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.

TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice.

Abnormal accumulation of the amyloid-beta peptide (Abeta) in the brain appears crucial to pathogenesis in all forms of Alzheimer disease (AD), but the underlying mechanisms in the sporadic forms of AD remain unknown. Transforming growth factor beta1 (TGF-beta1), a key regulator of the brain's responses to injury and inflammation, has been implicated in Abeta deposition in vivo. Here we demonstrate that a modest increase in astroglial TGF-beta1 production in aged transgenic mice expressing the human beta-amyloid precursor protein (hAPP) results in a three-fold reduction in the number of parenchymal amyloid plaques, a 50% reduction in the overall Abeta load in the hippocampus and neocortex, and a decrease in the number of dystrophic neurites. In mice expressing hAPP and TGF-beta1, Abeta accumulated substantially in cerebral blood vessels, but not in parenchymal plaques. In human cases of AD, Abeta immunoreactivity associated with parenchymal plaques was inversely correlated with Abeta in blood vessels and cortical TGF-beta1 mRNA levels. The reduction of parenchymal plaques in hAPP/TGF-beta1 mice was associated with a strong activation of microglia and an increase in inflammatory mediators. Recombinant TGF-beta1 stimulated Abeta clearance in microglial cell cultures. These results demonstrate that TGF-beta1 is an important modifier of amyloid deposition in vivo and indicate that TGF-beta1 might promote microglial processes that inhibit the accumulation of Abeta in the brain parenchyma.

Retention of the Alzheimer's amyloid precursor fragment C99 in the endoplasmic reticulum prevents formation of amyloid beta-peptide.

gamma-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimer's beta-amyloid precursor protein (APP), resulting in the release of amyloid beta-peptide (Abeta). The molecular identity of gamma-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that gamma-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of Abeta. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimer's disease resulted in a characteristic elevation of the Abeta(42)/Abeta(40) ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of gamma-secretase cleavage. Biogenesis of both Abeta(40) and Abeta(42) was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for gamma-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as gamma-secretase. The results suggest that presenilins may acquire the characteristics of gamma-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes.

Astroglial expression of human alpha(1)-antichymotrypsin enhances alzheimer-like pathology in amyloid protein precursor transgenic mice.

Proteases and their inhibitors play key roles in physiological and pathological processes. Cerebral amyloid plaques are a pathological hallmark of Alzheimer's disease (AD). They contain amyloid-ss (Ass) peptides in tight association with the serine protease inhibitor alpha(1)-antichymotrypsin.(1,2) However, it is unknown whether the increased expression of alpha(1)-antichymotrypsin found in AD brains counteracts or contributes to the disease. We used regulatory sequences of the glial fibrillary acidic protein gene(3) to express human alpha(1)-antichymotrypsin (hACT) in astrocytes of transgenic mice. These mice were crossed with transgenic mice that produce human amyloid protein precursors (hAPP) and Ass in neurons.(4,5) No amyloid plaques were found in transgenic mice expressing hACT alone, whereas hAPP transgenic mice and hAPP/hACT doubly transgenic mice developed typical AD-like amyloid plaques in the hippocampus and neocortex around 6 to 8 months of age. Co-expression of hAPP and hACT significantly increased the plaque burden at 7 to 8, 14, and 20 months. Both hAPP and hAPP/hACT mice showed significant decreases in synaptophysin-immunoreactive presynaptic terminals in the dentate gyrus, compared with nontransgenic littermates. Our results demonstrate that hACT acts as an amyloidogenic co-factor in vivo and suggest that the role of hACT in AD is pathogenic.

High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation.

Amyloid plaques are a neuropathological hallmark of Alzheimer's disease (AD), but their relationship to neurodegeneration and dementia remains controversial. In contrast, there is a good correlation in AD between cognitive decline and loss of synaptophysin-immunoreactive (SYN-IR) presynaptic terminals in specific brain regions. We used expression-matched transgenic mouse lines to compare the effects of different human amyloid protein precursors (hAPP) and their products on plaque formation and SYN-IR presynaptic terminals. Four distinct minigenes were generated encoding wild-type hAPP or hAPP carrying mutations that alter the production of amyloidogenic Abeta peptides. The platelet-derived growth factor beta chain promoter was used to express these constructs in neurons. hAPP mutations associated with familial AD (FAD) increased cerebral Abeta(1-42) levels, whereas an experimental mutation of the beta-secretase cleavage site (671(M-->I)) eliminated production of human Abeta. High levels of Abeta(1-42) resulted in age-dependent formation of amyloid plaques in FAD-mutant hAPP mice but not in expression-matched wild-type hAPP mice. Yet, significant decreases in the density of SYN-IR presynaptic terminals were found in both groups of mice. Across mice from different transgenic lines, the density of SYN-IR presynaptic terminals correlated inversely with Abeta levels but not with hAPP levels or plaque load. We conclude that Abeta is synaptotoxic even in the absence of plaques and that high levels of Abeta(1-42) are insufficient to induce plaque formation in mice expressing wild-type hAPP. Our results support the emerging view that plaque-independent Abeta toxicity plays an important role in the development of synaptic deficits in AD and related conditions.

A learning deficit related to age and beta-amyloid plaques in a mouse model of Alzheimer's disease.

Mice that overexpress the human mutant amyloid precursor protein (hAPP) show learning deficits, but the apparent lack of a relationship between these deficits and the progressive beta-amyloid plaque formation that the hAPP mice display is puzzling. In the water maze, hAPP mice are impaired before and after amyloid plaque deposition. Here we show, using a new water-maze training protocol, that PDAPP mice also exhibit a separate age-related deficit in learning a series of spatial locations. This impairment correlates with beta-amyloid plaque burden and is shown in both cross-sectional and longitudinal experimental designs. Cued navigation and object-recognition memory are normal. These findings indicate that A beta overexpression and/or A beta plaques are associated with disturbed cognitive function and, importantly, suggest that some but not all forms of learning and memory are suitable behavioural assays of the progressive cognitive deficits associated with Alzheimer's-disease-type pathologies.

Purification and cloning of amyloid precursor protein beta-secretase from human brain.

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.

Elimination of the class A scavenger receptor does not affect amyloid plaque formation or neurodegeneration in transgenic mice expressing human amyloid protein precursors.

The class A scavenger receptor (SR) is expressed on reactive microglia surrounding cerebral amyloid plaques in Alzheimer's disease (AD). Interactions between the SR and amyloid beta peptides (Abeta) in microglial cultures elicit phagocytosis of Abeta aggregates and release of neurotoxins. To assess the role of the SR in amyloid clearance and Abeta-associated neurodegeneration in vivo, we used the platelet-derived growth factor promoter to express human amyloid protein precursors (hAPPs) in neurons of transgenic mice. With increasing age, hAPP mice develop AD-like amyloid plaques. We bred heterozygous hAPP (hAPP(+/-)) mice that were wild type for SR (SR(+/+)) with SR knockout (SR(-/-)) mice. Crosses among the resulting hAPP(+/-)SR(+/-) offspring yielded hAPP(+/-) and hAPP(-/-) littermates that were SR(+/+) or SR(-/-). These second-generation mice were analyzed at 6 and 12 months of age for extent of cerebral amyloid deposition and loss of synaptophysin-immunoreactive presynaptic terminals. hAPP(-/-)SR(-/-) mice showed no lack of SR expression, plaque formation, or synaptic degeneration, indicating that lack of SR expression does not result in significant accumulation of endogenous amyloidogenic or neurotoxic factors. In hAPP(+/-) mice, ablation of SR expression did not alter number, extent, distribution, or age-dependent accumulation of plaques; nor did it affect synaptic degeneration. Our results do not support a critical pathogenic role for microglial SR expression in neurodegenerative alterations associated with cerebral beta amyloidosis.

Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models.

Autosomal dominant forms of familial Alzheimer's disease (FAD) are associated with increased production of the amyloid beta peptide, Abeta42, which is derived from the amyloid protein precursor (APP). In FAD, as well as in sporadic forms of the illness, Abeta peptides accumulate abnormally in the brain in the form of amyloid plaques. Here, we show that overexpression of FAD(717V-->F)-mutant human APP in neurons of transgenic mice decreases the density of presynaptic terminals and neurons well before these mice develop amyloid plaques. Electrophysiological recordings from the hippocampus revealed prominent deficits in synaptic transmission, which also preceded amyloid deposition by several months. Although in young mice, functional and structural neuronal deficits were of similar magnitude, functional deficits became predominant with advancing age. Increased Abeta production in the context of decreased overall APP expression, achieved by addition of the Swedish FAD mutation to the APP transgene in a second line of mice, further increased synaptic transmission deficits in young APP mice without plaques. These results suggest a neurotoxic effect of Abeta that is independent of plaque formation.

Presenilin-1 mutations associated with familial Alzheimer's disease do not disrupt protein transport from the endoplasmic reticulum to the Golgi apparatus.

Mutations in genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) have been linked to familial forms of Alzheimer's disease (AD). Cells expressing mutant presenilins produce elevated levels of Abeta42, the major amyloid peptide found in AD plaques. The mechanism whereby this occurs remains unknown, but the localization of presenilins to endoplasmic reticulum (ER) and Golgi compartments has suggested that they may function in intracellular trafficking pathways involved in processing beta-amyloid precursor proteins (APP). To test this possibility, we coexpressed PS1(wt), PS1(M146L), or PS1(L286V) in HEK293 cells together with the LDL receptor, a classic glycoprotein marker that undergoes post-translational O-glycosylation in the Golgi compartment. Pulse-chase analysis of the receptor indicated that mutant presenilins had no effect on ER-->Golgi transport. Similar results were obtained when the studies were carried out with cells expressing the Swedish variant of APP (SWAPP751) instead of the LDL receptor. Moreover, secretion of the soluble exodomain polypeptide fragments of SWAPP751 that arise from alpha-secretase and beta-secretase cleavage was not markedly affected by the PS1 mutants. Despite the lack of discernible effect of the PS1 mutants on trafficking of proteins through the Golgi apparatus, they caused a substantial increase in the proportion of Abeta42 relative to total Abeta in the culture medium. The results suggest that mutant forms of PS1 cause elevated production of Abeta42 by a mechanism that is independent of a major disruption of exocytic trafficking of APP.

Amyloidogenic role of cytokine TGF-beta1 in transgenic mice and in Alzheimer's disease.

Deposition of amyoid-beta peptide in the central nervous system is a hallmark of Alzheimer's disease and a possible cause of neurodegeneration. The factors that initiate or promote deposition of amyloid-beta peptide are not known. The transforming growth factor TGF-beta1 plays a central role in the response of the brain to injury, and increased TGF-beta1 has been found in the central nervous system of patients with Alzheimer's disease. Here we report that TGF-beta1 induces amyloid-beta deposition in cerebral blood vessels and meninges of aged transgenic mice overexpressing this cytokine from astrocytes. Co-expression of TGF-beta1 in transgenic mice overexpressing amyloid-precursor protein, which develop Alzheimer's like pathology, accelerated the deposition of amyloid-beta peptide. More TGF-beta1 messenger RNA was present in post-mortem brain tissue of Alzheimer's patients than in controls, the levels correlating strongly with amyloid-beta deposition in the damaged cerebral blood vessels of patients with cerebral amyloid angiopathy. These results indicate that overexpression of TGF-beta1 may initiate or promote amyloidogenesis in Alzheimer's disease and in experimental models and so may be a risk factor for developing Alzheimer's disease.

Individual isoforms of the amyloid beta precursor protein demonstrate differential adhesive potentials to constituents of the extracellular matrix.

The amyloid beta precursor protein (AbetaPP) can exist as a membrane-bound glycoprotein which modulates neural cell adhesion. The adhesion of clones of the AtT20 mouse pituitary cell line, transfected with cDNA coding for the 695 (AbetaPP695) and 751 (AbetaPP751) amino acid forms of the protein, to individual components of the extracellular matrix was determined using a centrifugal shear assay. On laminin, poly-L-lysine, fibronectin, and uncoated glass substrata, the cells transfected with AbetaPP695 (6A1 cells) demonstrated a 50% increase in adhesivity over nontransfected cells, while those transfected with AbetaPP751 (7A1 cells) showed a significant decrease in adhesion. There was, however, a significant increase in the adhesive strength of the 7A1 cells to collagen type IV with no change in the adhesivity of the 6A1 cells when compared with control. These changes in adhesivity could be attributed to changes in the levels of the membrane-bound protein and were not due to the interaction of soluble AbetaPP with elements of the extracellular matrix. These studies provide evidence for differential adhesivities of the constituent AbetaPP isoforms and the possible role of the Kunitz protease inhibitor (KPI) domain in influencing the adhesive properties of the protein backbone.

Amyloid precursor protein processing and A beta42 deposition in a transgenic mouse model of Alzheimer disease.

The PDAPP transgenic mouse, which overexpresses human amyloid precursor protein (APP717V-->F), has been shown to develop much of the pathology associated with Alzheimer disease. In this report, levels of APP and its amyloidogenic metabolites were measured in brain regions of transgenic mice between 4 and 18 months of age. While absolute levels of APP expression likely contribute to the rate of amyloid beta-peptide (Abeta) deposition, regionally specific factors also seem important, as homozygotic mice express APP levels in pathologically unaffected regions in excess of that measured in certain amyloid plaque-prone regions of heterozygotic mice. Regional levels of APP and APP-beta were nearly constant at all ages, while A beta levels dramatically and predictably increased in brain regions undergoing histochemically confirmed amyloidosis, most notably in the cortex and hippocampus. In hippocampus, A beta concentrations increase 17-fold between the ages of 4 and 8 months, and by 18 months of age are over 500-fold that at 4 months, reaching an average level in excess of 20 nmol of A beta per g of tissue. A beta1-42 constitutes the vast majority of the depositing A beta species. The similarities observed between the PDAPP mouse and human Alzheimer disease with regard to A beta42 deposition occurring in a temporally and regionally specific fashion further validate the use of the model in understanding processes related to the disease.

Beta-secretase processing of the beta-amyloid precursor protein in transgenic mice is efficient in neurons but inefficient in astrocytes.

Alzheimer's disease is characterized by the extracellular deposition of beta-amyloid peptide (Abeta) in cerebral plaques and evidence is accumulating that amyloid is neurotoxic. Abeta is derived from the beta-amyloid precursor protein (APP). Proteolytic processing of APP by the enzyme, beta-secretase, produces the N terminus of Abeta, and releases a secreted ectodomain of APP (beta-s-APP). To develop animal models for measuring beta-secretase activity in specific brain cells in vivo, we have targeted the expression of the full-length human APP to either neurons or astrocytes in transgenic mice using the neuron- specific enolase (NSE) promoter or a modified glial fibrillary acidic protein (GFAP) gene, respectively. The APP cDNAs expressed were mutated (KM to NL at 670/671) to encode amino acid substitutions that enhance amyloidogenic processing in vitro. Western analyses revealed abundant production of beta-s-APP in the brains of NSE-APP mice and enzyme-linked immunosorbent assay analyses showed production of Abeta in fetal primary mixed brain cultures and brain homogenates from these transgenic animals. Because the NSE promoter drives expression primarily in neurons, this provides in vivo evidence that the beta-secretase cleavage necessary for generation of beta-s-APP and Abeta is efficiently performed in neurons. In contrast, only little beta-s-APP was detected in brain homogenates of GFAP-APP mice, indicating that astrocytes show very little beta-secretase activity in vivo. This provides strong in vivo evidence that the major source of Abeta in brain is from neurons and not from astrocytes.

Differential effects of a Rab6 mutant on secretory versus amyloidogenic processing of Alzheimer's beta-amyloid precursor protein.

The Ras-related GTP-binding protein, Rab6, is localized in late Golgi compartments where it mediates intra-Golgi vesicular trafficking. Herein we report that coexpression of Alzheimer's beta-amyloid precursor protein (beta APP751) with a dominant-negative Rab6 mutant (Rab6N126I) in human embryonal kidney 293 cells causes an increase in secretion of the soluble amino-terminal exodomain (s-APP alpha) derived from non-amyloidogenic processing of beta-APP751 by alpha-secretase. The effect was specific to Rab6N126I, since the corresponding mutation in Rab8 (i.e. Rab8N121I), which has been implicated in protein transport to the plasma membrane, caused a modest reduction in s-APP alpha secretion. While Rab6N126I stimulated secretion of APP alpha, the accumulation of amyloid beta peptide (A beta) in the medium was either moderately reduced or unaffected. Similar differential effects of Rab6N126I on secretion of s-APP alpha versus A beta were observed in cell cultures that were overproducing A beta after transfection with a plasmid encoding Swedish variant of beta APP751. Moreover, assays of medium from the latter cultures revealed a marked increase in secretion of s-APP alpha relative to s-APP beta (the immediate product derived from cleavage of beta APP by beta-secretase). The results indicate that vesicular transport events controlled by Rab6 occur at or near a critical juncture in the trans-Golgi network where beta APP is sorted into either the constitutive alpha-secretase pathway or the amyloidogenic beta-secretase pathway.

Levels and alternative splicing of amyloid beta protein precursor (APP) transcripts in brains of APP transgenic mice and humans with Alzheimer's disease.

Abnormal expression of human amyloid precursor protein (hAPP) gene products may play a critical role in Alzheimer's disease (AD). Recently, a transgenic model was established in which platelet-derived growth factor (PDGF) promoter-driven neuronal expression of an alternatively spliced hAPP minigene resulted in prominent AD-type neuropathology (Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., Carr, T., Clemens, J., Donaldson, T., Gillespie, F., Guido, T., Hagopian, S., Johnson-Wood, K., Khan, K., Lee, M., Leibowitz, P., Lieberburg, I., Little, S., Masliah, E., McConlogue, L., Montoya-Zavala, M., Mucke, L., Paganini, L., and Penniman, E. (1995) Nature 373, 523-527). Here we compared the levels and alternative splicing of APP transcripts in brain tissue of hAPP transgenic and nontransgenic mice and of humans with and without AD. PDGF-hAPP mice showed severalfold higher levels of total APP mRNA than did nontransgenic mice or humans, whereas their endogenous mouse APP mRNA levels were decreased. This resulted in a high ratio of mRNAs encoding mutated hAPP versus wild-type mouse APP. Modifications of hAPP introns 6, 7, and 8 in the PDGF-hAPP construct resulted in a prominent change in alternative splice site selection with transcripts encoding hAPP770 or hAPP751 being expressed at substantially higher levels than hAPP695 mRNA. Frontal cortex of humans with AD showed a subtle increase in the relative abundance of hAPP751 mRNA compared with normal controls. These data identify specific intron sequences that may contribute to the normal neuronspecific alternative splicing of APP pre-mRNA in vivo and support a causal role of hAPP gene products in the development of AD-type brain alterations.

The Ras-related GTP-binding protein, Rab1B, regulates early steps in exocytic transport and processing of beta-amyloid precursor protein.

The role of the Ras-related GTP-binding protein, Rab1B, in intracellular trafficking of beta-amyloid precursor protein (beta APP) was studied in cultured 293 cells. beta APP is processed via one of two alternative routes. In the major secretory pathway, beta APP is cleaved by alpha-secretase within the region comprising the beta-amyloid peptide (A beta), resulting in release of a soluble NH2-terminal exodomain (APP alpha) and a 3-kDa peptide (p3) derived from the carboxyl-terminal tail. In the alternative amyloidogenic pathway, beta APP is cleaved by beta-secretase, with the release of a truncated exodomain (APP beta) and an intact A beta peptide. When beta APP751 was coexpressed with Rab1B(wt) or dominant-negative Rab1B mutants (Rab1BN121I or Rab1BS22N) there was a marked decrease in conversion of the immature Endo-H sensitive form of beta APP751 (108 kDa) to the mature O-glycosylated form of beta APP751 (130 kDa) in cells expressing the mutant forms of Rab1B. The block in Golgi-dependent processing of beta APP was accompanied by inhibition of secretion of APPS (APP alpha). A similar decrease in secretion of APPS (APP alpha+APP beta) was observed in cells that were coexpressing Rab1BN121I with the "Swedish" variant of beta APP751 (i.e. beta APPSW751), which undergoes increased amyloidogenic processing. Coincident with the decline in APPS secretion, the cells coexpressing beta APPSW751 with Rab1BN121I showed a 90% decrease in A beta secretion. The data indicate that Rab1B plays a key role in endoplasmic reticulum-->Golgi transport of beta APP, and that beta APP must pass through a late Golgi compartment before entering either the alpha-secretase or the amyloidogenic beta-secretase pathway. The results also suggest that mutant versions of other Rab proteins that function in different parts of the exocytic and endocytic pathways may be useful in defining the specific routes of beta APP transport involved in the biogenesis of A beta.