Molecular determination of alpha 5-subunit containing gamma-aminobutyric acid type A receptors in a rat model of Alzheimer’s disease
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Abstract
In the United States, 5.5 million individuals suffer with Alzheimer’s Disease (AD), which is characterized by a decline in cognition and eventually the lethal disruption of all brain functions. There is no cure for this debilitating disorder, and while current therapeutics aim to manage the symptoms in the early stages of the disease, they provide little relief for most patients. We postulate that discovering the molecular basis for the first signs of memory impairment in AD at a neural circuitry level may provide the key to restoring neural information processing even in the face of neuropathology.There is a delicate balance of excitation and inhibition in the brain that needs to be maintained for information flow and the synaptic plasticity that underlies dynamic processing of new information, its storage, and retrieval. Interestingly, while the brain’s inhibitory neurotransmitter receptor system, γ-aminobutyric acid type A receptors (GABAARs), play a crucial role in maintaining this balance, functional inhibition of a select group of GABAARs that contain the α5 subunit (α5GABAARs), or removal of α5 subunit synthesis, enhances spatial memory in rodents. In wild-type Fisher 344 rats (WtF344), treatment with the cognitive enhancer α5IA, a negative allosteric modulator with selectivity for α5GABAARs, markedly increases the amplitude of sharp wave ripples (SPW-Rs), which are oscillatory brain rhythms originating in the hippocampal trisynaptic circuit (HTC) that are essential for memory consolidation. Most interesting to us, the beta amyloid expressing transgenic F344 (TgF344) AD rat model has lost this α5IA modulation of SPW-Rs at 9 months of age, prior to evident neuropathology.
We hypothesized that the loss in α5IA responsivity of TgF344-AD rats reflects a region-specific downregulation in the levels of α5 subunits and/or the abundance, localization, and composition of α5GABAARs in the hippocampal formation, the most important structure in the brain for spatial and episodic memory. To test this possibility, we used RNAscope high resolution in situ hybridization, immunofluorescence, and confocal microscopy to interrogate the presence of specific molecular species in the dentate gyrus, the gatekeeper of the hippocampus, and the hippocampal CA1 pyramidal layer where the amplitude of SPW-Rs is modulated by α5IA in WtF344 rats.
Specific to CA1, our results show an age-dependent decrease in the average number of α5 transcripts per cell in TgF344-AD, which is not seen in WtF344 and could be predictive of lower α5 subunit levels in individual neurons. However, we see no change in the percentage of area stained with the α5 signal in tissue derived from TgF344-AD rats but rather an age-dependent increase in the average signal intensity of discrete α5-containing pixels regardless of genotype, most likely reflecting an increase in the presence of receptor clusters. We next turned to the presence of α5GABAARs at the membrane. The localization of these receptors is dominated by their role in tonic inhibition through their extrasynaptic presence, although recent studies suggest the importance of a smaller population of α5GABAARs at the synapse and their specific role in SPW-Rs and phasic inhibition.
We used immunofluorescence to measure the spatial overlap of α5 fluorescent signals with presynaptic (synaptophysin) and postsynaptic (gephyrin) markers to specifically detect extrasynaptic and postsynaptic receptor populations. Although we discovered an age-dependent downregulation of extrasynaptic receptor levels, it was present in both genotypes. To our surprise, however, we discovered a significant age-dependent increase in the levels of postsynaptic α5GABAARs in the dentate gyrus of WtF344 that is absent in their TgF344-AD littermates. These findings are most likely consistent with a similar loss in CA1 where SPW-Rs are recorded. Our results, taken together, suggest that the major alteration in the HTC of TgF344-AD rats, revealed by loss of α5IA ripple modulation, is due to a failure in α5GABAAR trafficking to the postsynaptic compartment in response to toxic beta amyloid burden, opening a new area of investigation and potential therapeutic intervention in the treatment of AD.
Description
2024
License
Attribution-NoDerivatives 4.0 International