Calcium Imaging; New way to identify AβO targets

Author: Endonita Hajzeraj

AβOs or soluble amyloid β oligomers are recognized by the public as neurotoxins that trigger divergent signaling in neurons. This signaling of neurons leads to an accumulation of neuronal damage and memory disorders in the disease that we all know about, Alzheimer’s (Xueying et al. 2018). Soluble amyloid β oligomers first showed signs of neurotoxicity about twenty years ago and today it is known as the leading cause of Alzheimer’s disease. AβOs are endogenously produced and are elevated in brain tissue from patients with Alzheimer’s (Chang et al. 2003). Common knowledge of Alzheimer’s suggests that synapse failure is a direct consequence of AβOs, and the strategy most commonly employed to combat this issue was to directly block AβOs, their formation, or their divergent signaling.
Over the years, research has been dedicated to this disease with a focus on anti-amyloid β immunotherapy, using antibodies that have completed clinical trials. Little clinical benefit has been reported (Bard et al. 2003). Among the therapies used to treat Alzheimer’s, aducanumab, a monoclonal antibody, holds promise for potential clinical success (Goure et al. 2014). The downfall of this monoclonal antibody is that it does not distinguish between AβOs and the many times more prevalent Aβ fibrils. Therefore, predicting that this antibody will effectively block AβO neurotoxicity, is a difficult task. The reason many therapeutic antibodies have failed is because of their lack of selectivity and binding for AβOs, which is the most common and potent form of Aβ, which cause neurotoxicity (Xueying et al. 2018).
Aducanumab treatment has reduced amyloid plaque levels, but no statistically significant improvements have been made to cognition function. The authors state that the missing piece of the therapeutic puzzle is the ability to block synaptic attack by AβOs or lower the levels of AβOs in order to reduce divergent signaling that amyloid oligomers trigger. One of the factors disrupted by divergent signaling is cytosolic calcium concentration ([Ca2+)], which is part of the pathway for pathogenic changes that result in neuronal death and cognitive impairment (Khachaturian et al. 1994). Because calcium homeostasis is regulated by [Ca2+], elevations and fluctuations in calcium can hinder neuronal functions. The researchers in this study have enabled a screening and identification of potential AβO-blocking drug candidates in neuronal cell cultures in vitro and on exposed brain surface cortical neurons in vivo. Using this method, the researchers can quantify the amyloid oligomer-induced divergent signaling by measuring [Ca+2] and then evaluate the binding sensitivity and selectivity of candidate antibodies that are directed toward specific amyloid species (Xueying et al. 2018).
Among the various drug candidates available for screening, the monoclonal antibody ACU3B3 was determined to be highly effective in blocking AβO-induced calcium elevations in neuronal cultures and wild type. To characterize the interaction of amyloid β oligomers, the authors employed array tomography to understand the interaction of AβOs with synaptic proteins in mouse and human AD brains (Xueying et al. 2018). These results provide evidence that AβOs provide potential benefit to Alzheimer’s diseased patients.
To assess the ability of anti-Aβ antibodies to specifically target AβOs, Xueying and colleagues designed an assay that induced neurotoxicity in neuronal cultures with [Ca+2] measured. The cultures were treated with either control normal AβO solutions or immunodepleted solutions of AβOs by various test antibodies. Among the eight antibodies tested, 3B3 along with 3D6 exhibited AβO-blocking potential. Subsequent experiments involved treatment conditions and finally measured the Aβ concentrations amongst these treatments. The data indicates that calcium levels in wild type neuronal cultures while the non-control saw an increase in calcium concentrations (Xueying et al. 2018).
3B3 protection from AβO dependent calcium dysregulation was validated in the experiment. 3B3 has the ability to block AβOs as it was reported in the first results section, and it was further tested to evaluate the AβO-mediated calcium increasing and the blocking ability. They measured neuronal calcium levels in adult wild type mice and treated mice. A one-hour acute exposure of naïve brains to a concentration of AβOs triggered dramatic calcium elevations and no elevations in baseline calcium were observed when 3B3 was applied for one hour prior to AβOs application (Xueying et al. 2018). This suggests that 3B3 might be important for regulating calcium levels as a therapy. In order to establish that 3B3 could bind to endogenous AβOs, the authors evaluated the distribution of AβOs in relation to plaques which were immunolabeled with 3B3 and a fluorescent secondary antibody. They found a dense core of amyloid plaques stained positive, suggesting a strong binding of 3B3 with plaques.
The more important area of study that the authors studied was targets of AβOs at synapses in human AD brains. They determined that in human Alzheimer’s tissue, synaptic density and pairing were negatively correlated with 3B3 burden which suggests that there is a consistent AβO binding and selectivity profile for 3B3. Evidence suggests that selective neuronal vulnerability manifest in Alzheimer’s disease comes from specific AβO binding to high affinity receptors, which will lead to a disruption of synaptic signaling pathways, which will lead to alterations in neurology. Therefore, targeting of AβOs to prevent synaptic binding and divergent signaling can mediate effective Alzheimer’s treatment (Xueying et al. 2018).
The authors sought out to develop a method for screening and characterizing potential AβO blocking antibodies based on calcium imaging. Changes in measured calcium reflect the extent of aberrant amyloid oligomer neuronal signaling or protection by the antibodies. Application of amyloid oligomers exposed to cortical surfaces resulted in an increase in calcium. This study provides great evidence that amyloid oligomer-immunotherapeutic such as 3B3 should be highly effective for treatment and prevention of Alzheimer’s. Previous studies and clinical trials over the years have only tested non-specific antibodies mostly targeting non-toxic Aβ monomers, which can be capable of binding to AβOs, but fail because brain levels of monomer fibril are higher than levels of therapeutic antibody. From this research provided here, future research can focus on creating drug therapies that can be created to specifically block AβOs.
In our class, we are continuously reading papers about new proteins that can be used to treat various diseases. Learning about proteins in class, allow us to understand the mechanisms of action that proteins take to express their function.

References:
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Xueying Wang, Ksenia V. Kastanenka, Michal Arbel-Ornath, Caitlin Commins, Akira Kuzuya, Amanda J. Lariviere, Grant A. Krafft, Franz Hefti, Jasna Jerecic & Brian J. Bacskai Scientific Reports
volume 8, Article number: 4634 (2018) doi:10.1038/s41598-018-22979-2

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http://www.nature.com/articles/s41598-018-22979-2

 

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