Optogenetic Stimulation of Astrocytes as Novel Approach for Modulating Neural Network Activity in Alzheimer’s disease

Abstract:

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder considered the most common form of dementia, affecting 6.9 million older American adults (Nitrini, 2023). There is an urgent need to develop effective treatments that target the underlying causes, as current therapies only aim to ameliorate the symptoms. This research proposal explores how optogenetic techniques can modulate neural network activity of astrocytes by expressing the opsin channelrhodopsin-2 (ChR2) using an adeno-associated virus carrying the glial fibrillary acidic protein (GFAP) promoter to ensure cell-type specificity, preserving the neural organization. Unlike previous approaches that have shown side effects and have limited effect in halting long-term disease progression, this method offers a safer and more precise way method to restore balance within neural circuits, enhancing neuronal connections.

 

Introduction:

AD is a prevalent neurodegenerative disorder associated with cognitive decline, where as it progresses, the accumulation of β-amyloid plaques and hyperphosphorylated tau neurofibrillary tangles (NFTs), accompanied by neuroinflammation and brain dysfunction, disrupts communication leading to neuronal death (Breijyeh & Karaman, 2020). This neurodegeneration affects regions such as the hippocampus and entorhinal cortex, resulting in deficits in memory, attention, and then, essential functions like language, behavior, and motor control (Igarashi, 2023). Despite ongoing research, no definitive cure exists for AD. Current pharmacological treatments, such as acetylcholinesterase inhibitors and NMDA receptor antagonists, only provide symptomatic relief or have shown limited efficacy and adverse side effects without modifying the underlying disease progression (Yiannopoulou & Papageorgiou, 2020). Optogenetics is an emerging field that allows researchers to control cellular activity using light-sensitive proteins called opsins, which are genetically introduced into specific cell types (Kim et al., 2017). One of the most studied is Channelrhodopsin-2 (ChR2), a light-gated cation channel, which after stimulation using specific wavelengths of light induces membrane depolarization, allowing researchers to activate or inhibit cells.

 

In this proposal, primary hippocampal cultures exposed to β-amyloid will provide an in vitro AD model to investigate the impact of astrocytic modulation using optogenetics, in which a GFAP-ChR2 viral construct will transduce and enable astrocytes to be stimulated. MEA (Microelectrode Arrays) recordings will collect network-level responses, and statistical testing will assess these effects. This study aims to identify the effects of optogenetic activation of astrocytes on neuronal activity and functional connectivity under β-amyloid conditions. This approach may provide novel insights into glial-neuronal interactions and uncover potential intervention points for AD.

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Literature Review:

Currently, there is no cure for AD; existing FDA-approved treatments, including acetylcholinesterase inhibitors (ChEIs; donepezil, galantamine, and rivastigmine) and NMDA receptor antagonists (memantine) that prevent neuronal (Long & Holtzman, 2019). However, they often cause side effects such as nausea, vomiting, headache, and sometimes worsening in early stages (Thangwaritorn et al., 2024). Astrocytes enter a reactive state in AD, which triggers NF-κB-mediated inflammatory responses and contributes to neuronal dysfunction and death (Wu et al., 2022). These reactive astrocytes release glutamate, ATP, and pro-inflammatory cytokines, exacerbating excitotoxicity and synaptic impairment (Price et al., 2021). This highlights the crucial role of these cells in maintaining synaptic homeostasis and modulating neuroinflammation, making them an attractive target of therapies for AD. In a notable study, astrocyte stimulation using various opsins such as ChR2 or Opto-a1AR was studied, achieving an increase in neuronal plasticity, a key element to recover neuronal connections and reverse the effects caused by AD (Gerasimov et al., 2021).

 

While there is accumulating evidence for astrocytic contributions to AD pathology, knowledge of the relationship between astrocytic activation, changes in neuronal network activity, and dysfunction has not been clearly established. This study addresses this gap by applying optogenetic modulation in a controlled AD model to determine astrocyte-driven effects on network dynamics.

 

Methodology:​ 

Preparation of Cell Cultures

Primary hippocampal cell cultures must be obtained from mouse embryos on embryonic day 18 (E18). After extraction, embryos should be transferred into sterile Hanks’ solution to preserve tissue viability (Fagundes et al., 2018). Isolate the hippocampi using 0.25% trypsin-EDTA at 37ºC, then suspend in Neurobasal Medium™ supplemented with 5% FBS, 1% B27, and 0.5% L-glutamine to support neuronal and glial survival (Tomassoni-Ardori et al., 2020). Seed the cells onto MEA60 multielectrode arrays to perform electrophysiological recordings (Vernekar & LaPlaca, 2020). Regular changes will be performed using the same supplement composition.

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Transduction and Optogenetic Stimulation

Implement the AAV-GFAP-ChR2-mCherry-WPRE viral vector for the transduction of the cultures, enabling selective expression of optogenetic opsin, Channelrhodopsin-2 in astrocytes. It was found that insertion of Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE) considerably increased transgene expression in neuron cultures (Borodinova et al., 2021) and mCherry is a fluorescent protein that allows the confirmation of a successful insertion. To ensure the specificity of ChR2 expression in astrocytes, implement immunocytochemistry using GFAP and NeuN markers (Gusel’nikova & Korzhevskiy, 2015). Experimental groups will include four conditions: condition one as a control group, condition two as amyloid-β-treated cultures, and conditions three and four as cultures receiving optogenetic stimulation with or without Aβ exposure after transduction. From 14 day of cultivation (DIV) to DIV21, stimulate the astrocytes daily using a micromanipulated optical fiber positioned above the electrode arrays (Ung & Arenkiel, 2012). Utilize 470 nm blue light, as previous studies have shown to be the ideal parameter for Chr2 (Yao et al., 2012).

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Data collection and statistical analysis

MEA60 system, in combination with MC_Rack Software, will be in charge of data collection of neural activity. To compare molecular and electrophysiological parameters across experimental groups, statistical analysis will be conducted. One-way ANOVA tests will be used to asses group differences in neural architecture, connectivity and neural communication. Then, Tukey’s post hoc test will be applied to identify specific group comparisons (Juarros-Basterretxea et al., 2024). Additionally, to quantify functional connectivity within the network, apply graph analysis using NeuroExplorer software. To further assess molecular changes associated with neuroinflammatory responses induced by Aβ exposure perform RT-PCR to quantify mRNA of inflammatory markers such as IL-1α or IL-6 (Park et al., 2020). These analyses will provide insights on how optogenetic modulation influences neuronal communication under pathological conditions.

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Conclusion:

In conclusion, optogenetic applications in the activation of astrocytes is a promising method for enhancing neuronal activity and preserving its functional architecture. While current treatments for AD provide relief for symptoms, they do not arrest the progression of the disease or target its underlying causes. Therefore, it is essential to explore the potential of this technique as it represents a non-invasive and precise approach for combating AD.

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