Date of Presentation
5-4-2023 12:00 AM
College
School of Osteopathic Medicine
Poster Abstract
Background: Increased blood-brain barrier (BBB) permeability is reported in both the neuropathological and in vivo studies in both Alzheimer’s Disease (AD) and age matched cognitively normal, no cognitive impairment (NCI), subjects. Impaired BBB allows various vascular components such as immunoglobulin G (IgG) to extravasate into the brain and specifically bind to various neuronal surface proteins (NSP), also known as brain reactive autoantibodies (BrABs). This interaction is predicted to further enhance deposition of amyloid plaques.
Hypothesis: Interaction between extravasated BrABs and its cognate NSPs lower the expression of that NSPs in AD patients.
Methods: We selected Western blotting technique to study the expression of various brain proteins and test our hypothesis. Fresh frozen brain samples of AD and NCI subjects were acquired, and total brain protein was extracted using protocol established in Acharya lab. We also identified various NSPs to study the impact of BrABs-NSPs interactions. Additionally, we investigated the expression of amyloid plaques ((amyloid precursor protein (APP)) and apoptosis (Caspase-3) markers. Specific NSPs examined included the alpha7 nicotinic acetylcholine receptor (α7nAChR) and anti-choline acetyltransferase (ChAT). To image the membranes, fluorescent imaging was used initially, which was later switched to chemiluminescence, after much troubleshooting.
Results: Most of the work done through these experiments was focused on establishing a thorough Western blot protocol that can be used to reliably perform these experiments. This involved determining the appropriate primary and secondary antibodies concentrations, loading concentrations, and testing different imaging settings to determine the most ideal image-acquisition conditions. Towards the end of the fellowship, we were successful in developing a protocol to further explore our investigation. Using this protocol, we were able to visualize bands for ChAT, α7nAChR, and caspase – 3.
Conclusions: Using this protocol further Western blot experiments can be run to study and compare the expression levels of various NSP in AD and control samples for testing our hypothesis
Keywords
Alzheimer Disease, Blood-Brain Barrier, Cell Surface Proteins, Neurons, Amyloid Plaques, Caspase 3
Disciplines
Biotechnology | Cell Biology | Geriatrics | Investigative Techniques | Laboratory and Basic Science Research | Medical Cell Biology | Medical Neurobiology | Medicine and Health Sciences | Nervous System Diseases | Neuroscience and Neurobiology | Translational Medical Research
Document Type
Poster
Included in
Biotechnology Commons, Cell Biology Commons, Geriatrics Commons, Investigative Techniques Commons, Laboratory and Basic Science Research Commons, Medical Cell Biology Commons, Medical Neurobiology Commons, Nervous System Diseases Commons, Neuroscience and Neurobiology Commons, Translational Medical Research Commons
Extravasated Brain-Reactive Autoantibodies Perturb Neuronal Surface Protein Expression in Alzheimer's Pathology
Background: Increased blood-brain barrier (BBB) permeability is reported in both the neuropathological and in vivo studies in both Alzheimer’s Disease (AD) and age matched cognitively normal, no cognitive impairment (NCI), subjects. Impaired BBB allows various vascular components such as immunoglobulin G (IgG) to extravasate into the brain and specifically bind to various neuronal surface proteins (NSP), also known as brain reactive autoantibodies (BrABs). This interaction is predicted to further enhance deposition of amyloid plaques.
Hypothesis: Interaction between extravasated BrABs and its cognate NSPs lower the expression of that NSPs in AD patients.
Methods: We selected Western blotting technique to study the expression of various brain proteins and test our hypothesis. Fresh frozen brain samples of AD and NCI subjects were acquired, and total brain protein was extracted using protocol established in Acharya lab. We also identified various NSPs to study the impact of BrABs-NSPs interactions. Additionally, we investigated the expression of amyloid plaques ((amyloid precursor protein (APP)) and apoptosis (Caspase-3) markers. Specific NSPs examined included the alpha7 nicotinic acetylcholine receptor (α7nAChR) and anti-choline acetyltransferase (ChAT). To image the membranes, fluorescent imaging was used initially, which was later switched to chemiluminescence, after much troubleshooting.
Results: Most of the work done through these experiments was focused on establishing a thorough Western blot protocol that can be used to reliably perform these experiments. This involved determining the appropriate primary and secondary antibodies concentrations, loading concentrations, and testing different imaging settings to determine the most ideal image-acquisition conditions. Towards the end of the fellowship, we were successful in developing a protocol to further explore our investigation. Using this protocol, we were able to visualize bands for ChAT, α7nAChR, and caspase – 3.
Conclusions: Using this protocol further Western blot experiments can be run to study and compare the expression levels of various NSP in AD and control samples for testing our hypothesis