Identification of neuronal plasma membrane microdomains that colocalize beta-amyloid and presenilin: implications for beta-amyloid precursor protein processing

Alzheimer's disease (AD) is associated with the accumulation of extracellular deposits of the beta-amyloid protein (Abeta). Abeta is a result of misprocessing of the beta-amyloid precursor protein (APP). Gamma-secretase is involved in APP misprocessing and one hypothesis holds that this secretase is identical to PS1. We tested this hypothesis by determining whether PS is co-localised with Abeta in situ. Using confocal analyses and a sensitive immunogold procedure we show that PS and Abeta are co-localised within discrete microdomains of neuronal plasma membranes in AD patients and in aged dogs, an established model of human brain aging. Our data indicate that APP misprocessing occurs in discrete plasma membrane domains of neurons and provide evidence that PS1 is critically involved in Abeta formation.     

beta-amyloid: friend or foe

The function of the amyloid precursor protein (APP) and its product, beta-amyloid, (Abeta) is at present unknown. The deposition of Abeta in senile plaques as well as meningeal and cerebral vessels has led many researchers to discount the possibility of a beneficial protective function for the protein. Thus it is generally believed that the aberrant processing of APP leads to increased beta-amyloid secretion that in turn leads to subsequent plaque formation and Alzheimer's disease. Here, a hypothesis is presented that the protein may indeed be protective and that a potential role for beta amyloid in innate immunity may exist.     

Interferon-gamma and tumor necrosis factor-alpha regulate amyloid-beta plaque deposition and beta-secretase expression in Swedish mutant APP transgenic mice

Reactive astrocytes and microglia in Alzheimer's disease surround amyloid plaques and secrete proinflammatory cytokines that affect neuronal function. Relationship between cytokine signaling and amyloid-beta peptide (Abeta) accumulation is poorly understood. Thus, we generated a novel Swedish beta-amyloid precursor protein mutant (APP) transgenic mouse in which the interferon (IFN)-gamma receptor type I was knocked out (APP/GRKO). IFN-gamma signaling loss in the APP/GRKO mice reduced gliosis and amyloid plaques at 14 months of age. Aggregated Abeta induced IFN-gamma production from co-culture of astrocytes and microglia, and IFN-gamma elicited tumor necrosis factor (TNF)-alpha secretion in wild type (WT) but not GRKO microglia co-cultured with astrocytes. Both IFN-gamma and TNF-alpha enhanced Abeta production from APP-expressing astrocytes and cortical neurons. TNF-alpha directly stimulated beta-site APP-cleaving enzyme (BACE1) expression and enhanced beta-processing of APP in astrocytes. The numbers of reactive astrocytes expressing BACE1 were increased in APP compared with APP/GRKO mice in both cortex and hippocampus. IFN-gamma and TNF-alpha activation of WT microglia suppressed Abeta degradation, whereas GRKO microglia had no changes. These results support the idea that glial IFN-gamma and TNF-alpha enhance Abeta deposition through BACE1 expression and suppression of Abeta clearance. Taken together, these observations suggest that proinflammatory cytokines are directly linked to Alzheimer's disease pathogenesis.     

Immunotherapy for Alzheimer's disease: attacking amyloid-beta from the inside

Although extracellular Abeta plaques are a hallmark of Alzheimer's disease, it remains to be determined whether extracellular or intracellular Abeta accumulation initiates the disease process. A recent paper from Gunnar Gouras' group showed that Abeta antibodies lead to reduced intracellular Abeta levels in neurons via antibody internalization after binding to the Abeta domain of amyloid precursor protein (APP) at the plasma membrane. This work suggests novel avenues for the immunotherapy of Alzheimer's disease.     

Neuronal and microglial involvement in beta-amyloid protein deposition in Alzheimer's disease.

This study was undertaken to localize amyloid precursor protein (APP) and to determine how APP might be released and proteolyzed to yield the beta-amyloid protein deposits found in senile plaques in the brains of Alzheimer's disease patients. We found that antibodies to recombinantly expressed APP labeled many normal neurons and neurites. In addition, dystrophic neurites in different types of senile plaques and degenerating neurons in the temporal cortex and hippocampus of Alzheimer's disease patients were immunostained. We also detected small clusters of dystrophic APP immunoreactive neurites that were not associated with beta-amyloid protein deposits. Microglia was involved in different types of senile plaques and often were associated closely with APP immunoreactive neurites and neurons. The greatest concurrence of APP immunoreactivity and reactive microglia was seen in the subiculum and area CA1, regions with a high density of congophilic plaques and subject to intense Alzheimer's pathology. Our findings suggest that neuronally derived APP is the source for senile plaque beta-amyloid protein, while microglia may act as processing cells.     

Beta-amyloid-induced activation of caspase-3 in primary cultures of rat neurons

It is known that beta-amyloid peptide (Abeta) contributes to the neurodegeneration in Alzheimer's disease (AD) and operates through activation of an apoptotic pathway. Apoptotic signal is driven by a family of cysteine proteases called caspases. The beta-amyloid precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated beta-amyloid peptide formation. Cerebellar neurons from rat pups were treated with the aged Abeta(25-35) at 1 and 5 microM and fluorescence assays of caspase activity performed over 4 days. We observed an increase in caspase activity after 48 h treatment in both 1 and 5 microM treated cells, then (72-96 h) caspase activity decreased to control values. The data presented support the hypothesis that Abeta(25-35)-induced apoptosis is mediated by the activation of Caspase-3 and that this is a transient effect.     

Co-occurrence of Alzheimer's disease ß-amyloid and τ pathologies at synapses

Although beta-amyloid (Abeta) plaques and tau neurofibrillary tangles are hallmarks of Alzheimer's disease (AD) neuropathology, loss of synapses is considered the best correlate of cognitive decline in AD, rather than plaques or tangles. How pathological Abeta and tau aggregation relate to each other and to alterations in synapses remains unclear. Since aberrant tau phosphorylation occurs in amyloid precursor protein (APP) Swedish mutant transgenic mice, and since neurofibrillary tangles develop in triple transgenic mice harboring mutations in APP, tau and presenilin 1, we utilized these well-characterized mouse models to explore the relation between Abeta and tau pathologies. We now report that pathological accumulation of Abeta and hyperphosphorylation of tau develop concomitantly within synaptic terminals.     

Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice

We have shown that interaction of CD40 with CD40L enables microglial activation in response to amyloid-beta peptide (Abeta), which is associated with Alzheimer's disease (AD)-like neuronal tau hyperphosphorylation in vivo. Here we report that transgenic mice overproducing Abeta, but deficient in CD40L, showed decreased astrocytosis and microgliosis associated with diminished Abeta levels and beta-amyloid plaque load. Furthermore, in the PSAPP transgenic mouse model of AD, a depleting antibody against CD40L caused marked attenuation of Abeta/beta-amyloid pathology, which was associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased circulating levels of Abeta. Conversely, in neuroblastoma cells overexpressing wild-type human APP, the CD40-CD40L interaction resulted in amyloidogenic APP processing. These findings suggest several possible mechanisms underlying mitigation of AD pathology in response to CD40L depletion, and validate the CD40-CD40L interaction as a target for therapeutic intervention in AD.     

Hippocampal neuron loss exceeds amyloid plaque load in a transgenic mouse model of Alzheimer's disease

According to the "amyloid hypothesis of Alzheimer's disease," beta-amyloid is the primary driving force in Alzheimer's disease pathogenesis. Despite the development of many transgenic mouse lines developing abundant beta-amyloid-containing plaques in the brain, the actual link between amyloid plaques and neuron loss has not been clearly established, as reports on neuron loss in these models have remained controversial. We investigated transgenic mice expressing human mutant amyloid precursor protein APP751 (KM670/671NL and V717I) and human mutant presenilin-1 (PS-1 M146L). Stereologic and image analyses revealed substantial age-related neuron loss in the hippocampal pyramidal cell layer of APP/PS-1 double-transgenic mice. The loss of neurons was observed at sites of Abeta aggregation and surrounding astrocytes but, most importantly, was also clearly observed in areas of the parenchyma distant from plaques. These findings point to the potential involvement of more than one mechanism in hippocampal neuron loss in this APP/PS-1 double-transgenic mouse model of Alzheimer's disease.     

Extracellular amyloid formation and associated pathology in neural grafts

Amyloid precursor protein (APP) processing and the generation of beta-amyloid peptide (Abeta) are important in the pathogenesis of Alzheimer's disease. Although this has been studied extensively at the molecular and cellular levels, much less is known about the mechanisms of amyloid accumulation in vivo. We transplanted transgenic APP23 and wild-type B6 embryonic neural cells into the neocortex and hippocampus of both B6 and APP23 mice. APP23 grafts into wild-type hosts did not develop amyloid deposits up to 20 months after grafting. In contrast, both transgenic and wild-type grafts into young transgenic hosts developed amyloid plaques as early as 3 months after grafting. Although largely diffuse in nature, some of the amyloid deposits in wild-type grafts were congophilic and were surrounded by neuritic changes and gliosis, similar to the amyloid-associated pathology previously described in APP23 mice. Our results indicate that diffusion of soluble Abeta in the extracellular space is involved in the spread of Abeta pathology, and that extracellular amyloid formation can lead to neurodegeneration.     

Platelet as a physiological model to investigate apoptotic mechanisms in Alzheimer beta-amyloid peptide production

Although neuronal apoptosis in Alzheimer's disease is generally interpreted as the consequence of the toxicity of extracellular beta-amyloid (Abeta) peptide aggregates, some experimental results provide evidence that the Abeta overproduction can be the result of a primary neuronal degeneration. As platelets are considered a good model where to study proteolytic processing of the amyloid precursor protein (APP), we exposed platelets to the proapoptotic agent ionomycin and analyzed Abeta40 and Abeta42 levels in the intracellular and extracellular compartments. The activation of apoptotic pathways in platelets has been verified by mitochondrial membrane depolarization, exposure of phosphatidylserine, protease activation and morphological changes. A significative increase in intraplatelet Abeta40, but not Abeta42, was observed after 10 min treatment with ionomycin. Thus, the activation of apoptotic pathways in platelets determines an altered processing of APP leading to elevated levels of intracellular Abeta40. The specific intracellular production of Abeta40 represents a potential threat to the cells since very high local Abeta40 concentration increases the risk of its aggregation and toxicity. As a result, Abeta40 might be dangerous even before it becomes secreted rendering neurons highly vulnerable.     

Overexpression of monocyte chemotactic protein-1/CCL2 in beta-amyloid precursor protein transgenic mice show accelerated diffuse beta-amyloid deposition

Microglia accumulation at the site of amyloid plaques is a strong indication that microglia play a major role in Alzheimer's disease pathogenesis. However, how microglia affect amyloid-beta peptide (Abeta) deposition remains poorly understood. To address this question, we developed a novel bigenic mouse that overexpresses both amyloid precursor protein (APP) and monocyte chemotactic protein-1 (MCP-1; CCL2 in systematic nomenclature). CCL2 expression, driven by the glial fibrillary acidic protein promoter, induced mononuclear phagocyte (MP; monocyte-derived macrophage and microglial) accumulation in the brain. When APP/CCL2 transgenic mice were compared to APP mice, a fivefold increase in Abeta deposition was present despite increased MP accumulation around hippocampal and cortical amyloid plaques. Levels of full-length APP, its C-terminal fragment, and Abeta-degrading enzymes (insulin-degrading enzyme and neprilysin) in APP/CCL2 and APP mice were indistinguishable. Sodium dodecyl sulfate-insoluble Abeta (an indicator of fibrillar Abeta) was increased in APP/CCL2 mice at 5 months of age. Apolipoprotein E, which enhances Abeta deposition, was also increased (2.2-fold) in aged APP/CCL2 as compared to APP mice. We propose that although CCL2 stimulates MP accumulation, it increases Abeta deposition by reducing Abeta clearance through increased apolipoprotein E expression. Understanding the mechanisms underlying these events could be used to modulate microglial function in Alzheimer's disease and positively affect disease outcomes.     

Cholesterol and Alzheimer's disease--is there a relation?

The predominating theory on the pathophysiology of Alzheimer's disease (AD) concerns the mis-metabolism of amyloid precursor protein (APP). As a result of this mis-metabolism, there is an increased production of the 42 amino acid form of beta-amyloid (Abeta42) that rapidly will form oligomers that initiates a cascade of events leading to the accumulation of amyloid plaques. Commonly recognised as vascular factors, hypertension, hypercholesterolemia and diabetes and the inheritance of the epsilon4 allele of the APOE gene, are also risk factors for AD. These risks have been found to promote the production of Abeta42. An association between cholesterol and the development of AD was suggested in the early 1990s and ever since, an increasing amount of research has confirmed that there is a link between cholesterol and the development of AD. A high cholesterol levels in mid-life is a risk for AD and statins, i.e., cholesterol-lowering drugs, reduce this risk. Statins may not only inhibit enzymes involved in the endogenous synthesis of cholesterol but also affect enzymes involved in Abeta metabolism, i.e., alpha-secretase and beta-secretase. This normalises the breakdown of APP thereby promoting the non-amyloidogenic pathway. In this review, investigations focusing on cholesterol and Alzheimer's disease are presented.     

Intracellular accumulation of amyloidogenic fragments of amyloid-beta precursor protein in neurons with Niemann-Pick type C defects is associated with endosomal abnormalities

Niemann-Pick type C disease (NPC) is characterized by neurodegeneration secondary to impaired cholesterol trafficking and excessive glycosphingolipid storage. Abnormal cholesterol and ganglioside metabolism may influence the generation and aggregation of amyloidogenic fragments (ie, C99 and Abeta) from amyloid-beta precursor protein (APP), crucial factors causing neurodegeneration in Alzheimer's disease. To reveal whether abnormal accumulation and aggregation of APP fragments also occurs in NPC, we studied their expression in cultured cortical neurons treated with U18666A, a compound widely used to induce NPC defects, and also in brain tissues from NPC patients. U18666A treatment resulted in increased intraneuronal levels of C99 and insoluble Abeta42, which were distributed among early and late endosomes, in compartments distinct from where endogenous cholesterol accumulates. Analyses of NPC brains revealed that C99 or other APP C-terminal fragments (APP-CTF), but not Abeta42, accumulated in Purkinje cells, mainly in early endosomes. In contrast, in hippocampal pyramidal neurons, the major accumulated species was Abeta42, in late endosomes. Similar to what has been shown in Alzheimer's disease, cathepsin D, a lysosomal hydrolase, was redistributed to early endosomes in NPC Purkinje cells, where it co-localized with C99/APP-CTF. Our results suggest that endosomal abnormalities related to abnormal lipid trafficking in NPC may contribute to abnormal APP processing and Abeta42/C99/APP-CTF deposition.     

Amyloid beta interacts with the amyloid precursor protein: a potential toxic mechanism in Alzheimer's disease

Amyloid beta protein (Abeta) deposition in the brain is a hallmark of Alzheimer's disease (AD). The fibrillar form of Abeta is neurotoxic, although the mechanism of its toxicity is unknown. We showed that conversion of Abeta to the fibrillar form markedly increased binding to specific neuronal membrane proteins, including amyloid precursor protein (APP). Nanomolar concentrations of fibrillar Abeta bound cell-surface holo-APP in cortical neurons. Reduced vulnerability of cultured APP-null neurons to Abeta neurotoxicity suggested that Abeta neurotoxicity involves APP. Thus Abeta toxicity may be mediated by the interaction of fibrillar Abeta with neuronal membrane proteins, notably APP. An Abeta-APP interaction reminiscent of the pathogenic mechanism of prions may thus contribute to neuronal degeneration in AD.     

Beta-site APP cleaving enzyme 1 (BACE1) is increased in remaining neurons in Alzheimer's disease brains

Alzheimer's disease (AD) is characterized by the extensive deposition of amyloid beta protein (Abeta) in the brain cortex. Abeta is produced from beta-amyloid precursor protein (APP) by beta-secretase and gamma-secretase. beta-Secretase has been identified as beta-site APP cleaving enzyme1 (BACE1). We produced rabbit polyclonal antibodies against the amino and the carboxyl terminals of BACE1. Using these antibodies, BACE1 was characterized in temporal lobe cortices by Western blotting and immunohistochemistry. Immunohistochemical studies employing anti-GFAP and anti-MAP2 antibodies as well as anti-BACE1 antibodies showed that BACE1 was expressed exclusively in neurons but not in glial cells. Brain samples were directly extracted by 0.5% SDS and analyzed by Western blotting and densitometer. Although the mean level of BACE1/mg protein in AD brains was not increased, the ratio of BACE1 to MAP2 or to NSE was significantly increased compared with that in control brains. Taken together, these findings suggest that those neurons that survive in AD brains might generate more BACE1 than normal neurons in control brains, indicating that increased BACE1 activity could be one of the causes of AD. This could justify the development of anti-BACE1 drugs for AD treatment.     

Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42 accumulation in a novel Alzheimer transgenic model

Alzheimer's disease (AD) is characterized by a substantial degeneration of pyramidal neurons and the appearance of neuritic plaques and neurofibrillary tangles. Here we present a novel transgenic mouse model, APP(SL)PS1KI that closely mimics the development of AD-related neuropathological features including a significant hippocampal neuronal loss. This transgenic mouse model carries M233T/L235P knocked-in mutations in presenilin-1 and overexpresses mutated human beta-amyloid (Abeta) precursor protein. Abeta(x-42) is the major form of Abeta species present in this model with progressive development of a complex pattern of N-truncated variants and dimers, similar to those observed in AD brain. At 10 months of age, an extensive neuronal loss (>50%) is present in the CA1/2 hippocampal pyramidal cell layer that correlates with strong accumulation of intraneuronal Abeta and thioflavine-S-positive intracellular material but not with extracellular Abeta deposits. A strong reactive astrogliosis develops together with the neuronal loss. This loss is already detectable at 6 months of age and is PS1KI gene dosage-dependent. Thus, APP(SL)PS1KI mice further confirm the critical role of intraneuronal Abeta(42) in neuronal loss and provide an excellent tool to investigate therapeutic strategies designed to prevent AD neurodegeneration.     

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.     

Environmental enrichment counteracts Alzheimer's neurovascular dysfunction in TgCRND8 mice

We and others have recently demonstrated that cognitive and physical stimulation in form of environmental enrichment reduces cerebral beta-amyloid (Abeta) deposition in transgenic mouse models of Alzheimer's disease. This effect was independent from amyloid precursor protein (APP) expression or processing and rather a consequence of enhanced clearance of Abeta. However, the detailed mechanisms remain unclear. In the present study, we show that environmental enrichment in TgCRND8 mice (carrying human APP(Swedish+Indiana)) affect the neurovascular unit by increased angiogenesis and differential regulation of Abeta receptor/transporter molecules, namely up-regulation of LRP1, ApoE and A2M as well as down-regulation of RAGE so that brain to blood Abeta clearance is facilitated. These results suggest a hitherto unknown effect of environmental enrichment counteracting the vascular dysfunction in Alzheimer diseased brain.     

Sequencing of exons 16 and 17 of the beta-amyloid precursor protein gene in 14 families with early onset Alzheimer's disease fails to reveal mutations in the beta-amyloid sequence.

A mutation within exon 17 at codon 717 of the beta-amyloid protein precursor (APP) gene is one cause of early onset familial Alzheimer's disease. Direct sequencing of exons 16 and 17 of the beta-amyloid precursor protein gene in 14 families with familial early onset Alzheimer's disease without the known pathogenic mutation (APP717) failed to reveal other mutations within the beta-amyloid sequence in this form of the disorder.