Showing 1 - 7 of 7 results
Dynamic Fas signaling network regulates neural stem cell proliferation and memory enhancement.
Activation of Fas (CD95) is observed in various neurological disorders and can lead to both apoptosis and prosurvival outputs, yet how Fas signaling operates dynamically in the hippocampus is poorly understood. The optogenetic dissection of a signaling network can yield molecular-level explanations for cellular responses or fates, including the signaling dysfunctions seen in numerous diseases. Here, we developed an optogenetically activatable Fas that works in a physiologically plausible manner. Fas activation in immature neurons of the dentate gyrus triggered mammalian target of rapamycin (mTOR) activation and subsequent brain-derived neurotrophic factor secretion. Phosphorylation of extracellular signal-regulated kinase (Erk) in neural stem cells was induced under prolonged Fas activation. Repetitive activation of this signaling network yielded proliferation of neural stem cells and a transient increase in spatial working memory in mice. Our results demonstrate a novel Fas signaling network in the dentate gyrus and illuminate its consequences for adult neurogenesis and memory enhancement.
Non-invasive optical control of endogenous Ca2+ channels in awake mice.
Optogenetic approaches for controlling Ca2+ channels provide powerful means for modulating diverse Ca2+-specific biological events in space and time. However, blue light-responsive photoreceptors are, in principle, considered inadequate for deep tissue stimulation unless accompanied by optic fiber insertion. Here, we present an ultra-light-sensitive optogenetic Ca2+ modulator, named monSTIM1 encompassing engineered cryptochrome2 for manipulating Ca2+ signaling in the brain of awake mice through non-invasive light delivery. Activation of monSTIM1 in either excitatory neurons or astrocytes of mice brain is able to induce Ca2+-dependent gene expression without any mechanical damage in the brain. Furthermore, we demonstrate that non-invasive Ca2+ modulation in neurons can be sufficiently and effectively translated into changes in behavioral phenotypes of awake mice.
Locally Activating TrkB Receptor Generates Actin Waves and Specifies Axonal Fate.
Actin waves are filamentous actin (F-actin)-rich structures that initiate in the somato-neuritic area and move toward neurite ends. The upstream cues that initiate actin waves are poorly understood. Here, using an optogenetic approach (Opto-cytTrkB), we found that local activation of the TrkB receptor around the neurite end initiates actin waves and triggers neurite elongation. During actin wave generation, locally activated TrkB signaling in the distal neurite was functionally connected with preferentially localized Rac1 and its signaling pathways in the proximal region. Moreover, TrkB activity changed the location of ankyrinG--the master organizer of the axonal initial segment-and initiated the stimulated neurite to acquire axonal characteristics. Taken together, these findings suggest that local Opto-cytTrkB activation switches the fate from minor to major axonal neurite during neuronal polarization by generating actin waves.
Noninvasive optical activation of Flp recombinase for genetic manipulation in deep mouse brain regions.
Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp-dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.
Optogenetic control of endogenous Ca(2+) channels in vivo.
Calcium (Ca(2+)) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis. However, study of Ca(2+) responses has been hampered by technological limitations of existing Ca(2+)-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca(2+) levels through activation of Ca(2+)-selective endogenous Ca(2+) release-activated Ca(2+) (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca(2+) levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca(2+)-associated processes and facilitate screening for drug candidates that antagonize Ca(2+) signals.
Light-inducible receptor tyrosine kinases that regulate neurotrophin signalling.
Receptor tyrosine kinases (RTKs) are a family of cell-surface receptors that have a key role in regulating critical cellular processes. Here, to understand and precisely control RTK signalling, we report the development of a genetically encoded, photoactivatable Trk (tropomyosin-related kinase) family of RTKs using a light-responsive module based on Arabidopsis thaliana cryptochrome 2. Blue-light stimulation (488 nm) of mammalian cells harbouring these receptors robustly upregulates canonical Trk signalling. A single light stimulus triggers transient signalling activation, which is reversibly tuned by repetitive delivery of blue-light pulses. In addition, the light-provoked process is induced in a spatially restricted and cell-specific manner. A prolonged patterned illumination causes sustained activation of extracellular signal-regulated kinase and promotes neurite outgrowth in a neuronal cell line, and induces filopodia formation in rat hippocampal neurons. These light-controllable receptors are expected to create experimental opportunities to spatiotemporally manipulate many biological processes both in vitro and in vivo.
Reversible protein inactivation by optogenetic trapping in cells.
We present a versatile platform to inactivate proteins in living cells using light, light-activated reversible inhibition by assembled trap (LARIAT), which sequesters target proteins into complexes formed by multimeric proteins and a blue light-mediated heterodimerization module. Using LARIAT, we inhibited diverse proteins that modulate cytoskeleton, lipid signaling and cell cycle with high spatiotemporal resolution. Use of single-domain antibodies extends the method to target proteins containing specific epitopes, including GFP.