The form(s) of amyloid-β peptide (Aβ) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Aβ accumulation is an ...issue of considerable controversy; even the existence of Aβ deposits within neurons has recently been challenged by Winton and co-workers. These authors purport that it is actually intraneuronal APP that is being detected by antibodies thought to be specific for Aβ. To further address this issue, an anti-Aβ antibody was developed (MOAB-2) that specifically detects Aβ, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal Aβ in transgenic mice with increased levels of human Aβ in 5xFAD and 3xTg mice.
MOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Aβ residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to Aβ residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK-APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice). Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for Aβ40 and Aβ42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunoreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal Aβ, distinct from Aβ associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues.
Both intraneuronal Aβ accumulation and extracellular Aβ deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular Aβ from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal Aβ with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical.
Abstract Existing data on the expression of H4 histamine receptor in the CNS are conflicting and inconclusive. In this report, we present the results of experiments that were conducted in order to ...elucidate H4 receptor expression and localization in the brain, spinal cord, and dorsal root ganglia (DRG). Here we show that transcripts of H4 receptor are present in all analyzed regions of the human CNS, including spinal cord, hippocampus, cortex, thalamus and amygdala, with the highest levels of H4 mRNA detected in the spinal cord. In rat, H4 mRNA was detected in cortex, cerebellum, brainstem, amygdala, thalamus and striatum. Very low levels of H4 mRNA were detected in hypothalamus, and no H4 signal was detected in the rat hippocampus. Fairly low levels of H4 mRNA were detected in examined peripheral tissues including spleen and liver. Interestingly, strong expression of H4 mRNA was detected in the rat DRG and spinal cord. Immunohistochemical analysis revealed expression of H4 receptors on neurons in the rat lumbar DRG and in the lumbar spinal cord. Our observations provide evidence of the H4 presence in both human and rodent CNS and offer some insight into possible role of H4 in itch and pain.
Genetic evidence predicts a causative role for amyloid-beta (A beta) in Alzheimer's disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of A beta are the active ...species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of A beta-(1-42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and approximately 40-fold more than unaggregated peptide, with oligomeric A beta-(1-42)-induced inhibition significant at 10 nm. Under A beta-(1-42) oligomer- and fibril-forming conditions, A beta-(1-40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of A beta-(1-42). We applied the aggregation protocols developed for wild type A beta-(1-42) to A beta-(1-42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type A beta-(1-42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type A beta-(1-42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar A beta-(1-42) are useful in distinguishing the structural and functional differences between A beta-(1-42) and A beta-(1-40) and genetic mutations of A beta-(1-42).
Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease in Caucasians caused by pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (
). ...Significant small molecule therapeutic advances over the past two decades have been made to target the defective CFTR protein and enhance its function. To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in CF, two biomolecular activities are required, namely, correctors to increase the amount of properly folded F508delCFTR levels at the cell surface and potentiators to allow the effective opening,
, function of the F508delCFTR channel. Combined, these activities enhance chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. To enhance clinical benefits to CF patients, a complementary triple combination therapy consisting of two corrector molecules, type 1 (C1) and type 2, with additive mechanisms along with a potentiator are being investigated in the clinic for maximum restoration of mutated CFTR function. We report the identification and in vitro biologic characterization of ABBV-2222/GLPG2222 (
),-a novel, potent, and orally bioavailable C1 corrector developed by AbbVie-Galapagos and currently in clinical trials-which exhibits substantial improvements over the existing C1 correctors. This includes improvements in potency and drug-drug interaction (DDI) compared with 3-(6-(1-(2,2-difluorobenzod1,3dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (VX-809, Lumacaftor) and improvements in potency and efficacy compared with 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-1-(2R)-2,3-dihydroxypropyl-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-ylcyclopropane-1-carboxamide (VX-661, Tezacaftor). ABBV-2222/GLPG2222 exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR with an EC
value <10 nM. SIGNIFICANCE STATEMENT: To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in cystic fibrosis, AbbVie-Galapagos has developed ABBV-2222/GLPG2222, a novel, potent, and orally bioavailable C1 corrector of this protein. ABBV-2222/GLPG2222, which is currently in clinical trials, exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR and substantial improvements over the existing C1 correctors.
Cystic fibrosis (CF) is a genetic disorder that affects multiple tissues and organs. CF is caused by mutations in the CFTR gene, resulting in insufficient or impaired cystic fibrosis transmembrane ...conductance regulator (CFTR) protein. The deletion of phenylalanine at position 508 of the protein (F508del-CFTR) is the most common mutation observed in CF patients. The most effective treatments of these patients employ two CFTR modulator classes, correctors and potentiators. CFTR correctors increase protein levels at the cell surface; CFTR potentiators enable the functional opening of CFTR channels at the cell surface. Triple-combination therapies utilize two distinct corrector molecules (C1 and C2) to further improve the overall efficacy. We identified the need to develop a C2 corrector series that had the potential to be used in conjunction with our existing C1 corrector series and provide robust clinical efficacy for CF patients. The identification of a pyrrolidine series of CFTR C2 correctors and the structure–activity relationship of this series is described. This work resulted in the discovery and selection of (2S,3R,4S,5S)-3-(tert-butyl)-4-((2-methoxy-5-(trifluoromethyl)pyridin-3-yl)methoxy)-1-((S)-tetrahydro-2H-pyran-2-carbonyl)-5-(o-tolyl)pyrrolidine-2-carboxylic acid (ABBV/GLPG-3221), which was advanced to clinical trials.
Autosomal dominant mutations that increase amyloid- beta (1-42) (A beta 42) cause familial Alzheimer's disease (AD), and the most common genetic risk factor for AD is the presence of the var epsilon4 ...allele of apolipoprotein E (apoE). Previously, we characterized stable preparations of A beta 42 oligomers and fibrils and reported that oligomers induced a 10-fold greater increase in neurotoxicity than fibrils in Neuro-2A cells. To determine the effects of apoE genotype on A beta 42 oligomer- and fibril-induced neurotoxicity in vitro, we co-cultured wild type (WT) neurons with glia from WT, apoE- knockout (apoE-KO), and human apoE2-, E3-, and E4-targeted replacement (TR) mice. Dose-dependent neurotoxicity was induced by oligomeric A beta 42 with a ranking order of apoE4-TR > KO = apoE2-TR = apoE3-TR > WT. Neurotoxicity induced by staurosporine or glutamate were not affected by apoE genotype, indicating specificity for oligomeric A beta 42-induced neurotoxicity. These in vitro data demonstrate a gain of negative function for apoE4, synergistic with oligomeric A beta 42, in mediating neurotoxicity.
Abstract Autosomal dominant mutations that increase amyloid-β(1–42) (Aβ42) cause familial Alzheimer's disease (AD), and the most common genetic risk factor for AD is the presence of the ε4 allele of ...apolipoprotein E (apoE). Previously, we characterized stable preparations of Aβ42 oligomers and fibrils and reported that oligomers induced a 10-fold greater increase in neurotoxicity than fibrils in Neuro-2A cells. To determine the effects of apoE genotype on Aβ42 oligomer- and fibril-induced neurotoxicity in vitro , we co-cultured wild type (WT) neurons with glia from WT, apoE-knockout (apoE-KO), and human apoE2-, E3-, and E4-targeted replacement (TR) mice. Dose-dependent neurotoxicity was induced by oligomeric Aβ42 with a ranking order of apoE4-TR > KO = apoE2-TR = apoE3-TR > WT. Neurotoxicity induced by staurosporine or glutamate were not affected by apoE genotype, indicating specificity for oligomeric Aβ42-induced neurotoxicity. These in vitro data demonstrate a gain of negative function for apoE4, synergistic with oligomeric Aβ42, in mediating neurotoxicity.
Activated glia, as a result of chronic inflammation, are associated with amyloid-β peptide (Aβ) deposits in the brain of Alzheimer's disease (AD) patients. In vitro, glia are activated by Aβ inducing ...secretion of pro-inflammatory molecules. Recent studies have focused on soluble oligomers (or protofibrils) of Aβ as the toxic species in AD. In the present study, using rat astrocyte cultures, oligomeric Aβ induced initial high levels of IL-1β decreasing over time and, in contrast, fibrillar Aβ increased IL-1β levels over time. In addition, oligomeric Aβ, but not fibrillar Aβ, induced high levels of iNOS, NO, and TNF-α. Our results suggest that oligomers induced a profound, early inflammatory response, whereas fibrillar Aβ showed less increase of pro-inflammatory molecules, consistent with a more chronic form of inflammation.
cis-4-(Piperazin-1-yl)-5,6,7a,8,9,10,11,11a-octahydrobenzofuro2,3-hquinazolin-2-amine, 4 (A-987306) is a new histamine H4 antagonist. The compound is potent in H4 receptor binding assays (rat H4, K i ...= 3.4 nM, human H4 K i = 5.8 nM) and demonstrated potent functional antagonism in vitro at human, rat, and mouse H4 receptors in cell-based FLIPR assays. Compound 4 also demonstrated H4 antagonism in vivo in mice, blocking H4-agonist induced scratch responses, and showed anti-inflammatory activity in mice in a peritonitis model. Most interesting was the high potency and efficacy of this compound in blocking pain responses, where it showed an ED50 of 42 μmol/kg (ip) in a rat post-carrageenan thermal hyperalgesia model of inflammatory pain.
Abnormalities in the processing of amyloid precursor protein to amyloid-beta (Abeta) are causal factors, and the presence of the epsilon4 allele of apolipoprotein E (apoE) is the primary risk factor ...for Alzheimer's disease (AD). Based, at least in part, on these genetics, the potential structural and functional interactions between these two proteins are the focus of our research. To understand the nature of the physical interactions between apoE and Abeta, we initially utilized gel-shift assays to demonstrate that native apoE2 and E3 (associated with lipid particles) form an SDS-stable complex with Abeta that is more abundant than the apoE4:Abeta complex. We further demonstrated that exogenous apoE3 but not E4 prevents Abeta-induced neurotoxicity by a process that requires apoE receptors. In addition, both exogenous apoE3 and E4 prevent Abeta-induced, glial-mediated inflammation, also via a process that requires apoE receptors. These functional effects all occur at a molar ratio of apoE to Abeta of 1:30. Because the biological activities for both apoE and Abeta are profoundly influenced by their isoform and conformation, respectively, we further investigated the idea that apoE3 and E4 differentially interact with particular aggregation species of Abeta1-42. Our overall hypothesis is that apoE has two general functions in relation to Abeta. First, apoE interacts with oligomeric Abeta via an apoE receptor-mediated process to inhibit neurotoxicity and neuroinflammation (apoE3 > apoE4) a process possibly related to binding and clearance of apoE3:oligomer complexes. Second, apoE facilitates the deposition of Abeta as amyloid (apoE4 > apoE3). We will continue to investigate the effect of apoE isoform and Abeta conformation on the structural and functional interactions between these two proteins in relation to the pathogenesis of AD.
PDF
