Neurology Central

Flash! An optogenetics approach to reinforcing memory through modulating Ca2+

A novel hybrid protein, engineered by researchers at the Korea Advanced Institute of Science and Technology and the Institute for Basic Science (both Daejeon, South Korea), has exhibited effective modulation of Ca2+ levels through regulation of Ca2+ -selective endogenous Ca2+ release-activated Ca2+ channels (CRACs). The hybrid protein resulted in both qualitative and quantitative control of intracellular Ca2+ levels in a variety of biological systems, including human stem cells and memory-forming cells in mice. The results were published recently in Nature Biotechnology.

Ca2+ is involved in a wide range of essential cellular processes such as excitation, contraction, growth, differentiation and apoptosis. Although other modulators of Ca2+ channels have been established through the use of drugs and electro-stimulation, these methods for exploring Ca2+ signaling are often not accurate enough to produce reliable results.

The hybrid protein is composed of the photoreceptive protein Cry2 – extracted from the plant Arabidopsis thaliana – and the widely distributed STIM1 protein, which is found in most animals and is responsible for the opening of cellular Ca2+ channels. The resulting hybrid, named OptoSTIM1, was introduced into target cells.

Following application of blue light to OptoSTIM1-expressing cells, there was an observed increase in intracellular Ca2+ levels, due to an influx from outside of the cell; an influx of a greater volume than observed in previous studies utilizing optogenetic molecules. The researchers believe this enhanced influx is due to the tendency of Cry2 to cluster under blue light. “Our method worked better because other plant proteins are not as efficient as Cry2 at clustering.” commented Taeyoon Kyung (Korea Advanced Institute of Science and Technology). This clustering resulted in five- to ten-times more Ca2+ being detected than in previous studies. Similar Ca2+ influx was observed after applying blue light to zebrafish embryos containing OptoSTIM1, indicating that this modulatory effect is also effective in living cells.

In a further effort to determine if OptoSTIM1 would have an effect on intercellular Ca2+ signaling, researchers used human stem cells engineered to contain OptoSTIM1 and applied blue light to only one cell within a cluster. A delayed Ca2+ response was observed in other non-illuminated cells within the cluster, suggesting a level of intercellular signaling originating from the light-exposed cell.

Ca2+ release and regulation is an important aspect of many brain cell functions, including those in the hippocampus that controls memory. Researchers first engineered and cultured OptoSTIM1-containing hippocampal cells in vitro, in which a similar effect on Ca2+ channel regulation and Ca2+ influx was observed as in the aforementioned experiments with living cells. The researchers then implanted these modified hippocampal cells into mice and examined the effect on memory, comparing illuminated mice with non-illuminated mice – both containing OptoSTIM1. After exposure to conditioning cues followed by a fear stimulus, illuminated mice displayed a greater fear response (almost twofold) without the conditioning cue than non-light-stimulated mice, indicating that the illuminated mice had developed an increased ability to develop stimulus–response memory.

Given the essential and widespread role Ca2+ channels play in a plethora of essential biological processes, this novel and effective optogenetics approach provides a potential new avenue for research into therapeutic Ca2+ channel modulation and screening of potential new Ca2+-signaling antagonist drug candidates, as well as providing a new means by which to understand Ca2+ and Ca2+ channel regulation. Kyung remarked “There are diseases that result from dysfunction in cellular Ca2+ regulation, such as Alzheimer’s disease, so we can apply our system to those areas and hopefully in the near future help people to recover from those diseases.”

Sources: Institute for Basic Science Press Release; Kyung T, Lee S,  Kim JE et al. Optogenetic control of endogenous Ca2 channels in vivo. Nat. Biotechnol. doi:10.1038/nbt.3350 (2015) (Epub ahead of print).