Proteomic identification of specifically carbonylated brain proteins in APP(NLh)/APP(NLh) × PS-1(P264L)/PS-1(P264L) human double mutant knock-in mice model of Alzheimer disease as a function of age

Alzheimer disease (AD) is the most common type of dementia and is characterized pathologically by the presence of neurofibrillary tangles (NFTs), senile plaques (SPs), and loss of synapses. The main component of SP is amyloid-beta peptide (Aβ), a 39 to 43 amino acid peptide, generated by the proteolytic cleavage of amyloid precursor protein (APP) by the action of beta- and gamma-secretases. The presenilins (PS) are components of the γ-secretase, which contains the protease active center. Mutations in PS enhance the production of the Aβ42 peptide. To date, more than 160 mutations in PS1 have been identified. Many PS mutations increase the production of the β-secretase-mediated C-terminal (CT) 99 amino acid-long fragment (CT99), which is subsequently cleaved by γ-secretase to yield Aβ peptides. Aβ has been proposed to induce oxidative stress and neurotoxicity. Previous studies from our laboratory and others showed an age-dependent increase in oxidative stress markers, loss of lipid asymmetry, and Aβ production and amyloid deposition in the brain of APP/PS1 mice. In the present study, we used APP (NLh)/APP(NLh) × PS-1(P246L)/PS-1(P246L) human double mutant knock-in APP/PS-1 mice to identify specific targets of brain protein carbonylation in an age-dependent manner. We found a number of proteins that are oxidatively modified in APP/PS1 mice compared to age-matched controls. The relevance of the identified proteins to the progression and pathogenesis of AD is discussed.     

Presenilins, the Endoplasmic Reticulum, and Neuronal Apoptosis in Alzheimer's Disease

Abstract: Many cases of autosomal dominant inherited forms of early-onset Alzheimer's disease are caused by mutations in the genes encoding presenilin-1 (PS-1; chromosome 14) and presenilin-2 (PS-2; chromosome 1). PSs are expressed in neurons throughout the brain wherein they appear to be localized primarily to the endoplasmic reticulum (ER) of cell bodies and dendrities. PS-1 and PS-2 show high homology and are predicted to have eight transmembrane domains with the C terminus, N terminus, and a loop domain all on the cytosolic side of the membrane; an enzymatic cleavage of PSs occurs at a site near the loop domain. The normal function of PSs is unknown, but data suggest roles in membrane trafficking, amyloid precursor protein processing, and regulation of ER calcium homeostasis. Homology of PSs to the C. elegans gene sel-12 , which is involved in Notch signaling, and phenotypic similarities of PS-1 and Notch knockout mice suggest a developmental role for PSs in the nervous system. When expressed in cultured cells and transgenic mice, mutant PSs promote increased production of a long form of amyloid β-peptide (Aβ1-42) that may possess enhanced amyloidogenic and neurotoxic properties. PS mutations sensitize cultured neural cells to apoptosis induced by trophic factor withdrawal, metabolic insults, and amyloid β-peptide. The mechanism responsible for the proapoptotic action of mutant PSs may involve perturbed calcium release from ER stores and increased levels of oxidative stress. Recent studies of apoptosis in many different cell types suggest that ER calcium signaling can modulate apoptosis. The evolving picture of PS roles in neuronal plasticity and Alzheimer's disease is bringing to the forefront the ER, an organelle increasingly recognized as a key regulator of neuronal plasticity and survival.     

Abeta42 is essential for parenchymal and vascular amyloid deposition in mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.     

Enzymatic characteristics of I213T mutant presenilin-1/gamma-secretase in cell models and knock-in mouse brains: familial Alzheimer disease-linked mutation impairs gamma-site cleavage of amyloid precursor protein C-terminal fragment beta

Presenilin (PS)/gamma-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid beta (Abeta) peptides in which Abeta species of different lengths are generated through multiple cleavages at the gamma-, zeta-, and epsilon-sites. An increased Abeta42/Abeta40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Abeta42/Abeta40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of gamma-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS(-/-)) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Abeta generation, suggesting that mutation impairs the total catalytic rate of gamma-secretase. Analysis of each Abeta species revealed that the FAD mutation specifically reduced Abeta40 levels more drastically than Abeta42 levels, leading to an increased Abeta42/Abeta40 ratio. By contrast, the FAD mutation increased the generation of longer Abeta species such as Abeta43, Abeta45, and >Abeta46. These results were confirmed by analyses of gamma-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Abeta generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Abeta42/Abeta40 ratio observed in cases of FAD mutations is related to the differential inhibition of gamma-site cleavage reactions, in which the reaction producing Abeta40 is subject to more inhibition than that producing Abeta42. Our results also provide novel insight into how enhancing the generation of longer Abetas may contribute to Alzheimer disease onset.     

Increased Phosphorylation of Tau and Synaptic Protein Loss in the Aged Transgenic Mice Expressing Familiar Alzheimer’s Disease-Linked Presenilin 1 Mutation

Mutations in presenilin 1 (PS-1) are associated with most early-onset familiar Alzheimer’s disease (AD). Previous studies have demonstrated that PS-1 mutations enhance the production of beta-amyloid (Aβ). In this study, we further examined the in vivo effects of PS-1 mutation on tau and synapse protein markers. The data showed that the phosphorylation of tau at Ser396, Ser404, Thr231 and Tau-1 (Ser198/199/202) epitopes was significantly increased in hippocampus of the aged (twenty-one and a half-month-old) transgenic mice expressing PS-1 (L235P) compared to that of the age-matched wild-type littermates (WTs). Concurrently, a significant decrease in the phosphorylation of glycogen synthase kinase (GSK)-3β at Ser9 was observed. The above changes were not observed in the young transgenic mice (6–8 months old). No significant changes in the levels of cyclin-dependent kinase (CDK)-5, its co-activator p35, and phosphorylation of protein phosphatase (PP)-2A catalytic subunit at Tyrosine 307 (Y307), a crucial site regulating the activity of PP-2A, were observed both in the young and aged transgenic mice compared to that of WTs. Furthermore, we also observed that the levels of presynaptic synaptophysin were significantly decreased but postsynaptic density protein (PSD)-95 were not significantly altered in hippocampus of the aged transgenic mice. No significant changes of synaptophysin or PSD-95 were observed in the brains of the young transgenic mice. Our data indicate that the L235P PS-1 mutation can induce Alzheimer-like tau hyperphosphorylation and synaptic protein loss, as well as increased production of Aβ.     

In vivo oxidative stress in brain of Alzheimer disease transgenic mice: Requirement for methionine 35 in amyloid β-peptide of APP

Numerous studies have demonstrated oxidative damage in the central nervous system in subjects with Alzheimer disease and in animal models of this dementing disorder. In this study, we show that transgenic mice modeling Alzheimer disease—PDAPP mice with Swedish and Indiana mutations in the human amyloid precursor protein (APP)—develop oxidative damage in brain, including elevated levels of protein oxidation (indexed by protein carbonyls and 3-nitrotyrosine) and lipid peroxidation (indexed by protein-bound 4-hydroxy-2-nonenal). This oxidative damage requires the presence of a single methionine residue at position 35 of the amyloid β-peptide (Aβ), because all indices of oxidative damage in brain were completely prevented in genetically and age-matched PDAPP mice with an M631L mutation in APP. No significant differences in the levels of APP, Aβ(1–42), and Aβ(1–40) or in the ratio Aβ(1–42)/Aβ(1–40) were found, suggesting that the loss of oxidative stress in vivo in the brain of PDAPP(M631L) mice results solely from the mutation of the Met35 residue to Leu in the Aβ peptide. However, a marked reduction in Aβ-immunoreactive plaques was observed in the M631L mice, which instead displayed small punctate areas of nonplaque immunoreactivity and a microglial response. In contrast to the requirement for Met at residue 35 of the Aβ sequence (M631 of APP) for oxidative damage, indices of spatial learning and memory were not significantly improved by the M631L substitution. Furthermore, a genetically matched line with a different mutation—PDAPP(D664A)—showed the reverse: no reduction in oxidative damage but marked improvement in memory. This is the first in vivo study to demonstrate the requirement for Aβ residue Met35 for oxidative stress in the brain of a mammalian model of Alzheimer disease. However, in this specific transgenic mouse model of AD, oxidative stress is neither required nor sufficient for memory abnormalities.     

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.     

Wild-type presenilin 1 protects against Alzheimer disease mutation-induced amyloid pathology

Mutations in presenilin 1 (PS1) lead to dominant inheritance of early onset familial Alzheimer disease (FAD). These mutations are known to alter the gamma-secretase cleavage of the amyloid precursor protein, resulting in increased ratio of Abeta42/Abeta40 and accelerated amyloid plaque pathology in transgenic mouse models. To investigate the factors that drive the Abeta42/Abeta40 ratio and amyloid pathogenesis and to investigate the possible interactions between wild-type and FAD mutant PS1, which are co-expressed in transgenic animals, we expressed the PS1 M146V knock-in allele either on wild-type PS1 (PS1M146V/+) or PS1 null (PS1M146V/-) background and crossed these alleles with the Tg2576 APP transgenic mice. Introduction of the PS1 M146V mutation on Tg2576 background resulted in earlier onset of plaque pathology. Surprisingly, removing the wild-type PS1 in the presence of the PS1 M146V mutation (PS1M146V/-) greatly exacerbated the amyloid burden; and this was attributed to a reduction of gamma-secretase activity rather than an increase in Abeta42. Our findings establish a protective role of the wild-type PS1 against the FAD mutation-induced amyloid pathology through a partial loss-of-function mechanism.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.