PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-6 (6)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Treatment Strategies Targeting Amyloid β-Protein 
With the advent of the key discovery in the mid-1980s that the amyloid β-protein (Aβ) is the core constituent of the amyloid plaque pathology found in Alzheimer disease (AD), an intensive effort has been underway to attempt to mitigate its role in the hope of treating the disease. This effort fully matured when it was clarified that the Aβ is a normal product of cleavage of the amyloid precursor protein, and well-defined proteases for this process were identified. Further therapeutic options have been developed around the concept of anti-Aβ aggregation inhibitors and the surprising finding that immunization with Aβ itself leads to reduction of pathology in animal models of the disease. Here we review the progress in this field toward the goal of targeting Aβ for treatment and prevention of AD and identify some of the major challenges for the future of this area of medicine.
There are three main ways to target amyloid β-protein for treating Alzheimer disease: inhibiting its production, preventing its aggregation (or promoting its disaggregation), and promoting its clearance.
doi:10.1101/cshperspect.a006387
PMCID: PMC3426815  PMID: 22951439
2.  Crystal structure reveals conservation of amyloid-β conformation recognized by 3D6 following humanization to bapineuzumab 
Introduction
Immunotherapy targeting amyloid-β peptide is under active clinical investigation for treatment of Alzheimer’s disease (AD). Among the hypotheses being investigated for impact on clinical outcome are the preferred epitope or conformation of amyloid-β to target for treatment, and the mechanism of action underlying immunotherapy. Bapineuzumab (humanized 3D6), a neo-epitope specific antibody recognizing amyloid-β1-5 with strong preference for an exposed Asp residue at the N-terminus of the peptide, has undergone advanced clinical testing for treatment of AD.
Methods
To gain further insight into the epitope conformation, we interrogated structural details of amino-terminal epitopes in amyloid-β using x-ray crystallography of 3D6Fab:amyloid-β complexes. Humanization of 3D6 was carried out using standard procedures integrating recombinant methods, sequence informatics, and homology modeling predictions to identify important mouse framework residues for retention in the finished humanized product.
Results
Here we report the crystal structure of a recombinant Fab fragment of 3D6 in complex with amyloid-β1-7 solved at 2.0 Å resolution. The N-terminus of amyloid-β is bound to 3D6 as a 310 helix. The amino-terminal Asp residue is buried deepest in the antibody binding pocket, with the Cβ atom of residue 6 visible at the entrance to the binding pocket near the surface of the antibody. We further evaluate homology model based predictions used to guide humanization of 3D6 to bapineuzumab, with actual structure of the Fab. The structure of the Fab:amyloid-β complex validates design of the humanized antibody, and confirms the amyloid-β epitope recognized by 3D6 as previously mapped by ELISA.
Conclusions
The conformation of amyloid-β antigen recognized by 3D6 is novel and distinct from other antibodies recognizing N-terminal epitopes. Our result provides the first report demonstrating structural conservation of antigen contact residues, and conformation of antigen recognized, between the parent murine antibody and its humanized version.
doi:10.1186/alzrt261
PMCID: PMC4095729  PMID: 25024748
3.  Accelerating drug discovery for Alzheimer's disease: best practices for preclinical animal studies 
Animal models have contributed significantly to our understanding of the underlying biological mechanisms of Alzheimer's disease (AD). As a result, over 300 interventions have been investigated and reported to mitigate pathological phenotypes or improve behavior in AD animal models or both. To date, however, very few of these findings have resulted in target validation in humans or successful translation to disease-modifying therapies. Challenges in translating preclinical studies to clinical trials include the inability of animal models to recapitulate the human disease, variations in breeding and colony maintenance, lack of standards in design, conduct and analysis of animal trials, and publication bias due to under-reporting of negative results in the scientific literature. The quality of animal model research on novel therapeutics can be improved by bringing the rigor of human clinical trials to animal studies. Research communities in several disease areas have developed recommendations for the conduct and reporting of preclinical studies in order to increase their validity, reproducibility, and predictive value. To address these issues in the AD community, the Alzheimer's Drug Discovery Foundation partnered with Charles River Discovery Services (Morrisville, NC, USA) and Cerebricon Ltd. (Kuopio, Finland) to convene an expert advisory panel of academic, industry, and government scientists to make recommendations on best practices for animal studies testing investigational AD therapies. The panel produced recommendations regarding the measurement, analysis, and reporting of relevant AD targets, th choice of animal model, quality control measures for breeding and colony maintenance, and preclinical animal study design. Major considerations to incorporate into preclinical study design include a priori hypotheses, pharmacokinetics-pharmacodynamics studies prior to proof-of-concept testing, biomarker measurements, sample size determination, and power analysis. The panel also recommended distinguishing between pilot 'exploratory' animal studies and more extensive 'therapeutic' studies to guide interpretation. Finally, the panel proposed infrastructure and resource development, such as the establishment of a public data repository in which both positive animal studies and negative ones could be reported. By promoting best practices, these recommendations can improve the methodological quality and predictive value of AD animal studies and make the translation to human clinical trials more efficient and reliable.
doi:10.1186/alzrt90
PMCID: PMC3218805  PMID: 21943025
4.  Amyloid precursor protein selective gamma-secretase inhibitors for treatment of Alzheimer's disease 
Introduction
Inhibition of gamma-secretase presents a direct target for lowering Aβ production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy.
Methods
In vitro assays monitoring inhibitor potencies at APP γ-site cleavage (equivalent to Aβ40), and Notch ε-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of Aβ production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain Aβ was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain Aβ reduction vs. Notch signaling endpoints in periphery.
Results
The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting Aβ production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain Aβ in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain Aβ was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested.
Conclusions
The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.
doi:10.1186/alzrt60
PMCID: PMC3031881  PMID: 21190552
5.  Polo-like Kinase 2 (PLK2) Phosphorylates α-Synuclein at Serine 129 in Central Nervous System*S⃞ 
The Journal of Biological Chemistry  2009;284(5):2598-2602.
Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in α-synuclein phosphorylation in central nervous system.
doi:10.1074/jbc.C800206200
PMCID: PMC2631975  PMID: 19004816
6.  Evidence that Enzyme Processivity Mediates Differential Aβ Production by PS1 and PS2 
Current Alzheimer Research  2013;10(1):4-10.
The γ-secretase complex cleaves the carboxy-terminal 99 residue (C99) fragment of the amyloid precursor protein (APP) to generate the amyloid-β (Aβ) peptide. The catalytic activity of this complex is mediated either by the presenilin-1 (PS1) or the presenilin-2 (PS2) subunit. In vitro and in vivo studies have demonstrated that PS1-containing complexes generate more total Aβ product than PS2-containing complexes, indicating greater cleavage activity by PS1-containing γ-secretase complexes at the APP γ-site. However, it remains untested whether γ-secretase cleavage at the APP ε-site, which precedes γ-site cleavage and produces the physiologically active APP intracellular domain (AICD), follows the same rule. Using a novel Swedish APP-GVP substrate to facilitate the parallel detection of Aβ and AICD products from PS1-/-/PS2-/- cells co-transfected with either PS1 or PS2, we observed that while PS1 generates more total Aβ product than PS2, consistent with published reports, PS1 and PS2 unexpectedly generate equal amounts of AICD product. We also observed that PS1 and PS2 produce equivalent amounts of Notch intracellular domain (NICD), indicating equal cleavage activity at the Notch S3-site (the corollary of the APP ε-site). Our findings suggest that processivity differences between PS1 and PS2 underlie the differential production of Aβ peptide. Taken together these findings offer novel insights into γ-secretase biology and have important implications for therapeutically targeting γ-secretase
doi:10.2174/156720513804871480
PMCID: PMC3617584  PMID: 22950863
Aβ; AICD; γ-secretase; NICD; PS1; PS2; processivity.

Results 1-6 (6)