Electrophysiological and pharmacological in vivo characterization of EEG fingerprints / biomarkers in AD mouse models

Abstract

Alzheimer’s disease (AD) is a major leading cause of deaths worldwide with underlying conditions of dementia and cognitive decline mostly in older age patients (≥ 65 years). Currently, there are approximately 50 million people worldwide suffering from dementia, and the prevalence is likely to increase up to 130 million in 2050 with 60-70% of cases due to AD. There is no medication available for AD, which can cure the disease or stop its progression. Electrophysiological biomarkers, most importantly the electroencephalographic (EEG) fingerprints, are very important tools to detect not only the incidence of dementia but also characterize the progression of the disease in later life, better than other diagnostic markers. The present study was designed to investigate the hippocampal (CA1) and cortical (M1) seizures activity and EEG frequency characteristics by a Fast Fourier Transform (FFT) based approach using implantable video EEG radiotelemetry in Cav3.2^-/- and APPswePS1dE9 mice under unrestrained conditions. In the first part of my studies, I investigated the role of Cav3.2 Ca2⁺ channels in CA1 oscillatory patterns, particularly in theta genesis. FFT based analysis of long-term baseline recordings revealed an enhanced relative hippocampal type II theta (4.1-12 Hz) and relative alpha power (8-12 Hz) in Cav3.2^-/- mice, predominately in the inactive phase of dark cycles. These findings correlated with our post-urethane analysis, which demonstrated the increased type theta II upon Cav3.2 ablation. RT-qPCR of Cav3.2^+/+ and Cav3.2^-/- mice was carried out to evaluate the potential role of highly expressed hippocampal genes in molecular mechanisms of theta genesis and memory formation. This analysis identified a reduced dynein light chain Tctex-Type 1 (dynlt1b) expression in Cav3.2 deficient mice. Furthermore, RT-qPCR analysis of the septohippocampal GABAergic system revealed a decrease in GABA A δ subunit (Gabrd) and GABA B1 (Gabbr1) receptor subunits. These finding strongly support the hypothesis that the Cav3.2 T-type VGCC ablation activates the tonic mode of action in septal GABAergic interneurons that leads to tonic inhibition of hippocampal GABAergic interneurons and disinhibition of pyramidal neurons with increased type II theta oscillatory activity. Moreover, these results suggest a significant role of T-type Cav3.2 Ca2⁺ channels in hippocampal theta related cognitive functions and memory formation. Thus, Cav3.2 T-type channels could serve as potential therapeutic drug target for different neuropsychiatric conditions. <br /> In the second part of my study, FFT based frequency and seizure analysis was performed in 3-6 months old APPswePS1dE9 AD mice after chronic administration of pantoprazole. Initially, the analysis was carried out in younger mice of both genders (age: 12-16 weeks) and later in older male mice (age: 17-21 weeks). FFT based frequency analysis in younger mice revealed a decrease in hippocampal and cortical theta (θ1: 4-8 Hz) relative power in APPswePS1dE9 male mice compared to controls during the dark cycle while no significant changes in theta frequency relative powers (θ1: 4-8 Hz, θ2: 4.1-12 Hz) were observed in both female genotypes. However, enhanced relative theta powers (θ1: 4-8 Hz, θ2: 4.1-12 Hz) were also detected in pantoprazole treated controls compared to untreated controls recorded during the dark cycle from the hippocampus and motor cortex. No significant changes in relative theta powers (θ1: 4-8 Hz, θ2: 4.1-12 Hz) were detected between pantoprazole treated and untreated APPswePS1dE9 mice. A significant higher hippocampal and cortical beta frequency relative powers (β112.1-30 Hz, β2: 16-24 Hz, β3: 16-30 Hz) were found in APPswePS1dE9 male mice during both dark and light cycles. Relative cortical beta powers were also higher in APPswePS1dE9 female mice during the light cycle. Furthermore, a significant increase in cortical relative gamma low power (γlow: 30-50 Hz) was identified in APPswePS1dE9 mice of both genders during dark and light cycles while cortical gamma high power (γmid: 50-70 Hz) was elevated in APPswePS1dE9 male mice during the dark cycle. Moreover, electroencephalographic seizure analysis revealed significantly increased cortical seizure parameters in APPswePS1dE9 mice of both genders compared to controls during dark and light cycles. Interestingly, reduced cortical seizures parameters were found in pantoprazole treated APPswePS1dE9 mice as compared to untreated APPswePS1dE9 mice of both genders at light cycle. Additionally, pantoprazole was detected by LC-MS/MS analysis of plasma and liver tissues from both genders. Later, the FFT based analysis of older male mice also revealed consistent findings of reduced hippocampal theta (θ1: 4-8 Hz) and enhanced cortical gamma mid relative powers (γmid: 50-70 Hz) in APPswePS1dE9 mice. Furthermore, urethane administration resulted in enhanced relative hippocampal theta powers in APPswePS1dE9 mice that were subsequently reduced following atropine administration. Similarly, enhanced seizures parameters were also detected in APPswePS1dE9 mice compared to controls but reduced seizure markers were observed in pantoprazole treated APPswePS1dE9 mice compared to untreated APPswePS1dE9 mice. Age and gender EEG oscillatory activity specific alterations in APPswePS1dE9 mice suggest a functional and diagnostic role of EEG. In the future, EEG could be recognized as diagnostic biomarker for AD. An increased theta activity in pantoprazole treated controls might be associated with enhanced theta related cognitive ability in healthy male controls. No positive or negative effects of chronic pantoprazole administration were observed in theta and gamma frequency parameters in APPswePS1dE9 mice. These findings suggest a neutral role of pantoprazole long-term administration in AD progression. <br /> In future studies, a gender and age specific pharmacokinetics, pharmacodynamics and pharmacogenetics approaches will be necessary to analyze the efficacy and safety profile of pantoprazole/PPIs in normal healthy controls, MCI and AD patients on individualized basis.