Development of an anti-Aβ monoclonal antibody for in vivo imaging of amyloid angiopathy in Alzheimer's disease

We evaluated the efficacy of murine monoclonal antibodies (MAbs) targeted to the Aβ amyloid of Alzheimer's disease for development of procedures for the in vivo identification of amyloid angiopathy (AA). MAbs to Aβ were prepared and screened for effectiveness in visualizing AA and neuritic plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with technetium-99m (^99mTc) using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity toward amyloid-laden blood vessels and neuritic plaques. A highly specific murine MAb, 10H3, was identified and characterized that fulfills criteria necessary for the development of an in vivo diagnostic imaging agent. Toxicity studies in rats showed the MAb to be safe. Biodistribution studies in mice demonstrated desirable properties for use as an imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.     

Mutant ubiquitin decreases amyloid β plaque formation in a transgenic mouse model of Alzheimer's disease

The mutant ubiquitin UBB(+1) is a substrate as well as an inhibitor of the ubiquitin-proteasome system (UPS) and accumulates in the neuropathological hallmarks of Alzheimer's disease (AD). A role for the UPS has been suggested in the generation of amyloid β (Aβ) plaques in AD. To investigate the effect of UBB(+1) expression on amyloid pathology in vivo, we crossed UBB(+1) transgenic mice with a transgenic line expressing AD-associated mutant amyloid precursor protein (APPSwe) and mutant presenilin 1 (PS1dE9), resulting in APPPS1/UBB(+1) triple transgenic mice. In these mice, we determined the Aβ levels at 3, 6, 9 and 11months of age. Surprisingly, we found a significant decrease in Aβ deposition in amyloid plaques and levels of soluble Aβ(42) in APPPS1/UBB(+1) transgenic mice compared to APPPS1 mice at 6months of age, without alterations in UBB(+1) protein levels or proteasomal chymotrypsin activity. These lowering effects of UBB(+1) on Aβ deposition were transient, as this relative decrease in plaque load was not significant in APPPS1/UBB(+1) mice at 9 and 11months of age. We also show that APPPS1/UBB(+1) mice exhibit astrogliosis, indicating that they may not be improved functionally compared to APPPS1 mice despite the Aβ reduction. The molecular mechanism underlying this decrease in Aβ deposition in APPPS1/UBB(+1) mice is more complex than previously assumed because UBB(+1) is also ubiquitinated at K63 opening the possibility of additional effects of UBB(+1) (e.g. kinase activation).     

Time sequence of oxidative stress in the brain from transgenic mouse models of Alzheimer's disease related to the amyloid-β cascade

Alzheimer's disease (AD) is a multifactorial disorder characterized by the presence of amyloid plaques and neurofibrillary tangles (NFTs). Rare early-onset forms of AD are associated with autosomal dominant mutations in the amyloid precursor protein gene, presenilin 1 gene, or presenilin 2 gene. The late-onset form of the disease (LOAD) is the most common form. The causes of LOAD are not yet clarified, but several environmental and genetic risk factors have been identified. Numerous studies have highlighted a role for free radical-mediated injury to brain regions of this illness. In addition, studies from mild cognitive impairment patients suggest that oxidative stress is an early event in the pathogenesis of AD. The associations between these markers of free radical damage and the pathogenic cascades involved in AD are complex. Over the past 2 decades, a number of mouse models have been created to recapitulate the major neuropathological hallmarks of AD, namely amyloid plaques and NFTs. These mice recapitulate many, although not all, of the key features of AD. Some strains of transgenic mice develop amyloid plaques, some accumulate NFTs, and some do both. Here we review the evidence for increased free radical-mediated damage to the brain with particular attention to the stage of the disease in various transgenic models of AD related to the amyloid-β cascade.     

Peripheral delivery of a CNS targeted, metalo-protease reduces aβ toxicity in a mouse model of Alzheimer's disease

Alzheimer's disease (AD), an incurable, progressive neurodegenerative disorder, is the most common form of dementia. Therapeutic options have been elusive due to the inability to deliver proteins across the blood-brain barrier (BBB). In order to improve the therapeutic potential for AD, we utilized a promising new approach for delivery of proteins across the BBB. We generated a lentivirus vector expressing the amyloid β-degrading enzyme, neprilysin, fused to the ApoB transport domain and delivered this by intra-peritoneal injection to amyloid protein precursor (APP) transgenic model of AD. Treated mice had reduced levels of Aβ, reduced plaques and increased synaptic density in the CNS. Furthermore, mice treated with the neprilysin targeting the CNS had a reversal of memory deficits. Thus, the addition of the ApoB transport domain to the secreted neprilysin generated a non-invasive therapeutic approach that may be a potential treatment in patients with AD.     

Ganglioside metabolism in a transgenic mouse model of Alzheimer's disease: expression of Chol-1α antigens in the brain

The accumulation of Aβ (amyloid β-protein) is one of the major pathological hallmarks in AD (Alzheimer''s disease). Gangliosides, sialic acid-containing glycosphingolipids enriched in the nervous system and frequently used as biomarkers associated with the biochemical pathology of neurological disorders, have been suggested to be involved in the initial aggregation of Aβ. In the present study, we have examined ganglioside metabolism in the brain of a double-Tg (transgenic) mouse model of AD that co-expresses mouse/human chimaeric APP (amyloid precursor protein) with the Swedish mutation and human presenilin-1 with a deletion of exon 9. Although accumulation of Aβ was confirmed in the double-Tg mouse brains and sera, no statistically significant change was detected in the concentration and composition of major ganglio-N-tetraosyl-series gangliosides in the double-Tg brain. Most interestingly, Chol-1α antigens (cholinergic neuron-specific gangliosides), such as GT1aα and GQ1bα, which are minor species in the brain, were found to be increased in the double-Tg mouse brain. We interpret that the occurrence of these gangliosides may represent evidence for generation of cholinergic neurons in the AD brain, as a result of compensatory neurogenesis activated by the presence of Aβ.     

Depletion of CXCR2 inhibits γ-secretase activity and amyloid-β production in a murine model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to progressive cognitive decline. Recent studies from our group and others have suggested that certain G-protein coupled receptors (GPCRs) can influence the processing of the amyloid precursor protein (APP). Earlier, we demonstrated that stimulation of a chemokine receptor, CXCR2, results in enhanced γ-secretase activity and in increased amyloid-beta (Aβ) production. Taken together, results obtained from in vitro studies indicate that therapeutic targeting of CXCR2 might aid in lowering Aβ levels in the AD brain. To better understand the precise function and to predict the consequences of CXCR2 depletion in the AD brain, we have crossed CXCR2 knockout mice with mice expressing presenilin (PS1 M146L) and APPsw mutations (PSAPP). Our present study confirms that CXCR2 depletion results in reduction of Aβ with concurrent increases of γ-secretase substrates. At the mechanistic level, the effect of CXCR2 on γ-secretase was not found to occur via their direct interaction. Furthermore, we provide evidence that Aβ promotes endocytosis of CXCR2 via increasing levels of CXCR2 ligands. In conclusion, our current study confirms the regulatory role of CXCR2 in APP processing, and poses it as a potential target for developing novel therapeutics for intervention in AD.     

The role of metallobiology and amyloid-β peptides in Alzheimer's disease

The biggest risk factor for Alzheimer's disease is the process of ageing, but the mechanisms that lead to the manifestation of the disease remain to be elucidated. Why age triggers the disease is unclear but an emerging theme is the inability for a cell to efficiently maintain many key processes such as energy production, repair, and regenerative mechanisms. Metal ions are essential to the metabolic function of every cell. This review will explore the role and reported changes in metal ions in Alzheimer disease, particularly the brain, blood and cerebral spinal fluid, emphasizing how iron, copper and zinc may be involved through the interactions with amyloid precursor protein, the proteolytically cleaved peptide amyloid-beta (Aβ), and other related metalloproteins. Finally, we explore the monomeric makeup of possible Aβ dimers, what a dimeric Aβ species from Alzheimer's disease brain tissue is likely to be composed of, and discuss how metals may influence Aβ production and toxicity via a copper catalyzed dityrosine cross-link.     

Chronic intranasal treatment with an anti-Aβ(30-42) scFv antibody ameliorates amyloid pathology in a transgenic mouse model of Alzheimer's disease

Amyloid-beta peptide (Aβ)-directed active and passive immunization therapeutic strategies reduce brain levels of Aβ, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aβ-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aβ, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aβ-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aβ fibril formation and Aβ-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aβ from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aβ-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aβ-directed immunotherapy can reduce Aβ deposition in brain vessels in transgenic mice.     

Qingyangshen mitigates amyloid-β and Tau aggregate defects involving PPARα-TFEB activation in transgenic mice of Alzheimer's disease

BackgroundAlzheimer's disease (AD) is the most common neurodegenerative disease. Deposition of amyloid β plaques (Aβ) and neurofibrillary tangles (NFTs) is the key pathological hallmark of AD. Accumulating evidence suggest that impairment of autophagy-lysosomal pathway (ALP) plays key roles in AD pathology.PurposeThe present study aims to assess the neuroprotective effects of Qingyangshen (QYS), a Chinese herbal medicine, in AD cellular and animal models and to determine its underlying mechanisms involving ALP regulation.MethodsQYS extract was prepared and its chemical components were characterized by LC/MS. Then the pharmacokinetics and acute toxicity of QYS extract were evaluated. The neuroprotective effects of QYS extract were determined in 3XTg AD mice, by using a series of behavioral tests and biochemical assays, and the mechanisms were examined in vitro.ResultsOral administration of QYS extract improved learning and spatial memory, reduced carboxy-terminal fragments (CTFs), amyloid precursor protein (APP), Aβ and Tau aggregates, and inhibited microgliosis and astrocytosis in the brains of 3XTg mice. Mechanistically, QYS extract increased the expression of PPARα and TFEB, and promoted ALP both in vivo and in vitro.ConclusionQYS attenuates AD pathology, and improves cognitive function in 3XTg mice, which may be mediated by activation of PPARα-TFEB pathway and the subsequent ALP enhancement. Therefore, QYS may be a promising herbal material for further anti-AD drug discovery.     

High tryptophan diet reduces CA1 intraneuronal β-amyloid in the triple transgenic mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that impairs mnesic functions. The histopathology of the disease is manifested by the accumulation of intracellular β‐amyloid (Aβ) and subsequent formation of neuritic plaques as well as the presence neurofibrillary tangles in specific brain regions associated with learning and memory including the hippocampus. Here, we analysed the effect of chronic (1 month) food diets containing low (LTrP), normal (NTrP) and high tryptophan (HTrP), 0.04, 0.20 and 0.40 g/100 g, respectively, on CA1 serotonin transporter (SERT) fibre density, intraneuronal Aβ deposition and total number of serotonergic (5‐HT) neurons in an AD triple transgenic (3xTg‐AD) mouse model. Nontransgenic (non‐Tg) animals fed with HTrP displayed increased SERT fibre density in CA1 (35%) and in stratum lacunosum moleculare (S.Mol) (48%) compared to LTrP diet. Transgenic animals showed increased SERT fibre density in CA1 S.Mol compared to diet‐matched non‐Tg irrespective of dietary tryptophan content (104% for LTrP, 74% for NTrP and 35% for HTrP); no differences were observed in the total number of 5‐HT neurons neither in the dorsal nor in the median raphe nuclei. However, and more relevant to AD, HTrP diet reduced intraneuronal Aβ density (by a 17%) in transgenic animals compared to transgenic animals fed with NTrP diet. Our results show that increased dietary TrP intake reduces intraneuronal Aβ load in the 3xTg‐AD mouse model of AD, suggesting that enhanced TrP intake and in consequence a potential increase in 5‐HT neurotransmission may be effective in reducing plaque pathology in AD.     

Proteomic profiling of brain cortex tissues in a Tau transgenic mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) involves regionalized neuronal death, synaptic loss, and an accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. Although there have been numerous studies on tau proteins and AD in various stages of neurodegenerative disease pathology, the relationship between tau and AD is not yet fully understood. A transgenic mouse model expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE-htau23), which displays some of the typical Alzheimer-associated pathological features, was used to analyze the brain proteome associated with tau tangle deposition. Two-dimensional electrophoresis was performed to compare the cortex proteins of transgenic mice (6- and 12-month-old) with those of control mice. Differentially expressed spots in different stages of AD were identified with ESI-Q-TOF (electrospray ionization quadruple time-of-flight) mass spectrometry and liquid chromatography/tandem mass spectrometry. Among the identified proteins, glutathione S-transferase P 1 (GSTP1) and carbonic anhydrase II (CAII) were down-regulated with the progression of AD, and secerin-1 (SCRN1) and V-type proton ATPase subunit E 1 (ATP6VE1) were up-regulated only in the early stages, and down-regulated in the later stages of AD. The proteins, which were further confirmed by RT-PCR at the mRNA level and with western blotting at the protein level, are expected to be good candidates as drug targets for AD. The study of up- and down-regulation of proteins during the progression of AD helps to explain the mechanisms associated with neuronal degeneration in AD.     

Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer’s disease

Gene-targeting technology using mouse embryonic stem (ES) cells has become the “gold standard” for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer’s disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409–421, 2003) harboring 3 mutated genes (APP_swe, Tau_P301L, and PS1_M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F₁ mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.     

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.     

Versatile near-infrared fluorescent probe for in vivo detection of Aβ oligomers

Amyloid-β oligomers (AβOs) enrichment in brain is highly related to Alzheimer’s pathogenesis, but tracing them in the brain by imaging technique is still a great challenge due to their heterogeneity and metastability. Herein, a new near-infrared (NIR) fluorescent probe, namely, PTO-41, was designed and synthesized to specifically target AβOs. PTO-41 possesses excellent functional properties including optimal fluorescent properties (emission maxima at 680 nm upon interacting with AβOs), high affinity (K_d = 349 nM), low cell toxicity, desirable lipophilicity (log P = 2.24), and fast wash out from the brain (brain_{2 min}/brain_{60 min} = 5.0). Furthermore, PTO-41 exhibits a high sensitivity toward AβOs in vitro phantom imaging experiments. More importantly, PTO-41 shows great capacity to differentiate between 4-month-old APP/PS1 model mice from age-matched control mice using in vivo imaging. In summary, PTO-41 almost meets all the requirements as a versatile NIR fluorescent probe for the detection of AβOs both in vitro and in vivo.