Chloroquine-induced endocytic pathway abnormalities: Cellular model of GM1 ganglioside-induced Abeta fibrillogenesis in Alzheimer's disease

Endocytic pathway abnormalities were previously observed in brains affected with Alzheimer’s disease (AD). To clarify the pathological relevance of these abnormalities to assembly of amyloid β-protein (Aβ), we treated PC12 cells with chloroquine, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment induced accumulation of GM1 ganglioside (GM1) in Rab5-positive enlarged early endosomes and on the cell surface. Notably, an increase in GM1 level on the cell surface was sufficient to induce Aβ assembly. Our results suggest that endocytic pathway abnormalities in AD brain induce GM1 accumulation on the cell surface, leading to amyloid fibril formation in brain.     

The Neuroendocrine Protein 7B2 Suppresses the Aggregation of Neurodegenerative Disease-related Proteins

Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded β-sheet-rich proteins, including amyloid-β (Aβ)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aβ_1–42, Aβ_1–40, and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aβ_1–42 and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aβ_1–42-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aβ plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.     

Oxidative Modification of Glutamine Synthetase by Amyloid Beta Peptide

β-Amyloid peptide (Aβ), the main constituent of senile plaques and diffuse amyloid deposits in Alzheimer's diseased brain, was shown to initiate the development of oxidative stress in neuronal cell cultures. Toxic lots of Aβ form free radical species in aqueous solution. It was proposed that Aβ-derived free radicals can directly damage cell proteins via oxidative modification. Recently we reported that synthetic Aβ can interact with glutamine synthetase (GS) and induce inactivation of this enzyme. In the present study we present the evidence that toxic Aβ(25-35) induces the oxidation of pure GS in vitro. It was found that inactivation of GS by Aβ, as well as the oxidation of GS by metal-catalyzed oxidation system, is accompanied by an increase of protein carbonyl content. As it was reported previously by our laboratory, radicalization of Aβ is not iron or peroxide-dependent. Our present observations consistently show that toxic Aβ does not need iron or peroxide to oxidize GS. However, treatment of GS with the peptide, iron and peroxide together significantly stimulates the protein carbonyl formation. Here we report also that Aβ(25-35) induces carbonyl formation in BSA. Our results demonstrate that P-peptide, as well as other free radical generators, induces carbonyl formation when brought into contact with different proteins.     

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer''s disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer''s disease. The central component of neuritic plaques is a small filamentous protein called amyloid β protein (Aβ)¹, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. The amyloid hypothesis entails that Aγ-containing plaques as the underlying toxic mechanism in AD pathology². The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of Aγ neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.     

Existence of different types of senile plaques between brain and spinal cord of TgCRND8 mice

Conflicting findings exist regarding the formation of diffuse and dense-core β-amyloid (Aβ) plaques in Alzheimer’s disease (AD). In the present study, we characterized Aβ plaque types in the brain and spinal cord of TgCRND8 mice, which express a transgene incorporating both the Indiana mutation (V717F) and the Swedish mutations (K670N/M671L) in the human amyloid-β protein precursor (APP) gene. By combining immunohistochemistry and thioflavin S staining, we were able to define dense-core and diffuse plaques in neocortex of the brain and spinal cord of 9 week-, 5 month-, 10 month- and 20-month-old TgCRND8 mice. The senile plaques in the neocortex were predominantly dense-core plaques, even in the youngest mice. However, diffuse plaques were instead detected in spinal cord of the mice, regardless of age. Our results that relative predominance of dense-core plaques in the neocortex and diffuse plaques in the spinal cord of TgCNRD8 mice of all disease durations argue against the notion that diffuse plaques may represent an early stage in the evolution of dense-core plaques. Furthermore, we also found that the ratio of Aβ42/Aβ40 of the brain was much higher than that of the spinal cord by Aβ ELISA assay. Our findings strongly indicate that diffuse and dense-core plaques may form via independent processes in AD and Aβ42 is more prone to form dense-core plaques than is Aβ40.     

��-Amyloid1�C42 Gene Transfer Model Exhibits Intraneuronal Amyloid, Gliosis, Tau Phosphorylation, and Neuronal Loss

Alzheimer disease is characterized by extracellular β-amyloid (Aβ) plaques and intracellular inclusions containing neurofibrillary tangles of phospho-Tau and intraneuronal Aβ associated with neuronal cell death. We generated a novel gene transfer animal model using lentiviral Aβ_1–42 that resulted in intracellular but not extracellular Aβ accumulations in the targeted rat primary motor cortex. Expression of intracellular Aβ_1–42 led to pathological changes seen in human Alzheimer disease brains, including cell death, inflammatory signs, activation of two Tau kinases, and Tau hyperphosphorylation. Promoting clearance of lentiviral Aβ_1–42 reversed these effects, demonstrating that intraneuronal Aβ_1–42 is a toxic peptide that lies upstream of Tau modification. These studies reveal the role of intracellular Aβ_1–42 in a novel gene transfer animal model, which is a useful tool to study intraneuronal Aβ_1–42-induced pathology in the absence of extracellular plaques. Targeted delivery of Aβ will allow speedy delineation of pathological mechanisms associated with specific neurodegenerative lesions.     

In vitro and in vivo degradation of Abeta peptide by peptidases coupled to erythrocytes

It is generally believed that amyloid β peptides (Aβ) are the key mediators of Alzheimer's disease. Therapeutic interventions have been directed toward impairing the synthesis or accelerating the clearance of Aβ. An equilibrium between blood and brain Aβ exists mediated by carriers that transport Aβ across the blood–brain barrier. Passive immunotherapy has been shown to be effective in mouse models of AD, where the plasma borne antibody binds plasma Aβ causing an efflux of Aβ from the brain. As an alternative to passive immunotherapy we have considered the use of Aβ-degrading peptidases to lower plasma Aβ levels. Here we compare the ability of three Aβ-degrading peptidases to degrade Aβ. Biotinylated peptidases were coupled to the surface of biotinylated erythrocytes via streptavidin. These erythrocyte-bound peptidases degrade Aβ peptide in plasma. Thus, peptidases bound to or expressed on the surface of erythroid cells represent an alternative to passive immunotherapy.     

MAP2 IHC detection: a marker of antigenicity in CNS tissues

Immunohistochemistry (IHC) is used to detect antibody-specific antigens in tissues; the results depend on the ability of the primary antibodies to bind to their antigens. Therefore, results depend on the quality of preservation of the specimen. Many investigators have overcome the deleterious effects of over-fixation on the binding of primary antibodies to specimen antigens using IHC, but if the specimen is under-fixed or fixation is delayed, false negative results could be obtained despite certified laboratory practices. Microtubule-associated protein 2 (MAP2) is an abundant microtubule-associate protein that participates in the outgrowth of neuronal processes and synaptic plasticity; it is localized primarily in cell bodies and dendrites of neurons. MAP2 immunolabeling has been reported to be absent in areas of the entorhinal cortex and hippocampus of Alzheimer’s disease brains that were co-localized with the dense-core type of amyloid plaques. It was hypothesized that the lack of MAP2 immunolabeling in these structures was due to the degradation of the MAP2 antigen by the neuronal proteases that were released as the neurons lysed leading to the formation of these plaques. Because MAP2 is sensitive to proteolysis, we hypothesized that changes in MAP2 immunolabeling may be correlated with the degree of fixation of central nervous system (CNS) tissues. We detected normal MAP2 immunolabeling in fixed rat brain tissues, but MAP2 immunolabeling was decreased or lost in unfixed and delayed-fixed rat brain tissues. By contrast, two ubiquitous CNS-specific markers, myelin basic protein and glial fibrillary acidic protein, were unaffected by the degree of fixation in the same tissues. Our observations suggest that preservation of various CNS-specific antigens differs with the degree of fixation and that the lack of MAP2 immunolabeling in the rat brain may indicate inadequate tissue fixation. We recommend applying MAP2 IHC for all CNS tissues as a pre-screen to assess the quality of the tissue preservation and to avoid potentially false negative IHC results.     

The Large Hydrophilic Loop of Presenilin 1 Is Important for Regulating ��-Secretase Complex Assembly and Dictating the Amyloid �� Peptide (A��) Profile without Affecting Notch Processing

γ-Secretase is an enzyme complex that mediates both Notch signaling and β-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid β peptide (Aβ), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of γ-secretase gives rise to Aβ peptides of different lengths, where Aβ42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320–374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of γ-secretase, for γ-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, γ-secretase complex formation, and had a differential effect on Aβ-peptide production. Although the production of Aβ38, Aβ39, and Aβ40 was severely impaired, the effect on Aβ42 was affected to a lesser extent, implying that the production of the AD-related Aβ42 peptide is separate from the production of the Aβ38, Aβ39, and Aβ40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the ϵ/S3 and γ sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Aβ peptides without affecting Notch processing, two parameters of significance when considering γ-secretase as a target for pharmaceutical intervention in AD.     

Increased 5-Lipoxygenase Immunoreactivity in the Hippocampus of Patients With Alzheimer's Disease

The proinflammatory enzyme 5-lipoxygenase (5-LOX) is upregulated in Alzheimer''s disease (AD), but its localization and association with the hallmark lesions of the disease, β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs), is unknown. This study examined the distribution and cellular localization of 5-LOX in the medial temporal lobe from AD and control subjects. The spatial relationship between 5-LOX immunoreactive structures and AD lesions was also examined. We report that, in AD subjects, 5-LOX immunoreactivity is elevated relative to controls, and its localization is dependent on the antibody-targeted portion of the 5-LOX amino acid sequence. Carboxy terminus–directed antibodies detected 5-LOX in glial cells and neurons, but less frequently in neurons with dystrophic (NFT) morphology. In contrast, immunoreactivity observed using 5-LOX amino terminus–directed antibodies was virtually absent in neurons and abundant in NFTs, neuritic plaques, and glia. Double-labeling studies showed a close association of 5-LOX–immunoreactive processes and glial cells with Aβ immunoreactive plaques and vasculature and also detected 5-LOX in tau immunoreactive and amyloid containing NFTs. Different immunolabeling patterns with antibodies against carboxy vs amino terminus of 5-LOX may be caused by post-translational modifications of 5-LOX protein in Aβ plaques and NFTs. The relationship between elevated intracellular 5-LOX and hallmark AD pathological lesions provides further evidence that neuroinflammatory pathways contribute to the pathogenesis of AD. (J Histochem Cytochem 56:1065–1073, 2008)     

Alzheimer's Disease Related Markers,Cellular Toxicity and Behavioral Deficits Induced Six Weeks after Oligomeric Amyloid-β Peptide Injection in Rats

Alzheimer’s disease (AD) is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-β protein (Aβ). Recently, we characterized the effects of a single intracerebroventricular (icv) injection of Aβ fragment (25–35) oligomers (oAβ_25–35) for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAβ_25–35 and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAβ_25–35 was still present in the brain after 6 weeks. oAβ_25–35 injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation), mitochondrial (caspase-9 levels) and reticulum stress (caspase-12 levels), astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAβ_25–35 injection resulted in increased APP expression, Aβ_1–42 generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aβ, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.     

An N-terminal Fragment of the Prion Protein Binds to Amyloid-β Oligomers and Inhibits Their Neurotoxicity in Vivo

A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-β (Aβ) peptide in the brain. Considerable evidence suggests that soluble Aβ oligomers are responsible for the synaptic dysfunction and cognitive deficit observed in AD. However, the mechanism by which these oligomers exert their neurotoxic effect remains unknown. Recently, it was reported that Aβ oligomers bind to the cellular prion protein with high affinity. Here, we show that N1, the main physiological cleavage fragment of the cellular prion protein, is necessary and sufficient for binding early oligomeric intermediates during Aβ polymerization into amyloid fibrils. The ability of N1 to bind Aβ oligomers is influenced by positively charged residues in two sites (positions 23–31 and 95–105) and is dependent on the length of the sequence between them. Importantly, we also show that N1 strongly suppresses Aβ oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aβ-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aβ oligomer toxicity and represent an entirely new class of therapeutic agents for AD.     

Aurones serve as probes of beta-amyloid plaques in Alzheimer's disease

A novel series of aurone derivatives for in vivo imaging of beta-amyloid plaques in the brain of Alzheimer's disease (AD) were synthesized and characterized. When in vitro binding studies using Abeta(1-42) aggregates were carried out with aurone derivatives, they showed high binding affinities for Abeta(1-42) aggregates at the K(i) values ranging from 1.2 to 6.8 nM. When in vitro plaque labeling was carried out using double transgenic mice brain sections, the aurone derivatives intensely stained beta-amyiloid plaques. Biodistribution studies in normal mice after i.v. injection of the radioiodinated aurones displayed high brain uptake (1.9-4.6% ID/g at 2 min) and rapid clearance from the brain (0.11-0.26% ID/g at 60 min), which is highly desirable for amyloid imaging agents. The results in this study suggest that novel radiolabeled aurones may be useful amyloid imaging agents for detecting beta-amyloid plaques in the brain of AD.     

Immunomodulation targeting abnormal protein conformation reduces pathology in a mouse model of Alzheimer's disease

Many neurodegenerative diseases are characterized by the conformational change of normal self-proteins into amyloidogenic, pathological conformers, which share structural properties such as high β-sheet content and resistance to degradation. The most common is Alzheimer''s disease (AD) where the normal soluble amyloid β (sAβ) peptide is converted into highly toxic oligomeric Aβ and fibrillar Aβ that deposits as neuritic plaques and congophilic angiopathy. Currently, there is no highly effective treatment for AD, but immunotherapy is emerging as a potential disease modifying intervention. A major problem with most active and passive immunization approaches for AD is that both the normal sAβ and pathogenic forms are equally targeted with the potential of autoimmune inflammation. In order to avoid this pitfall, we have developed a novel immunomodulatory method that specifically targets the pathological conformations, by immunizing with polymerized British amyloidosis (pABri) related peptide which has no sequence homology to Aβ or other human proteins. We show that the pABri peptide through conformational mimicry induces a humoral immune response not only to the toxic Aβ in APP/PS1 AD transgenic mice but also to paired helical filaments as shown on AD human tissue samples. Treated APP/PS1 mice had a cognitive benefit compared to controls (p<0.0001), associated with a reduction in the amyloid burden (p = 0.0001) and Aβ40/42 levels, as well as reduced Aβ oligomer levels. This type of immunomodulation has the potential to be a universal β-sheet disrupter, which could be useful for the prevention or treatment of a wide range of neurodegenerative diseases.     

Amyloid β protein activates PKC-δ and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia

The increased accumulation of activated microglia containing amyloid β protein (Aβ) around senile plaques is a common pathological feature in subjects with Alzheimer's disease (AD). Much less is known, however, of intracellular signal transduction pathways for microglial activation in response to Aβ. We investigated intracellular signaling in response to Aβ stimulation in primary cultured rat microglia. We found that the kinase activity of PKC-δ but not that of PKC- or - is increased by stimulation of microglia with Aβ, with a striking tyrosine phosphorylation of PKC-δ. In microglia stimulated with Aβ, tyrosine phosphorylation of PKC-δ was evident at the membrane fraction without an overt translocation of PKC-δ. PKC-δ co-immunoprecipitated with MARCKS from microglia stimulated with Aβ. Aβ induced translocation of MARCKS from the membrane fraction to the cytosolic fraction. Immunocytochemical analysis revealed that phosphorylated MARCKS accumulated in the cytoplasm, particularly at the perinuclear region in microglia treated with Aβ. Taken together with our previous observations that Aβ-induced phosphorylation of MARCKS and chemotaxis of microglia are inhibited by either tyrosine kinase or PKC inhibitors, our results provide evidence that Aβ induces phosphorylation and translocation of MARCKS through the tyrosine kinase-PKC-δ signaling pathway in microglia.     

Methylene Blue Modulates β-Secretase,Reverses Cerebral Amyloidosis,and Improves Cognition in Transgenic Mice

Amyloid precursor protein (APP) proteolysis is required for production of amyloid-β (Aβ) peptides that comprise β-amyloid plaques in the brains of patients with Alzheimer disease (AD). Here, we tested whether the experimental agent methylene blue (MB), used for treatment of methemoglobinemia, might improve AD-like pathology and behavioral deficits. We orally administered MB to the aged transgenic PSAPP mouse model of cerebral amyloidosis and evaluated cognitive function and cerebral amyloid pathology. Beginning at 15 months of age, animals were gavaged with MB (3 mg/kg) or vehicle once daily for 3 months. MB treatment significantly prevented transgene-associated behavioral impairment, including hyperactivity, decreased object recognition, and defective spatial working and reference memory, but it did not alter nontransgenic mouse behavior. Moreover, brain parenchymal and cerebral vascular β-amyloid deposits as well as levels of various Aβ species, including oligomers, were mitigated in MB-treated PSAPP mice. These effects occurred with inhibition of amyloidogenic APP proteolysis. Specifically, β-carboxyl-terminal APP fragment and β-site APP cleaving enzyme 1 protein expression and activity were attenuated. Additionally, treatment of Chinese hamster ovary cells overexpressing human wild-type APP with MB significantly decreased Aβ production and amyloidogenic APP proteolysis. These results underscore the potential for oral MB treatment against AD-related cerebral amyloidosis by modulating the amyloidogenic pathway.     

The p75NTR extracellular domain: A potential molecule regulating the solubility and removal of amyloid-��

Senile plaque composed of amyloid-beta (Aβ) in the brain is one of the hallmarks of Alzheimer disease (AD). Removal of Aβ from the brain is the most important therapeutic strategy for AD. The solubility of Aβ is critical for its endocytosis, transcytosis and removal from the brain. Our recent study has found that the extracellular domain of p75NTR, the neurotrophin receptor, plays an important role in the solubility of Aβ and might be one of the endogenous mechanisms in the regulation of Aβ plaque formation. The physiologically shedded extracellular domain of p75NTR is able to inhibit Aβ aggregation and diasggregate preformed Aβ fibrils, while the full p75NTR expressed on neurites, endothelial cells and smooth muscle cells in blood-brain barrier (BBB) might initiate Aβ endocytosis and degradation, and/or remove Aβ from the brain via BBB. Understanding the roles of p75NTR in the solubility and clearance of Aβ may allow targetting p75NTR as a unique opportunity to develop therapeutic drugs for the prevention and treatment of AD.Key words: Alzheimer disease, amyloid-β, p75NTR, extracellular domain, blood-brain barrier, clearanceSenile plaque in the brain is one of the hallmarks of Alzheimer disease (AD). The main component of the senile plaques is amyloid-beta (Aβ), which is a metabolic product of amyloid precursor protein (APP). The steady-state level of Aβ in the normal brain is maintained by the balance between its production and clearance. However in the AD brain this balance is broken due to either over-production of Aβ or a reduction in Aβ clearance;^1,2 thus Aβ accumulates in the brain and forms amyloid plaques which cause dementia and neurodegeneration in patients. Based on the Aβ hypothesis proposed a decade ago, Aβ plays a causal and pivotal role in the development of AD.³ Therefore, removal of Aβ from the brain is the most important therapeutic strategy for AD.⁴ To reach this goal, it is essential to understand how the Aβ metabolism is regulated in the AD brain. Despite the dramatic progress has been made in the understanding of how Aβ is produced from APP, the mechanisms of Aβ aggregation, metabolism and clearance from the brain remain unclear so far. Only 5% of AD (familial cases) is due to the over-production of Aβ because of mutations in the APP gene or in the APP processing enzymes, while the majority (95%) of so-called sporadic or late-onset AD (LOAD) are likely caused by dysfunctions in Aβ solubility or aggregation, endocytosis, degradation, transcytosis and removal.The solubility of Aβ is critical for its endocytosis, transcytosis and removal from the brain. In AD patients, one of the most consistent biomarkers found so far is the reduction of Aβ level in the cerebral spinal fluid (CSF).⁵ This is the most convincing evidence that there is a reduction in the solubility of Aβ and an increase in the Aβ aggregation and beta-sheet formation in AD patients. In sporadic cases of AD, the polymorphism of the Aβ-binding protein ApoE4 is highly associated with AD. It is known that other variants of ApoE proteins have a higher binding affinity to Aβ than ApoE4. It is likely that ApoE protein plays a critical role in the solubility of Aβ.⁶ The reduction in the Aβ binding ability of ApoE4 may reduce the solubility of Aβ. Thus ApoE protein may act on Aβ keeping it soluble and preventing its aggregation, and ApoE4 variant may reduce Aβ solubility and increase aggregation in the brain. It is not fully understood at this time, what other proteins that might regulate Aβ solubility and prevent its aggregation. Understanding the endogenous mechanism of suppressing Aβ aggregation and enhancing its removal will help to target Aβ for developing disease-modifying drugs.The neurotrophin receptor p75NTR may be such the protein which plays critical roles in the Aβ solubility and prevents Aβ aggregation and deposition in the brain. During the investigation into the functions of p75NTR in the development of AD in a recent study, we have found that the extracellular domain of p75NTR regulates the deposition of Aβ in a mouse model of AD.⁷ In p75NTR gene-knockout APPswe/PS1dE9 mice, soluble Aβ which reflects the steady-state level of Aβ production, is reduced in the brain. The serum Aβ level, which is associated with the level of soluble Aβ in the brain, is also reduced in p75NTR-knockout animals. In comparison, we found that p75NTR knockout increases the insoluble Aβ as reflected by the increased amyloid plaques and formic acid-extracted Aβ levels. Our results indicate that p75NTR may play critical roles in the solubility of Aβ in the brain of AD mice. To test the hypothesis, we have made recombinant extracellular domain-fused with human immunoglobulin Fc fragment and tested its effects in the solubility of Aβ in vitro. We have found that the recombinant extracellular domain of p75NTR has a very strong effect on the solubility of Aβ. It reduces Aβ oligomerization and fibrillization, solubilizes fibrilized Aβ. Most interestingly, when injected into the hippocampus of AD mice, it reduces the number and size of Aβ plaques. Thus, we have clearly demonstrated that the extracellular domain plays an important role in the solubility of Aβ and might be one of endogenous mechanisms in the regulation of Aβ plaque formation in patients.How might p75NTR play a role in the Aβ plaque formation or deposition in AD brain? One of the features of the Aβ pathology is that Aβ exclusively deposits in the neocortex, hippocampus and vessel walls. These areas are also the projection area of p75NTR positive fibers. The close anatomical association between p75NTR expression and Aβ deposition strongly suggests that p75NTR is involved in the initiation and development of Aβ deposition in the brain. Interestingly, we have observed there is a spatial relationship between p75NTR fibers and Aβ plaques in the brain of AD mice. We have found that p75NTR positive neurites locates in the center of compact senile plaques, while p75NTR negative degenerative neurites locate in the outer region of Aβ plaques.⁷ This phenomenon suggests that p75NTR positive neurodegenerative fibers may play a seeding role to initiate the Aβ aggregation and plaque formation. It is known that Aβ can bind to the extracellular domain of p75NTR.⁸ Normally, Aβ-bound p75NTR is likely endocytosed and degraded in the lysosomes, but the degenerated neurites may be abnormal in the endocytosis of the Aβ-p75NTR complex. Thus Aβ which binds to p75NTR on the cell surface may act as seeds to initiate the cascade of Aβ aggregation and beta-sheet formation.On the other hand, the extracellular domain of p75NTR after enzymatic shedding may play a different role than the membranous p75NTR. It is known that the p75NTR extracellular domain is physiologically shedded by TACE to generate a soluble and diffusible factor.⁹ The physiological function of the shedded diffusible extracellular domain of p75NTR remains unknown. During the aging process and during the development of AD, p75NTR expression is upregulated,^10,11 and presumably the production of the diffusible extracellar domain of p75NTR is also increased. The increased extracellular domain of the p75NTR is likely a critical factor to maintain the solubility of Aβ, acting in concert with ApoE and other Aβ-binding proteins such as low-density lipoprotein receptor-related protein-1 (LRP1).¹² Indeed, in our animal experiment, we have found that knockout of p75NTR significantly increases the insoluble Aβ, even though the production of Aβ is reduced in p75NTR knockout neurons.⁷ Our data have provided strong evidence that the brain Aβ deposition and amyloid plaque formation may be mainly due to the decreased Aβ solubility or decreased Aβ clearance.The solubility of Aβ goes hand in hand with the clearance of Aβ because only the solubilized Aβ can be endocytosed and degraded in lysosomes by neurons, microglia and astrocytes, and be transported from the brain to the blood for degradation and clearance.⁴ Whether p75NTR plays any roles in the endocytosis of Aβ and degradation is unclear. Our data of increased Aβ deposition in the brain of p75NTR knockout mice may also be explained by the decreased endocytosis of Aβ via p75NTR. It is likely that p75NTR may play a role in Aβ endocytosis, as p75NTR is a receptor of Aβ and mediates its toxicity in neurons.⁸ p75NTR ligands can trigger a clathrin-dependent endocytosis of both p75NTR and its ligands.¹³ If p75NTR normally mediates Aβ endocytosis, the knockout of p75NTR would reduce the removal of Aβ by endocytosis and lead to the increased deposition in the brain.Normally, p75NTR is also expressed in endothelial cells and smooth muscle cells of blood vessels, vessel-innervating sympathetic and sensory neurons and choroid plexus in the brain.^14,15 This raises the possibility that p75NTR within blood vessels may play a role in transport and trafficking of Aβ from the brain to the blood. It is well known that LRP1 plays important roles in the transport and transcytosis of Aβ and clears Aβ from the brain.¹² LRP1 on the cell-surface of the blood-brain barrier (BBB) can bind, transcytose and transport Aβ from the brain to the blood. p75NTR expressed on the BBB may play similar roles in the removal of Aβ in a similar manner to the Aβ binding proteins, LRP1 and G-glycoprotein, expressed on the BBB. Future studies should test roles of p75NTR in the endocytosis, transcytosis and clearance of Aβ by different cells such as neurons, endothelial cells and smooth muscle cells. The levels of shedded diffusible p75NTR in the brain and blood of AD patients should be determined as a biomarker and correlated with Aβ levels or Aβ plaques to reveal its potential roles in the solubility and clearance of Aβ in AD patients.In summary, our studies have provided strong evidence that p75NTR is an essential molecule to keep the solubility of Aβ during development of AD. We speculate that p75NTR might also play many vital roles in removing Aβ from brain (Fig. 1). However, it is still far from certain how p75NTR regulates the solubility of Aβ and suppresses its deposition in the AD brain. Understanding the roles of p75NTR in the solubility and clearance of Aβ may help using p75NTR as a target to develop therapeutic drugs for the prevention and treatment of AD.Open in a separate windowFigure 1Schematic diagram depicting functions of p75NTR in Aβ solubility and clearance. p75NTR locates on the neurites, epithelial cells and smooth muscle cells of the blood-brain barrier (BBB). Binding of Aβ to p75NTR on neurites may initiate the endocytosis of Aβ and its degradation in the neurons. Shedding of p75NTR from the cell membrane releases the soluble extracellular domain (p75NTR-ECD), which is capable of inhibiting Aβ aggregation and disaggregating preformed Aβ fibrils. p75NTR at BBB might be able to transport Aβ from the brain to blood.     

Beta-amyloid precursor polypeptide in SAMP8 mice affects learning and memory

Senescence accelerated (SAMP8 [P8]) mice develop age-related deficits in memory and learning. We show that increased expression of amyloid precursor protein (APP) and its mRNA in the hippocampus are also age-related. Immunocytochemical data suggest that a critical amount of APP expression may be needed to generate amyloid (Aβ) protein plaques in the hippocampus. Deficits in acquisition and retention test performance were alleviated by administration of antibody to Aβ protein into the cerebral ventricles. This reversal of cognitive deficits provides a link between increased expression of both APP and Aβ protein and learning and memory loss in these mice.