A New Target for Amyloid Beta Toxicity Validated by Standard and High-Throughput Electrophysiology

Soluble oligomers of amyloid beta (Abeta) are considered to be one of the major contributing factors to the development of Alzheimer's disease. Most therapeutic development studies have focused on toxicity directly at the synapse.Patch clamp studies detailed here have demonstrated that soluble Abeta can also cause functional toxicity, namely it inhibits spontaneous firing of hippocampal neurons without significant cell death at low concentrations. This toxicity will eventually lead to the loss of the synapse as well, but may precede this loss by a considerable amount of time. In a key technological advance we have reproduced these results utilizing a fast and simple method based on extracellular electrophysiological recording of the temporal electrical activity of cultured hippocampal neurons using multielectrode arrays (MEAs) at low concentrations of Abeta (1-42). We have also shown that this functional deficit can be reversed through use of curcumin, an inhibitor of Abeta oligomerization, using both analysis methods.The MEA recording method utilized here is non-invasive, thus long term chronic measurements are possible and it does not require precise positioning of electrodes, thus it is ideal for functional screens. Even more significantly, we believe we have now identified a new target for drug development for AD based on functional toxicity of hippocampal neurons that could treat neurodegenerative diseases prior to the development of mild cognitive impairment

Regeneration And Characterization Of Adult Mouse Hippocampal Neurons In A Defined In Vitro System

Although the majority of human illnesses occur during adulthood, most of the available in vitro disease models are based upon cells obtained from embryonic/fetal tissues because of the difficulties involved with culturing adult cells. Development of adult mouse neuronal cultures has a special significance because of the abundance of transgenic disease models that use this species. In this study a novel cell culture method has been developed that supports the long-term survival and physiological regeneration of adult mouse hippocampal cells in a serum-free defined environment. In this well-defined, controlled system, adult mouse hippocampal cells survived for up to 21 days in culture. The cultured cells exhibited typical hippocampal neuronal morphology and electrophysiological properties after recovery from the trauma of dissociation, and stained positive for the expected neuronal markers. This system has great potential as an investigative tool for in vitro studies of adult diseases, the aging brain or transgenic models of age-associated disorders. © 2008 Elsevier B.V. All rights reserved

03. Immunogenicity and Safety of a Quadrivalent Meningococcal Conjugate Vaccine (MenACYW-TT) Administered as a Booster Dose in Adults and Adolescents Vaccinated Against Meningococcal Disease 3 - 6 Years Earlier

BACKGROUND: Booster doses of meningococcal conjugate vaccines may induce long-term protection against invasive meningococcal disease. MenACYW-TT [MenQuadfi®] is a quadrivalent meningococcal conjugate vaccine, licensed for use in ages 2 years and older in USA. The vaccine is also licensed in ages 12 months and older in EU and other countries. METHODS: A phase IIIb study (NCT04084769) was conducted to evaluate the persistence of immune response in adults and adolescents primed 3-6 years earlier with either MenACYW-TT or MCV4-CRM (Menveo®) and, safety and immunogenicity of MenACYW-TT when administered as a booster dose with or without concomitant administration with MenB vaccines (Bexsero® and Trumenba®). Serum bactericidal assays with human complement (hSBA) and baby rabbit complement (rSBA) were used to measure antibodies against vaccine serogroups at baseline (Day 0 [D0]), D06 (in a subset) and 30 days post-vaccination (D30). Safety data were collected up to 6 months post-vaccination. RESULTS: At D0, the GMTs were higher in subjects primed with MenACYW-TT vs MCV4-CRM for serogroups C, Y and W, and were comparable for serogroup A. At D0, all hSBA GMTs were higher than those observed pre-priming dose, suggesting persistence of immunity. Sufficiency of hSBA seroresponse ( >75%) was demonstrated following administration of MenACYW-TT booster dose regardless of the priming vaccine administered 3-6 years earlier. Vaccine seroresponse in a subset of participants at D06 ranged from 77.8% (95%CI 62.9%; 88.8%) for serogroup A to 97.8% (88.5%; 99.9%) for serogroup W suggesting a quick onset of immune response post-booster. Post-vaccination (D30) hSBA GMTs were comparable for serogroups A, Y and W regardless of the nature of the priming vaccine and were higher for serogroup C in subjects primed with MenACYW-TT vaccine. The MenACYW-TT booster dose was well-tolerated and had similar safety profiles regardless of the priming vaccine. The safety profiles were comparable regardless of the MenB vaccine co-administered with MenACYW-TT vaccine. CONCLUSION: MenACYW-TT used as priming vaccine was able to demonstrate persistence of immune response 3-6 years later. MenACYW-TT elicits robust booster responses in adults and adolescents primed with MenACYW-TT or MCV4-CRM DISCLOSURES: Betzana Zambrano, MD, Sanofi Pasteur (Employee) Germán Áñez, MD, Sanofi Pasteur (Other Financial or Material Support, Former employee) Sue Jiayuan, MSc, Sanofi Pasteur (Independent Contractor) Judy Pan, PhD, Sanofi Pasteur (Employee) Habiba Arroum, MD, Sanofi Pasteur (Employee) Kucku Varghese, PhD, Sanofi Pasteur (Employee) Emilia Jordanov, MD, Sanofi Pasteur (Employee, Shareholder) Mandeep S. Dhingra, MD, Sanofi Pasteur (Employee, Shareholder

Time course of the application of 20 uM Aβ on spontaneous activity of cultured embryonic rat hippocampal cells on MEAs.

<p>Spontaneous firing observed before administration of 20 uM Aβ (A). Spontaneous firing observed 45 minutes after administration of 20 uM Aβ (B), 90 minutes after administration of 20 uM Aβ (C) and 150 minutes after administration of 20 uM Aβ (D).</p

Immunoblot of Aβ oligomers.

<p>From left to right: Lane 1 is the monomer. Lane 2 indicates the apparent inhibition of Aβ oligomerization in the presence of Curcumin. Lane 3 indicates Aβ oligomers.</p

Reversal of the effect of Aβ on firing frequency by curcumin applied together with Aβ or after Aβ exposure.

<p>Time course of the effect of curcumin on spontaneous firing frequency of embryonic hippocampal neurons when coadministered with Aβ (N = 5) (A). Time course of curcumin effect on spontaneous firing frequency of embryonic hippocampal neurons when administered after cells were exposed to Aβ for 24 h (N = 5) (B). 100% implies baseline values before exposure to Aβ.</p

Effect of 24 h exposure to 100 nM Aβ on cell functionality measured using whole cell patch clamp electrophysiology.

<p>Changes after exposure to Aβ for 24 hr. in spontaneous firing frequency; ***: p = 0.00007 (A), K⁺ ion channel currents; **: p = 0.008 (B), and membrane potential; *: p = 0.035 (C). Cell survival after administration of 100 nM Aβ at 24 hours and 7 days; *: p = 0.04 (D). Data is presented as mean±SEM; N = 30 utilizing a two-sample Student's t-test.</p

Time course of the effect of Aβ on spontaneous activity of cultured embryonic rat hippocampal cells at various concentrations.

<p>Concentrations of 100 nM and 5 µM Aβ caused a complete cessation of firing activity with different time delays (N = 5) (A). Composite logarithmic graph of the time taken for cells to stop firing at the various Aβ concentrations tested (B).</p