Does dysregulation of the Notch and wingless/Wnt pathways underlie the pathogenesis of Alzheimer's disease?

Alzheimer's disease is characterized by the presence of neurofibrillary tangles and senile neuritic plaques in the brain. Tangles are aggregates of paired helical filaments composed of the microtubule-associated protein, tau, in a hyperphosphorylated state. Senile plaques have a core of amyloid beta-peptide derived by proteolysis of the amyloid precursor protein. A major hurdle in defining the pathogenic mechanisms in Alzheimer's disease is to understand how both amyloid beta-peptide deposition and paired helical filament formation are biochemically linked. Recent genetic discoveries provide some clues, suggesting that components of two developmentally important signalling pathways, Notch and wingless, or the vertebrate homologue of wingless, Wnt, are involved.     

Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease

One hallmark of Alzheimer disease is the accumulation of amyloid beta-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid beta precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid beta-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid beta-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid beta-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood-brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.     

Partial purification and characterization of gamma-secretase from post-mortem human brain

One characteristic feature of Alzheimer's disease is the deposition of amyloid beta-peptide (Abeta) as amyloid plaques within specific regions of the human brain. Abeta is derived from the amyloid beta-peptide precursor protein (beta-APP) by the intramembranous cleavage activity of gamma-secretase. Studies in cells have revealed that gamma-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of gamma-secretase is as yet to be fully elucidated, and no investigations have analyzed gamma-secretase in human brain. To address this we have developed a novel in vitro gamma-secretase activity assay using detergent-solubilized cell membranes and a beta-APP-derived fluorescent probe. We report that human brain-derived gamma-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived gamma-secretase are similar to those described in cells, and proteolysis occurs at the Abeta40- and Abeta42-generating cleavage sites. The ability to isolate gamma-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of beta-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.     

The cell biology of Alzheimer's disease: uncovering the secrets of secretases

Progress has been made in characterizing the secretases involved in endoproteolytic processing of the beta-amyloid precursor protein - the precursor of the amyloid beta-peptide (Abeta), which is the main constituent of amyloid plaques that form in the brains of patients with Alzheimer's disease. It is now thought that Abeta is pivotal in the pathogenesis of Alzheimer's disease, and that reducing brain Abeta levels may help to treat or prevent the disease. Two essential factors for the proteolytic generation of Abeta have been identified, beta-secretase and the presenilins, which might aid the design of drugs against this disease.     

Amyloid precursor protein cross-linking stimulates beta amyloid production and pro-inflammatory cytokine release in monocytic lineage cells

Beta amyloid peptide-containing neuritic plaques are a defining feature of Alzheimer's disease pathology. Beta amyloid are 38-43 residue peptides derived by proteolytic cleavage of amyloid precursor protein. Although much attention has focused on the proteolytic events leading to beta amyloid generation, the function of amyloid precursor protein remains poorly described. Previously, we reported that amyloid precursor protein functions as a pro-inflammatory receptor on monocytic lineage cells and defined a role for amyloid precursor protein in adhesion by demonstrating that beta(1) integrin-mediated pro-inflammatory activation of monocytes is amyloid precursor protein dependent. We demonstrated that antibody-induced cross-linking of amyloid precursor protein in human THP-1 monocytes and primary mouse microglia stimulates a tyrosine kinase-based pro-inflammatory signaling response leading to acquisition of a reactive phenotype. Here, we have identified pro-inflammatory mediators released upon amyloid precursor protein-dependent activation of monocytes and microglia. We show that amyloid precursor protein cross-linking stimulated tyrosine kinase-dependent increases in pro-inflammatory cytokine release and a tyrosine kinase-independent increase in beta amyloid 1-42 generation. These data provide much needed insight into the function of amyloid precursor protein and provide potential therapeutic targets to limit inflammatory changes associated with the progression of Alzheimer's disease.     

A pathogenic presenilin-1 deletion causes abberrant Abeta 42 production in the absence of congophilic amyloid plaques

Familial Alzheimer's disease (FAD) is frequently associated with mutations in the presenilin-1 (PS1) gene. Almost all PS1-associated FAD mutations reported so far are exchanges of single conserved amino acids and cause the increased production of the highly amyloidogenic 42-residue amyloid beta-peptide Abeta42. Here we report the identification and pathological function of an unusual FAD-associated PS1 deletion (PS1 DeltaI83/DeltaM84). This FAD mutation is associated with spastic paraparesis clinically and causes accumulation of noncongophilic Abeta-positive "cotton wool" plaques in brain parenchyma. Cerebral amyloid angiopathy due to Abeta deposition was widespread as were neurofibrillary tangles and neuropil threads, although tau-positive neurites were sparse. Although significant deposition of Abeta42 was observed, no neuritic pathology was associated with these unusual lesions. Overexpressing PS1 DeltaI83/DeltaM84 in cultured cells results in a significantly elevated level of the highly amyloidogenic 42-amino acid amyloid beta-peptide Abeta42. Moreover, functional analysis in Caenorhabditis elegans reveals reduced activity of PS1 DeltaI83/DeltaM84 in Notch signaling. Our data therefore demonstrate that a small deletion of PS proteins can pathologically affect PS function in endoproteolysis of beta-amyloid precursor protein and in Notch signaling. Therefore, the PS1 DeltaI83/DeltaM84 deletion shows a very similar biochemical/functional phenotype like all other FAD-associated PS1 or PS2 point mutations. Since increased Abeta42 production is not associated with classical senile plaque formation, these data demonstrate that amyloid plaque formation is not a prerequisite for dementia and neurodegeneration.     

Review: Alzheimer's amyloid beta-peptide-associated free radical oxidative stress and neurotoxicity

Alzheimer's disease, the major dementing disorder of the elderly that affects over 4 million Americans, is related to amyloid beta-peptide, the principal component of senile plaques in Alzheimer's disease brain. Oxidative stress, manifested by protein oxidation and lipid peroxidation, among other alterations, is a characteristic of Alzheimer's disease brain. Our laboratory united these two observations in a model to account for neurodegeneration in Alzheimer's disease brain, the amyloid beta-peptide-associated oxidative stress model for neurotoxicity in Alzheimer's disease. Under this model, the aggregated peptide, perhaps in concert with bound redox metal ions, initiates free radical processes resulting in protein oxidation, lipid peroxidation, reactive oxygen species formation, cellular dysfunction leading to calcium ion accumulation, and subsequent neuronal death. Free radical antioxidants abrogate these findings. This review outlines the substantial evidence from multiidisciplinary approaches for amyloid beta-peptide-associated free radical oxidative stress and neurotoxicity and protection against these oxidative processes and cell death by free radical scavengers. In addition, we review the strong evidence supporting the notion that the single methionine residue of amyloid beta-peptide is vital to the oxidative stress and neurotoxicological properties of this peptide. Further, we discuss studies that support the hypothesis that aggregated soluble amyloid beta-peptide and not fibrils per se are necessary for oxidative stress and neurotoxicity associated with amyloid beta-peptide.     

Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695

We have created early-onset transgenic (Tg) models by exploiting the synergistic effects of familial Alzheimer's disease mutations on amyloid beta-peptide (Abeta) biogenesis. TgCRND8 mice encode a double mutant form of amyloid precursor protein 695 (KM670/671NL+V717F) under the control of the PrP gene promoter. Thioflavine S-positive Abeta amyloid deposits are present at 3 months, with dense-cored plaques and neuritic pathology evident from 5 months of age. TgCRND8 mice exhibit 3,200-4,600 pmol of Abeta42 per g brain at age 6 months, with an excess of Abeta42 over Abeta40. High level production of the pathogenic Abeta42 form of Abeta peptide was associated with an early impairment in TgCRND8 mice in acquisition and learning reversal in the reference memory version of the Morris water maze, present by 3 months of age. Notably, learning impairment in young mice was offset by immunization against Abeta42 (Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T. J., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St. George-Hyslop, P., and Westaway, D. (2000) Nature 408, 979-982). Amyloid deposition in TgCRND8 mice was enhanced by the expression of presenilin 1 transgenes including familial Alzheimer's disease mutations; for mice also expressing a M146L+L286V presenilin 1 transgene, amyloid deposits were apparent by 1 month of age. The Tg mice described here suggest a potential to investigate aspects of Alzheimer's disease pathogenesis, prophylaxis, and therapy within short time frames.     

Amyloid Angiopathy of Alzheimer''s Disease: Amino Acid Composition and Partial Sequence of a 4,200-Dalton Peptide Isolated from Cortical Microvessels

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intramembrane proteolysis by presenilins

Many neurodegenerative diseases involve the deposition of insoluble amyloid molecules. In Alzheimer's disease, for example, the amyloid beta-peptide (A beta) is the main component of the characteristic senile plaques. Proteolytic enzymes called secretases are involved in generating A beta, and one of these may have been identified as presenilin--a discovery that paves the way for a more complete understanding of presenilin structure and function.     

The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice

Accumulation of cerebral amyloid beta-protein (Abeta) is believed to be part of the pathogenic process in Alzheimer's disease. Abeta is derived by proteolytic cleavage from a precursor protein, the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein, and its carboxyl-terminal intracellular domain binds to X11beta, a neuronal adaptor protein. X11beta has been shown to inhibit the production of Abeta in transfected non-neuronal cells in culture. However, whether this is also the case in vivo in the brain and whether X11beta can also inhibit the deposition of Abeta as amyloid plaques is not known. Here we show that transgenic overexpression of X11beta in neurons leads to a decrease in cerebral Abeta levels in transgenic APPswe Tg2576 mice that are a model of the amyloid pathology of Alzheimer's disease. Moreover, overexpression of X11beta retards amyloid plaque formation in these APPswe mice. Our findings suggest that modulation of X11beta function may represent a novel therapeutic approach for preventing the amyloid pathology of Alzheimer's disease.     

Strategies to alter the progression of Alzheimer's disease

Recent advances in the genetics of familial Alzheimer's disease provide direction for therapeutic strategies to alter the progressive neurodegeneration. The rationale is particularly strong for targeting the deposition of amyloid into neuritic plaques, but attention has also turned to abnormalities in apoptosis and signal-transduction processes.     

Stable insertion of Alzheimer Abeta peptide into the ER membrane strongly correlates with its length

Alzheimer's disease is characterized by the deposition of amyloid beta-peptide (Abeta) plaques in the brain. Full-length amyloid-beta precursor protein (APP) is processed by alpha- and beta-secretases to yield soluble APP derivatives and membrane-bound C-terminal fragments, which are further processed by gamma-secretase to a non-amyloidogenic 3 kDa product or to Abeta fragments. As different Abeta fragments contain different parts of the APP transmembrane helix, one may speculate that they are retained more or less efficiently in the membrane. Here, we use the translocon-mediated insertion of different APP-derived polypeptide segments into the endoplasmic reticulum membrane to assess the propensities for membrane retention of Abeta fragments. Our results show a strong correlation between the length of an Abeta-derived segment and its ability to integrate into the microsomal membrane.     

Mechanical manipulation of Alzheimer's amyloid beta1-42 fibrils

The 39- to 42-residue-long amyloid beta-peptide (Abeta-peptide) forms filamentous structures in the neuritic plaques found in the neuropil of Alzheimer's disease patients. The assembly and deposition of Abeta-fibrils is one of the most important factors in the pathogenesis of this neurodegenerative disease. Although the structural analysis of amyloid fibrils is difficult, single-molecule methods may provide unique insights into their characteristics. In the present work, we explored the nanomechanical properties of amyloid fibrils formed from the full-length, most neurotoxic Abeta1-42 peptide, by manipulating individual fibrils with an atomic force microscope. We show that Abeta-subunit sheets can be mechanically unzipped from the fibril surface with constant forces in a reversible transition. The fundamental unzipping force (approximately 23 pN) was significantly lower than that observed earlier for fibrils formed from the Abeta1-40 peptide (approximately 33 pN), suggesting that the presence of the two extra residues (Ile and Ala) at the peptide's C-terminus result in a mechanical destabilization of the fibril. Deviations from the constant force transition may arise as a result of geometrical constraints within the fibril caused by its left-handed helical structure. The nanomechanical fingerprint of the Abeta1-42 is further influenced by the structural dynamics of intrafibrillar interactions.     

Pin1 in Alzheimer's disease: multiple substrates, one regulatory mechanism?

Presence of neuritic plaques and neurofibrillary tangles in the brain are two neuropathological hallmarks of Alzheimer's disease (AD), although the molecular basis of their coexistence remains elusive. The neurofibrillary tangles are composed of microtubule binding protein Tau, whereas neuritic plaques consist of amyloid-beta peptides derived from amyloid precursor protein (APP). Recently, the peptidyl-prolyl cis/trans isomerase Pin1 has been identified to regulate the function of certain proteins after phosphorylation and to play an important role in cell cycle regulation and cancer development. New data indicate that Pin1 also regulates the function and processing of Tau and APP, respectively, and is important for protecting against age-dependent neurodegeneration. Furthermore, Pin1 is the only gene known so far that, when deleted in mice, can cause both Tau and Abeta-related pathologies in an age-dependent manner, resembling many aspects of human Alzheimer's disease. Moreover, in the human AD brain Pin1 is downregulated or inhibited by oxidative modifications and/or genetic changes. These results suggest that Pin1 deregulation may provide a link between formation of tangles and plaques in AD.     

Expression of beta-amyloid precursor protein in reactive astrocytes following neuronal damage

Although the beta-amyloid peptide is an established core component of neuritic plaques that accumulate in Alzheimer's disease, the mechanisms responsible for its deposition are not well understood. We now report that lesions of rat hippocampal neurons cause a time-dependent, long-lasting elevation of immunoreactivity for the beta-amyloid precursor protein (APP) in neighboring astrocytes, a cell type not normally containing the protein. The increase represents astroglial expression of the protein rather than a scavenging of APP released by damaged neurons. Immunoelectron microscopy confirmed that APP-containing cells are reactive astroglia, both surrounding capillaries and within the neuropil. These results demonstrate that neuronal damage stimulates APP expression in adult brain and suggest that reactive astrocytes may be a source of the beta-amyloid that forms neuropathological plaques in Alzheimer's disease.     

Up-regulation of the Kv3.4 potassium channel subunit in early stages of Alzheimer's disease

Gene expression throughout the different stages of Alzheimer's disease was analysed in samples from cerebral cortex. The gene encoding the voltage-gated potassium channel Kv3.4 was already overexpressed in early stages of the disease, and in advanced stages Kv3.4 was present at high levels in neurodegenerative structures. This subunit regulates delayed-rectifier currents, which are primary determinants of spike repolarization in neurones. In unique samples from a patient with Alzheimer's disease whose amount of amyloid plaques was decreased by beta amyloid immunization, Kv3.4 was overexpressed. The channel subunit was expressed in the neuropil, in the remaining conventional plaques in the frontal cortex and in collapsed plaques in the orbitary cortex. Therefore, amyloid deposition in plaques does not seem to be responsible for the increase in Kv3.4 levels. Nevertheless, Kv3.4 up-regulation is related to amyloid pathology, given that transgenic mice with the Swedish mutation of amyloid precursor protein showed increased expression of Kv3.4. Up-regulation of voltage-gated potassium channel subunits alters potassium currents in neurones and leads to altered synaptic activity that may underlie the neurodegeneration observed in Alzheimer's disease. Thus, Kv3.4 likely represents a novel therapeutic target for the disease.     

Identification of a Trypsin-Like Site Associated with Acetylcholinesterase by Affinity Labelling with [3H]Diisopropyl Fluorophosphate

Neuritic plaque core amyloid protein in Alzheimer's disease brain tissue was investigated for the extent of amino acid racemization. Long-lived human proteins exhibit racemization of certain amino acids over the course of a human lifetime. Purified core amyloid was found to contain relatively large proportions of D-aspartate and D-serine, suggesting that neuritic plaque amyloid is derived from a long-lived precursor protein. Alternatively, racemization of protein amino acids may be abnormally accelerated in Alzheimer's disease.     

The presence of astrocytes enhances beta amyloid-induced neurotoxicity in hippocampal cell cultures.

A characteristic feature of neuritic plaques in Alzheimer's disease is represented by the presence of activated astrocytes, surrounding dystrophic neurons and beta-amyloid deposition. To explore the role of astrocytes in in vitro beta-amyloid neurotoxicity, we studied the effect of beta-amyloid treatment in hippocampal neurons in two different cell models: pure cultures, where neurons were grown in absence of astrocytes and mixed cultures, where neurons were seeded on a confluent layer of astrocytes. We evaluated two characteristic aspects of in vitro beta-amyloid neurotoxicity: reduction of cell viability and degeneration of the neuritic tree. We demonstrated that neurons growing on astrocytes were more prone to the detrimental effect of the amyloid peptide, with respect to neurons grown in absence of the glial component. Our results support the hypothesis that beta-amyloid-astrocyte interaction can adversely condition neurons and contribute to neuronal damage in Alzheimer's disease.     

In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase-3β

Abstract: The two pathological lesions found in the brains of Alzheimer's disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule-associated protein τ. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of β-amyloid protein, a 4-kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline-directed protein kinases, glycogen synthase kinase-3β (GSK-3β) and mitogen-activated protein kinase (MAPK), were able to hyperphosphorylate τ, GSK-3β but not MAPK phosphorylated recombinant APP_cyt. The sole site of phosphorylation in APP_cyt by GSK-3β was determined by phosphoamino acid analysis and phosphorylation of APP_cyt mutant peptides to be Thr⁷⁴³ (numbering as for APP₇₇₀). This site was confirmed by endoproteinase Glu-C digestion of APP_cyt and peptide sequencing. The ability of GSK-3β to phosphorylate APP_cyt and τ provides a putative link between the two lesions and indicates a critical role of GSK-3β in the pathogenesis of Alzheimer's disease.