Genetic Constructs for the Control of Astrocytes' Activity

Abstract

In the current review, we aim to discuss the principles and the perspectives of using the genetic constructs based on AAV vectors to regulate astrocytes' activity. Practical applications of optogenetic approaches utilizing different genetically encoded opsins to control astroglia activity were evaluated. The diversity of astrocytic cell-types complicates the rational design of an ideal viral vector for particular experimental goals. Therefore, efficient and sufficient targeting of astrocytes is a multiparametric process that requires a combination of specific AAV serotypes naturally predisposed to transduce astroglia with astrocyte-specific promoters in the AAV cassette. Inadequate combinations may result in off-target neuronal transduction to different degrees. Potentially, these constraints may be bypassed with the latest strategies of generating novel synthetic AAV serotypes with specified properties by rational engineering of AAV capsids or using directed evolution approach by searching within a more specific promoter or its replacement with the unique enhancer sequences characterized using modern molecular techniques (ChIP-seq, scATAC-seq, snATAC-seq) to drive the selective transgene expression in the target population of cells or desired brain regions. Realizing these strategies to restrict expression and to efficiently target astrocytic populations in specific brain regions or across the brain has great potential to enable future studies.

Keywords: AAV; GFAP; astrocytes; glia; opsins; promoter; serotype; viral vector.

Figure 1

Schematic representation of characterized promoter isoforms for astrocytic targeting constructed based on human GFAP gene (for details, see [99,134]). Letters A–D indicate enhancer regions, BP–basal promoter. Relative distances from the transcriptional start site (TSS) are provided in bp (base pairs). Arrows show the direction of transcription.

Figure 2

High-resolution confocal images of mouse hippocampal astrocytes at low ((A), scale bar: 500 µm) and high magnification ((B), scale bar: 30 µm), transduced by AAV5 viruses with a reporter gene (opto-a1AR, green) driven from the astrocytic gfaABC₁D promoter. Nuclei are labeled with DAPI (blue).

Figure 3

Schematic representation of putative promoter isoforms for astrocytic targeting constructed based on rat Aldh1l1 gene (for details, see [142]); mouse Slc1a3 gene (for details, see [148]), and human GJB6 gene (for details, see [149). Relative distances from the transcriptional start site (TSS) are provided in bp (base pairs). Arrows show the direction of transcription. Yellow boxes represent important regulatory regions. Ex1-the initial part of the first exon.