KR102578795B1 - Optogenetically Activatable Fas Receptor and Use Thereof - Google Patents

Abstract

The present invention relates to a fusion protein containing an optogenetically activatable Fas receptor and its use. Specifically, the present invention relates to a fusion protein containing an optogenetically activatable Fas receptor by light irradiation by combining a photo-induced dimer forming protein. It relates to a protein and its use for regulating the activity of the Fas protein, improving cognitive or memory function, and improving neurogenesis in the brain.

Description

Optogenetically Activatable Fas Receptor and Use Thereof}

The present invention relates to a fusion protein containing an optogenetically activatable Fas receptor and its use. Specifically, the present invention relates to a fusion protein containing an optogenetically activatable Fas receptor by light irradiation by combining a photo-induced dimer forming protein. It relates to a protein and its use for regulating the activity of the Fas protein, improving cognitive or memory function, and improving neurogenesis in the brain.

Cell signaling networks can exhibit complex responses to various external stimuli. Recent advances in optogenetics have made it possible to manipulate the components of the signaling pathway in a spatio-temporal manner, making it possible to determine how signal transduction is dynamically regulated. Each component of the signaling pathway is known to respond differently to different modes of stimulation. Optogenetic studies of signaling networks can demonstrate not only spatiotemporal regulation of signaling pathways, but also signaling dysfunction seen in many diseases, in addition to consequences at the cellular and organ levels (L. J. Bugaj et al., Cancer mutations and targeted drugs can disrupt dynamic signal encoding by the Ras-Erk pathway. Science 361, eaao3048 (2018)).

Fas receptor (Fas/CD95/APO-1) signaling exhibits a complex network with diverse downstream outputs. Fas receptors belong to the TNF receptor superfamily, each of which acts as a death receptor involved in the initiation of the extrinsic apoptotic pathway. Activation of Fas not only induces apoptosis but can also induce survival-promoting signals such as JNK, NFκ, and AKT-mTOR, depending on the type and situation of the cell. Pro-survival signaling induced by Fas is frequently confirmed in the brain in various lesion states, and overexpression of Fas is confirmed in various neurological diseases such as inflammatory diseases, neurodegenerative diseases, and cerebral ischemia.

However, there is controversy regarding the mode of activation of Fas downstream components in these disease states. The involvement of hippocampal Fas activation in cognitive function and memory has long been controversial, because both cognitive improvements and mental impairments have been observed in experiments performed using Fas deletion or knockout mice.

In particular, it has been known that Fas is involved in nerve regeneration in the cerebral hippocampus in cases of brain disease, but the detailed mechanism is not yet known in detail due to limitations in research methods. Additionally, there has been controversy about how spatial memory governed by the hippocampus in the diseased brain is affected by Fas.

Under this technical background, the inventors of the present application developed an optogenetically activatable Fas receptor and confirmed that it can regulate the activity of Fas protein, improve cognitive or memory function, and enhance neurogenesis in the brain. The invention was completed.

The purpose of the present invention is to provide a fusion protein containing an optogenetically activatable Fas receptor.

The purpose of the present invention is to provide a polynucleotide encoding a fusion protein containing the optogenetically activatable Fas receptor.

The object of the present invention is to provide a composition for regulating the activity of Fas protein, a composition for improving cognitive or memory function, or a brain fusion protein containing the optogenetically activatable Fas receptor or a polynucleotide encoding the same. The object is to provide a composition for improving neurogenesis.

In order to achieve the above object, the present invention includes a membrane-anchoring sequence (lyn); Fas receptor (Fas cell surface death receptor) or its cytoplasmic domain; and a fusion protein that is optogenetically activatable by light irradiation, including a light-induced dimer forming protein.

The present invention also provides a polynucleotide encoding the fusion protein.

The present invention also provides a composition for controlling the activity of Fas protein, which includes the fusion protein or a polynucleotide encoding the same, and irradiates light that can induce oligomerization of the light-induced dimer formation protein.

The present invention also provides repetitive activation of the optogenetically activatable fusion protein, which includes the fusion protein or a polynucleotide encoding the same, and repeatedly irradiates light that can induce oligomerization of the light-induced dimer formation protein. Provides a composition for improving cognition or memory.

The present invention also provides repetitive activation of the optogenetically activatable fusion protein, which includes the fusion protein or a polynucleotide encoding the same, and repeatedly irradiates light that can induce oligomerization of the light-induced dimer formation protein. Provides a composition for improving neurogenesis in the brain.

Figure 1 shows the results of development and verification of an optogenetically activable Fas receptor.
Figure 2 shows the results demonstrating a dynamic signaling network in the dentate gyrus by optogenetic activation of Fas.
Figure 3 shows the results demonstrating that optogenetic stimulation of Fas in immature neurons induces BDNF secretion and can act as a paracrine mediator.
Figure 4 shows results showing that repetitive activation of Fas can induce hippocampal neurogenesis in adults.
Figure 5 shows results showing that repetitive activation of the Fas signaling network can induce an increase in temporary memory ability.
Figure 6 shows the results of Fas signaling molecules and cellular dynamics in the adult hippocampal dentate gyrus (DG) according to long-term repetitive activation state.
Figure 7 shows results showing that optoFAS can cause sensitive and specific cell death in various types of cells.
Figure 8 shows the time-dependent expression results of the nestin promoter in the dentate gyrus.
Figure 9 shows results showing that OptoFAS can specifically induce the activation of mTORC1.
Figure 10 shows results showing that different modes of stimulation can induce different responses in the Fas signaling network.
Figures 11A to 11C show results showing that Fas, AKT-mTOR, and ERK pathways are activated in human disease samples and animal models.
Figure 12 shows the results of immunocytochemical marker expression for differentiated neurons in vitro .
Figure 13 shows qRT-PCR results of genes known to be affected by Fas activation.
Figure 14 shows the results of blocking the Fas signaling network in the dentate gyrus by inhibitor treatment.
Figure 15 shows the results of in vitro confirmation of the mTOR-BDNF-ERK axis between immature neurons and neural stem cells.
Figure 16 shows the results of analysis of locomotor function and anxiety index by open field test.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

Activation of Fas (CD95) is observed in a variety of neurological diseases and can induce both apoptosis and pro-survival outputs, but nevertheless it is not well known how Fas signaling operates dynamically in the hippocampus. didn't

The inventors of the present application developed an optogenetically activatable Fas module (optoFAS) comprising a membrane anchoring sequence (lyn), the cytoplasmic domain of Fas (cyFAS), the PHR domain of cryptochrome 2 (CRY2PHR) and EGFP. Optogenetic dissection of signaling networks allows molecular-level elucidation of cellular responses or cell fates, including signaling dysfunctions seen in many diseases. Through this, there are no side effects that may occur when treating drugs or using genetically modified animal models, and the activity of Fas protein can be controlled only by light stimulation without interfering with physiological phenomena.

Through the optogenetically activatable Fas module (optoFAS), it was confirmed that several signaling pathways are activated sequentially in the cerebral hippocampus by providing light stimulation to the cerebrum and controlling the activity of Fas protein in a spatiotemporal manner. Specifically, activation of Fas in immature neurons of the dentate gyrus induced activation of mTOR and subsequent secretion of brain-derived neurotrophic factor (BDNF). Under long-term Fas activation, phosphorylation of Erk was induced in neural stem cells.

Repetitive activation of the signaling network resulted in proliferation of neural stem cells and a transient increase in spatial working memory in mice. These research results demonstrated the Fas signaling network in the dentate gyrus and confirmed the results of adult neurogenesis and memory enhancement.

Based on this, the present invention, in one aspect, includes a membrane-anchoring sequence (lyn); Fas receptor (Fas cell surface death receptor) or its cytoplasmic domain; and a fusion protein that is optogenetically activatable by light irradiation, including a light-induced dimer forming protein.

The present invention also relates to a polynucleotide encoding a fusion protein from another aspect.

The present invention also, from another aspect, relates to a composition comprising a fusion protein or a polynucleotide encoding the same and for controlling the activity of Fas protein by light irradiation.

The present invention also provides, from another perspective, a composition comprising the fusion protein or a polynucleotide encoding the same and improving cognition or memory through repeated activation of the optogenetically activatable fusion protein by repeated irradiation of light. It's about.

In another aspect, the present invention includes the fusion protein or a polynucleotide encoding the same, and improves neurogenesis in the brain through repeated activation of the optogenetically activatable fusion protein by repeated irradiation of light. It relates to a composition for doing so.

In one embodiment, the membrane anchoring sequence is a sequence included for myristoylation, a lipid specific to the N terminus of the Fas receptor, and may include, for example, the amino acid sequence of SEQ ID NO: 1 (MGCIKSKGKDSAGADSAGSAGS) .

In one embodiment, with respect to the Fas receptor or the cytoplasmic domain thereof, the Fas receptor may include, for example, the amino acid sequence of SEQ ID NO: 2, and the cytoplasmic domain of the Fas receptor may include, for example, the amino acid sequence of SEQ ID NO: 3. It may contain an amino acid sequence.

The light-induced dimer forming protein may refer to a protein that forms a homodimer or a heterodimer with a partner protein when light is stimulated. “Heterodimer” may mean that two different proteins interact to form a complex. “Homodimer” may mean that two identical proteins interact with each other to form a complex.

The light-induced dimer forming proteins include, for example, CIB (cryptochrome-interacting basic-helix-loop-helix protein), CIBN (N-terminal domain of CIB), Phy (phytochrome), PIF (phytochrome interacting factor), FKF1 ( It may be Flavin-binding, Kelch repeat, F-box 1), GIGANTEA, CRY (chryptochrome), or PHR (phytolyase homolgous region), but is not limited thereto. The light-induced dimer forming protein may specifically be the PHR domain (CRY2PHR), which is the N-terminal region of CRY (chryptochrome) 2, a cryptochrome protein, and is a photolyase homology region.

For example, the light-induced dimer forming protein may include the amino acid sequence of SEQ ID NO: 4.

In some cases, it may additionally contain a fluorescent protein. The fluorescent protein is, for example, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and red fluorescent protein (red fluorescent protein). , RFP), orange fluorescent protein (OFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), far-red fluorescent protein (far-red fluorescent protein) or tetra It may be a cysteine motif (tetracystein motif), but is not limited thereto.

The present invention refers to a polynucleotide encoding a fusion protein, which is used interchangeably with “nucleotide”, “nucleotide sequence”, “nucleic acid”, and “oligonucleotide”. It may include polymeric forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The polynucleotide is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units, include not only natural nucleotides but also analogues with modified sugar or base sites. The sequence of the polynucleotide may be modified. The modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

The present invention relates to a vector containing the above polynucleotide. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

As used herein, the term “vector” refers to a means for expressing a gene of interest in a host cell, including a plasmid vector; cosmid vector; Includes viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated viral vectors. In the vector, the polynucleotide is operably linked to a promoter.

“Operably linked” means a functional linkage between an expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites) and another polynucleotide, whereby the control sequence is linked to the other polynucleotide. It regulates the transcription and/or translation of the sequence.

When a prokaryotic cell is used as a host, a strong promoter capable of advancing transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. Additionally, for example, in the case of eukaryotic cells as hosts, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human heroglobin promoter, and human muscle creatine promoter) or mammalian cell genomes Promoters derived from animal viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV , the promoter of Moloney virus, the promoter of Epstein-Barr virus (EBV), and the promoter of Roux Sarcoma virus (RSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The vector contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. There is a resistance gene.

The present invention relates to cells transformed with the above-mentioned vectors. The cells may be, but are not limited to, prokaryotic, yeast, or higher eukaryotic cells.

Bacillus genus strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringensis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus Prokaryotic host cells such as Proteus mirabilis and Staphylococcus (e.g., Staphylocus carnosus) can be used.

However, animal cells are of greatest interest, and examples of useful host cell lines include COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0 , U20S, or HT1080, but is not limited thereto.

The cells can be cultured in various media. Any commercially available medium can be used as a culture medium without limitation. All other necessary supplements known to those skilled in the art may also be included in suitable concentrations. Culture conditions, such as temperature, pH, etc., are already used with host cells selected for expression and will be clear to those skilled in the art.

The fusion protein may have impurities removed by, for example, centrifugation or ultrafiltration, and the resulting product may be purified using, for example, affinity chromatography. Additional other purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc.

The composition of the present invention includes a fusion protein or a polynucleotide encoding the same, and may additionally include a pharmaceutically acceptable carrier for administration of the composition.

The polynucleotide encoding the fusion protein can be delivered through viral delivery means or non-viral delivery means. The viral delivery vehicle may include a viral vector. Viral vectors may include, but are not limited to, for example, Adeno-Associated Viral Vector (AAV), Adenoviral Vector (AdV), Lentiviral Vector (LV), or Retroviral Vector (RV).

Non-viral delivery vehicles may include, for example, episomal vectors containing viral replicons. Non-viral delivery means may include delivery in the form of mRNA, RNP (Ribonucleoprotein), or delivery by carrier. Delivery in the form of mRNA may include synthetic modified mRNA or self-replicating RNA (srRNA). As a carrier, it may include nanoparticles, cell penetrating peptides (CPP) or polymers.

Delivery means usable in the present invention may include, for example, vectors. “Vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. For example, nucleic acid molecules that are single-stranded, double-stranded or partially double-stranded; A nucleic acid molecule comprising one or more free, non-free (e.g., circular) ends; Nucleic acid molecules, including DNA, RNA, or both; and various other polynucleotides known in the art.

For example, the vector is a “plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.

The vectors can be delivered in vivo or into cells through electroporation, lipofection, viral vectors, nanoparticles, as well as PTD (Protein translocation domain) fusion protein methods, respectively.

The compositions, methods and uses of the present invention can be administered in sufficient or effective amounts to a subject in need thereof. An ‘effective amount’ or ‘sufficient amount’ is an amount that, in single or multiple doses, alone or in combination with one or more other therapeutic compositions, protocols, or treatment regimens, benefits the subject for any period of time or provides an expected or desired result in the subject. Indicates quantity. It can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, administration time, administration route, excretion rate, and response sensitivity.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

1. Manufacturing example and experimental method

(1) Plasmid construction

To construct optoFas, the myristoylation sequence (Lyn) and the PHR domain of cryptochrome 2 were amplified by polymerase chain reaction (PCR) and inserted into pEGFP-N1 (Clontech) using NheI/XhoI and BamHI/AgeI digestions. The cytoplasmic domain of Fas was PCR amplified from HeLa cell cDNA and inserted into the Lyn-PHR-EGFP construct using XhoI/EcoRI digestion. The following primers were used: F: 5'-GTAGCTAGCCACC ATGGGATGTATAAAATCAAAAG-'3, R: 5'-GTACTCGAGCGCACTACCAGCACTACCAG-'3 Lyn; F: 5'-GTAGGATCCCATGAAGATGGACAAAAAGACCA-'3, R: 5'-GTAACCGGTGCGTACACGGCAGCACCGATC-'3 PHR; F: 5'-GTACTCGAGAAGAGAAAGGAAGTACAGAAAACATGCAGA-'3, R: 5'-GTAGAATTCTGACCAAGCTTTGGATTTCATTT-'3 Fas. AAV-MAG-optoFAS was constructed by Gibson Assembly Cloning (New England Biolabs) and used to replace EGFP with PCR-amplified optoFas in the pAAV-MAG-EGFP vector (provided by M. Klugmann, University of New South Wales). AAV-Nestin-Cre was generated by PCR amplification of the Nestin promoter from pNestin-EGFP (Addgene #38777) and Cre from pCAG-iCre (Addgene #89573). The products were digested with MluI/XbaI and EcoRI/AgeI, respectively, and inserted into the pAAV-CaMKIIa-EGFP vector (Addgene #50469). The following primers were used: F: 5'-GTAACGCGTGGAGCAGGAGAAACAGGGCC-'3, R: 5'-GTATCTAGAAAGTCTTGGAGCCACCGC-'3 (Nestin). F: 5'-ATGCAGAATTCTTAGTCCCCATCCTCGAGCAG-'3, R: 5'-ATGCAACCGGTGCCACCATGCCCAAGAAGAAG-'3 Cre. AAV-hSyn-DIO-optoFAS was constructed by replacing EGFP with optoFas using PCR amplification and NheI/AscI digestion in the pAAV-hSyn-DIO-EGFP vector (Addgene #50457). The following primers were used: F: 5'-GTAGCTAGCCACCATGGGATGTATAAAATCAAAAGG-'3, R: 5'-GTAGGCGCGCCCGGCCGCTTTACTTGTACAGC-'3. AAV-CAG-optoFas and AAV-CAG-EGFP were constructed by Gibson Assembly Cloning, replacing the XbaI/EcoRV sequence in the AAV-CAG-FLEX-EGFP vector (Addgene #28304) with optoFas or EGFP in the pEGFP-N1 vector It was used to The following primers were used: F: 5'-GTGTGACCGGCGGCTCTAGAGCTAGCCACCATGGGATGTATAAAATCAA-'3, R: 5'-AGGTTGATTCCGGAGATATCGCGGCCGCTTTTACTT-'3 (optoFAS). F: 5'-GTGTGACCGGCGGCTCTAGAGCTAGCCACCATGGTGAGCAAGGGCGAGG-'3, R: 5'-AGGTTGATTCCGGAGATATCGCGGCCGCTTTTACTT-'3 (EGFP). Lenti-Nestin-EGFP was generated by Gibson Assembly Cloning. The fragment was PCR amplified from pNestin-EGFP and inserted into pLenti-CamKIIa-optoSTIM1 using PacI/EcoRI digestion. The following primers were used: F: 5'-CAGAGATCCAGTTTGGTTAATTAACTGCAGGTCGACGGAGC-'3, R: 5'-GATTATCGATAAGCTTGATATCGAATTCGCGGCCGCTTTACTTG-'3.

(2) qRT-PCR

Cultured neurons were incubated in 12-well plates. AAV was applied to the plate at MOI20,000. After light stimulation or treatment with soluble Fas ligand, neurons were recovered, centrifuged, and stored at -80°C until RNA was isolated. Hippocampal tissue was dissected on ice with a razor in the dark immediately after light stimulation. The collected tissues were quickly frozen and stored at -80°C until RNA isolation.

RNA was isolated and purified using the PureLink RNA Mini Kit (cat.No.12183018A; Ambion) according to the manufacturer's protocol. The obtained mRNA (1 μg) was used to prepare cDNA with the SuperScript III First-Strand Synthesis System (cat. No. 18080-051; Invitrogen). qRT-PCR was performed using Solg 2X Real-Time PCR Smart Mix (cat. No. SRH72-M40h; SolGent) and EvaGreenTM dye. A three-step cycling protocol recommended by the manufacturer was used for 50 cycles. The following primers were used: rat BDNF, F: 5'-GGTTCGAGAGGTCTGACGAC-'3, R: 5'-CAAAGGCACTTGACTGCTGA-'3; Rat IGF-1, F: 5'-CCTGGGCTTTGTTTTCACTTCGG-'3, R: 5'-TTTGTAGGCTTCAGCGGAGCAC-'3; Rat IL-6, F: 5'-CAGGAACGAAAGTCAACTCCA-'3, R: 5'-ATCAGTCCCAAGAAGGCAACT-'3; Rat NeuroD, F: 5'-GTTCCACGTCAAGCCGCCGC-'3, R: 5'-AGCGGCACCCGAGGAGAAGA-'3; Rat Hes5, F: 5'-CGGCACCAGCCCAACTCCAA-'3, R: 5'-GGAACTGCACCGCCTCCTGC-'3. Mouse BDNFI, F: 5'-CCTGCATCTGTTTGGGGAGAC-'3, R: 5'-CGCCTTCATGCAACCGAAGTAT-'3; Mouse BDNFII, F: 5'-ACCTTTTCCTCCTCCTGCG-'3, R: 5'-TGGATGAAGTACTACCACCTCGG-'3; Mouse BDNFIII, F: 5'-TGAGACTGCGCTCCACTCCC-'3, R: 5'-CGCCTTCATGCAACCGAAGTAT-'3; Mouse BDNFIV, F: 5'-CAGAGCAGCTGCCTTGATGTTT-'3, R: 5'-CGCCTTCATGCAACCGAAGTAT-'3; Mouse BDNFV, F: 5'-CTCTGTGTAGTTTCATTGTGTGTTC-'3, R: 5'-GAAGTGTACAAGTCCCGCGTCCTTA-'3; Mouse BDNFVI, F: 5'-ACAATGTGACTCCACTGCCGG-'3, R: 5'-CGCCTTCATGCAACCGAAGTAT-'3; Mouse BDNFVII, F: 5'-ACTTACAGGTCCAAGGTCAACG-'3, R: 5'-GGACAGAGGGTCGGATACAG-'3; Mouse BDNFVIII, F: 5'-ATGACTGTGCATCCCAGGAGAAA-'3, R: 5'-CGCCTTCATGCAACCGAAGTAT-'3; Mouse BDNFIXa, F: 5'-CCCAAAGCTGCTAAAGCGGGAGGAAG-'3, R: 5'-GAAGTGTACAAGTCCCGCGTCCTTA-'3; Mouse total BDNF, F: 5'-TCGTTCCTTTCGAGTTAGCC-'3, R: 5'-TTGGTAACGGCACAAAAC-'3.

(3) LED stimulation and cell survival assay.

Light stimulation of HeLa cells and oligodendrocytes was performed using the TouchBright W-96 LED Excitation System (Live Cell Instrument), and cell viability was tested.

Light stimulation of HeLa cells and oligodensites was tested for cell viability. By adjusting the power intensity of the LED system, cells were irradiated with light of 0~20μW/mm2 and 470nm. In most experiments, a light intensity of 5 μW/mm2 and a duty cycle of 33% (1 second light, 2 seconds dark) were used. To induce apoptosis in oligodendrocytes, they were light stimulated for 12 hours.

Cell Counting Kit-8 (cat. No. CK04-01; Dojindo Molecular Technologies) was used to analyze the viability of 96-well plate cells. Light-stimulated cells were cultured in medium containing 10% Cell Counting Kit-8 solution according to the manufacturer's instructions, and absorbance was measured at 450 nm using a VERSA max microplate reader (Molecular Devices).

(4) Live cell imaging and photoactivation

Live cell imaging was performed using a Nikon A1R confocal microscope equipped with a CFI PlanApo objective at 60x magnification. Multi-color images were retrieved using lasers of 488 nm, 561 nm and 647 nm. The microscope stage is equipped with a Chamlide TC system (Live Cell Instrument) used to maintain temperature (37°C) and CO ₂ concentration (10%).

For JNK-KTR and ERK-KTR experiments and pS6 immunocytochemistry experiments, neurons were incubated in Ringer's solution (145mM NaCl, 2.5mM KCl, 10mM glucose, 10mM HEPES, 2mM CaCl2 and 1mM MgCl2) for 1-3 hours. were left to starve. For light activation, a 488 nm laser emitted from a Galvano scanner was integrated into a hybrid confocal scan head equipped with a high-speed selector (Nikon). Most in vitro experiments used a light intensity of 5 μW/mm2 and a frequency of 1 second every 3 minutes (duty cycle 0.55%) for activation, unless specifically specified. Local stimulation of HeLa cells used a laser intensity of 5 μW/mm2 and a duration of 11 seconds every 5 minutes. The stimulation area was adjusted based on the information obtained using Nikon imaging software (NIS-elements AR 64-bit version 4.10, Laboratory Imaging).

(5) Mouse light stimulation.

Blue light was irradiated in vivo using an optical fiber coupled to a blue diode 473 nm laser (MBL-III-473m; CNI). Optical stimulation of the mouse brain was performed at an intensity of 5 mW/mm2 and a frequency of 500 mHz (33% duty cycle). In the repetitive stimulation experiment, light was irradiated for 4 hours per day for 3-5 days.

(6) Behavioral experiment

To physiologically induce BDNF secretion, mice were placed on a Mouse RotaRod (Ugo Basile) and exercised for 1 hour.

Before behavioral testing, all mice were handled for 10 minutes per day for 3 days.

Hippocampus-dependent spatial working memory was assessed by measuring spontaneous alternation performance during the Y maze task. The Y maze chamber is made of acrylic and has three arms spaced 120 degrees apart. Each arm measures 40 cm long, 17 cm high, 4 cm wide (bottom) and 13 cm wide (top). Mice were placed at the end of one maze arm and allowed to freely explore the maze for 15 minutes. The movement order and total items for each arm were analyzed in the recorded video. Invasion was considered to have occurred when the mouse's hind limbs were placed on the arm. The rate of spontaneous alterations is the number of triads containing entries into all three arms/maximum possible alterations (the total number of triads containing entries - 2) It was calculated as total number of arms entered - 2)

The open field test was conducted in an acrylic chamber measuring 42 x 42 x 42 cm. Each mouse was placed in the center of the chamber and allowed to move freely for 30 minutes. By analyzing the video recording data using OptiMouse, the total distance traveled and the time spent in the central area (20 x 20 cm) were determined. Body position was detected using OptiMouse software algorithm 6 with search parameters. Average speed was calculated as total distance/time. Anxiety index was calculated as follows: (total time - time spent in central zone) / time spent in central zone.

2. Example

(1) Development and validation of an optogenetically activatable Fas receptor (Figure 1)

(A) Schematic diagram of optoFAS and downstream signaling pathways. (B) Representative confocal images of apoptosis induced in optoFAS transfected HeLa cells exposed to light stimulation and Lyn-cyFAS-EGFP transfected cells exposed to light stimulation, compared to optoFAS transfected cells cultured in dark conditions. . Scale bar, 50 μm. (C) Quantification of apoptosis induction in HeLa cells over time. Labels: OptoFAS Light, cells expressing OptoFAS exposed to continuous blue light at 5 μW/mm2; OptoFAS Dark Identical cells cultured in dark conditions; FKBP/FRB + Rap, cells transfected with Lyn-cyFAS-FKBP-EGFP and Lyn-cyFAS-FRB-mCherry treated with 500 nM rapamycin. FKBP/FRB-Rap, same cells without rapamycin treatment. SFasL, treated with 100 ng/ml of soluble Fas ligand. Cisplatin, treated with cisplatin 10 μg/ml (n≥80 cells per group). (D) Representative confocal images of HeLa cells transfected with apoptotic optoFAS and caspase-3 biosensors, showing activation of caspase-3. Scale bar, 20 μm. (E) Representative confocal image of HeLa cells transfected with apoptotic optoFAS and caspase-8 biosensors, showing activation of caspase-8. Scale bar, 20 μm. (F) Quantification of caspase-3 biosensor activity for cells shown in (D). (G) Quantification of caspase-8 biosensor activity for cells shown in (E). (H) Activation of JNK in the presence and absence of illumination in optoFAS transfected DIV 7 cultured cerebral hippocampal neurons as determined by the JNK-KTR sensor (n = 20 cells included in both light and dark groups). (I) Representative immunocytochemical staining images of optoFAS transfected cells with or without light stimulation showing expression of pS6. Scale bar, 50 μm. (J) Quantification of data shown in (I). Data are expressed as mean ± SEM. n≥100 cells per each group. Statistical analysis used two-way ANOVA (ANOVA). ****P<0.0001; ns not significant.

(2) Optogenetic activation of Fas reveals a dynamic signaling network in the dentate gyrus (Figure 2)

(A) Schematic and timeline representing the steps of the virus injection experiment. (B) Representative images showing the expression of AAV-Nestin-Cre and AAV-hSyn-DIO-optoFAS in the dentate gyrus. Scale bar, 200 μm. (C) Representative images of changes in pS6 and pErk levels in the optoFAS transduced dentate gyrus when illumination duration was increased. The inset shows a representative focal area. Scale bar, 100 μm (inset, 50 μm). (D) Quantification of pS6-positive cells in (C). Data are expressed as mean ± SEM. n≥4 mice were included in each condition. One-way ANOVA was used for statistical analysis. ****P<0.0001;*P<0.05; ns not significant. (E) Quantification of pErk-positive cells in (C). Data are expressed as mean ± SEM. n≥4 mice were included in each condition. One-way ANOVA was used for statistical analysis. ns is not significant. ****P<0.0001;*P<0.05. (F) Representative image showing colocalization of GFP + cells and pS6 + cells. Scale bar, 20 μm. (G) Quantification of (F). Data are expressed as mean ± SEM. n = 6 mice. One section per mouse was randomly selected. n≥20 pS6 + cells were included in each section. (H) Representative image showing the lack of colocalization of GFP + cells and pErk + cells. Scale bar, 20 μm. (I) Quantification of data shown in (H). Data are expressed as mean ± SEM. n = 6 mice. One section per mouse was randomly selected. n≥20 pErk + cells were included in each section. (J) pErk-positive cells in the subgranular zone counterstained for neural stem cell markers SOX2 (top) and DCX (bottom). Arrows show cells with colocalizing signals. Scale bar, 50 μm. (K) Proportion of SOX2-positive or DCX-positive cells in all pErk-positive cells. Data are expressed as mean ± SEM. n = 5 mice. One section per mouse was randomly selected. n≥20 pErk + cells were included in each section. (L) Schematic and timeline showing rapamycin-induced blockade of the mTOR pathway in vivo . (M) Representative images of the effect of mTOR blockade on pS6 and pErk levels. Scale bar, 100 μm. (N) Quantification of pS6 positive cells in (M) top row. Data are expressed as mean ± SEM. n = 5 mice per group, 4 sections per mouse were randomly selected. Unpaired two-tailed t-test was used for statistical analysis. ****P<0.0001. (O) (M) Quantification of pErk-positive cells in the bottom row. Data are expressed as mean ± SEM. n = 5 mice per group, 4 sections per mouse were randomly selected. Unpaired two-tailed t-test was used for statistical analysis. ****P<0.0001.

(3) Optogenetic stimulation of Fas in immature neurons induces BDNF secretion, acting as a paracrine mediator (Figure 3)

(A) Schematic and timeline of viral transduction and light stimulation of cultured hippocampal neurons. (BD) Levels of BDNF (B), IGF-1 (C), and IL-6 (D) in optoFAS-transduced neurons exposed to light stimulation, GFP-transduced neurons exposed to light stimulation, and GFP-transduced neurons treated with soluble Fas ligand. qRT-PCR results. Data are expressed as mean ± SEM. n = 3 per group. One-way analysis of variance was used for statistical analysis. ns not significant. **P<0.01;***P<0.001;****P<0.0001. (E) Schematic and timeline of viral transduction, rapamycin treatment, and light stimulation of cultured hippocampal neurons. (FG) qRT-PCR results of BDNF (F) and IGF-1 (G) in optoFAS transduced neurons exposed to rapamycin-treated light stimulation and GFP-transduced neurons treated with soluble Fas ligand. Data are expressed as mean ± SEM. n = 3 per group. Statistical analysis used two-way ANOVA. ns not significant. **P<0.01. (H) BDNF ELISA results for supernatants of optoFAS transduced neurons exposed to light stimulation, GFP transduced neurons exposed to light stimulation, and GFP transduced neurons treated with Fas ligand. Data are expressed as mean ± SEM. n≥3 per group. Statistical analysis used two-way ANOVA. ns not significant. ****P<0.0001. (I) Schematic showing how immature neurons in Bdnf ^flox/flox mice are targeted to acquire defects in BDNF transcription through transduction of AAV-Nestin-Cre and AAV-hSyn-DIO-optoFAS. (J) Representative images of pS6 (top) and pErk (bottom) immunohistochemical staining in light-exposed Bdnf ^flox/flox mice transduced with AAV-Nestin-Cre and AAV-hSyn-DIO-optoFAS. The insets show representative focal areas. Scale bar, 100 μm (inset, 50 μm). (K) Quantification of pS6 positive cells in (J) and comparison with cells from Bdnf ^+/+ mice. Data are expressed as mean ± SEM. n = 5 mice. Four sections were randomly selected per mouse. Unpaired two-tailed t-test was used for statistical analysis. ns not significant. (L) Quantification of pErk-positive cells from (J) and comparison with cells from Bdnf ^+/+ mice. Data are expressed as mean ± SEM. n = 5 mice. Four sections were randomly selected per mouse. An unpaired two-tailed t-test was used for statistical analysis. ***P<0.001. (M) Schematic diagram and timeline of virus injection and qRT-PCR experimental procedures on mouse hippocampal tissue. (N) Total BDNF mRNA expression levels in the brain in optoFAS transduced mice exposed to light stimulation and in exercised mice. Data are expressed as mean ± SEM. n = 3 per group. One-way analysis of variance was used for statistical analysis. ****P<0.0001; ns not significant. ***P<0.001. (O) mRNA expression levels for splicing variants of BDNF in the brains of optoFAS-transduced and exercised mice. Data are expressed as mean ± SEM. n = 3 per group. (P) Timeline of viral injection and qRT-PCR experimental procedures on mouse hippocampal tissue after exposure to repetitive stimulation. (Q) Total BDNF mRNA expression levels in optoFAS transduced hippocampus subjected to single or repeated light stimulation. Data are expressed as mean ± SEM. n = 3 per group. One-way analysis of variance was used for statistical analysis. *P<0.05, **P<0.01. (R) mRNA expression levels of BDNF II, IV, VI, and VIII in optoFAS transduced hippocampus subjected to single or repeated light stimulation. Data are expressed as mean ± SEM. n = 3 per group. One-way analysis of variance was used for statistical analysis. ****P<0.0001.

(4) Repetitive activation of Fas induces adult hippocampal neurogenesis (Figure 4)

(A) Schematic diagram and timeline of experiments used to evaluate proliferation of neural stem cells after single or repeated light stimulation of the brains of mice transduced with optoFAS. (B) Representative images of neural stem cell proliferation upon light stimulation assessed by BrdU immunohistochemical staining (left column; insets; representative focal areas) and images of optoFAS expression in each case (right column). Scale bar, 100 μm (inset, 50 μm). (C) BrdU-positive cells in the subgranular area after 5 rounds of light stimulation in the section counterstained for SOX2 and DCX (the inset shows the area surrounded by a square, and arrows indicate cells with colocalization signals). Scale bar, 100 μm (inset, 20 μm). (D) Quantification of BrdU + SOX2 + or BrdU + DCX + cells in the dentate gyrus after single or repeated light stimulation of optoFAS-transduced mouse brain. Data expressed as mean ± SEM. n = 5 mice in each condition. One-way analysis of variance was used for statistical analysis. ns is not significant. ***P<0.001; ****P<0.0001. (E) Timeline of experiments to assess adult neurogenesis by repetitive optoFAS stimulation. (F) Representative image of BrdU-positive cells counterstained for calretinin. The right window shows a magnification of the image box area on the left. Arrows show cells with co-localizing signals. Scale bar, 50 μm (left), 20 μm (right). (G) Quantification of data shown in (F). Data are expressed as mean ± SEM. n = 5 mice in each condition. Unpaired two-tailed t-test was used for statistical analysis. ***P<0.001. (H) Timeline of experiments used to evaluate neural stem cell proliferation upon blockade of the mTOR pathway in mouse brains transduced with optoFAS. (I) Representative image of BrdU-positive cells in the optoFAS transduced dentate gyrus treated with rapamycin (the inset shows the area enclosed by a square). Scale bar, 100 μm (inset, 50 μm). (J) Quantification of data shown in (I). Data expressed as mean ± SEM. n = 5 mice in each condition. One-way analysis of variance was used for statistical analysis. ****P <0.0001 ***P<0.001.

(5) Repeated activation of the Fas signaling network induces an increase in temporary memory function (Figure 5)

(A) Schematic and timeline of behavioral tests used to evaluate light-stimulated optoFAS transduced mice. (B) Spontaneous changes in the Y maze test in optoFAS transgenic mice exposed to single or repeated light stimulation. Data expressed as mean ± SEM. n = 5–6 mice per group. One-way analysis of variance was used for statistical analysis. ns is not significant. **P<0.01. (C) Total entries of the Y maze test between optoFAS transduced mice exposed to single or repeated light stimulation. Data expressed as mean ± SEM. n = 5–6 mice per group. One-way analysis of variance was used for statistical analysis. ns is not significant. (D) Timeline of behavioral tests in mice transduced with optoFAS or GFP after prolonged incubation after light stimulation. (E) Spontaneous alterations on the Y maze test between optoFAS or GFP transduced mice with varying incubation periods after five rounds of light stimulation. Data expressed as mean ± SEM. n = 5 mice per group. Statistical analysis used one-way analysis of variance. ns is not significant. *P<0.05. (F) Total entries in the Y maze test between optoFAS or GFP transduced mice with varying incubation periods after five rounds of light stimulation. Data expressed as mean ± SEM. n = 5 mice per group. One-way analysis of variance was used for statistical analysis. ns is not significant. (G) Timeline of behavioral testing of optoFAS transgenic mice exposed to light stimulation while mTOR pathway is blocked. (H) Spontaneous alterations on the Y maze test between repetitively stimulated and rapamycin-treated optoFAS transgenic mice. Data expressed as mean ± SEM. n = 5-6 mice per group. One-way analysis of variance was used for statistical analysis. ns is not significant. **P<0.01. (I) Total entries of the Y maze test between repetitively stimulated and rapamycin-treated optoFAS transduced mice. Data expressed as mean ± SEM. n = 5–6 mice per group. One-way analysis of variance was used for statistical analysis. ns is not significant. (J) Timeline of behavioral tests in optoFAS transduced mice exposed to light stimulation in the presence of BDNF/TrkB blockade. (K) Spontaneous alterations in the Y maze test between repeated stimulation and ANA-12 treated optoFAS transgenic mice. Data are expressed as mean ± SEM. n = 5 mice per group. One-way analysis of variance was used for statistical analysis. ns is not significant. *P<0.05. (L) Total entries of the Y maze test between repeated stimulation and ANA-12 treated optoFAS transgenic mice. Data expressed as mean ± SEM. n = 5 mice per group. One-way analysis of variance was used for statistical analysis. ns is not significant.

(6) Summary of Fas signaling molecules and cellular dynamics in the adult hippocampal dentate gyrus (DG) following long-term repetitive activation states (Figure 6)

(A) Summary of the molecular dynamics of Fas signaling in the DG upon optogenetic Fas activation. Transient Fas activation in immature neurons of the DG induces mTOR pathway activation in these cells. As the result of activation is prolonged, pErk of neural stem cells increases through BDNF secreted from Fas-activated immature neurons. NSC neural stem cells; IN immature neurons; MN mature neurons. (B) Summary of histological and behavioral outcomes observed after repeated activation of Fas signaling in the DG. Repeated activation of the Fas-mTOR (immature neuron)-BDNF VI-pErk (neural stem cell) paracrine pathway in the DG induces proliferation of neural stem cells and temporarily increases spatial working memory ability. IN immature neurons; NSC neural stem cells.

(7) Sensitive and specific induction of apoptosis by optoFAS in various types of cells (Figure 7)

(A) Representative confocal images showing apoptosis of optoFAS-transfected HeLa cells exposed to increasing light intensity by LED illumination. Scale bar, 50 μm. (B) Quantification of apoptosis assessed using a cell counting kit. Data are expressed as mean ± SEM. n = ^1x104 cells per well. 3 wells per group. One-way analysis of variance was used for statistical analysis. **P<0.01;*P<0.05;***P<0.001;****P<0.0001. (C) Representative confocal images of stimulation of the center of the field over time, showing selective induction of apoptosis in HeLa cells transfected with optoFAS. Scale bar, 50 μm. (D) Quantification of apoptosis induction in stimulated and non-stimulated areas. Data are expressed as mean ± SEM. n = 5 randomly selected image fields. n≥20 cells are included in each field. Unpaired two-tailed t-test was used for statistical analysis. ****P<0.0001. (E) Representative confocal images of selective apoptosis of optoFAS-transfected HeLa cells co-cultured with Lyn-mCherry-transfected cells and photostimulated. Scale bar, 20 μm. (F) Quantification of data shown in (E). Data are expressed as mean ± SEM. Four imaging fields were randomly selected in each group. n≥20 cells are included in each field. Unpaired two-tailed t-test was used for statistical analysis. ****P<0.0001. (G) Cell death induced in cultured rat hippocampal neurons (HcNs) and cortical astrocytes transfected with optoFAS by light stimulation. Data are expressed as mean ± SEM. n≥20 cells/experimental group (indicated by dots). One-way analysis of variance was used for statistical analysis. ****P<0.0001; ns not significant. (H) Apoptosis induced in DIV7 optoFAS transfected cultured hippocampal neurons exposed to increasing light intensity. Data are expressed as mean ± SEM. n≥20 cells/experimental group (indicated by dots). One-way analysis of variance was used for statistical analysis. ns not significant. (I) Apoptosis induced in optoFAS-transfected cultured astrocytes exposed to increasing light intensity. Data are expressed as mean ± SEM. n≥20 cells/experimental group (indicated by dots). One-way analysis of variance was used for statistical analysis. ns not significant. (J) optoFAS transfected cultured oligodendrocytes exposed to light stimulation, optoFAS transfected oligodendrocytes incubated in dark conditions, GFP transfected oligodendrocytes exposed to light stimulation, and soluble Fas ligand ( Levels of induced apoptosis confirmed by cell counting kit analysis in GFP-transfected oligodendrocytes treated with 100 ng/ml). Data are expressed as mean ± SEM. n = 1x ¹⁰ cells per well, n≥7 wells per group. One-way analysis of variance was used for statistical analysis. ****P<0.0001; ns not significant. *P<0.05. (K) qRT-PCR results showing mRNA levels of c-FLIP in cultured rat hippocampal neurons, cortical astrocytes, and oligodendrocytes. Data are expressed as mean ± SEM. n≥3 per group. One-way analysis of variance was used for statistical analysis. **P<0.01;***P<0.001.

(8) Time-dependent expression of the nestin promoter in the dentate gyrus (Figure 8)

(A) Schematic diagram and timeline of virus injection and expression analysis. (B) Representative images of Lenti-Nestin-GFP expression (left) and co-immunohistochemical staining images of GFP and DCX (right) at days 7, 14, 21, and 28. Scale bar, 100 μm. (C) Quantification of co-localization of GFP + cells and DCX + cells in (B). Data are expressed as mean ± SEM. n = 3 mice per group. Four slices 200 μm apart were selected from the brain of each mouse. One-way analysis of variance was used for statistical analysis. ****P<0.0001.

(9) OptoFAS specifically induces the activation of mTORC1 (Figure 9)

(A) Representative images of pS6 levels confirmed by pS6 S240/244 immunohistochemical staining in the optoFAS transduced dentate gyrus with increasing light duration. The inset shows representative foci. Scale bar, 100 μm (inset, 50 μm). (B) Comparison of the number of pS6 positive cells confirmed by immunostaining using pS6 S235/236 and pS6S240/244 antibodies. Data are expressed as mean ± SEM. n≥4 mice were included in each condition of pS6S235/236. n = 3 mice were included in each condition on pS6 S240/244. Unpaired two-tailed t test was used for statistical analysis. ns not significant.

(10) Different modes of stimulation induce different responses in the Fas signaling network (Figure 10)

(A) pErk (left), pS6 (center), and optoFAS expression in response to 0.5 h of light stimulation followed by 3.5 h of dark condition incubation (top) and 0.25 h of light stimulation followed by 3.75 h of dark condition incubation (bottom). Representative image for (right). Scale bar, 100 μm. (B) Quantification of pErk-positive cells in (A). Data expressed as mean ± SEM. n more than 5 mice per group. One-way analysis of variance was used for statistical analysis. *P<0.05; ****P<0.0001. (C) Quantification of pS6-positive cells in (A). Data expressed as mean ± SEM. n more than 5 mice per group. One-way analysis of variance was used for statistical analysis. **P<0.01; ****P<0.0001. (D) Representative images of pErk (left) and pS6 (right) in response to single or repeated stimulation after 12 h of incubation in dark conditions. The inset shows representative foci. Scale bar, 100 μm (inset, 50 μm). (E) Quantification of pErk-positive cells in (D). Data expressed as mean ± SEM. n = 3 mice per group. One-way analysis of variance was used for statistical analysis. ***P<0.001; ****P<0.0001. (F) (D) Quantification of pS6 positive cells. Data expressed as mean ± SEM. n = 3 mice per group. One-way analysis of variance was used for statistical analysis. ns not significant.

(11) Activation of Fas, AKT-mTOR and ERK pathways in human disease samples and animal models (FIGS. 11A to 11C)

(A) Fas pathway (M9503) gene signature derived from mild Alzheimer's patient group and healthy control group (GSE84422). (B) Fas pathway (M9503) gene signature derived from a mild Alzheimer's patient group and a severe Alzheimer's group (GSE84422). (C) Fas pathway (M9503) gene signature from severe Alzheimer's patient group and healthy control group (GSE84422). (D) AKT1_SIGNALING_VIA_MTOR-upregulated (M15377) gene signature from mild Alzheimer's patient group and healthy control group (GSE84422). (E) AKT1_SIGNALING_VIA_MTOR-upregulated (M15377) gene signature derived from mild and severe Alzheimer's patient group (GSE84422). (F) AKT1_SIGNALING_VIA_MTOR (M15377) gene signature derived from a group of severe Alzheimer's patients and a healthy control group (GSE84422). (G) MAPK upregulated (M13863) gene signature from mild Alzheimer's patient group and healthy control group (GSE84422). (H) MAPK upregulated (M13863) gene signature from mild and severe Alzheimer's group (GSE84422). (I) MAPK pathway (M13863) gene signature from severe Alzheimer's patient group and healthy control group (GSE84422). (J) Representative images showing Fas immunohistochemical staining of wild-type mouse brain (left), mouse brain injected intracerebroventricularly with lipopolysaccharide (LPS) (center), and 5XFAD mouse brain (right). (K) Representative histological images from LPS (top) and normal saline (NS bottom) injected mice co-stained for pErk and DCX. The inset shows an enlarged view of the boxed area. Scale bar, 100 μm (inset, 50 μm). (L) Representative histological images of 5XFAD (top) and wild-type (WT bottom) mice costained for pErk and SOX2. The inset shows an enlarged view of the boxed area. Scale bar, 100 μm (inset, 50 μm). (M) Quantification of data shown in (K). Data are expressed as mean ± SEM. n = 5 mice per group, 4 sections per mouse were randomly selected. An unpaired two-tailed t-test was used for statistical analysis. *P<0.05. (N) Quantification of data shown in (L). Data are expressed as mean ± SEM. n = 4 mice per group, 4 sections per mouse were randomly selected. An unpaired two-tailed t-test was used for statistical analysis. **P<0.01.

(12) Expression of immunocytochemical markers for differentiated neurons in vitro (Figure 12)

(A) Representative blanks of immunocytochemical staining of SOX2 (left column), DCX (center column), and calretinin (right column) in cultured hippocampal neurons at DIV1 (column 1), DIV7 (column 2), and DIV14 (column 3). Focus image. Scale bar, 50 μm. (B) Quantification of SOX2 (red), DCX (blue) and calretinin (green) intensities in the studied neurons. Data expressed as mean ± SEM. n = 4 wells in each group (all wells contain at least 20 cells). One-way analysis of variance was used for statistical analysis. ****P<0.0001; *P<0.05.

(13) qRT-PCR results of genes known to be affected by Fas activation (Figure 13)

(A) Schematic diagram and timeline of viral transduction and light stimulation of cultured hippocampal neurons. (B, C) qRT-PCR for NeuroD (B) and Hes5 (C) in optoFAS transduced neurons exposed to light stimulation, GFP transduced neurons exposed to light stimulation, and GFP transduced neurons treated with soluble Fas ligand. result. Data expressed as mean ± SEM. n = 3 per group. One-way analysis of variance was used for statistical analysis. ns not significant. **P<0.01; ***P<0.001; ****P<0.0001.

(14) Blockade of the Fas signaling network in the dentate gyrus by inhibitor treatment (Figure 14)

(A) Schematic diagram and timeline of viral transduction, rapamycin treatment, and light stimulation of cultured hippocampal neurons for blockade of the mTOR signaling pathway. (B) Representative immunocytochemical staining images for pS6 in rapamycin-treated and optoFAS-transduced neurons exposed to light, optoFAS-transduced neurons incubated in dark conditions, and GFP-transduced neurons treated with soluble Fas ligand. Scale bar, 50 μm. (C) Quantification of data shown in (B). Data expressed as mean ± SEM. n≥91 cells/group. One-way analysis of variance was used for statistical analysis. ns not significant. **P<0.01; ****P<0.0001. (D) Timeline of a blocking experiment of BDNF/TrkB in the mouse dentate gyrus transduced with optoFAS. (E) Representative images of pErk levels in optoFAS-transduced dentate gyrus treated with ANA-12 and exposed to light stimulation. Scale bar, 100 μm. (F) Quantification of pErk-positive cells in (E). Data expressed as mean ± SEM. n = 5 mice, 4 sections per mouse randomly selected. An unpaired two-tailed t-test was used for statistical analysis. *P<0.05

(15) In vitro confirmation of the mTOR-BDNF-ERK axis between immature neurons and neural stem cells (Figure 15)

(A) Representative images showing signal changes from the ERK-KTR sensor among DIV1-2 neurons in response to BDNF in the presence and absence of rapamycin. Scale bar, 20 μm. (B) Quantification of the normalized cytoplasmic to nuclear ratio of the ERK-KTR sensor under conditions (A). Data expressed as mean ± SEM. n = 18,18,23 and 20 cells in each group. Unpaired two-tailed t-test was used for statistical analysis. ****P<0.0001. (C) Representative images showing signal changes from the ERK-KTR sensor among DIV1-2 neurons in response to supernatants concentrated from Fas-stimulated DIV7 neurons. Scale bar, 20 μm. (D) Quantification of data shown in (C). Data expressed as mean ± SEM. n = 10 cells per group. Unpaired two-tailed t-test was used for statistical analysis. ****P<0.0001. (E) Representative images showing signal changes in the DIV1-2 neuronal ERK-KTR sensor in response to concentrated supernatants from Fas-stimulated DIV7 neurons preincubated in the presence and absence of rapamycin and subsequent exogenous BDNF treatment. Scale bar, 20 μm. (F) Quantification of data shown in (E). Data expressed as mean ± SEM. n = 7,8,7 and 7 cells in each group.

(16) Analysis of locomotor function and anxiety index by open field test (Figure 16)

(A-D) Total distance traveled (A), average speed (B), and time spent in the central area of the open field chamber (C) anxiety 1 day after stimulation in mice transduced with optoFAS or GFP exposed to single or repetitive light stimulation. Exponential (D) analysis. Data expressed as mean ± SEM. n = 5-6 per group. One-way analysis of variance was used for statistical analysis. ns not significant. (E-H) Total distance moved (E), average speed (F), time spent in the central region of the open field chamber (G), and 1 week after stimulation in mice transduced with optoFAS or GFP exposed to single or repetitive light stimulation. Anxiety Index (H) Analysis. Data expressed as mean ± SEM. n = 5 per group. One-way ANOVA used for statistical analysis. ns not significant. (I-L) Total distance traveled (I), average speed (J), time spent in the central region of the open field chamber (K), and 2 weeks after stimulation in mice transduced with optoFAS or GFP exposed to single or repetitive light stimulation. Anxiety Index (L) Analysis. Data expressed as mean ± SEM. n = 5 per group. One-way analysis of variance was used for statistical analysis. ns is not significant. (M-P) Total distance traveled (M), average speed (N), and time spent in the central area of the open field chamber (O) 4 weeks after stimulation in mice transduced with optoFAS or GFP exposed to single or repetitive light stimulation. and anxiety quotient (P) analysis. Data expressed as mean ± SEM. n = 5 per group. One-way analysis of variance was used for statistical analysis. ns not significant. (Q-T) Total distance traveled (Q), average speed (R), time spent in the central area of the open field chamber (S), and 1 day after stimulation in optoFAS transgenic mice treated with rapamycin and exposed to repetitive light stimulation. Anxiety quotient (T) analysis. Data expressed as mean ± SEM. n = 5-6 per group. One-way analysis of variance was used for statistical analysis. ns is not significant. (U-X) Total distance traveled (U), average velocity (V), time spent in the central area of the open field chamber (W), and anxiety index ( X) Analysis. Data expressed as mean ± SEM. n = 5 mice per group. One-way analysis of variance was used for statistical analysis. ns not significant.

3. Results

(1) Optogenetically activatable Fas receptor

We developed an optogenetically activatable Fas module (optoFAS) containing a membrane anchoring sequence (lyn), the cytoplasmic domain of Fas (cyFAS), the PHR domain of cryptochrome 2 (CRY2PHR), and EGFP (Figure 1A). The receptor can be activated using CRY2PHR, which homooligomerizes in response to blue light (wavelength 488 nm). When transfected into HeLa cells, optoFAS induced apoptosis in response to light (Figure 1B). Cell death cannot be induced by transfection of a CRY2PHR-deficient module (Lyn-cyFAS-EGFP), indicating that activation of optoFAS depends on the oligomerization properties of CRY2PHR. We also confirmed that optoFAS functions with high sensitivity and specificity (Figure 7A-F). The level of apoptosis induced was superior to that obtained through conventional chemical induction systems or treatment with soluble Fas ligand (100 ng/ml) or cisplatin (10 μg/ml, Figure 1C). Additionally, upon light irradiation, the activation of downstream caspases could be visualized through real-time imaging using a fluorescent protein exchange (FPX) biosensor (Figure 1D-G).

Neurons and astrocytes are known to resist apoptosis by expressing inhibitory molecules at various stages of the Fas-induced apoptosis pathway. One such molecule is c-FLIP, whose function is a decoy molecule for activated Fas receptor complexes to block subsequent caspase activity. We confirmed that 7 days in vitro (DIV) neurons and astrocytes did not undergo apoptosis upon Fas activation and showed high levels of c-FLIP expression, as expected (Figure 7G, H, I, K). Instead, light stimulation induced activation of non-apoptotic signaling components in optoFAS-transfected cultured hippocampal neurons. Activation of JNK was observed in optoFAS-transfected DIV 7 neurons upon light irradiation using the JNK-KTR sensor (Figure 1H). Activation of the AKT-mTOR pathway can be confirmed by pS6 immunocytochemical (ICC) staining (Figure 1I, J). On the other hand, oligodendrocytes, which are susceptible to apoptosis upon Fas activation with low levels of c-FLIP expression, showed reduced cell survival upon optoFas activation (Fig. 7J,K). Taken together, these results show that the newly developed optoFAS can induce apoptotic and non-apoptotic signals in appropriate cell types and thus reflect a physiological mechanism of action.

(2) Dynamic Fas signaling network in the dentate gyrus identified by optogenetic activation.

To investigate the dynamics of Fas signaling in the brain in relation to cognition and memory, we injected adeno-associated viral vector (AAV) into the hippocampal dentate gyrus (DG) of 8-week-old mice. By mixing the Nestin-Cre vector and the hSyn-DIO vector, we were able to target expression in immature neurons of the DG approximately 3 weeks after injection (Figures 2A, B, Figure 8A-C). Optical stimulation of the optoFAS transfected area can be used to induce various activation modes for downstream signaling constructs. pS6, a downstream signaling component of Akt-mTOR, was induced immediately after light irradiation, specifically at 0.5 h (Fig. 2C, D), but pErk was activated later, with maximum levels seen after 4 h of irradiation (Fig. 2C, E). Considering that pS6-positive cells were identified by immunohistochemical (IHC) staining of pS6S240/S244 and pS6S235/236, specific induction of mTORC1 by optoFAS activation can be suggested (Figures 9A,B). pS6 positive cells express GFP and represent immature neurons transduced with optoFAS (Figure 2F,G). However, pErk-positive cells observed in the subgranular zone (SGZ) lacked optoFAS (Figures 2H,I). IHC staining of SOX2 and DCX (doublecortin) revealed that pErk-positive cells in the SGZ were in the developmental stage of neural stem cells or progenitor cells ( Fig. 2J,K ). The 0.5 hour light condition followed by the 3.5 hour dark condition did not induce pErk to levels comparable to those seen after 4 hours of stimulation. This means that continuous stimulation of 4 h is required to induce maximal pErk levels in the SGZ (Fig. 10A,B). Maintaining mice under 0.5 hour light conditions followed by 3.5 hour dark conditions resulted in a decrease in pS6 levels, suggesting that pS6 levels decreased after peak activation at 0.5 hours. 0.25 hours of light stimulation did not induce pS6 to levels comparable to those seen after 0.5 hours of light stimulation. This shows that at least 0.5 hours of stimulation is required to maximally activate the mTOR pathway (Figure 10A,C). When incubated under dark conditions for 12 hours after light irradiation, the elevated pS6 and pErk levels dropped to the levels of unstimulated samples. However, when light stimulation was repeated (4 hours, 3 to 5 times per day), the number of pErk-positive cells in the SGZ rose to a certain level, despite incubation in the dark for 12 hours (Figure 10D, E). , indicating that repeated stimulation of Fas in immature neurons induces prolonged ERK activation of neural stem cells. Meanwhile, when accompanied by repeated stimulation and dark incubation, pS6 levels were similar to those of unstimulated samples (Figures 10D, F). Intraperitoneal (ip) injection of rapamycin aimed at blocking the mTOR pathway in optoFAS transduced mice inhibited pS6 in the granule cell layer (GCL) of the hippocampal DG and pErk in the SGZ (Figure 2L-O). These results imply that Fas-mediated activation of the mTOR pathway in immature neurons is associated with a subsequent elevation of pErk levels among neural stem cells.

Evidence for activation of mTOR and Erk can be found in human disease samples and mouse models showing overexpression of Fas. Gene set enrichment analysis (GSEA) was performed on the hippocampus of Alzheimer's patients (GEO: GSE84422) using a public database (https://www.ncbi.nlm.nih.gov/geo). Alzheimer's patients were classified into mild (clinical dementia grade, CDR 0.5 and 1) and severe (CDR 3-5) cases, and analysis of the FAS-PATHWAY gene signature (M9503) showed that mild Alzheimer's cases compared with healthy controls and severe cases. An increase in Fas expression was confirmed in the cases (Figure 11a-c). The gene signature in which AKT-MTOR was upregulated was enriched in cases of mild Alzheimer's disease compared to healthy controls and severe cases (Figure 11A). Additionally, MAPK upregulated signatures were enriched in cases of mild Alzheimer's disease compared to healthy controls and severe cases (Figure 11B). The upregulated gene signatures of Akt-mTOR and MAPK pathways in Fas-overexpressing human hippocampal samples are consistent with the identified results. Additionally, activation of pErk in the SGZ was observed in the brains of mice exposed to intracerebroventricular injection of lipopolysaccharide (LPS) and in the brains of Alzheimer's model (5XFAD) mice, showing high levels of Fas expression (Figure 11c).

(3) BDNF secretion from immature neurons upon Fas activation

To identify some mediating factors between mTOR activation in immature neurons and Erk activation in neural stem cells, we investigated three candidate molecules known to contribute to the regulation of neural stem cell populations. In the cell culture system, DIV 7 hippocampal neurons showed a rapid decrease in the neural stem cell markers SOX2 and DCX, but an increase in calretinin expressed in differentiating immature neurons (Figure 12A, B). Expression of brain-derived neurotrophic factor (BDNF) mRNA gradually increased in optoFAS transduced DIV 6 neurons after light irradiation ( Fig. 3A,B ). Another candidate, IGF-1 (insulin-like growth factor 1), steadily increased, and another candidate, IL-6, decreased (Figure 3C, D). To confirm the activation of Fas by light irradiation, we analyzed transcriptional changes in NeuroD (Figure 13A,B) and Hes5 (Figure 13C) by light stimulation. Treatment of cultured cells with rapamycin, an mTOR pathway inhibitor, inhibited both pS6 levels (Figures 14A-C) and mRNA expression of BDNF (Figures 3E, F). This is consistent with a previous report that BDNF transcription is downstream of Fas network mTOR signaling (21). Increased transcription of IGF-1 by optoFAS activation was not affected by rapamycin treatment (Figure 3G). Secretion of BDNF upon light irradiation by optoFAS-transduced neurons was confirmed by performing BDNF ELISA using supernatants from cultured neural plates (Figure 3H). Administration of ANA-12, a BDNF/TrkB inhibitor, to optoFAS-injected mice significantly reduced the number of pErk-positive cells in the SGZ after light stimulation, supporting the hypothesis that BDNF induces activation of pErk in neural stem cells in immature neurons ( Figure 14D-F). Culture of neural stem cells at the neuron or neural progenitor stage (DIV 1-2) induced ERK activation in response to exogenous BDNF treatment (200 ng/ml), regardless of whether they were pre-incubated with rapamycin (Figure 15A,B). Treatment with concentrated supernatants from light-stimulated immature neurons (DIV 7) induced ERK activation in DIV1-2 neurons. Considering that activation of ERK did not result from DIV 7 neurons pretreated with ANA-12, it is likely that BDNF contained in the stimulated supernatant induced the observed changes in ERK levels (Figure 15C, D). Additionally, although recipient DIV 1 neurons still responded to exogenous BDNF, supernatants from DIV 7 neurons pretreated with rapamycin were unable to induce ERK activation (Fig. 15E,F), and ERK activation in neural stem cells was impaired in immature neurons. This means that it is a result of mTOR-induced BDNF secretion by Fas activation. To further investigate whether secretion of BDNF from immature neurons triggers pErk activation of neural stem cells in vivo , AAV-Nestin-Cre and AAV-hSyn-DIO-optoFAS were injected into the DG of BDNF-floxed mice (Bdnfflox/flox, Figure 3I). Although the number of pS6-positive cells in the GCL did not change significantly (Fig. 3J,K), Bdnfflox/flox mice had significantly fewer pErk-positive cells in the SGZ upon light irradiation compared with identical maternal mice (Fig. 3J,L). From these observations, we conclude that there is a paracrine signaling network in the DG through which prolonged Fas activation in immature neurons induces the release of BDNF and activates pErk in neural stem cells.

(4) BDNF VI transcription upon optogenetic activation of Fas in immature neurons

We confirmed how BDNF secreted from immature neurons can induce the activation of pErk, especially in neural stem cells. qRT-PCR revealed that total BDNF transcription was elevated by light stimulation in optoFAS transduced hippocampus tissue, reaching a level similar to that seen in the exercised hippocampus ( Fig. 3M, N ). However, unlike exercise, activation of optoFAS preferentially increased transcription of BDNF VI among BDNF splicing variants (Figure 3O). In the DG, repeated optoFAS stimulation (4 h each round) significantly increased BDNF VI transcript levels without significantly changing the total transcript levels of BDNF, regardless of the number of stimulation rounds (Fig. 3P–R). Considering that BDNF splicing variants show distinct subcellular localization and spatially restricted roles, it is likely that BDNF VI encodes spatial information and directs pErk activation only in the SGZ.

(5) Adult hippocampal neurogenesis induced by repetitive activation of Fas

Because ERK signaling plays an important role in cell proliferation, we investigated whether proliferation of neural stem cells could be induced through the discovered Fas signaling network. Eight-week-old mice transfected with OptoFAS or GFP were i.p. BrdU was injected and exposed to light stimulation (Figure 4A). Repeated light stimulation (4 hours per round) of the optoFAS-transduced DG induced proliferation of neural stem cells, which was confirmed by an increase in BrdU-positive cells (Figure 4B). IHC staining results for SOX-2 and DCX confirmed that the cells were neural stem cells or progenitor cells (Figures 4C, D). After 1 week of culture, optoFAS-stimulated mice showed a statistically significant increase in BrdU and calretinin double-positive cells in the DG, indicating that the proliferated stem cells differentiated and induced adult neurogenesis ( Fig. 4E-G ). Administration of rapamycin, which blocks the mTOR pathway, significantly reduced the number of BrdU-positive cells upon light stimulation, confirming that adult neurogenesis was induced by a novel Fas signaling network ( Fig. 4H-J ).

(6) Increased spatial memory ability by repetitive activation of the Fas signaling network

We investigated whether the Fas signaling network could influence hippocampus-dependent memory-related behavior. Since the DG is well known to be involved in spatial working memory, mice transduced with optoFAS or GFP were subjected to the Y maze test and changes in spontaneous alteration behavior by light stimulation were measured. monitored. When tested 1 day after stimulation, a single 4-hour light stimulation did not change spontaneous alterations in optoFAS-injected mice, but repeated stimulation (5 rounds) significantly increased the rate of spontaneous alterations at any given time point (Figure 5A , B). In repeatedly stimulated optoFAS transgenic mice, the elevation of spontaneous alterations was not observed in samples obtained at days 7, 14, and 28 after stimulation, indicating that the phenomenon was transient (Figure 5D,E). To confirm that the behavioral changes were mediated through the novel Fas signaling network, we blocked the mTOR pathway or BDNF/TrkB signaling using rapamycin (Figure 5G) or ANA-12 (Figure 5J), respectively. Treatment inhibited the elevation of spontaneous alterations in optoFAS-activated mice (Figures 5H,K). Total entries into the Y maze arm did not differ significantly between experimental groups (Figure 5C, F, I, L), and neither locomotor function nor anxiety levels, as determined through open field testing in mice, differed between experimental groups. (Figure 16A-X). Therefore, it was confirmed that repetitive activation of the identified Fas signaling network can cause a transient increase in mouse spatial working memory.

Despite the well-known inducible expression of Fas in brain diseases, it is not yet understood how Fas signaling works and whether it affects the phenotype. Here, by examining Fas signals in the brain using optogenetically activable Fas, it was possible to control the signaling network in space and time. Regarding the interaction between immature neurons and neural stem cells, we discovered a novel Fas signaling network in the DG. Transient activation of Fas in immature neurons activates only the mTOR pathway, whereas long-term activation induces both the mTOR pathway in Fas-expressing immature neurons and the ERK pathway (via secreted BDNF) in neural stem cells lacking Fas expression (Figure 6A) . Additionally, repetitive activation of the Fas signaling network can result in neural stem cell proliferation and transient increases in spatial working memory (Figure 6B).

Fas is overexpressed rather than absent in most brain diseases, reflecting the actual mechanism of action of Fas. In addition to the discovery of the Fas signaling network that induces adult neurogenesis, it was confirmed that neurogenesis is caused by repetitive activation rather than single transmission of the signaling network. This means that neural stem cell proliferation is the result of repeated warning signals in the Fas-induced diseased brain. Given that neural stem cells contribute to improved cognition through BDNF in the Alzheimer's brain, and given that BDNF is preferentially elevated in mice showing increased spontaneous alterations, BDNF VI improves spatial working memory in Fas-activated brains. It is assumed that there is a possibility of inducing . Levels of adult neurogenesis are known to be increased in acute neurological diseases such as stroke. There is some discussion about changes in adult neurogenesis in chronic diseases. We suggest that the identified Fas signaling network may be responsible for the increased adult neurogenesis observed in these diseases. Evidence suggests that Akt-mTOR and MAPK-up-signatures were observed only in mild Alzheimer's patients with increased Fas expression. Furthermore, problems with cognition and memory are rarely identified in the early stages of chronic neurological disease. In Alzheimer's patients, clinical stages of mild cognitive impairment (i.e., mildly affected memory levels) are usually seen early in the disease course. The same results have been reported for other chronic neurological diseases. Given that repeated activation of optoFAS resulted in increased episodic memory performance, repeated activation of Fas in the early course of the disease may help protect cognitive and memory functions.

Using an optogenetically activatable Fas receptor, we discovered a mechanism linking Fas activation in immature neurons to ERK activation in neural stem cells. We were able to see how the signal transduction network induces adult neurogenesis through spatiotemporal and repetitive activation realized only by optogenetics. We confirmed that the proposed Fas signaling network can temporarily increase spatial working memory through BDNF as a behavioral consequence of the network.

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Institute for Basic Science <120> Optogenetically Activatable Fas Receptor and Use Thereof <130> 063 <150> KR 20/046,780 <151> 2020-04-17 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> membrane-anchoring sequence <400> 1 Met Gly Cys Ile Lys Ser Lys Gly Lys Asp Ser Ala Gly Ala Asp Ser 1 5 10 15 Ala Gly Ser Ala Gly Ser 20 <210> 2 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> Fas Receptor <400> 2 Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala 1 5 10 15 Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser 20 25 30 Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn 35 40 45 Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro 50 55 60 Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro 65 70 75 80 Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95 Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110 Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120 125 Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 130 135 140 Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr 145 150 155 160 Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp 165 170 175 Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg 180 185 190 Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly 195 200 205 Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu 210 215 220 Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met 225 230 235 240 Thr Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu 245 250 255 Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu 260 265 270 Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys 275 280 285 Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys 290 295 300 Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser 305 310 315 320 Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val 325 330 335 <210> 3 <211> 145 <212> PRT <213> Artificial Sequence <220> <223> cytoplasmic domain of Fas Receptor <400> 3 Lys Arg Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn 1 5 10 15 Gln Gly Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val Ala Ile 20 25 30 Asn Leu Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly 35 40 45 Val Met Thr Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val 50 55 60 Asn Glu Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr 65 70 75 80 Ala Glu Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly 85 90 95 Lys Lys Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn 100 105 110 Leu Cys Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile 115 120 125 Thr Ser Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu 130 135 140 Val 145 <210> 4 <211> 498 <212> PRT <213> Artificial Sequence <220> <223> CRY2PHR <400> 4 Met Lys Met Asp Lys Lys Thr Ile Val Trp Phe Arg Arg Asp Leu Arg 1 5 10 15 Ile Glu Asp Asn Pro Ala Leu Ala Ala Ala Ala His Glu Gly Ser Val 20 25 30 Phe Pro Val Phe Ile Trp Cys Pro Glu Glu Glu Gly Gln Phe Tyr Pro 35 40 45 Gly Arg Ala Ser Arg Trp Trp Met Lys Gln Ser Leu Ala His Leu Ser 50 55 60 Gln Ser Leu Lys Ala Leu Gly Ser Asp Leu Thr Leu Ile Lys Thr His 65 70 75 80 Asn Thr Ile Ser Ala Ile Leu Asp Cys Ile Arg Val Thr Gly Ala Thr 85 90 95 Lys Val Val Phe Asn His Leu Tyr Asp Pro Val Ser Leu Val Arg Asp 100 105 110 His Thr Val Lys Glu Lys Leu Val Glu Arg Gly Ile Ser Val Gln Ser 115 120 125 Tyr Asn Gly Asp Leu Leu Tyr Glu Pro Trp Glu Ile Tyr Cys Glu Lys 130 135 140 Gly Lys Pro Phe Thr Ser Phe Asn Ser Tyr Trp Lys Lys Cys Leu Asp 145 150 155 160 Met Ser Ile Glu Ser Val Met Leu Pro Pro Pro Trp Arg Leu Met Pro 165 170 175 Ile Thr Ala Ala Ala Glu Ala Ile Trp Ala Cys Ser Ile Glu Glu Leu 180 185 190 Gly Leu Glu Asn Glu Ala Glu Lys Pro Ser Asn Ala Leu Leu Thr Arg 195 200 205 Ala Trp Ser Pro Gly Trp Ser Asn Ala Asp Lys Leu Leu Asn Glu Phe 210 215 220 Ile Glu Lys Gln Leu Ile Asp Tyr Ala Lys Asn Ser Lys Lys Val Val 225 230 235 240 Gly Asn Ser Thr Ser Leu Leu Ser Pro Tyr Leu His Phe Gly Glu Ile 245 250 255 Ser Val Arg His Val Phe Gln Cys Ala Arg Met Lys Gln Ile Ile Trp 260 265 270 Ala Arg Asp Lys Asn Ser Glu Gly Ala Glu Ser Ala Asp Leu Phe Leu 275 280 285 Arg Gly Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile Cys Phe Asn Phe 290 295 300 Pro Phe Thr His Glu Gln Ser Leu Leu Ser His Leu Arg Phe Phe Pro 305 310 315 320 Trp Asp Ala Asp Val Asp Lys Phe Lys Ala Trp Arg Gln Gly Arg Thr 325 330 335 Gly Tyr Pro Leu Val Asp Ala Gly Met Arg Glu Leu Trp Ala Thr Gly 340 345 350 Trp Met His Asn Arg Ile Arg Val Ile Val Ser Ser Phe Ala Val Lys 355 360 365 Phe Leu Leu Leu Pro Trp Lys Trp Gly Met Lys Tyr Phe Trp Asp Thr 370 375 380 Leu Leu Asp Ala Asp Leu Glu Cys Asp Ile Leu Gly Trp Gln Tyr Ile 385 390 395 400 Ser Gly Ser Ile Pro Asp Gly His Glu Leu Asp Arg Leu Asp Asn Pro 405 410 415 Ala Leu Gln Gly Ala Lys Tyr Asp Pro Glu Gly Glu Tyr Ile Arg Gln 420 425 430 Trp Leu Pro Glu Leu Ala Arg Leu Pro Thr Glu Trp Ile His His Pro 435 440 445 Trp Asp Ala Pro Leu Thr Val Leu Lys Ala Ser Gly Val Glu Leu Gly 450 455 460 Thr Asn Tyr Ala Lys Pro Ile Val Asp Ile Asp Thr Ala Arg Glu Leu 465 470 475 480 Leu Ala Lys Ala Ile Ser Arg Thr Arg Glu Ala Gln Ile Met Ile Gly 485 490 495 Ala Ala

Claims (13)

Cytoplasmic domain of the Fas receptor (Fas cell surface death receptor);
A membrane-anchoring sequence (lyn) comprising the amino acid sequence of SEQ ID NO: 1, bound to the cytoplasmic domain; and
A fusion protein comprising a light-induced dimer forming protein and capable of being optogenetically activated by light irradiation. delete delete The method of claim 1, wherein the light-induced dimer forming protein is CIB (cryptochrome-interacting basic-helix-loop-helix protein), CIBN (N-terminal domain of CIB), Phy (phytochrome), PIF (phytochrome interacting factor), Fusion proteins that are FKF1 (Flavin-binding, Kelch repeat, F-box 1), GIGANTEA, CRY (cryptochrome), or PHR (photolyase homolgous region). The fusion protein according to claim 1, wherein the light-induced dimer forming protein is the PHR domain of CRY (chryptochrome) 2 (CRY2PHR). The fusion protein according to claim 1, further comprising a fluorescent protein. The method of claim 6, wherein the fluorescent protein is green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and red fluorescent protein (red fluorescent protein). fluorescent protein (RFP), orange fluorescent protein (OFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), far-red fluorescent protein Or a fusion protein with a tetracysteine motif. The fusion protein according to claim 1, wherein the light can induce oligomerization of the light-induced dimer forming protein. A polynucleotide encoding the fusion protein according to any one of claims 1 and 4 to 8. A composition comprising the fusion protein of claim 1 or a polynucleotide encoding the same, and for controlling the activity of Fas protein by light irradiation. A composition comprising the fusion protein of claim 1 or a polynucleotide encoding the same, and for improving cognition or memory through repeated activation of the optogenetically activatable fusion protein by repeated irradiation of light. A composition comprising the fusion protein of claim 1 or a polynucleotide encoding the same, and for improving neurogenesis in the brain through repeated activation of the optogenetically activatable fusion protein by repeated irradiation of light. The composition according to claim 11 or 12, wherein the fusion protein is activated by repeated irradiation of blue light with an intensity of 5 mW/mm2 and a frequency of 500 mHz for 4 hours per day for 3-5 days.