Pyroglutamate-Aβ: Role in the natural history of Alzheimer's disease

The accumulation of amyloid-beta (Aβ) peptides is believed to be a central contributor to the neurodegeneration typically seen in Alzheimer's disease (AD) brain. Aβ extracted from AD brains invariably possesses extensive truncations, yielding peptides of differing N- and C-terminal composition. Whilst Aβ is often abundant in the brains of cognitively normal elderly people, the brains of AD patients are highly enriched for N-terminally truncated Aβ bearing the pyroglutamate modification. Pyroglutamate-Aβ (pE-Aβ) has a higher propensity for oligomerisation and aggregation than full-length Aβ, potentially seeding the accumulation of neurotoxic Aβ oligomers and amyloid deposits. In addition, pE-Aβ has increased resistance to clearance by peptidases, causing these peptides to persist in biological fluids and tissues. The extensive deposition of pE-Aβ in human AD brain is under-represented in many transgenic mouse models of AD, reflecting major differences in the production and processing of Aβ peptides in these models compared to the human disease state.     

APP mouse models for Alzheimer's disease preclinical studies

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimer's disease (AD) research community has historically used first‐generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD. Second‐generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aβ accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD. In this review, we evaluate different APP mouse models of AD, and review recent studies using the second‐generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study.     

Glutathione Peroxidase 4 is associated with Neuromelanin in Substantia Nigra and Dystrophic Axons in Putamen of Parkinson's brain

Fibrillar amyloid β (fAβ) peptide is the major component of Aβ plaques in the brains of Alzheimer's disease (AD) patients. Inflammatory mediators have previously been proposed to be drivers of Aβ pathology in AD patients by increasing amyloidogenic processing of APP and promoting Aβ accumulation, but recent data have shown that expression of various inflammatory cytokines attenuates Aβ pathology in mouse models. In an effort to further study the role of different inflammatory cytokines on Aβ pathology in vivo, we explored the effect of murine Tumor Necrosis Factor α (mTNFα) in regulating Aβ accumulation. Recombinant adeno-associated virus serotype 1 (AAV2/1) mediated expression of mTNFα in the hippocampus of 4 month old APP transgenic TgCRND8 mice resulted in significant reduction in hippocampal Aβ burden. No changes in APP levels or APP processing were observed in either mTNFα expressing APP transgenic mice or in non-transgenic littermates. Analysis of Aβ plaque burden in mTNFα expressing mice showed that even after substantial reduction compared to EGFP expressing age-matched controls, the Aβ plaque burden levels of the former do not decrease to the levels of 4 month old unmanipulated mice. Taken together, our data suggests that proinflammatory cytokine expression induced robust glial activation can attenuate plaque deposition. Whether such an enhanced microglial response actually clears preexisting deposits without causing bystander neurotoxicity remains an open question.     

Interactions between amyloid-β and hemoglobin: implications for amyloid plaque formation in Alzheimer's disease

Accumulation of amyloid-β (Aβ) peptides in the brain is one of the central pathogenic events in Alzheimer's disease (AD). However, why and how Aβ aggregates within the brain of AD patients remains elusive. Previously, we demonstrated hemoglobin (Hb) binds to Aβ and co-localizes with the plaque and vascular amyloid deposits in post-mortem AD brains. In this study, we further characterize the interactions between Hb and Aβ in vitro and in vivo and report the following observations: 1) the binding of Hb to Aβ required iron-containing heme; 2) other heme-containing proteins, such as myoglobin and cytochrome C, also bound to Aβ; 3) hemin-induced cytotoxicity was reduced in neuroblastoma cells by low levels of Aβ; 4) Hb was detected in neurons and glial cells of post-mortem AD brains and was up-regulated in aging and APP/PS1 transgenic mice; 5) microinjection of human Hb into the dorsal hippocampi of the APP/PS1 transgenic mice induced the formation of an envelope-like structure composed of Aβ surrounding the Hb droplets. Our results reveal an enhanced endogenous expression of Hb in aging brain cells, probably serving as a compensatory mechanism against hypoxia. In addition, Aβ binds to Hb and other hemoproteins via the iron-containing heme moiety, thereby reducing Hb/heme/iron-induced cytotoxicity. As some of the brain Hb could be derived from the peripheral circulation due to a compromised blood-brain barrier frequently observed in aged and AD brains, our work also suggests the genesis of some plaques may be a consequence of sustained amyloid accretion at sites of vascular injury.     

Beta-amyloid peptide variants in brains and cerebrospinal fluid from amyloid precursor protein (APP) transgenic mice: comparison with human Alzheimer amyloid

In this study, we report a detailed analysis of the different variants of amyloid-β (Aβ) peptides in the brains and the cerebrospinal fluid from APP23 transgenic mice, expressing amyloid precursor protein with the Swedish familial Alzheimer disease mutation, at different ages. Using one- and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry, we identified the Aβ peptides Aβ(1-40), -(1-42), -(1-39), -(1-38), -(1-37), -(2-40), and -(3-40) as well as minor amounts of pyroglutamate-modified Aβ (Aβ(N3pE)) and endogenous murine Aβ in brains from 24-month-old mice. Chemical modifications of the N-terminal amino group of Aβ were identified that had clearly been introduced during standard experimental procedures. To address this issue, we additionally applied amyloid extraction in ultrapure water. Clear differences between APP23 mice and Alzheimer disease (AD) brain samples were observed in terms of the relative abundance of specific variants of Aβ peptides, such as Aβ(N3pE), Aβ(1-42), and N-terminally truncated Aβ(2/3-42). These differences to human AD amyloid were also noticed in a related mouse line transgenic for human wild type amyloid precursor protein. Taken together, our findings suggest different underlying molecular mechanisms driving the amyloid deposition in transgenic mice and AD patients.     

Seeded strain‐like transmission of β‐amyloid morphotypes in APP transgenic mice

The polymorphic β‐amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral β‐amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop β‐amyloid depositions that differ in morphology, binding of amyloid conformation‐sensitive dyes, and Aβ40/Aβ42 peptide ratio. To determine the nature of such β‐amyloid morphotypes, β‐amyloid‐containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced β‐amyloid deposition with the morphological, conformational, and Aβ40/Aβ42 ratio characteristics of β‐amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced β‐amyloid deposits with the characteristics of β‐amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced β‐amyloid deposits, although less prominent, and the induced deposits were similar to the β‐amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated Aβ in APP transgenic mice can be maintained by seeded conversion.     

APP transgenic mice and their application to drug discovery

The development of transgenic mice expressing mutated forms of the human amyloid precursor protein (APP) and presenilin-1 (PS1), proteins associated with familial forms of Alzheimer's disease (AD), has provided a backbone for translational studies of potential novel drug therapies. Such mice model some aspects of AD pathology in that they develop senile plaque-like deposits of the amyloid beta-protein (Aβ) together with inflammatory pathology and some degree of neurodegeneration. Aβ deposition is considered to be a potentially pathogenic feature of AD and drug discovery programmes utilising such mice and associated with drugs now reaching the clinic have been largely directed towards decreasing the deposition. This goal has been achieved in the mouse models, although the agents developed have not, to date, shown evidence of efficacy in AD sufferers and, in some cases, have worsened the clinical state. Nevertheless, reducing the pathological features of the disease continues to be the objective of pharmacological intervention and ongoing programmes continue to use transgenic mice expressing mutated APP and PS1 transgenes in attempts to overcome issues and difficulties arising from the initial clinical trials and to explore new approaches to AD treatment.     

Tannic acid is a natural β-secretase inhibitor that prevents cognitive impairment and mitigates Alzheimer-like pathology in transgenic mice

Amyloid precursor protein (APP) proteolysis is essential for production of amyloid-β (Aβ) peptides that form β-amyloid plaques in brains of Alzheimer disease (AD) patients. Recent focus has been directed toward a group of naturally occurring anti-amyloidogenic polyphenols known as flavonoids. We orally administered the flavonoid tannic acid (TA) to the transgenic PSAPP mouse model of cerebral amyloidosis (bearing mutant human APP and presenilin-1 transgenes) and evaluated cognitive function and AD-like pathology. Consumption of TA for 6 months prevented transgene-associated behavioral impairment including hyperactivity, decreased object recognition, and defective spatial reference memory, but did not alter nontransgenic mouse behavior. Accordingly, brain parenchymal and cerebral vascular β-amyloid deposits and abundance of various Aβ species including oligomers were mitigated in TA-treated PSAPP mice. These effects occurred with decreased cleavage of the β-carboxyl-terminal APP fragment, lowered soluble APP-β production, reduced β-site APP cleaving enzyme 1 protein stability and activity, and attenuated neuroinflammation. As in vitro validation, we treated well characterized mutant human APP-overexpressing murine neuron-like cells with TA and found significantly reduced Aβ production associated with less amyloidogenic APP proteolysis. Taken together, these results raise the possibility that dietary supplementation with TA may be prophylactic for AD by inhibiting β-secretase activity and neuroinflammation and thereby mitigating AD pathology.     

Transsynaptic progression of amyloid-β-induced neuronal dysfunction within the entorhinal-hippocampal network

The entorhinal cortex (EC) is one of the earliest affected, most vulnerable brain regions in Alzheimer's disease (AD), which is associated with amyloid-β (Aβ) accumulation in many brain areas. Selective overexpression of mutant amyloid precursor protein (APP) predominantly in layer II/III neurons of the EC caused cognitive and behavioral abnormalities characteristic of mouse models with widespread neuronal APP overexpression, including hyperactivity, disinhibition, and spatial learning and memory deficits. APP/Aβ overexpression in the EC elicited abnormalities in synaptic functions and activity-related molecules in the dentate gyrus and CA1 and epileptiform activity in parietal cortex. Soluble Aβ was observed in the dentate gyrus, and Aβ deposits in the hippocampus were localized to perforant pathway terminal fields. Thus, APP/Aβ expression in EC neurons causes transsynaptic deficits that could initiate the cortical-hippocampal network dysfunction in mouse models and human patients with AD.     

Brains of rhesus monkeys display Aβ deposits and glial pathology while lacking Aβ dimers and other Alzheimer's pathologies

Cerebral amyloid beta (Aβ) deposits are the main early pathology of Alzheimer's disease (AD). However, abundant Aβ deposits also occur spontaneously in the brains of many healthy people who are free of AD with advancing aging. A crucial unanswered question in AD prevention is why AD does not develop in some elderly people, despite the presence of Aβ deposits. The answer may lie in the composition of Aβ oligomer isoforms in the Aβ deposits of healthy brains, which are different from AD brains. However, which Aβ oligomer triggers the transformation from aging to AD pathogenesis is still under debate. Some researchers insist that the Aβ 12‐mer causes AD pathology, while others suggest that the Aβ dimer is the crucial molecule in AD pathology. Aged rhesus monkeys spontaneously develop Aβ deposits in the brain with striking similarities to those of aged humans. Thus, rhesus monkeys are an ideal natural model to study the composition of Aβ oligomer isoforms and their downstream effects on AD pathology. In this study, we found that Aβ deposits in aged monkey brains included 3‐mer, 5‐mer, 9‐mer, 10‐mer, and 12‐mer oligomers, but not 2‐mer oligomers. The Aβ deposits, which were devoid of Aβ dimers, induced glial pathology (microgliosis, abnormal microglia morphology, and astrocytosis), but not the subsequent downstream pathologies of AD, including Tau pathology, neurodegeneration, and synapse loss. Our results indicate that the Aβ dimer plays an important role in AD pathogenesis. Thus, targeting the Aβ dimer is a promising strategy for preventing AD.     

TUDCA, a Bile Acid, Attenuates Amyloid Precursor Protein Processing and Amyloid-β Deposition in APP/PS1 Mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid-β (Aβ) peptide in the hippocampus and frontal cortex of the brain, leading to progressive cognitive decline. The endogenous bile acid tauroursodeoxycholic acid (TUDCA) is a strong neuroprotective agent in several experimental models of disease, including neuronal exposure to Aβ. Nevertheless, the therapeutic role of TUDCA in AD pathology has not yet been ascertained. Here we report that feeding APP/PS1 double-transgenic mice with diet containing 0.4 % TUDCA for 6 months reduced accumulation of Aβ deposits in the brain, markedly ameliorating memory deficits. This was accompanied by reduced glial activation and neuronal integrity loss in TUDCA-fed APP/PS1 mice compared to untreated APP/PS1 mice. Furthermore, TUDCA regulated lipid-metabolism mediators involved in Aβ production and accumulation in the brains of transgenic mice. Overall amyloidogenic APP processing was reduced with TUDCA treatment, in association with, but not limited to, modulation of γ-secretase activity. Consequently, a significant decrease in Aβ(1-40) and Aβ(1-42) levels was observed in both hippocampus and frontal cortex of TUDCA-treated APP/PS1 mice, suggesting that chronic feeding of TUDCA interferes with Aβ production, possibly through the regulation of lipid-metabolism mediators associated with APP processing. These results highlight TUDCA as a potential therapeutic strategy for the prevention and treatment of AD.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Regulatory region of human amyloid precursor protein (APP) gene promotes neuron-specific gene expression in the CNS of transgenic mice.

The accumulation of beta-amyloid protein in specific brain regions is a central pathological feature of Alzheimer's disease (AD). The 4 kd beta-amyloid protein derives from a larger amyloid precursor protein (APP) by as yet unknown mechanisms. In the absence of a laboratory animal model of AD, transgenic mice expressing various APP gene products may provide new insights into the relationship between APP and beta-amyloid formation and the pathogenesis of AD. beta-amyloid accumulation in AD brain may result from interactions between APP and other molecules. Such interactions are likely to be developmentally regulated and tissue-specific. A transgenic mouse model of AD, therefore, would aim for APP transgene expression that mimics the endogenous APP gene. As an initial step in developing an animal model, we have identified a 4.5 kb DNA fragment from the 5' end of the human APP gene, which mediates neuron-specific gene expression in the CNS of transgenic mice, using E. coli lacZ as a reporter gene. Detectable levels of transgene expression are found in most neurons but not in glial and vascular endothelial cells. The expression pattern of this reporter gene closely resembles the distribution of endogenous APP mRNA in both the human and mouse CNS.     

APP transgenic mice Tg2576 accumulate Abeta peptides that are distinct from the chemically modified and insoluble peptides deposited in Alzheimer's disease senile plaques.

The amyloid (Abeta) peptides generated in Hsiao's APP Tg2576 transgenic (Tg) mice are physically and chemically distinct from those characteristic of Alzheimer's disease (AD). Transgenic mouse Abeta peptides were purified using sequential size-exclusion and reverse-phase chromatographic systems and subjected to amino acid sequencing and mass spectrometry analyses. The mouse Abeta peptides lacked the extensive N-terminal degradations, posttranslational modifications, and cross-linkages abundant in the stable Abeta peptide deposits observed in AD. Truncated Abeta molecules appear to be generated in vivo by hydrolysis at multiple sites rather than by post-mortem C-terminal degradation. In contrast to AD amyloid cores, the Tg mice peptides were soluble in Tris-SDS-EDTA solutions, revealing both monomeric and SDS-stable oligomeric species of Abeta. In contrast to our report on Novartis Pharma APP23 Tg mice [Kuo et al. (2001) J. Biol. Chem. 276, 12991], which maintain high levels of soluble Abeta early on with later development of extensive vascular amyloid, Tg2576 mice exhibited an age-related elevation of soluble Abeta with relatively limited vascular amyloid deposition. The transgenic mouse levels of carboxy-terminal (CT) APP fragments were nearly 10-fold greater than those of human brains, and this condition may contribute to the unique pathology observed in these animals. Immunization of transgenic mice may act to prevent the pathological effects of betaAPP overproduction by binding CT molecules or halting their processing to toxic forms, in addition to having any effects on Abeta itself. Thus, differences in disease evolution and biochemistry must be considered when using transgenic animals to evaluate drugs or therapeutic interventions intended to reduce the Abeta burden in Alzheimer's disease.     

Is the mitochondrial outermembrane protein VDAC1 therapeutic target for Alzheimer's disease?

Mitochondrial dysfunction and synaptic damage have been described as early events in Alzheimer's disease (AD) pathogenesis. Recent research using AD postmortem brains, and AD mouse and cell models revealed that amyloid beta (Aβ) and tau hyperphosphorylation are involved in mitochondrial dysfunction and synaptic damage in AD. Further, recent research also revealed that the protein levels of mitochondrial outer membrane protein, voltage-dependent anion channel 1 (VDAC1), are elevated in the affected regions of AD postmortem brains and cortical tissues from APP transgenic mice. In addition, emerging research using AD postmortem brains and AD mouse models revealed that VDAC1 is linked to Aβ and phosphorylated tau, blocks the mitochondrial permeability transition (MPT) pores, disrupts the transport of mitochondrial proteins and metabolites, impairs gating of VDAC, and causes defects in oxidative phosphorylation, leading to mitochondrial dysfunction in AD neurons. The purpose of this article is to review research that has investigated the relationship between VDAC1 and the regulation of MPT pores in AD progression.     

Three-dimensional analysis of abnormal ultrastructural alteration in mitochondria of hippocampus of APP/PSEN1 transgenic mouse

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. The deterioration of subcellular organelles, including the mitochondria, is another major ultrastructural characteristic of AD pathogenesis, in addition to amyloid plaque deposition. However, the three-dimensional (3-D) study of mitochondrial structural alteration in AD remains poorly understood. Therefore, ultrastructural analysis, 3-D electron tomography, and immunogold electron microscopy were performed in the present study to clarify the abnormal structural alterations in mitochondria caused by the progression of AD in APP/PSEN1 transgenic mice, expressing human amyloid precursor protein, as a model for AD. Amyloid β (Aβ) plaques accumulated and dystrophic neurites (DN) developed in the hippocampus of transgenic AD mouse brains. We also identified the loss of peroxiredoxin 3, an endogenous cytoprotective antioxidant enzyme and the accumulation of Aβ in the hippocampal mitochondria of transgenic mice, which differs from those in age-matched wild-type mice. The mitochondria in Aβ plaque-detected regions were severely disrupted, and the patterns of ultrastructural abnormalities were classified into three groups: disappearance of cristae, swelling of cristae, and bulging of the outer membrane. These results demonstrated that morpho-functional alterations of mitochondria and AD progression are closely associated and may be beneficial in investigating the function of mitochondria in AD pathogenesis.     

Exogenous seeding of cerebral β-amyloid deposition in βAPP-transgenic rats

Deposition of the amyloid-β (Aβ) peptide in senile plaques and cerebral Aβ angiopathy (CAA) can be stimulated in Aβ-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aβ seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aβ itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aβ seeding have employed animal models that, as they age, eventually will generate Aβ lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aβ within the course of its median lifespan (30?months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aβ. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aβ-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aβ.     

The emerging role of cystatins in Alzheimer's disease

Recently opposing effects of cysteine protease inhibitors, the human cystatins, on neurodegeneration have been reported. Human cystatin C is a risk factor for late‐onset Alzheimer's disease (AD), whereas human stefin B (cystatin B) has no direct involvement in AD. Conflicting data show that their target protease, cathepsin B, might be anti‐amyloidogenic, helping in amyloid‐beta (Aβ) clearance or, instead, might be involved in Aβ production. Some reports claim that cystatin C binds soluble Aβ, making transgenic animals healthier, others, in contrast, that deleting cystatins genes may contribute to amyloid pathology in animal models of AD.