Amyloid Precursor Protein (APP)/APP-like Protein 2 (APLP2) Expression Is Required to Initiate Endosome-Nucleus-Autophagosome Trafficking of Glypican-1-derived Heparan Sulfate

Anhydromannose (anMan)-containing heparan sulfate (HS) derived from the proteoglycan glypican-1 is generated in endosomes by an endogenously or ascorbate-induced S-nitrosothiol-catalyzed reaction. Processing of the amyloid precursor protein (APP) and APP-like protein 2 (APLP2) by β- and γ-secretases into amyloid β (Aβ) and Aβ-like peptides also takes place in these compartments. Moreover, anMan-containing HS suppresses the formation of toxic Aβ assemblies in vitro. We showed by using deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody as well as ³⁵S labeling experiments that expression of APP/APLP2 is required for ascorbate-induced transport of HS from endosomes to the nucleus. Nuclear translocation was observed in wild-type mouse embryonic fibroblasts (WT MEFs), Tg2576 MEFs, and N2a neuroblastoma cells but not in APP^−/− and APLP2^−/− MEFs. Transfection of APP^−/− cells with a vector encoding APP restored nuclear import of anMan-containing HS. In WT MEFs and N2a neuroblastoma cells exposed to β- or γ-secretase inhibitors, nuclear translocation was greatly impeded, suggesting involvement of APP/APLP2 degradation products. In Tg2576 MEFs, the β-inhibitor blocked transport, but the γ-inhibitor did not. During chase in ascorbate-free medium, anMan-containing HS disappeared from the nuclei of WT MEFs. Confocal immunofluorescence microscopy showed that they appeared in acidic, LC3-positive vesicles in keeping with an autophagosomal location. There was increased accumulation of anMan-containing HS in nuclei and cytosolic vesicles upon treatment with chloroquine, indicating that HS was degraded in lysosomes. Manipulations of APP expression and processing may have deleterious effects upon HS function in the nucleus.     

Defective nitric oxide-dependent, deaminative cleavage of glypican-1 heparan sulfate in Niemann-Pick C1 fibroblasts

Exit of recycling cholesterol from late endosomes is defective in Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (NPC2) diseases. The traffic route of the recycling proteoglycan glypican-1 (Gpc-1) may also involve late endosomes and could thus be affected in these diseases. During recycling through intracellular compartments, the heparan sulfate (HS) side chains of Gpc-1 are deaminatively degraded by nitric oxide (NO) derived from preformed S-nitroso groups in the core protein. We have now investigated whether this NO-dependent Gpc-1 autoprocessing is active in fibroblasts from NPC1 disease. The results showed that Gpc-1 autoprocessing was defective in these cells and, furthermore, greatly depressed in normal fibroblasts treated with U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), a compound widely used to induce cholesterol accumulation. In both cases, autoprocessing was partially restored by treatment with ascorbate which induced NO release, resulting in deaminative cleavage of HS. However, when NO-dependent Gpc-1 autoprocessing is depressed and heparanase-catalyzed degradation of HS remains active, a truncated Gpc-1 with shorter HS chains would prevail, resulting in fewer NO-sensitive sites/proteoglycan. Therefore, addition of ascorbate to cells with depressed autoprocessing resulted in nitration of tyrosines. Nitration was diminished when heparanase was inhibited with suramin or when Gpc-1 expression was silenced by RNAi. Gpc-1 misprocessing in NPC1 cells could thus contribute to neurodegeneration mediated by reactive nitrogen species.     

Constitutive and vitamin C-induced, NO-catalyzed release of heparan sulfate from recycling glypican-1 in late endosomes

The recycling heparan sulfate (HS)-containing proteoglycan glypican-1 (Gpc-1) is processed by nitric oxide (NO)-catalyzed deaminative cleavage of its HS chains at N-unsubstituted glucosamines. This generates anhydromannose (anMan)-containing HS degradation products that can be detected by a specific antibody. Here we have attempted to identify the intracellular compartments where these products are formed. The anMan-positive degradation products generated constitutively in human bladder carcinoma cell line (T24) or fibroblasts appeared neither in caveolin-1-associated vesicles nor in lysosomes. In Niemann-Pick C-1 (NPC-1) fibroblasts, where deaminative degradation is abrogated, formation of anMan-positive products can be restored by ascorbate. These products colocalized with Rab7, a marker for late endosomes. When NO-catalyzed degradation of HS was depressed in mouse neuroblastoma cell line (N2a) by using 3-beta[2(diethylamino) ethoxy]androst-5-en-17-one (U18666A), both ascorbate and dehydroascorbic acid restored formation of anMan-positive products that colocalized with Rab7. In T24 cells, constitutively generated anMan-positive products colocalized with both Rab7 and Rab9, whereas Gpc-1 colocalized with Rab9, a marker for transporting endosomes. Inhibition of endosomal acidification, which blocks transfer from early (Rab5) to late (Rab7) endosomes, abrogated deaminative degradation of HS. This could also be overcome by the addition of ascorbate, which induced formation of anMan-positive degradation products that colocalized with Rab7. After (35)S-sulfate labeling, similar degradation products were recovered in Rab7-positive vesicles. We conclude that NO-catalyzed degradation of HS may begin in early endosomes but is mainly taking place in late endosomes.     

Characterization of oligomer formation of amyloid-beta peptide using a split-luciferase complementation assay

Amyloid-β peptide (Aβ) is the amyloid component of senile plaques in Alzheimer disease (AD) brains. Recently a soluble oliomeric form of Aβ in Aβ precursor protein transgenic mouse brains and AD brains was identified as a potential causative molecule for memory impairment, suggesting that soluble Aβ oligomers cause neurodegeneration in AD. Further characterization of this species has been hampered, however, because the concentrations are quite small and it is difficult to monitor Aβ oligomers specifically. Here we developed a novel method for monitoring Aβ oligomers using a split-luciferase complementation assay. In this assay, the N- and C-terminal fragments of Gaussia luciferase (Gluc) are fused separately to Aβ. We found that conditioned media from both N- and C-terminal fragments of Gluc-tagged Aβ1-42 doubly transfected HEK293 cells showed strong luminescence. We used gel filtration analyses to analyze the size of oligomers formed by the luciferase complementation assay, and found that it matched closely with oligomers formed by endogenous Aβ in Tg2576 neurons. Large oligomers (24-36-mers), 8-mers, trimers, and dimers predominate. In both systems, Aβ formed oligomers intracellularly, which then appear to be secreted as oligomers. We then evaluated several factors that might impact oligomer formation. The level of oligomerization of Aβ1-40 was similar to that of Aβ1-42. Homodimers formed more readily than heterodimers. The level of oligomerization of murine Aβ1-42 was similar to that of human Aβ1-42. As expected, the familial AD-linked Arctic mutation (E22G) significantly enhanced oligomer formation. These data suggest that Gluc-tagged Aβ enables the analysis of Aβ oligomers.     

7-Deoxy-trans-dihydronarciclasine Reduces β-Amyloid and Ameliorates Memory Impairment in a Transgenic Model of Alzheimer’s Disease

The critical pathological feature of Alzheimer’s disease (AD) is the accumulation of β-amyloid (Aβ), the main constituent of amyloid plaques. β-amyloid precursor protein (APP) undergoes amyloidogenic cleavage by β- and γ-secretase generating Aβ at endosomes or non-amyloidogenic processing by α-secretase precluding the production of Aβ at the plasma membrane. Recently, several natural products have been widely researched on the prevention of Aβ accumulation for AD treatment. We previously reported that Lycoris chejuensis K. Tae et S. Ko (CJ), which originated from Jeju Island in Korea, improved the disrupted memory functions and reduced Aβ production in vivo. Here, we further explored the effect of its active component, 7-deoxy-trans-dihydronarciclasine (coded as E144), on Aβ generation and the underlying mechanism. Our results showed that E144 reduced the level of APP, especially its mature form, in HeLa cells overexpressing human APP with the Swedish mutation. Concomitantly, E144 decreased the levels of Aβ, sAPPβ, sAPPα, and C-terminal fragment. In addition, administration of E144 normalized the behavioral deficits in Tg2576 mice, an APP transgenic mouse model of AD. E144 also decreased the Aβ and APP levels in the cerebral cortex of Tg2576 mice. Thus, we propose that E144 could be a potential drug candidate for an anti-amyloid disease-modifying AD therapy.     

Up-regulation of calsyntenin-3 by β-amyloid increases vulnerability of cortical neurons

β-Amyloid (Aβ) may play an important role in the pathogenesis of Alzheimer's disease. However, a causal relationship between Aβ oligomers and layer-specific neurodegeneration has not been clarified. Here we show up-regulation of calsyntenin (Cst)-3 in cultured neurons treated with Aβ oligomers and in Tg2576 mice. Cst-3 is distributed in large neurons in layers 2-3 and 5 of the cerebral cortex, and accumulated in dystrophic neurites surrounding Aβ-plaques. Overexpression of Cst-3 accelerates neuronal death. These results indicate that up-regulation of Cst-3 in cortical neurons in layers 2-3 and 5 by Aβ oligomers may lead to increase in vulnerability of neurons.     

In vivo hippocampal microdialysis reveals impairment of NMDA receptor–cGMP signaling in APPSW and APPSW/PS1L166P Alzheimer’s transgenic mice

Transgenic (Tg) mice overexpressing human amyloid precursor protein (APP) mutants reproduce features of early Alzheimer’s disease (AD) including memory deficit, presence of β-amyloid (Aβ) oligomers, and age-associated formation of amyloid deposits. In this study we used hippocampal microdialysis to characterize the signaling of N-methyl-d-aspartic acid receptors (NMDA-Rs) in awake and behaving AD Tg mice. The NMDA-R signaling is central to hippocampal synaptic plasticity underlying memory formation and several lines of evidence implicate the role of Aβ oligomers in effecting NMDA-R dysfunction. CA1 NMDA-Rs were stimulated by NMDA infused through reverse microdialysis while changes in the cyclic guanosine monophosphate (cGMP) concentration in the brain interstitial fluid (ISF) were used to determine NMDA-Rs responsiveness. While 4 months old wild type C57BL/6 mice mounted robust cGMP response to the NMDA challenge, the same stimulus failed to significantly change the cGMP level in 4 and 15 months old APP_SW and 4 months old APP_SW/PS1_L166P Tg mice, which were all on C57BL/6 background. Lack of response to NMDA in AD Tg mice occurred in the absence of changes in expression levels of several synaptic proteins including synaptophysin, NR1 NMDA-R subunit and postsynaptic density protein 95, which indicates lack of profound synaptic degeneration. Aβ oligomers were detected in all three AD Tg mice groups and their concentration in the hippocampus ranged from 40.5 ± 3.6 ng/g in 4 months old APP_SW mice to 60.8 ± 15.9 ng/g in 4 months old APP_SW/PS1_L166P mice. Four months old APP_SW mice had no Aβ amyloid plaques, while the other two AD Tg mice groups showed evidence of incipient Aβ amyloid plaque formation. Our studies describes a novel approach useful to study the function of NMDA-Rs in awake and behaving AD Tg mice and demonstrate impairment of NMDA-R response in the presence of endogenously formed Aβ oligomers but predating onset of Aβ amyloidosis.     

Promotion of β-amyloid production by C-reactive protein and its implications in the early pathogenesis of Alzheimer's disease

C-reactive protein (CRP) and β-amyloid protein (Aβ) are involved in the development of Alzheimer's disease (AD). However, the relationship between CRP and Aβ production is unclear. In vitro and in vivo experiments were performed to investigate the association of CRP with Aβ production. Using the rat adrenal pheochromocytoma cell line (PC12 cells) to mimic neurons, cytotoxicity was evaluated by cell viability and supernatant lactate dehydrogenase (LDH) activity. The levels of amyloid precursor protein (APP), beta-site APP cleaving enzyme (BACE-1), and presenilins (PS-1 and PS-2) were investigated using real-time polymerase chain reaction and Western blotting analysis. Aβ1-42 was measured by enzyme-linked immunosorbent assay. The relevance of CRP and Aβ as well as potential mechanisms were studied using APP/PS1 transgenic (Tg) mice. Treatment with 0.5-4.0 μM CRP for 48 h decreased cell viability and increased LDH leakage in PC12 cells. Incubation with CRP at a sub-toxic concentration of 0.2 μM increased the mRNA levels of APP, BACE-1, PS-1, and PS-2, as well as Aβ1-42 production. CRP inhibitor reversed the CRP-induced upregulations of the mRNA levels of APP, BACE-1, PS-1, and PS-2, and the protein levels of APP, BACE-1, PS-1, and Aβ1-42, but did not reversed Aβ1-42 cytotoxicity. The cerebral levels of CRP and Aβ1-42 in APP/PS1 Tg mice were positively correlated, accompanied with the elevated mRNA expressions of serum amyloid P component (SAP), complement component 1q (C1q), and tumor necrosis factor-α (TNF-α). These results suggest that CRP cytotoxicity is associated with Aβ formation and Aβ-related markers expressions; CRP and Aβ were relevant in early-stage AD; CRP may be an important trigger in AD pathogenesis.     

Deletion of Irs2 reduces amyloid deposition and rescues behavioural deficits in APP transgenic mice

As impaired insulin signalling (IIS) is a risk factor for Alzheimer’s disease we crossed mice (Tg2576) over-expressing human amyloid precursor protein (APP), with insulin receptor substrate 2 null (Irs2^−/−) mice which develop insulin resistance. The resulting Tg2576/Irs2^−/− animals had increased tau phosphorylation but a paradoxical amelioration of Aβ pathology. An increase of the Aβ binding protein transthyretin suggests that increased clearance of Aβ underlies the reduction in plaques. Increased tau phosphorylation correlated with reduced tau-phosphatase PP2A, despite an inhibition of the tau-kinase glycogen synthase kinase-3. Our findings demonstrate that disruption of IIS in Tg2576 mice has divergent effects on pathological processes—a reduction in aggregated Aβ but an increase in tau phosphorylation. However, as these effects are accompanied by improvement in behavioural deficits, our findings suggest a novel protective effect of disrupting IRS2 signalling in AD which may be a useful therapeutic strategy for this condition.     

Effects of NK-4 in a transgenic mouse model of Alzheimer's disease

Beta-amyloid (Aβ) peptides are considered to play a major role in the pathogenesis of Alzheimer's disease (AD) and molecules that can prevent pathways of Aβ toxicity may be potential therapeutic agents for treatment of AD. We have previously reported that NK-4, a cyanine photosensitizing dye, displays neurotrophic and antioxidant activities. In this study, we report the effects of NK-4 on the toxicity of Aβ and on cognitive function and Aβ concentration in a transgenic mouse model of AD (Tg2576). In vitro, NK-4 effectively protected neuronal cells from toxicity induced by Aβ. In addition, it displayed profound inhibitory activities on Aβ fibril formation. In vivo, Tg2576 mice received an intraperitoneal injection at 100 or 500 μg/kg of NK-4 once a day, five times a week for 9 months. Administration of NK-4 to the mice attenuated impairment of recognition memory, associative memory, and learning ability, as assessed by a novel object recognition test, a passive avoidance test, and a water maze test, respectively. NK-4 decreased the brain Aβ concentration while increasing the plasma amyloid level in a dose-dependent manner. NK-4 also improved memory impairments of ICR mice induced by direct intracerebroventricular administration of Aβ. These lines of evidence suggest that NK-4 may affect multiple pathways of amyloid pathogenesis and could be useful for treatment of AD.     

Rapamycin promotes β-amyloid production via ADAM-10 inhibition

Rapamycin is a well known immunosuppressant drug for rejection prevention in organ transplantation. Numerous clinical trials using rapamycin analogs, involving both children and adults with various disorders are currently ongoing worldwide. Most recently, rapamycin gained much attention for what appears to be life-span extending properties when administered to mice. The risk for Alzheimer disease (AD) is strongly and positively correlated with advancing age and is characterized by deposition of β-amyloid peptides (Aβ) as senile plaques in the brain. We report that rapamycin (2.5 μM), significantly increases Aβ generation in murine neuron-like cells (N2a) transfected with the human “Swedish” mutant amyloid precursor protein (APP). In concert with these observations, we found rapamycin significantly decreases the neuroprotective amino-terminal APP (amyloid precursor protein) cleavage product, soluble APP-α (sAPP-α) while increasing production of the β-carboxyl-terminal fragment of APP (β-CTF). These cleavage events are associated with decreased activation of a disintegrin and metallopeptidase domain-10 (ADAM-10), an important candidate α-secretase which opposes Aβ generation. To validate these findings in vivo, we intraperitoneal (i.p.) injected Tg2576 Aβ-overproducing transgenic mice with rapamycin (3 mg/kg/day) for 2 weeks. We found increased Aβ levels associated with decreased sAPP-α at an average rapamycin plasma concentration of 169.7 ± 23.5 ng/mL by high performance liquid chromatography (HPLC). These data suggest that although rapamycin may increase the lifespan in some mouse models, it may not decrease the risk for age-associated neurodegenerative disorders such as AD.     

Vaccination with a non-human random sequence amyloid oligomer mimic results in improved cognitive function and reduced plaque deposition and micro hemorrhage in Tg2576 mice

ABSTRACT: BACKGROUND: It is well established that vaccination of humans and transgenic animals against fibrillar amyloid beta (Abeta) prevents amyloid accumulation in plaques and preserves cognitive function in transgenic mouse models. However, autoimmune side effects have halted the development of vaccines based on full length human Abeta. Further development of an effective vaccine depends on overcoming these side effects while maintaining an effective immune response. RESULTS: We have previously reported that the immune response to amyloid oligomers is largely directed against generic epitopes that are common to amyloid oligomers of many different proteins and independent of a specific amino acid sequence. Here we have examined whether we can exploit this generic immune response to develop a vaccine that targets amyloid oligomers using a non-human random sequence amyloid oligomer. In order to study the effect of vaccination against generic oligomer epitopes, a random sequence oligomer (3A) was selected as it forms oligomers that react with the oligomer specific A11 antibody. Oligomer mimics from 3A peptide, Abeta, islet amyloid polypeptide (IAPP), and Abeta fibrils were used to vaccinate Tg2576 mice, which develop a progressive accumulation of plaques and cognitive impairment. Vaccination with the 3A random sequence antigen was just as effective as vaccination with the other antigens in improving cognitive function and reducing total plaque load (Abeta burden) in the Tg2576 mouse brains. CONCLUSION: These results show that the amyloid Abeta sequence is not necessary to produce a protective immune response that specifically targets generic amyloid oligomers. Using a non-human, random sequence antigen may facilitate the development of a vaccine that avoids autoimmune side effects.     

Clathrin-mediated endocytic proteins are upregulated in the cortex of the Tg2576 mouse model of Alzheimer's disease-like amyloid pathology

Amyloid-β (Aβ) is cleaved from amyloid precursor protein (APP) predominantly after APP has trafficked through the secretory pathway and then become re-internalised by endocytosis. Clathrin-mediated and, more recently, clathrin-independent endocytosis have both been implicated in this process. Furthermore, endocytic abnormalities have been identified in cases of Alzheimer’s disease (AD), however, the relevance of these changes to the aetiology of the disease remains unclear. We therefore examined the expression of proteins related to these endocytic processes in the cortex of Tg2576 mice that overexpress the Swedish mutation in APP, and consequently overexpress Aβ, to determine if there were any changes in their associated pathways. We identified significant increases in the levels of clathrin, dynamin and PICALM, all proteins intimately involved with the clathrin-mediated endocytic pathway, in the transgenic animals. However, levels of proteins associated with flotillin or caveolin-mediated endocytic pathways remained unchanged. These results emphasise the importance of clathrin-mediated endocytosis in the aetiology of AD and reinforce the results of the recent GWAS studies that identified genes for clathrin-mediated endocytosis as susceptibility genes for AD. Such studies in transgenic mice will allow us to learn more about the role of clathrin-mediated endocytosis in AD.     

Neuroprotective role of γ‐enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X

γ‐Enolase is a neurotrophic‐like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C‐terminal end of the molecule. We have investigated the expression and colocalization of γ‐enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ‐enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C‐terminally cleaved form of γ‐enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ‐enolase in microglial cells in response to amyloid‐β peptide (Aβ) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ‐enolase proved to be neuroprotective against Aβ toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ‐enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid‐β‐related neurodegeneration.     

Reduced levels of the tyrosine phosphatase STEP block β amyloid-mediated GluA1/GluA2 receptor internalization

Striatal-Enriched protein tyrosine Phosphatase of MW 61 kDa (STEP(61)) is a protein tyrosine phosphatase recently implicated in the pathophysiology of Alzheimer's disease (AD). STEP(61) is elevated in human AD prefrontal cortex and in the cortex of several AD mouse models. The elevated levels of active STEP(61) down-regulate surface expression of GluN1/GluN2B (formerly NR1/NR2B) receptor complexes, while genetically reducing STEP levels rescues both the biochemical and cognitive deficits in a triple transgenic AD mouse model (3xTg-AD). Here, we show that increased STEP(61) also plays a role in beta amyloid (Aβ)-mediated internalization of the α-amino-3-hydroxy-5-methyl-4-(AMPA) receptor (AMPAR) subunits GluA1/GluA2 (formerly GluR1/GluR2). We purified Aβ oligomers and determined that oligomers, but not monomers, lead to endocytosis of GluA1/GluA2 receptors in cortical cultures. The decrease in GluA1/GluA2 receptors is reversed in the progeny of STEP knock-out (KO) mice crossed with Tg2576 mice, despite elevated levels of Aβ. These results provide strong support for the hypothesis that STEP(61) is required for Aβ-mediated internalization of GluA1/GluA2 receptors.     

Amelioration of cognitive deficits in plaque‐bearing Alzheimer's disease model mice through selective reduction of nascent soluble A42 without affecting other A pools

Given that amyloid‐β 42 (Aβ42) is believed to be a culprit in Alzheimer's disease (AD), reducing Aβ42 production should be a potential therapeutic approach. γ‐Secretase modulators (GSMs) cause selective reduction of Aβ42 or both reduction of Aβ42 and Aβ40 without affecting total Aβ through shifting the γ‐cleavage position in amyloid precursor protein. We recently reported on GSM‐2, one of the second‐generation GSMs, that selectively reduced brain Aβ42 level and significantly ameliorated cognitive deficits in plaque‐free 5.5‐month‐old Tg2576 AD model mice. Here, we investigated the effects of GSM‐2 on 10‐, 14‐, and 18‐month‐old mice which had age‐dependent increase in amyloid plaques. Eight‐day treatment with GSM‐2 significantly ameliorated cognitive deficits measured by Y‐maze task in the mice of any age. However, GSM‐2 reduced brain soluble Aβ42 only in 10‐month‐old mice. In contrast, GSM‐2 markedly reduced newly synthesized soluble Aβ42 in both 10‐ and 18‐month‐old mice with similar efficacy when measured using the stable isotope‐labeling technique, suggesting that nascent Aβ42 plays a more significant role than plaque‐associated soluble Aβ42 in the cognitive deterioration of Tg2576 mice. These findings further indicate the potential utility of approach to reducing Aβ42 synthesis in AD therapeutic regimens.     

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ_(1–40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3 × Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB.     

The interaction of beta-amyloid protein with cellular membranes stimulates its own production

Gradual changes in steady-state levels of beta amyloid peptides (Aβ) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Aβ is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Aβ perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Aβ is initiated. Therefore, we tested effects of oligomeric Aβ on membrane fluidity of whole living cells, the impact of exogenous and cellular Aβ on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAβ_(1-40) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Aβ may start a vicious circle, where endogenous Aβ stimulates its own production. Based on our novel findings, we propose that oligoAβ_(1-40) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.     

Size and sulfation are critical for the effect of heparin on APP processing and Aβ production

Alzheimer's disease is associated with abnormal accumulation of Aβ, which is produced from the β‐amyloid precursor protein (APP) by the β‐site APP‐cleaving enzyme (BACE1) and γ‐secretase. Our previous studies showed that heparin can decrease APP processing by decreasing the levels of BACE1 and ADAM10. In this study, we examined the effects of glycosaminoglycans (GAGs) on APP processing and Aβ production with the aim of understanding the specificity of the effects. Various GAG analogs were incubated with primary cortical cells derived from APP (SW)Tg2576 mice and the level of APP, proteolytic products of APP and APP‐cleavage enzymes were measured. The effect of GAGs on APP processing was both size‐ and sulfation‐dependent. 6‐O‐Sulfation was important for the effect on APP processing as heparin lacking 6‐O sulfate were less potent than native heparin. However, deletion of carboxyl groups on heparin had no significant effect on APP processing. Our studies suggest that there is structural specificity to the effect of GAGs on APP processing and that certain GAGs have a greater effect on Aβ production than others. This suggests that it might be possible to alter the structure of GAGs to achieve more specific inhibitors of APP processing that can cross the blood–brain barrier.