Blocking the apolipoprotein E/Amyloid β interaction in triple transgenic mice ameliorates Alzheimer's disease related amyloid β and tau pathology

Inheritance of the apolipoprotein E4 (apoE4) genotype has been identified as the major genetic risk factor for late‐onset Alzheimer's disease (AD). Studies have shown that the binding between apoE and amyloid‐β (Aβ) peptides occurs at residues 244–272 of apoE and residues 12–28 of Aβ. ApoE4 has been implicated in promoting Aβ deposition and impairing clearance of Aβ. We hypothesized that blocking the apoE/Aβ interaction would serve as an effective new approach to AD therapy. We have previously shown that treatment with Aβ12‐28P can reduce amyloid plaques in APP/PS1 transgenic (Tg) mice and vascular amyloid in TgSwDI mice with congophilic amyloid angiopathy. In the present study, we investigated whether the Aβ12‐28P elicits a therapeutic effect on tau‐related pathology in addition to amyloid pathology using old triple transgenic AD mice (3xTg, with PS1_M146V, APP_Swe and tau_P30IL transgenes) with established pathology from the ages of 21 to 26 months. We show that treatment with Aβ12‐28P substantially reduces tau pathology both immunohistochemically and biochemically, as well as reducing the amyloid burden and suppressing the activation of astrocytes and microglia. These affects correlate with a behavioral amelioration in the treated Tg mice.

     

Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β-Protein 1–42 and Its N-Terminally Truncated Variant(s) Which Are Generated at Distinct Sites

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β-protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro-blastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N-terminally truncated variant(s) Aβx-42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx-42, whereas extracellular levels of Aβ1–42 and Aβx-42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1 -induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx-42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx-42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific γ-secretase cleavage that occurs in the normal β-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β-secretase cleavage or (b) in the distinct sites where Aβx-42 and Aβ1–42 are generated.     

Identification of new Presenilin‐1 phosphosites: implication for γ‐secretase activity and Aβ production

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.

     

Differential physiologic responses of α7 nicotinic acetylcholine receptors to β‐amyloid1–40 and β‐amyloid1–42

The β‐amyloid peptides (Aβ), Aβ_1–40 and Aβ_1–42, have been implicated in Alzheimer's disease (AD) pathology. Although Aβ_1–42 is generally considered to be the pathological peptide in AD, both Aβ_1–40 and Aβ_1–42 have been used in a variety of experimental models without discrimination. Here we show that monomeric or oligomeric forms of the two Aβ peptides, when interact with the neuronal cation channel, α7 nicotinic acetylcholine receptors (α7nAChR), would result in distinct physiologic responses as measured by acetylcholine release and calcium influx experiments. While Aβ_1–42 effectively attenuated these α7nAChR‐dependent physiology to an extent that was apparently irreversible, Aβ_1–40 showed a lower inhibitory activity that could be restored upon washings with physiologic buffers or treatment with α7nAChR antagonists. Our data suggest a clear pharmacological distinction between Aβ_1–40 and Aβ_1–42. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 25–30, 2003     

Expression Pattern of Synucleins (Non-Aβ Component of Alzheimer's Disease Amyloid Precursor Protein/α-Synuclein) During Murine Brain Development

Abstract: The non-Aβ component of Alzheimer's disease amyloid precursor protein (NACP) is predominantly a neuron-specific presynaptic protein that may play a central role in neurodegeneration because NACP fragments are found in Alzheimer's disease amyloid and a mutation in the NACP gene is associated with familial Parkinson's disease. In addition, NACP may play an important role during synaptogenesis and CNS development. To understand better the patterns of NACP expression during development, we analyzed the levels of this protein as well as the levels of another synaptic protein (synaptophysin) by ribonuclease protection assay, western blotting, and immunocytochemistry in fetal, juvenile, and adult mouse brain. From embryonic day 12 to 15, there was a slight increase, which was then followed by a more dramatic increase at later time points. Immunocytochemical staining for NACP increases throughout these stages as well. Although NACP appeared early in CNS development, synaptophysin levels started to rise at a later stage. These findings support the contention that NACP might be important for CNS development. Furthermore, the cytosolic component of NACP precedes the particulate component in development, indicating that a redistribution of the protein to the membrane fraction may be important for events later in neuronal development and in synaptogenesis.     

Structural Determinants of the Alzheimer's Amyloid β-Peptide

Abstract: The hallmark event of Alzheimer's disease (AD) is the deposition of amyloid as insoluble fiber masses in extracellular neuritic plaques and around the walls of cerebral blood vessels. The main component of amyloid is a hydrophobic peptide, named amyloid β-peptide (βA4), which results from the processing of a much longer membrane amyloid precursor protein (APP). This review focuses on the structural features of βA4 and the factors that determine βA4 insolubilization. Theoretical and experimental studies of the primary structure of βA4 have shown that it is composed of a completely hydrophobic C-terminal domain, which adopts β-strand structure, and an N-terminal region, whose sequence permits different secondary structures. In fact, this region can exist as an α-helical or β-strand conformation depending on the environmental condition (pH and hydrophobicity surrounding the molecule). The effects of pH and hydrophobicity on βA4 structure may elucidate the mechanisms determining its aggregation and amyloid deposition in AD.     

Wild‐type,Flemish, and Dutch amyloid‐β exhibit different cytotoxicities depending on Aβ40 to Aβ42 interaction time and concentration ratio

Addition of amyloid β (Aβ) peptide Aβ40 to Aβ42 can delay Aβ42 aggregation, but consequent cytotoxicity has been reported to be enhanced or diminished. In the present study, we found that cytotoxicity was enhanced when human neuroblastoma SH‐SY5Y cells were incubated in a mixture of wt Aβ42 and Aβ40wt at a ratio of 1 : 10–20 (0.1 : 1–2 μM) for 24–36 h, whereas the enhancement was detected in cells incubated for longer times (48–60 h) with the less amyloidogenic Flemish Aβ40 variant or in cells incubated for as short as 12 h with the more amyloidogenic Dutch variant. Reductions in cytotoxicity by Aβ40 were most prominently observed in the Flemish and wt Aβ40/Aβ42 mixture at ratio 1 : 20 incubated for a short time (~12 h). The most cytotoxic Aβ40/Aβ42 mixtures were enriched in Aβ protofibril‐like structures, implying a strong correlation between cytotoxicity and this structure, the formation of which was dependent on amyloidogenic properties and incubation time. The consequences of the interactions were probably because of the different amyloidogenic properties of the Aβ40 variants, rather than to those of Aβ42, because aggregation rates of Aβ40 variants were highly dependent on sequence, whereas those of Aβ42 variants were not. These studies highlight a potential role for Aβ40 in cytotoxicity and provide novel mechanistic insights into the pathogenesis of each familial Alzheimer's disease‐associated Aβ40 variant. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Functional analysis and purification of a Pen‐2 fusion protein for γ‐secretase structural studies

The 19‐transmembrane, multisubunit γ‐secretase complex generates the amyloid β‐peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β‐amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen‐2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high‐resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen‐2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N‐terminal maltose binding protein tag on Pen‐2 still permits incorporation into the complex and subsequent presenilin‐1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen‐2 and the γ‐secretase complex.

     

Effect of agitation on the peptide fibrillization: Alzheimer's amyloid‐β peptide 1‐42 but not amylin and insulin fibrils can grow under quiescent conditions

Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides – Alzheimer's amyloid‐β (Aβ) 1‐40 and 1‐42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Aβ₄₀ substantially decreased, whereas the fibrillization of Aβ₄₂ peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Amyloid beta1–42 (Aβ42) up‐regulates the expression of sortilin via the p75NTR/RhoA signaling pathway

Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co‐receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6‐month‐old swedish‐amyloid precursor protein/PS1dE9 transgenic mice. Aβ₄₂ enhanced the protein and mRNA expression levels of sortilin in a dose‐ and time‐dependent manner in SH‐SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75^NTR (P75ECD‐FC), or the antibody against the extracellular domain of p75^NTR, blocked the up‐regulation of sortilin induced by Amyloid‐β protein (Aβ), suggesting that Aβ₄₂ increased the expression level of sortilin and mRNA in SH‐SY5Y via the p75^NTR receptor. Inhibition of ROCK, but not Jun N‐terminal kinase, suppressed constitutive and Aβ₄₂‐induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aβ₄₂ oligomer increases sortilin gene and protein expression through p75^NTR and RhoA signaling pathways, suggesting a potential physiological interaction of Aβ₄₂ and sortilin in Alzheimer's disease.

     

TGF-β1 is an organizer of responses to neurodgeneration

TGF-β1 mRNA and protein were recently found to increase in animal brains after experimental lesions that cause local deafferentation or neuron death. Elevations of TGF-β1 mRNA after lesions are prominent in microglia but are also observed in neurons and astrocytes. Moreover, TGF-β1 mRNA autoinduces its own mRNA in the brain. These responses provide models for studying the increases of TGF-β1 protein observed in βA/amyloid-containing extracellular plaques of Alzheimer's disease (AD) and Down's syndrome (DS) and in brain cells of AIDS victims. Involvement of TGF-β1 in these human brain disorders is discussed in relation to the potent effects of TGF-β1 on wound healing and inflammatory responses in peripheral tissues. We hypothesize that TGF-β1 and possibly other TGF-β peptides have organizing roles in responses to neurodegeneration and brain injury that are similar to those observed in non-neural tissues. Work from many laboratories has shown that activities of TGF-β peptides on brain cells include chemotaxis, modification of extracellular matrix, and regulation of cytoskeletal gene expression and of neurotrophins. Similar activities of the TGF-β's are well established in other tissues.     

New mechanism of nerve injury in Alzheimer’s disease: β‐amyloid‐induced neuronal pyroptosis

The present study was designed to investigate the role of β‐amyloid (Aβ_1‐42) in inducing neuronal pyroptosis and its mechanism. Mice cortical neurons (MCNs) were used in this study, LPS + Nigericin was used to induce pyroptosis in MCNs (positive control group), and Aβ_1‐42 was used to interfere with MCNs. In addition, propidium iodide (PI) staining was used to examine cell permeability, lactate dehydrogenase (LDH) release assay was employed to detect cytotoxicity, immunofluorescence (IF) staining was used to investigate the expression level of the key protein GSDMD, Western blot was performed to detect the expression levels of key proteins, and enzyme‐linked immunosorbent assay (ELISA) was utilized to determine the expression levels of inflammatory factors in culture medium, including IL‐1β, IL‐18 and TNF‐α. Small interfering RNA (siRNA) was used to silence the mRNA expression of caspase‐1 and GSDMD, and Aβ_1‐42 was used to induce pyroptosis, followed by investigation of the role of caspase‐1‐mediated GSDMD cleavage in pyroptosis. In addition, necrosulfonamide (NSA), an inhibitor of GSDMD oligomerization, was used for pre‐treatment, and Aβ_1‐42 was subsequently used to observe the pyroptosis in MCNs. Finally, AAV9‐siRNA‐caspase‐1 was injected into the tail vein of APP/PS1 double transgenic mice (Alzheimer's disease mice) for caspase‐1 mRNA inhibition, followed by observation of behavioural changes in mice and measurement of the expression of inflammatory factors and pyroptosis‐related protein. As results, Aβ_1‐42 could induce pyroptosis in MCNs, increase cell permeability and enhance LDH release, which were similar to the LPS + Nigericin‐induced pyroptosis. Meanwhile, the expression levels of cellular GSDMD and p30‐GSDMD were up‐regulated, the levels of NLRP3 inflammasome and GSDMD‐cleaved protein caspase‐1 were up‐regulated, and the levels of inflammatory factors in the medium were also up‐regulated. siRNA intervention in caspase‐1 or GSDMD inhibited Aβ_1‐42‐induced pyroptosis, and NSA pre‐treatment also caused the similar inhibitory effects. The behavioural ability of Alzheimer's disease (AD) mice was relieved after the injection of AAV9‐siRNA‐caspase‐1, and the expression of pyroptosis‐related protein in the cortex and hippocampus was down‐regulated. In conclusion, Aβ_1‐42 could induce pyroptosis by GSDMD protein, and NLRP3‐caspase‐1 signalling was an important signal to mediate GSDMD cleavage, which plays an important role in Aβ_1‐42‐induced pyroptosis in neurons. Therefore, GSDMD is expected to be a novel therapeutic target for AD.     

Pre‐synaptic localization of the γ‐secretase‐inhibiting protein p24α2 in the mammalian brain

Dysregulated metabolism and consequent extracellular accumulation of amyloid‐β (Aβ) peptides in the brain underlie the pathogenesis of Alzheimer's disease. Extracellular Aβ in the brain parenchyma is mainly secreted from the pre‐synaptic terminals of neuronal cells in a synaptic activity‐dependent manner. The p24 family member p24α₂ reportedly attenuates Aβ generation by inhibiting γ‐secretase processing of amyloid precursor protein; however, the pattern of expression and localization of p24α₂ in the brain remains unknown. We performed immunohistochemical staining and subcellular fractionation for p24α₂ in the mouse brain. Immunostaining showed that p24α₂ is broadly distributed in the gray matter of the central nervous system and is predominantly localized to synapses. Subcellular fractionation revealed prominent localization of p24α₂ in the pre‐synaptic terminals. Immunoisolation of synaptic vesicles (SV) indicated that p24α₂ is condensed at active zone‐docked SV. During development, p24α₂ expression is highest in the post‐natal period and gradually decreases with age. We also confirmed that amyloid precursor protein and γ‐secretase components are localized at active zone‐docked SV. Our results suggest a novel functional role for p24α₂ in the regulation of synaptic transmission and synaptogenesis, and provide evidence for the participation of p24α₂ in the regulation of Aβ generation and secretion in the brain.

     

Comparison of neurotoxicity of different aggregated forms of Aβ40, Aβ42 and Aβ43 in cell cultures

The abnormal deposition of amyloid‐β (Aβ) peptides in the brain is the main neuropathological hallmark of Alzheimer's disease (AD). Amyloid deposits are formed by a heterogeneous mixture of Aβ peptides, among which the most studied are Aβ40 and Aβ42. Aβ40 is abundantly produced in the human brain, but the level of Aβ42 is remarkably increased in the brain of AD patients. Aside from Aβ40 and Aβ42, recent data have raised the possibility that Aβ43 peptides may be instrumental in AD pathogenesis. Besides its length, whether the Aβ aggregated form accounts for the neurotoxicity is also particularly controversial. Aβ fibrils are generally considered as key pathogenic substances in AD pathogenesis. Nevertheless, recent data implicated soluble Aβ oligomers as the main cause of synaptic dysfunction and memory loss in AD. To further address this uncertainty, we analyzed the neurotoxicity of different Aβ species and Aβ forms at the cellular level. The results showed that Aβ42 could form oligomers significantly faster than Aβ40 and Aβ43 and Aβ42 oligomers showed the greatest level of neurotoxicity. Regardless of the length of Aβ peptides, Aβ oligomers induced significantly higher cytotoxicity compared with the other two Aβ forms. Surprisingly, the neurotoxicity of fibrils in PC12 cells was only marginally but not significantly stronger than monomers, contrary to previous reports. Altogether, our findings demonstrate the high pathogenicity of Aβ42 among the three Aβ species and support the idea that Aβ42 oligomers contribute to the pathological events leading to neurodegeneration in AD. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

The Ca2+ influx induced by β-amyloid peptide 25–35 in cultured hippocampal neurons results from network excitation

Although a neurotoxic role has been postulated for the β-amyloid protein (βAP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (βAP25-35), in particular, has been reported to be toxic in cultured neurons. We report that βAP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), as measured by fura 2 fluorimetry. Furthermore, βAP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The βAP25-35–induced [Ca²⁺]_i elevations and electrical activity were enhanced by removal of extracellular Mg²⁺, and they could be blocked by tetrodotoxin, by non-N-methyl-D -aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca²⁺ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca²⁺]_i increases were evoked by βAP1-40. Responses to βAP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca²⁺]_i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of βAP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by βAP25-35 in voltage-clamp studies. We conclude that βAP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca²⁺ influx. This property may explain the toxicity of βAP25–35. © 1995 John Wiley & Sons, Inc.     

Spectroscopic detection of β ‐sheet structure in nascent Aβ oligomers

Deep‐UV resonance Raman (UVRR) spectroscopy and circular dichroism (CD) were employed to study the secondary structure of Aβ(1–42) in fresh samples with increasing fractions of oligomeric peptide. A feature with a minimum at ～217 nm appeared in CD spectra of samples containing oligomeric Aβ(1–42). UVRR spectra more closely resembled those of disordered proteins. The primary difference between UVRR spectra was the ratio of the 1236 cm^–1 to 1260 cm^–1 amide III peak intensities, which shifted in favor of the 1236 cm^–1 band as the fraction of oligomeric peptide increased. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)