, and their plaques have a different chemical composition, making them far more soluble and easier to remove. Furthermore, the consequences of vaccination are different. Vaccination of transgenic mice removes superfluous human Abeta while leaving endogenous mouse Abeta intact, whereas in humans the immune response is directed against an endogenous target that occurs naturally and is present in healthy brain tissue. The most important lesson to be learned from the AN 1792 trials is that new strategies for treating AD should not be tested on humans until they have been extensively tested on non-murine species.     

Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis

The E693Q mutation in the amyloid beta precursor protein (APP) leads to cerebral amyloid angiopathy (CAA), with recurrent cerebral hemorrhagic strokes and dementia. In contrast to Alzheimer disease (AD), the brains of those affected by hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) show few parenchymal amyloid plaques. We found that neuronal overexpression of human E693Q APP in mice (APPDutch mice) caused extensive CAA, smooth muscle cell degeneration, hemorrhages and neuroinflammation. In contrast, overexpression of human wild-type APP (APPwt mice) resulted in predominantly parenchymal amyloidosis, similar to that seen in AD. In APPDutch mice and HCHWA-D human brain, the ratio of the amyloid-beta40 peptide (Abeta40) to Abeta42 was significantly higher than that seen in APPwt mice or AD human brain. Genetically shifting the ratio of AbetaDutch40/AbetaDutch42 toward AbetaDutch42 by crossing APPDutch mice with transgenic mice producing mutated presenilin-1 redistributed the amyloid pathology from the vasculature to the parenchyma. The understanding that different Abeta species can drive amyloid pathology in different cerebral compartments has implications for current anti-amyloid therapeutic strategies. This HCHWA-D mouse model is the first to develop robust CAA in the absence of parenchymal amyloid, highlighting the key role of neuronally produced Abeta to vascular amyloid pathology and emphasizing the differing roles of Abeta40 and Abeta42 in vascular and parenchymal amyloid pathology.     

Pediatric respiratory and systemic effects of chronic air pollution exposure: nose, lung, heart, and brain pathology

Exposures to particulate matter and gaseous air pollutants have been associated with respiratory tract inflammation, disruption of the nasal respiratory and olfactory barriers, systemic inflammation, production of mediators of inflammation capable of reaching the brain and systemic circulation of particulate matter. Mexico City (MC) residents are exposed to significant amounts of ozone, particulate matter and associated lipopolysaccharides. MC dogs exhibit brain inflammation and an acceleration of Alzheimer's-like pathology, suggesting that the brain is adversely affected by air pollutants. MC children, adolescents and adults have a significant upregulation of cyclooxygenase-2 (COX2) and interleukin-1beta (IL-1beta) in olfactory bulb and frontal cortex, as well as neuronal and astrocytic accumulation of the 42 amino acid form of beta -amyloid peptide (Abeta 42), including diffuse amyloid plaques in frontal cortex. The pathogenesis of Alzheimer's disease (AD) is characterized by brain inflammation and the accumulation of Abeta 42, which precede the appearance of neuritic plaques and neurofibrillary tangles, the pathological hallmarks of AD. Our findings of nasal barrier disruption, systemic inflammation, and the upregulation of COX2 and IL-1beta expression and Abeta 42 accumulation in brain suggests that sustained exposures to significant concentrations of air pollutants such as particulate matter could be a risk factor for AD and other neurodegenerative diseases.     

Intraneuronal Abeta42 accumulation in human brain

Alzheimer's disease (AD) is characterized by the deposition of senile plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are composed of aggregated beta-amyloid (Abeta) 40/42(43) peptides. Evidence implicates a central role for Abeta in the pathophysiology of AD. Mutations in betaAPP and presenilin 1 (PS1) lead to elevated secretion of Abeta, especially the more amyloidogenic Abeta42. Immunohistochemical studies have also emphasized the importance of Abeta42 in initiating plaque pathology. Cell biological studies have demonstrated that Abeta is generated intracellularly. Recently, endogenous Abeta42 staining was demonstrated within cultured neurons by confocal immunofluorescence microscopy and within neurons of PS1 mutant transgenic mice. A central question about the role of Abeta in disease concerns whether extracellular Abeta deposition or intracellular Abeta accumulation initiates the disease process. Here we report that human neurons in AD-vulnerable brain regions specifically accumulate gamma-cleaved Abeta42 and suggest that this intraneuronal Abeta42 immunoreactivity appears to precede both NFT and Abeta plaque deposition. This study suggests that intracellular Abeta42 accumulation is an early event in neuronal dysfunction and that preventing intraneuronal Abeta42 aggregation may be an important therapeutic direction for the treatment of AD.     

Reduced effectiveness of Abeta1-42 immunization in APP transgenic mice with significant amyloid deposition.

Vaccinations with Abeta1-42 have been shown to reduce amyloid burden in transgenic models of Alzheimer's disease (AD). We have further tested the efficacy of Abeta1-42 immunization in the Tg2576 mouse model of AD by immunizing one group of mice with minimal Abeta deposition, one group of mice with modest Abeta deposition, and one group with significant Abeta deposition. The effects of immunization on Abeta deposition were examined using biochemical and immunohistochemical methods. In Tg2576 mice immunized prior to significant amyloid deposition, Abeta1-42 immunization was highly effective. Biochemically extracted Abeta40 and Abeta42 levels were significantly reduced and immunohistochemical plaque load was also reduced. Immunization of mice with modest amounts of pre-existing Abeta deposits selectively reduced Abeta42 without altering Abeta40, although plaque load was reduced. In contrast, in Tg2576 mice with significant pre-existing Abeta loads, Abeta1-42 immunization only minimally decreased Abeta42 levels, whereas no alteration in Abeta40 levels or in plaque load was observed. These results indicate that in Tg2576 mice, Abeta1-42 immunization is more effective at preventing additional Abeta accumulation and does not result in significant clearance of pre-existing Abeta deposits.     

How well does the CSF inform upon pathology in the brain in Creutzfeldt-Jakob and Alzheimer's diseases?

Analysis of lumbar cerebrospinal fluid (CSF) plays a major role in the investigation of central nervous system disease, but how well do the changes in the CSF reflect pathology within the brain and spinal cord parenchyma? Both Creutzfeldt-Jakob (CJD) and Alzheimer's disease (AD) are characterized by the deposition of insoluble beta-pleated sheet peptides [prion protein (PrP) and beta-amyloid (Abeta), respectively] in the extracellular spaces of grey matter in the brain, but there is discordance in both diseases between the peptide levels in the brain and in the CSF. Experimental studies using tracers have shown that interstitial fluid (ISF) drains through very narrow intercellular spaces within grey matter into bulk flow perivascular channels that surround penetrating arteries. ISF then flows to the surface of the brain and joins CSF to drain to cervical lymph nodes. Such drainage of ISF and CSF to regional lymph nodes in the rat plays a significant role in B-cell and T-cell immune reactions within the brain. In man, the pia mater separates the periarterial ISF drainage pathways from the CSF in the subarachnoid space. The almost complete lack of insoluble protease-resistant PrP entering the CSF from the brain in patients with CJD, reported by Wong et al. in this issue of the Journal of Pathology, illustrates the limitations of ISF drainage pathways for the elimination of insoluble peptides from brain tissue. Insoluble Abeta accumulates in the extracellular spaces as plaques in AD and in periarterial ISF drainage pathways as cerebral amyloid angiopathy. Soluble Abeta appears to become entrapped by the insoluble Abeta in the ISF drainage pathways; thus, as the level of soluble Abeta in the brain rises in AD, the level in the CSF falls. Thus, the changes in the CSF do not accurately reflect the accumulation of the abnormal peptides in the brain parenchyma in either CJD or AD. In both diseases, facilitation of ISF drainage and elimination of PrP and Abeta peptides from the extracellular spaces of the brain may lead to practical therapeutic strategies for these devastating disorders.     

BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.