Olfactory Bulb Neuronal Network Chip-Based Biosensor for Analysis of Dysosmia Model in Alzheimer's Disease

Abstract

Alzheimer disease (AD) is a chronic neurodegenerative disease threating the health of human being. The clinical symptoms mainly consist of severe cognitive impairment, comprehensive dementia, memory loss and other mental behavioral problems. According to data that collected in 2018, AD has become the sixth fatal disease in the worldwide. Nevertheless, the pathogenesis of AD has not been clarified, except for two main pathological features: from extracellular, the amyloid plagues appear in the dendrites which is mainly caused by the formation of the toxic amyloid-β oligomers (AβOs); from intracellular, it appears the accumulation of neurofibrillary tangles (NFTs) which is formed by hyperphosphorylated tau protein. Recent years, olfactory dysfunction has been reported to be an early symptom of AD. From the research of clinical experiment, olfactory dysfunction happens much earlier than other clinical symptoms. Besides, there have been plentiful methods for olfactory dysfunction diagnosis, which indicates olfactory test for disease diagnosis has been paid more and more attention. Many researches have indicated olfactory bulb is the first region of the olfactory system to be damaged in AD. AβOs has been confirmed in early olfactory dysfunction in AD, but how the AβOs lead to the olfactory dysfunction is still not exactly realized. Consequently, we chose the OB region as the research object and proposed to establish an olfactory dysfunction model of AD, by the means of a directly electrophysiology method.AβOs-induced olfactory bulb neural network cultured on MEA as dysosmia model of AD at early-stage in vitro is established to investigate that AβOs is detrimental to the processing of olfactory information over time. Firstly, olfactory bulb tissues were extracted and then were digested so that we got the single cell suspension. On the one hand, OB neurons were plated on the surface of MEA chip so that we can detect the extracellular signal in time after AβOs induction. On the other hand, they were cultured on 24-well plate for immunofluorescence to observe the changes of the neuron morphology and cellular proteins. When the OB neural network grew to 13 days, the typical extracellular signals of OB neurons in spontaneous firing state can be recorded. After induced by AβOs, the firing rate of OB neuron disappeared temporarily. After 1 h, the firing rate increased gradually, but after 2 h, the firing rates neurons decreased significantly. Moreover, the firing rate of OB neuron disappeared completely after 3 h.To observe the changes of the cell morphology and cellular proteins after treated with AβOs, we used immunofluorescence method to verify the validity of the dysosmia model in AD. After being induced by AβOs for 3 hours, the neuron plaques increase significantly. This indicates the fact that neurites degeneration and dendrites impairment. As previously described in the mechanics section, phosphorylated tau protein(p-tau) is hyperphosphorylated and abnormally accumulates in Alzheimer’s disease. We performed the staining of p-tau antibody, and analyzed the mean fluorescence intensity to quantify p-tau protein levels. The statistical graph shows the great significant difference between the control and AβOs-treated group. Our results validated that the AβOs can make tau protein phosphorylate, which is similarly to those observed in the degenerating neurons of AD. This also verifies the effectiveness of the model established in this study.Then we try to explain why the spike firing changes after the AβOs induction. When the AβOs was added, it caused N-methyl-D-aspartate (NMDA) receptor and L-type calcium channels opening so that the calcium flew into the cell largely. Eventually, the intracellular calcium overloaded, which then resulted in abnormally improving spontaneous activity. However, the large amount of calcium caused potassium channels opening, and impaired mitochondrial and endoplasmic reticulum (ER) metabolism. Eventually, the whole cellular metabolism disordered, and the neurons became inactive till death.The study will contribute to understanding the cellular and synaptic mechanisms of the early-stage olfactory dysfunction in AD and providing a potential strategy for diagnosis and treatment. And then discuss what it is for and what will we do next by the means of dysosmia model of AD established in the research. Recent study shows that intranasal drug administration is expected to be a targeted therapy method for treating the cerebral diseases. Because it is close to the central nervous system so that the drugs can be transmitted to the cerebral area and be absorbed quickly. Therefore, applying the early-stage dysosmia model of AD to screening the intranasal drug of treating AD is of great significance.