Abstract
Uncovering the physiological role of individual proteins that are part of the intricate process of cellular signaling is often a complex and challenging task. A straightforward strategy of studying a protein’s function is by manipulating the expression rate of its gene. In recent years, the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-based technology was established as a powerful gene-editing tool for generating sequence specific changes in proliferating cells. However, obtaining homogeneous populations of transgenic post-mitotic neurons by CRISPR/Cas9 turned out to be challenging. These constraints can be partially overcome by CRISPR interference (CRISPRi), which mediates the inhibition of gene expression by competing with the transcription machinery for promoter binding and, thus, transcription initiation. Notably, CRISPR/Cas is only one of several described approaches for the manipulation of gene expression. Here, we targeted neurons with recombinant Adeno-associated viruses to induce either CRISPRi or RNA interference (RNAi), a well-established method for impairing de novo protein biosynthesis by using cellular regulatory mechanisms that induce the degradation of pre-existing mRNA. We specifically targeted hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, which are widely expressed in neuronal tissues and play essential physiological roles in maintaining biophysical characteristics in neurons. Both of the strategies reduced the expression levels of three HCN isoforms (HCN1, 2, and 4) with high specificity. Furthermore, detailed analysis revealed that the knock-down of just a single HCN isoform (HCN4) in hippocampal neurons did not affect basic electrical parameters of transduced neurons, whereas substantial changes emerged in HCN-current specific properties.
Highlights
Different short hairpin RNA (shRNA) molecules were designed complementary to the target hyperpolarization-activated and cyclic nucleotide-gated (HCN) channelencoding mRNA molecules
We examined two independent experimental approaches, i.e., RNA interference (RNAi) and CRISPR interference (CRISPRi), in order to reduce the expression of HCN channel-encoding genes at the mRNA level without altering the gene’s nucleotide sequence
The main finding of our study was that both procedures reduced hcn transcript levels in transgenic cell-lines, primary hippocampal neurons (PHNs), and organotypic hippocampal slice cultures (OHSCs) for each of the three targeted HCN isoforms
Summary
Information processing in the nervous system relies on the coordinated activity of electrical and chemical signals. Many ion channels that actively contribute to the electrical properties of neurons have been intensively studied by electrophysiological, biochemical, pharmacological, as well as molecular biological methods (for reviews see: [1,2,3,4]). One gene family has been uncovered encoding ion channels with exceptional activation properties, i.e., hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels (for reviews, see: [5,6]). These channels are referred to as ‘pacemaker’ channels, since they are known to largely control the repetitive beating of the heart and to contribute to rhythmic neuronal activity [7]
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