Targeting the Brain's Astrocytes using Diverse Small Molecule Cargo

Abstract

Astrocytes are the most abundant glial cell in the central nervous system. In the brain specifically, astrocytes control or contribute to metabolism, blood flow, and water flux, as well as dynamic processes like synaptic formation, maintenance, and pruning. Astrocytes are well established as important players in understanding homeostatic brain function and disease, albeit, there are few methods to visualize or target these cells, greatly hindering our capability to fully characterize and study them. New therapeutic methods or tools to target astrocytes have the potential to combat neurodegenerative and neuropsychiatric conditions like depression, epilepsy, or Parkinson's. Each condition is characterized by either an imbalance in ion, neurotransmitter, or protein clearance— processes in the brain all modulated by astrocytes. Current methods utilizing genetic markers or fluorescent small molecules to target astrocytes suffer from a need for fixation, specificity concerns, gap junction diffusion, or an incapability for chemical modification. We have combated a few of these caveats by synthesizing an array of small molecule probes, recently demonstrating that we can deliver a variety of fluorophores to astrocytes. By leveraging organic chemistry to attach permanently positive, pyridinium-like moieties to a variety of fluorescent small molecules, we have successfully labelled astrocytes without targeting neurons or other glia. Here, we explore the delivery of diverse small molecule cargo like transcriptional activators, calcium sensors, or drugs when modified with our astrocyte-selective probes. Leveraging organic chemistry to attach permanently positive N-heterocyclic amines to biologically interesting molecules will allow us to further probe astrocyte basic function and biology, all while informing the development of future tools in chemical biology. Here, we assay a modified small molecule calcium indicator and a doxycycline-modified probe. The former will serve to mark activated astrocytes to characterize their response to various brain stimuli. Additionally, the calcium probe will help identify astrocytes that may be in direct communication with subsets of neurons. Painting a picture of the live calcium response in astrocytes could inform therapeutic development by revealing disease mechanisms in epilepsy or seizure, for example. Alternatively, the latter doxycycline probe will function as an astrocyte-specific, transcription activator in the tetracycline-inducible gene expression system. Despite the continued development of gene therapies, there are few that utilize regulated gene expression systems. The doxycycline probe will be used to selectively activate transcription of genes of interest in astrocytes in order to control when and how the gene product is produced in a given model. We hope to employ these, and similar molecules therapeutically where current constitutive gene therapies for Parkinson's or glioblastoma, for example, could benefit from regulated gene expression. These probes demonstrate the diversity of cargo that we can deliver to astrocytes while allowing us to visualize and/or modulate neuron-glia connectivity and cell-to-cell communication.