glial differentiation

glial differentiation

Biomaterials 225 (2019) 119539 Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials Nongen...

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Biomaterials 225 (2019) 119539

Contents lists available at ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Nongenetic optical modulation of neural stem cell proliferation and neuronal/glial differentiation

T

Meitian Wanga,1, Zhiliang Xua,1, Qiao Liua, Wenjie Suna, Baichun Jianga, Kun Yanga, Jiangxia Lia, Yaoqin Gonga, Qiji Liua, Duo Liub, Xi Lia,c,∗ a

Key Laboratory of Experimental Teratology, Ministry of Education Department of Medical Genetics, School of Basic Medical Sciences, Shandong University Jinan, Shandong, 250012, China b State Key Laboratory of Crystal Materials, Shandong University Jinan, Shandong, 250100, PR China c Advanced Medical Research Institute, Shandong University Jinan, Shandong, 250012, China

HIGHLIGHTS

stem cells constitutionally express opsins for light responsiveness. • Neural LED modulates NSC behavior in vitro and in vivo by nongenetic stimulation. • Blue is responsible for the light-induced neuromodulation. • Melanopsin/TRPC6/Jab1 • UCNP-mediated NIR light noninvasively modulates in vivo NSC behavior. ARTICLE INFO

ABSTRACT

Keywords: Neural stem cell Photosensitive Modulation Proliferation Differentiation Upconversion nanoparticle-mediated

Photostimulation has been widely used in neuromodulation. However, existing optogenetics techniques require genetic alternation of the targeted cell or tissue. Here, we report that neural stem cells (NSCs) constitutionally express blue/red light-sensitive photoreceptors. The proliferation and regulation of NSCs to neuronal or glial cells are wavelength-specific. Our results showed a 4.3-fold increase in proliferation and 2.7-fold increase in astrocyte differentiation for cells under low-power blue monochromatic light exposure (455 nm, 300 μW/cm2). The melanopsin (Opn4)/transient receptor potential channel 6 (TRPC6) non-visual opsin serves as a key photoreceptor response to blue light irradiation. Two-dimensional gel electrophoresis coupled with mass spectrometry further highlighted the Jun activation domain-binding protein 1 (Jab1) as a novel and specific modulator in phototransduction pathways induced by blue light exposure. Quiescent adult NSCs reside in specific regions of the mammalian brain. Therefore, we showed that melanopsin/TRPC6 expressed in these regions and blue light stimulation through optical fibers could directly stimulate the NSCs in vivo. Upconversion nanoparticles (UCNPs) converted deep-penetrating near-infrared (NIR) light into specific wavelengths of visible light. Accordingly, we demonstrated that UCNP-mediated NIR light could be used to modulate in vivo NSC differentiation in a less invasive manner. In the future, this light-triggered system of NSCs will enable nongenetic and noninvasive neuromodulation with therapeutic potential for central nervous system diseases.

1. Introduction The cellular basis of neurogenesis consists of neural stem/progenitor cells (NSCs/NPCs). NSCs are undifferentiated precursors that retain the ability to proliferate and self-renew while being able to differentiate into the three major neural lineages: neurons, astrocytes, and

oligodendrocytes [1]. Accumulative evidence has supported the critical roles of NSC proliferation and differentiation in learning, memory, and mood regulation [2]. Recently, considerable efforts have been made in regulating the fate of NSCs for therapeutic applications in regenerative medicine [3]. There is also evidence that the dysregulation of NSCs may contribute to various brain disorders and the development of brain

Corresponding author. Department of Medical Genetics, School of Basic Medical Sciences, Shandong University, 44 Wen Hua Xi Road, Jinan, Shandong, 250012, PR China. E-mail address: [email protected] (X. Li). 1 These authors contributed equally to this work. ∗

https://doi.org/10.1016/j.biomaterials.2019.119539 Received 24 April 2019; Received in revised form 1 October 2019; Accepted 7 October 2019 Available online 08 October 2019 0142-9612/ © 2019 Elsevier Ltd. All rights reserved.

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tumors [4–6]. In summary, these findings suggest that the proliferation and neuronal/glial differentiation of NSCs are controlled by intricate molecular networks. However, despite intensive attempts to understand NSC functions in neurogenesis, treatments to influence NSC behavior are limited. Thus, non-pharmacological and nongenetic approaches to neuromodulation of NSCs are highly desirable. It would be particularly relevant to extensively explore the molecular regulation underlying the influence of such new approaches on the behavior of stem cells. Light is an invaluable tool for controlling the activity of cells. Existing optogenetics provides precise control over neuron cells and brain tissue. However, it is limited because most mature neural cells do not have photoreceptors and require over-expression of optogenetic proteins (such as ChR2 or NpHR) via lentivirus transfection [7]. In contrast to the vast amount of information related to genetically engineered photostimulation in mature cells, there is limited information about the response of stem cells to light stimulation and the developmental process that leads to neurogenesis in mammalian brains. Moreover, emerging evidence demonstrates that physiological sensory stimuli could alter microenvironment interactions during the developmental process of stem cells [8,9]. Therefore, it is essential to develop new optogenetic manipulation techniques to extend the range of responses of stem cells to light-based sensory stimulation. In this study, we explicitly addressed the issues of NSC behaviors and nongenetic optical modulation. First, we characterized NSCs’ constitutional expression of blue- and red-sensitive opsins, which are photoreceptors present within mammalian retinas [10]. Then, we used blue (455 nm) and red (635 nm) monochromatic light exposure to investigate the processing of stimuli that preferentially trigger cell proliferation and neuronal or glial (astrocyte) differentiation, respectively. We hypothesized that such light exposure would induce a sustained modulation of NSC behaviors and that these modulations would be wavelength-dependent. This would give insight into the establishment of nongenetic light modulation of NSC behaviors related to the neurogenesis process. Our results support the hypothesis and demonstrate that illumination can directly activate NSC activity in vitro and in vivo. Importantly, lanthanide-doped upconversion nanoparticles (UCNPs) could convert highly penetrating NIR light in vitro to visible light in vivo [11]. Furthermore, we developed a UCNP-mediated NIR light modulation technique for NSCs in adult mouse brains, whereby NIR light is locally converted to blue light to noninvasively activate NSC differentiation in vivo. Moreover, we identified melanopsin/TRPC6/Jab1 as a novel and specific modulator that directs light-induced NSC behavior and functional changes. Hence, our nongenetic optical modulation system is expected to have triggered in vitro and in vivo neurogenesis through the melanopsin/TRPC6/Jab1 signaling pathway for nervous function rescue and repair.

For NSC differentiation, the neurospheres were dissociated into single cells. Next, the cells were plated at 200,000 cells per well in 6well plates with coverslips in differentiation medium (Neurobasal medium and B27 supplement, Thermo Fisher) containing 1% FBS (Thermo Fisher) without bFGF and EGF. Both the 6-well plates and coverslip were coated with laminin and polylysine before use. Cells were fixed using 4% paraformaldehyde in PBS and immunostained 4–8 days after induction. A statistical analysis of NSC differentiation was performed across ten randomly selected view-fields of the tested samples using Image J software (the National Institutes of Health Open Resources). To knock down Jab1 expression, we used shRNA expression vectors (GenePharma, China) packaged in lentiviral particles to infect cells. The NSCs were infected with lentivirus for 60–72 h before they were subjected to differentiation according to the manufacturer's instruction. The RNAi oligonucleotide sequence used to knock down the Jab1 expression was 5′-CCA GAC UAU UCC ACU UAA UTT-3′. 2.2. Photostimulation systems The NSCs (200,000 cells per well in 6-well plates) were exposed to blue (455 nm) or red (635 nm) monochromatic LED (Yuanming Lasever, Ningbo, China) light at a distance of 12 cm from the light source. The daily irradiation duration was 45 min or 90 min over 5 consecutive days. The full power density of the LED irradiated on cells was 300 μW/cm2, and the power density could be reduced to 180 and 100 μW/cm2 for blue and red light, respectively. Time-matched control cells were kept in darkness during the same period. 2.3. Immunostaining and immunoblotting assays The following protein antibodies were purchased: anti-Jab1 (ab124720, monoclonal antibody produced in rabbits), anti-GFAP (ab7260, polyclonal antibody produced in rabbits), anti-Tuj1 (ab78078, monoclonal antibody produced in mice), anti-MAP2 (ab16228, monoclonal antibody produced in mice), anti-PAX6 (ab5790, polyclonal antibody produced in rabbits), anti-melanopsin (ab19383, polyclonal antibody produced in rabbits), and anti-p27 (ab92741, monoclonal antibody produced in rabbits) purchased from Abcam, USA; anti-Smad1 (#9743, polyclonal antibody produced in rabbits) and anti-phosphoSmad1/Smad5/Smad8 (#9511, polyclonal antibody produced in rabbits) purchased from Cell Signaling Technology, USA; anti-Smad7 (SC11392, polyclonal antibody produced in rabbits), anti-S100β (sc136061, monoclonal antibody produced in mice), anti-β-actin (SC8432, monoclonal antibody produced in mice), and anti-GAPDH (SC365062, monoclonal antibody produced in mice) purchased from Santa Cruz Biotechnology, USA; anti-O4 (O7139, monoclonal antibody produced in mice) purchased from Sigma, USA; and anti-TRPC6 (#182361-AP) purchased from Proteintech, China. Fluo-3 AM (F14201 Thermo Fisher) was used as a Calcium indicator. The mouse brains were first fixed in 4% paraformaldehyde in PBS overnight at room temperature at 25 °C. They were sectioned at 4 μm using a vibratome (Thermo Scientific, USA). Immunohistochemistry, immunofluorescence, and immunoblotting assays were performed as described previously [13].

2. Materials and methods 2.1. NSC isolation and culture Animal care and experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Shandong University School of Basic Medical Sciences. The NSC isolation and culture were performed as previously described [12]. In brief, C57 BL/6 mice (Model Animal Research Center of Shandong University, Jinan, China) forebrains were dissected from E13.5 embryos, and the cells were mechanically dissociated by pipetting. The samples were resuspended in growth medium (Neurobasal medium and B27 supplement, Thermo Fisher, USA) containing 20 ng/ml bFGF and EGF (Thermo Fisher) and were cultured at 250,000–500,000 cells per 60mm dish at 37 °C with 5% CO2. During each passage, neurospheres were enzymatically and mechanically dissociated using 0.05% TrypLE (Thermo Fisher) and then reseeded at the same density. For each animal and passage, cells were seeded in three separate dishes, and the number of neurospheres was calculated as mean ± SD.

2.4. Reverse transcription and real-time PCR Total RNA was isolated using a TRIzol reagent (Invitrogen, USA) and treated with RQ1 RNase-Free DNase (Promega, USA) to prevent genomic DNA contamination. Freshly isolated RNA was reverse transcribed to generate cDNA using the Super Script first-strand synthesis system (Invitrogen) following the manufacturer's recommendations. The mRNA levels were measured with LightCycler 480 SYBR Green I Master mix (Roche, USA) using a LightCycler 480 II instrument (Roche). The primer sequences are listed in Supplementary Table 1S. Four independent measurements were performed per sample. The 2

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quantified individual RNA expression levels were normalized to those of Gapdh.

at a pH of 7.2.1-oleoyl-2-acetyl-sn-glycerol (OAG) (10 μM) was used as an agonist for TRPC6 activation. The currents were induced by a 500ms voltage ramp protocol (from −80 to 80 mV) every 2 s from a holding potential of 0 mV. Once the current was stable, 10 μM OAGinduced TRPC6 was recorded. The analysis and display of the data were performed with Clampfit 10.6, and average current amplitudes at −80 and 80 mV were compared using the Student's t-test.

2.5. 5-ethynyl-2-deoxyuridine incorporation EdU (Cell-LightTM EdU Cell Proliferation Detection Kit, Guangzhou RiboBio, China) was added at 50 μM, and the cells were cultured for an additional 2 h. After removal of the EdU-containing medium, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, washed with glycine (2 mg/ml) for 5 min in a shaker, treated with 0.2% Trion X-100 for 10 min, and washed with PBS twice. Click reaction buffer (Tris-HCl, pH 8.5, 100 mM; CuSO4, 1 mM; Apollo 550 fluorescent azide, 100 μM; ascorbic acid, 100 mM) was then added. After 20 min, the cells were washed three times with 0.5% Triton X-100, stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature, washed five times with 0.5% Triton X-100, and finally immersed in 150 μl PBS and examined under a fluorescence microscope (Olympus).

2.9. In vivo optical stimulation of endogenous NSCs within the subgranular zone Experiments were performed on C57BL/6 mice (n = 4, 8-week-old, provided by Model Animal Research Center of Shandong University, Jinan, China). Animals were housed for at least 7 days prior to experiments in a ventilated and temperature-controlled room and had access to ad libitum water. The mice were anaesthetized using 500 mg/ kg pentobarbital sodium unless mentioned otherwise. A small skin incision was first made to expose the skull. After being head-fixed with a procedure described previously [16], craniotomies were made over the motor, and the tip of a 455-nm transmitting optical fiber (200 μm in diameter) was positioned at the DG (AP: −2 mm, ML: ± 1 mm, DV: −1.82 mm). A customized laser apparatus with a blue-laser source (MDL-C-488, Changchun New Industries CNI Laser, China) was positioned above the mouse head. The left subgranular zone (SGZ) with only the optical fiber was used as a control after light stimulation of the stem cells. Blue light pulses with 25-mW power and 1.5-h duration at 10 Hz (50 ms pulses) were delivered onto the optic fiber for photostimulation of the NSCs in the brain. Fluorescence images were acquired on an automatic digital slide scanner (3DHISTECH Pannoramic MIDI). For statistics, sections (n = 4 animals, 2 sections per animal) were obtained, imaged and analyzed.

2.6. Sphere counts and diameter measurements Self-renewal capacity was measured by plating 10,000 cells per well (6-well plate) in NSC proliferation media containing EGF and bFGF. The number of neurospheres that formed subsequently per well was quantified after 5–7 days and relative sphere formation was plotted versus indicated control. Three replicate wells were performed for each group. A minimum cutoff diameter of 50 μm was used when defining neurospheres as spheres, and neurospheres below this cutoff were not considered as reliably multipotent. Image J software (the National Institutes of Health Open Resources) was used to measure neurosphere diameters. All experiments were conducted at cell passage < 5. 2.7. Two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis

2.10. Characterization of UCNPs

Approximately 300 μg of protein was resuspended in a rehydration solution (8 M urea, 2% CHAPS, 65 mM DTT, 0.2% pharmalyte (pH range 3–10), and 0.2% bromphenol blue) and applied to 18-cm linear IPG strips (GE Healthcare, USA) at pHs of 3–10 for isoelectrofocusing (IEF) [14]. IEF was performed using an Ettan IPGphor instrument (GE Healthcare), and the proteins in the IPG strips were subsequently placed on a 12% uniform sodium dodecyl sulfate (SDS)-polyacrylamide gel. The gels were Coomassie brilliant blue-stained and scanned using an image scanner in transmission mode, after which image analysis was conducted with a 2-D PDquest (Bio-Rad, USA). The 2-DE was repeated three times using independently grown cells. The peptide mass analysis was performed using a liquid chromatography (Shimadzu Prominence Nano 2D) MicrOTOF-QII (BrukerDaltonics, USA) mass spectrometer (LC-MS/MS). Based on the NCBI and SWISSPROT databases, the mass spectra were analyzed with a 50 ppm mass tolerance using GPS Explorer V.2.0.1 and Mascot V1.9 (Matrix Sciences, London).

Transmission electron microscopy (TEM) measurements were carried out with a JEM-2100F Field Emission Electron Microscope. The hydrodynamic sizes of UCNPs were measured by dynamic light scattering means on a Malvern Zetasizer Nano ZS Particle Sizer. Upconversion luminescence spectra were recorded at 25 °C with an AvaSpec-2048TEC-USB2 Thermo-Electric Cooled Fiber Optic Spectrometer in conjunction with a 980 nm NIR light. 2.11. In vivo NIR stimulation of endogenous NSCs within the SGZ UCNPs with a chemical composition of NaYF4: 20% Yb/0.5% Tm @ NaYF4 were purchased from Micro Era Company, Changchun, China. The 8 week-old C57BL/6 mice were randomly divided into three groups: UCNP (−) NIR (+), UCNP (+) NIR (−) and UCNP (+) NIR (+) to assess NIR stimulation effects (n = 4 mice per group). 2 μL of 50 mg/mL NaYF4: Yb/[email protected] UCNPs were injected into the SGZ (AP: −2 mm, ML: ± 1 mm, DV: −1.82 mm) of mice under anesthesia. For UCNP (−) controls, the same volume of saline was injected instead of UCNPs. The tip of an NIR optic fiber (200 μm in diameter) was placed 5 mm above the skull at an angle to target the NIR laser (Yuanming Lasever, Ningbo, China) at the UCNP injection site in the SGZ. Irradiation was carried out daily by the NIR laser (980 nm, 150 mw) with 62.5 ms pulses at 8 Hz over 90 min for 5 consecutive days. The mice were sacrificed 7 days later to examine the NIR-induced immunostaining for astrocyte and neuronal differentiation. Fluorescence images were acquired on an automatic digital slide scanner (3DHISTECH Pannoramic MIDI). For statistics, sections (n = 4 mice per group, 2 sections per mouse) were obtained, imaged and analyzed.

2.8. Electrophysiological analysis The procedures for cell-attached patch-clamp recording of the TRPC6 channel currents from the NSCs were performed using the methods described previously [15]. In brief, a capillary glass tube (BF150-86-10, Sutter Instruments, USA) was pulled into a recording electrode using a microelectrode puller (P97, Sutter Instruments). A microelectrode manipulator (Sutter Instruments, MP285) under an upright microscope (Olympus BX50WI, Japan) was used, and data were recorded with data acquisition software (pCLAMP 10.6, Clampex & Clampfit, Molecular Devices, USA). The extracellular fluid consisted of 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM D-glucose monohydrate, and 10 mM HEPES at a pH of 7.4. The intracellular fluid consisted of 120 mM Cs-aspartate, 20 mM CsCl, 0.4 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 2 mM Na2-ATP, 10 mM glucose, and 1 mM EGTA

2.12. UCNPs distributed in the SGZ tissue 2 μL of 50 mg/mL NaYF4: Yb/[email protected] UCNPs were injected into 3

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cm2 (Fig. 1e and f). Compared to the time-matched dark controls, the number of NSC spheres demonstrated a significant 5.4-fold increase in the 300 μW/cm2 LED-treated culture dishes (Fig. 1f, middle). The quantification of neurosphere size also revealed that the NSC spheres in the blue-light-stimulated group were significantly larger than the control (dark) spheres (Fig. 1f, right). To further investigate the role of wavelength in such a monochromatic-induced cell self-renewal, blue light (455 nm) was replaced with red light (635 nm), whose wavelength spectrum does not overlap with that of blue light. Under the same illumination conditions to the blue light irradiation, red light could not significantly affect the proliferation and self-renewal of NSCs in the photosystem (data not shown). Because thermal stress has been reported to promote NSC differentiation [18], we monitored the temperature in the culture medium under light exposure and in the dark controls. Light irradiation did not significantly raise the local temperature after illumination. On average, the medium temperature under LED irradiation was less than 0.1 °C higher than that of the dark control. Collectively, these data indicate that blue light is more effective at triggering NSC proliferation and self-renewal than red light.

the SGZ (AP: −2 mm, ML: ± 1 mm, DV: −1.82 mm) of 8 week-old C57 BL/6 mice under anesthesia. 24 h after UCNP injection, mice were transcardially perfused with 4% paraformaldehyde (PFA)/0.25% glutaraldehyde in 0.1 M sodium phosphate buffer. Brain tissues were fixed with 3% glutaraldehyde at 4 °C for 2 h and postfixed in 1% osmiumtetroxide phosphate buffer for 2 h. Next, tissues were dehydrated in a graded ethanol series, permeated and embedded in EPON (EMS, Fort Washington, PA). Ultrathin sections (70 nm) were cut with an ultramicrotome (Leica UC-6, Leica Microsystems, Nussloch, Germany), stained with uranyl acetate and lead citrate, and examined via TEM (JEM-1200EX, JEOL Transmission Electron Microscope, Tokyo, Japan). 2.13. Fiber photometry for confirming in vivo upconversion emission 2 μL of 50 mg/mL NaYF4: Yb/[email protected] UCNPs were injected into the SGZ (AP: −2 mm, ML: ± 1 mm, DV: −1.82 mm) of 8 week-old C57 BL/6 mice under anesthesia. The tip of a 980 nm NIR optic fiber was placed 5 mm above the skull at an angle to target the NIR laser on the UCNP injection site in the SGZ. An AvaSpec-2048TEC-USB2 ThermoElectric Cooled Fiber Optic Spectrometer was inserted into the SGZ for the detection of upconversion luminescence spectra.

3.2. Monochromatic light differentially modulates neuronal/glial differentiation in NSCs

2.14. Statistical analysis

NSCs are functionally described as cells with the capacity to proliferate, self-renew, and differentiate into three different cell types: neurons, astrocytes, and oligodendrocytes [1]. We used blue (455 nm) and red (635 nm) monochromatic light exposure of equal power densities to investigate the process whereby illumination preferentially triggers neuron or glial (astrocyte, the main subtype of glial cells) differentiation, respectively. For cell differentiation analysis, we assessed protein and mRNA levels of neuron marker Tuj1 and astrocyte marker GFAP using immunofluorescence, real-time PCR, and western-blotting assays. The number of Tuj1-positive (+, green) cells decreased compared to that in the control group (Fig. 2a). The GFAP-positive (+, red) cells increased in the blue light treated group compared to the control group, indicating that blue light triggered astrocyte differentiation (Fig. 2b). A statistical analysis of NSC differentiation was performed across ten randomly selected view-fields of the tested samples (Fig. 2c). As shown in Fig. 2d and e, protein and mRNA levels of GFAP were increased in blue-light-treated group than in the control group. Again, Tuj1 decreased in NSCs under blue light exposure. Interestingly, red light exposure under the same illumination conditions as the blue light exposure experiment (irradiation time of 45 min daily for 5 consecutive days) stimulated neuronal differentiation (Fig. 2f). As shown in Fig. 2g and h, we found that the numbers of Tuj1positive cells increased significantly in the red light group, and there were less GFAP-positive cells in the red light group than in the control group. The quantitative summary of the replicated experiments indicates that neuron percentage (Tuj1-positive/MAP2-positive) was decreased in the blue light group compared to the control group, whereas in the (blue + red) group, the percentage was partially restored to the level of the negative control group (Fig. 2h). However, the astrocyte percentage (GFAP-positive) in the (blue + red) group was not significantly different to that in the red group, possibly due to additional unknown photo-stimulated regulators towards neuron differentiation. Collectively, these data suggest that blue light promoted astrocyte differentiation, whereas red light stimulated differentiation to neurons.

The data are presented as mean ± SD. Data from two groups were evaluated statistically by the two-tailed unpaired t-test for any significant differences. Data were evaluated statistically by ANOVA to test for any differences among multiple groups. If significant differences were found by ANOVA, the Bonferroni method of multiple comparisons was used to determine which groups were significantly different from each other. P < 0.05 was considered to indicate statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001). GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) was used for all statistical analyses. All photographic images of immunoblotting, Edu incorporation assays, immunofluorescence, and immunohistochemical staining are representative of at least three independent experiments. 3. Results 3.1. Monochromatic light affects proliferation and self-renewal in NSCs The characterization of the NSCs isolated from the E13.5 mouse forebrains was studied using the specific stem cell markers Nestin and Sox2 [17]. As shown in Fig. 1a, the majority of NSCs were Nestin-immunoreactive, and they expressed the NSC-specific transcription factor Sox2. To test whether the NSCs express photopigment molecules that could respond to light-based stimulation, real-time PCR was first used to explore the basal expression level of the photoreceptors in the NSCs [10]. As shown in Supplementary Table 1S, which lists the identified mouse photosensitizers, gene expression of Opn1SW, Opn3, Opn4 (melanopsin), Rho, and Rgr was detected (Fig. 1b). In basic terms, mice have dichromatic color vision in blue and red, and their photoreceptors are blue and red light sensitive. To explore whether light exposure affects the phenotype and function of NSCs, we explored the response of NSCs to a blue (455 nm)/red (635 nm) light photosystem. A schematic of the monochromatic photosystem is shown in Fig. 1c. We measured the blue light effect by measuring EdU incorporation monitored by immunofluorescence microscopy (Fig. 1d). The proliferation was enhanced in the blue light treated group (14.1 ± 1.4% EdU-positive for 45 min daily and 8.1 ± 1.1% EdU-positive for 90 min daily for 5 consecutive days of irradiation) compared to that in the time-matched dark control group (3.2 ± 0.3% and 3.6 ± 0.5% EdUpositive, respectively). Then, to confirm the role of light exposure in NSC proliferation and self-renewal, we quantified the number and size of neurospheres with the following settings: irradiation time of 45 min daily for 5 consecutive days and power densities of 100 and 300 μW/

3.3. Photosensitive mechanisms of nongenetic optical modulation of neural activities in NSCs We demonstrated that the behavior of NSCs is more sensitive to blue light than red light. Therefore, we looked for the putative photosensitive mechanism that modulated the blue light-induced NSC proliferation and differentiation. Among all identified opsins listed in Fig. 1b, one nonvisual opsin, namely melanopsin (Opn4)/transient receptor potential 4

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Fig. 1. Expression of photopigment and monochromatic light irradiation-promoted NSC proliferation and self-renewal. (a) Representative micrographs of Nestin and Sox2 immunostained NSCs in culture. Nuclei were stained using DAPI (blue staining) and Nestin/Sox2 (green staining) immunostaining, as described in the experimental procedures. (b) Real-time PCR screening of the basal expression level of opsins. (Rrh constitutional expression level was selected as the control group, fold-change = 1). (c) Schema of the LED photosystem. (d) Photoimages of EdU incorporation assay of NSCs under blue LED exposure. (e) Representative micrographs of NSC sphere formation under 45 min exposure of 455-nm blue light at 0, 100, and 300 μW/cm2. (f) Quantitation data of NSC proliferation (left), sphere counts (middle) and sphere diameters (right) under illumination. The bulbs represent the results from each experiment. Values are given as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001.

channel 6 (TRPC6), served as a key photoreceptor response to the optical modulation in NSCs. Opn4, a non-visual opsin best characterized in intrinsically photo-sensitive retinal ganglion cells [19,20], forms a pigment maximally sensitive to approximately 450–480 nm blue light [21]. The immunofluorescence results showed the melanopsin staining in NSCs (Fig. 3a and b). Then, we performed an immunoblotting assay and confirmed the presence of melanopsin in mouse NSCs, especially between passages 3, 4, and 5 of the NSCs (Fig. 3c). Because melanopsin, which is the key protein for blue light responsiveness, is coupled to the TRPC6 channel isoform [22], we tested whether blue light exposure could alter the TRPC6 channel currents in NSCs. We performed an electrophysiological analysis using the wholecell patch-clamp recording method. The cells were subjected to 45 min exposure of 455-nm blue light at a 300 μW/cm2 intensity. Upon

application of OAG, an increase in cation currents was observed in the light-treated group compared to the control (dark) group (Fig. 3d). As shown in Fig. 3e, the blue light-sensitive currents triggered a significant increase in both positive and negative currents (3.5- and 2.5-fold, respectively). Moreover, we observed an induced intracellular calcium flux from the NSCs under 300 μW/cm2 blue light stimulation (Fig. 3f). Considered together, these data indicate that melanopsin/TRPC6 served as a photoreceptor response to optical-modulated NSC proliferation and differentiation. 3.4. Phototransduction modulator for light irradiation-induced NSCs To further identify the phototransduction target for the altered phenotype and function in NSCs, we conducted a 2-DE coupled with 5

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Fig. 2. Monochromatic light differentially modulates neuronal/glial differentiation in NSCs. (a) Immunofluorescence staining showing the numbers of cells positive for Tuj1 under blue light irradiation. (b) Immunofluorescence staining showing the numbers of cells positive for GFAP under blue light irradiation. (c) Statistical analysis of Fig. 2a and b was performed across ten randomly selected view-fields of tested samples using Image-J software. *P < 0.05, **P < 0.01 and ***P < 0.001. (d, e) Proteins and mRNAs were extracted from the P4 passage of NSCs with blue light illumination and subjected to western-blotting and real-time PCR, respectively. (f) Schema of red and blue LED photosystem. (g) Immunofluorescence staining showing the numbers of cells positive for Tuj1, GFAP and MAP2 in a wavelength-dependent illumination manner. (h) Statistical analysis of NSC differentiation was performed across 10 randomly selected view-fields of tested samples using Image-J software. *P < 0.05, **P < 0.01 and ***P < 0.001; control cells compared to NSCs induced by the indicated light exposure.

mass spectrometry to compare the NSCs in the control sample with those under blue LED exposure for changes in protein levels. Fig. 4a shows Coomassie brilliant blue-stained 2-DE IPG standard maps from

one representative experiment with the two cell types. The spot analysis using 2-D PDquest (Bio-Rad) detected 1989 ± 72 spots in the control NSCs and 2029 ± 65 spots in LED-irradiated NSCs. The expression of a 6

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Fig. 3. Blue light-induced activities are gated through a melanopsin/TRPC6 channel. (a, b) Immunofluorescence staining of melanopsin in NSCs. Nuclei were stained using DAPI (blue staining), melanopsin (red staining), and Nestin (green staining) immunostaining, as described in the experimental procedures. (c) Proteins were extracted from the P2, P3, P4, P5, and P6 passages of NSCs and subjected to immunoblotting. (d) Representative OAG-induced TRPC6-mediated whole-cell currents measured from the NSCs under 0 and 300 μW/cm2 blue light exposure. (e) Average normalized current amplitude measured at −80 mV (hatched bars) and 80 mV (filled bars) from these cells. **P < 0.01 and ***P < 0.001 (n = 6). (f) Immunofluorescence images showing intracellular calcium flux (in green) measure by Fluo3 AM probe under 300 μW/cm2 blue light exposure.

protein is considered to have changed if the percentage volume of its spots on the gels shows a two-fold or greater difference (P < 0.05). We excised 30 differentially expressed proteins from the 2-DE gels, digested these in the gel, and applied the proteins to a sample template for LCMS/MS mass spectrometry. We successfully identified 21 protein spots using the internet-based program Mascot. The protein names, NCBI accession numbers, theoretical molecular weights, and pI values are listed in Table 1. Of the 21 proteins identified, 7, including Jab1, were

up-regulated, while 14 were down-regulated. According to their known and postulated functions, all 21 identified proteins were further classified into several categories based on signaling pathways, cell cycle, transcription modulation, and energy metabolism (Supplementary Table 2S). Among all identified proteins, Jab1 was of particular interest because of its essential role in intracellular signaling modulation [23], as its accumulation and associated enhanced BMP signaling pathway are involved in the determination of 7

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Fig. 4. Identification and characterization of phototransduction modulator in NSCs. (a) 2-DE electrophoretograms. (b) Proteins were extracted from NSCs under exposures and analyzed by immunoblotting. (c) Immunofluorescence assay of Jab1 upon differentiation. (d) Proteins were extracted from the NSCs after differentiation and analyzed by immunoblotting. (e) NSCs were infected with lentivirus particles carrying shRNA to knock down Jab1 expression. Proteins were extracted from the NSCs and analyzed by immunoblotting with antibodies specific for the indicated proteins. (f) Immunofluorescence staining showing the numbers of cells positive for Tuj1, GFAP, MAP2 and S100β. (g) Statistical analysis of immmunosignals was performed across 10 randomly selected view-fields of tested samples using Image-J software. *P < 0.05, **P < 0.01 and ***P < 0.001.

NSC differentiation, and it decreased thereafter. To confirm that the Jab1 derived from the phototransduction pathway is necessary for the neuronal differentiation of NSCs, the NSCs were infected with lentiviral particles carrying shRNA specific for Jab1 (Fig. 4e). Both control and siJab1 NSCs could be induced for differentiation into neurons and astrocytes, as assessed by these markers; however, siJab1 suppression resulted in an increased generation of Tuj1-positive neurons compared with the control NSCs (Fig. 4f and g, P < 0.001). Similar results were obtained for the immunostaining of MAP2, which is another marker for neurons (Fig. 4f and g, P < 0.01). The depletion of Jab1 resulted in a decreased generation of GFAP-positive cells compared with that in the control NSCs (Fig. 4f and g, P < 0.001). Similar results were obtained for the immunostaining of S100β, which is another marker for astrocytes (Fig. 4f and g, P < 0.001). Furthermore, cells treated with shRNA specific for Jab1 or with control shRNAs were stimulated by blue light exposure. Immunofluorescence results indicated brighter signals for GFAP/S100β staining and darker signals for Tuj1/MAP2 in shJab1 cells stimulated with blue light compared with that observed in shJab1 cells (Fig. 4f and g). Importantly, the changes in Tuj1/MAP2 and GFAP/S100β levels caused by shJab1 were offset in the blue light treated cells (Fig. 4f and g), demonstrating that the elevation of Jab1 caused by blue LED irradiation leads to proliferative and differentiation-associated morphological changes in NSCs. Thus, these findings collectively demonstrate that the blue lightinduced effects in NSCs were mediated through the melanopsin/ TRPC6/Jab1 phototransduction pathway and that the induced Jab1 abundance contributed to the aggressive characteristic and enhanced differentiation into astrocytes of the NSCs.

Table 1 Identification of differentially expressed proteins under blue LED irradiation. NO.

Name

Upregulated proteins 1 Heat shock cognate 71 kDa protein 2 Keratin, type II cytoskeletal 1 3 Heat shock cognate 71 kDa protein 4 Elongation factor 1-alpha 1 5 Elongation factor 1-alpha 1 6 Far upstream element-binding protein 2 19 Jun activation domain-binding protein Jab1/CSN5 Downregulated proteins 7 Creatine kinase B-type 8 Creatine kinase B-type 9 Creatine kinase B-type 10 Proliferating cell nuclear antigen 11 40S ribosomal protein S3 12 Heat shock cognate 71 kDa protein 13 Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial 14 Eukaryotic translation initiation factor 3 subunit I 15 Serine/arginine-rich splicing factor 1 16 Aconitate hydratase, mitochondrial 17 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial 18 Heat shock cognate 71 kDa protein 20 Septin-11 21 Phosphoglycerate kinase 1

MW

pI

NCBI accession number

70871.07 65605.89 70871.07 50113.84 50113.84 76775.48

5.37 8.39 5.37 9.10 9.10 6.90

31981690 126116585 31981690 126032329 126032329 163954948

37548.78

6.12

3152934

42713.26 42713.26 42713.26 28784.87 26674.29 70871.07 39638.71

5.40 5.40 5.40 4.66 9.68 5.37 6.27

10946574 10946574 10946574 7242171 6755372 31981690 18250284

36460.79

5.38

9055370

133841.65

9.49

56605682

85463.51 79776.79

8.08 5.51

18079339 229892322

70871.07 49341.20 44550.47

5.37 6.36 8.02

31981690 39963577 70778976

neural cells’ fates [24,25]. To verify the proteomic analysis results, we conducted an immunoblotting assay using whole-cell extracts prepared from the cell lines. Notably, the results showed that the Jab1 protein level was increased under blue light exposure (Fig. 4b). Next, we decided to test whether the dysregulated Jab1 abundance could regulate the downstream p27 and BMP signaling inhibitor Smad7. As shown in Fig. 4b, we observed the corresponding reduced p27 and Smad7 abundance stimulated by blue light exposure. Moreover, the activated regulation of Jab1 by melanopsin/TRPC6 within the phototransduction complex was supported by the experiments involving cells under blue LED irradiation. Such blue light exposure increased the expression of melanopsin and TRPC6 proteins (Fig. 4b). To better delineate the role of Jab1 proteins in modulating the characteristics of NSCs induced by blue LED exposure, we determined the expression of Jab1 with regard to NSC differentiation. As shown in Fig. 4c, the expression of Jab1 at six time points during NSC differentiation, including 0, 12, 24, 48, 72, and 96 h, was detected by immunofluorescence. Therefore, we predicted that the decreased expression of Jab1 during differentiation would be associated with changes in the BMP signaling pathway. To test this hypothesis, we performed an immunoblotting analysis and observed that the NSC differentiation at 0, 12, 24 h, 48, 72, and 96 h resulted in decreased Jab1 and increased Smad7 levels (Fig. 4d). The levels of phosphorylated Smad1/5/8 (pSmad1/5/8) and total Smad1/5/8 (Smad1) were also measured and recorded using the immunoblotting assays. The quantitative summary indicated that the BMP signaling (ratio: p-Smad/total Smad) was approximately 2.2-fold higher at 12 h compared with that at the onset of

3.5. Blue light affects neuronal/glial differentiation of NSCs in vivo by nongenetic stimulation Next, we performed the in vivo photostimulation experiment using optical fiber illuminations. Adult neurogenesis occurs in the SGZ of the hippocampal dentate gyrus and the ventricular-subventricular zone (SVZ) of the lateral ventricle [26]. Before the in vivo stimulation study, the expression of the photosensitizer was validated in the SGZ and SVZ regions of the mouse brain. The expression of melanopsin was observed from E13.5–2 months, and the presence of the TRPC6 channel was also observed in the tissues, indicating that there was constitutional expression of photoreceptors in endogenous NSCs (Fig. 5a and Supplementary Fig. S1). To investigate the influence of in vivo blue light on NSC behavior, we used an optical fiber (455 nm, ~25 mW, 10 Hz) to illuminate the SGZ for 60 min (Fig. 5b). The left SGZ with only the optical fiber was used as a control after light stimulation of the stem cells. As shown in Fig. 5b and c, we found that GFAP immunosignals (astrocyte marker) in the blue light group increased significantly compared with the control group. To detect the neuronal differentiation in the SGZ, Tuj1 (neuron marker) and PAX6 (neural progenitor marker) immunofluorescences were also performed after light stimulation (Fig. 5b). The Tuj1 and PAX6 immunosignals in the irradiated group were not significantly different when compared with those in the control groups (Fig. 5c). Furthermore, the influence of blue light on the endogenous NSC differentiation was verified by western-blotting and real-time PCR assays. Notably, in the NSCs with blue light exposure, GFAP protein and mRNA levels were significantly higher than those observed in the control cells, 9

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Fig. 5. Blue light via an optical fiber affects NSCs in vivo by nongenetic stimulation. (a) Immunofluorescence staining of melanopsin and TRPC6 (red staining) in endogenous NSCs (Nestin +, green staining) located in SGZ as described in the experimental procedures. (b) Schematic flowchart of nongenetic in vivo NSC stimulation experiment. Representative images of immunofluorescence staining of GFAP, Tuj1 and PAX6 in the non-stimulated and stimulated SGZ regions as described in the experimental procedures. (c) Statistical analysis of immunosignals using Image-J software (n = 4 mice, the left SGZ with only the optical fiber was used as a control after light stimulation for each animal, 2 sections/left SGZ/per mouse, 2 sections/right SGZ/per mouse). (d) Equivalent amounts (25 μg) of tissue lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for the indicated proteins. GAPDH was used as a loading control (n = 4 mice per group). (e) The mRNA levels of Gfap, Tuj1 and Pax6 in the SGZ were measured by real-time PCR. Compiled data were produced from three independent experiments (n = 4 mice per group). Columns, mean; Bars, ± S.D.; **P < 0.01.

respectively (Fig. 5d and e). Considered together, these data demonstrate that blue light irradiation could directly activate quiescent NSCs in vivo and significantly stimulate glial differentiation in mouse brains.

4. Discussion Quiescent NSCs give rise to newborn neurons and astrocytes in the mammalian deep brain [26]. The characterization and modulation of NSCs have been influential in understanding the brain's regenerative capabilities for severe injuries as well as neurodegenerative diseases. Light processing has emerged as an innovative and powerful approach to modulate cell behavior owing to the advantages of multiple colors with broad-spectrum wavelengths [28]. Light-derived therapy has been reported to control seasonal affective disorder (a severe depression condition) [29], modulate acute nociception and chronic neuropathic pain [30], and attenuate amyloid load in Alzheimer's disease [31] with the use of appropriate irradiation wavelength, intensity, and duration. Additional evidence shows that light can directly influence learning and mood functions independently on the modulation, sleep, and circadian pathways [32]. However, the molecular mechanisms underlying the photo-stimulated effects of illumination on stem cell behavior are only beginning to be understood. In the current study, monochromatic light exposure (455 nm/ 635 nm) could influence the stem cells' aggressiveness and neuronal/ glial differentiation, thus promoting NSCs into neural lineages for therapeutic strategies for nervous function rescue and repair. Notably, NSCs are more sensitive to blue (455 nm) monochromatic light, and blue light irradiation promoted the proliferation of NSCs more than red (635 nm) monochromatic illumination. Furthermore, blue (455 nm) light irradiation enhanced stem cell differentiation into glial cells, while red light exposure increased neuronal differentiation. Moreover, we targeted optical fiber illumination of NSCs in the SGZ, which a critical region in the neurogenesis network [27]. For the blue light-gated melanopsin/TRPC6 activation, our results demonstrate that blue light irradiation could directly activate quiescent NSCs in vivo and significantly enhance glial differentiation in mouse brains. This would require the insertion of optical fibers into the deep brain hippocampal region for light-gated ion channel stimulation. NIR light with greater tissue penetration and less absorption and scattering for organisms has been considered as an alternative way to reduce invasiveness [33]. Finally, we used a UCNP-mediated NIR modulation approach that could noninvasively utilize the melanopsin/TRPC6 stimulator in vivo, enabling the modulation of NSC neuronal/glial differentiation. NSCs have been used in various neurodegenerative disease and brain injury models, such as Parkinson's disease, Huntington's disease, and spinal cord injury. Due to the low integration efficiency of NSC proliferation and differentiation alone, studies have begun to focus on the use of physiological sensory stimuli to alter microenvironment interactions on the differentiation potential of stem cells. To the best of our knowledge, this is the first study to effectively modulate neural stem cell differentiation into neuronal/glial cells in vitro and in vivo using nongenetic light exposure. Glial cells are a major cell type in the central nervous system. Astrocytes (GFAP immunofluorescence positive), which are the main sub-type of glial cells, provide metabolic and nutritional support to their partner neurons. Astrocytes are also critical during neurogenesis and integration into brain circuitry [34]. Moreover, astrocytes become activated during brain injury and form a glial scar [35]. However, emerging evidence supports the hypothesis that brain tumors, especially glioblastoma multiforme (GBM), may derive from the

3.6. NIR-triggered neuronal/glial differentiation of NSCs in vivo by the upconversion nanosystem UCNP-mediated optogenetics was first proposed in 2011, and it was first used for neural stimulation in mature rodent neurons by engaging photoreceptors in transgenetic mice [11]. We used the minimally invasive deep brain stimulation approach for in vivo neuromodulation of quiescent NSCs in the adult SGZ. For blue light-gated melanopsin/ TRPC6 channel activation, we used blue light-emitting NaYF4 nanocrystals codoped with Yb3+/Tm3+ (Fig. 6a and b and Supplementary Fig. S2a). The resulting core-shell UCNP (Fig. 6a) exhibited a characteristic upconversion emission spectrum peaking at 455 and 474 nm upon excitation at 980 nm (Supplementary Fig. S2b). Quiescent NSCs in the adult SGZ give rise to new neurons and astrocytes [27]; the expression of melanopsin/TRPC6 was validated in the SGZ (Fig. 5a and Supplementary Fig. S1). Therefore, we targeted the invasive NIR stimulation of NSCs in the SGZ, as shown in Fig. 6c. The UCNPs were first injected into the hippocampal SGZ and awake mice were then exposed to NIR irradiation (980 nm, 150 mW, 62.5 ms pulses at 8 Hz) delivered from an optical fiber (200 μm in diameter) placed 5 mm above the skull (Fig. 6d). The mice with no UCNP injection or no NIR illumination were used as control groups. Electron microscopy (Fig. 6e) showed that the UCNPs were localized in the injection SGZ area. The left image showed the distribution of UCNPs in the extracellular space, and the right image showed the uptake of UCNPs by a neural cell (Fig. 6e). We further performed an in vivo fiber photometry to examine NIR upconversion by UCNPs in the SGZ of the mouse brain. The tip of a NIR optic fiber was positioned above the skull to deliver excitation NIR light, and a fiber optic spectrometer was inserted into the zone to detect upconversion emission spectrum (Fig. 6f). We estimated that the local upconversion would generate ˃ 2.0 × 103 μW/cm2 blue light emission at a depth of 2 mm in the deep brain, which is sufficient to activate the melanopsin/TRPC6 for cation influx [11]. Next, the neuromodulation for NSC differentiation upon NIR illumination in this experiment was observed 7 days after the injection of UCNPs, indicating their long-term in vivo utility. GFAP, PAX6 and MAP2 immunofluorescences were performed to demonstrate the NIR neuromodulation of NSC differentiation. As shown in Fig. 6g and h, we found that the GFAP level in the UCNP-mediated NIR-stimulated group increased significantly compared with the control group. The PAX6 and MAP2 immunosignals in the UCNP-mediated NIR-stimulated group were not significantly different when compared with those in the control groups (Fig. 6h). Furthermore, the NIR-triggered neuronal/glial differentiation of NSCs was verified by western-blotting and real-time PCR assays (Fig. 6i and j). Notably, in the NSCs, upon NIR illumination, GFAP protein and mRNA levels were significantly higher than those observed in the control groups (Fig. 6i and j). In summary, we used UCNP-mediated neuromodulation for glial differentiation by targeting NIR excitation of NSCs in the deep brain hippocampal region. The results demonstrate that NIR light modulation is suitable as a minimally invasive approach for nongenetic optical control of in vivo NSC differentiation. 11

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Fig. 6. (a) Schematic diagram of a blue light emitting NaYF4: Yb/Tm @ NaYF4 nanoparticle. (b) TEM images of the UCNPs. (c) Schematic principle of UCNPmediated NIR gating melanopsin/TRPC6 channel in NSCs. (d) In vivo experimental scheme for UCNP-assisted NIR stimulation of the NSCs in adult mouse brains. (e) Electron micrographs of UCNPs distributed in the SGZ tissue. Red arrows indicate clusters of UCNPs. Black arrow indicates mitochondria. (f) Upconversion emission spectrum at the SGZ upon 980-nm NIR irradiation. (g) Representative images of immunofluorescence staining of GFAP, PAX6 and MAP2 after UCNP-mediated NIR stimulation under different conditions. (h) Statistical analysis of immunosignals using Image-J software (n = 4 mice per group, n = 2 sections per mouse). (i) Equivalent amounts (25 μg) of tissue lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for the indicated proteins. GAPDH was used as a loading control (n = 4 mice per group). (j) The mRNA levels of Gfap, Pax6 and Map2 in the SGZ were measured by real-time PCR. Compiled data were produced from three independent experiments (n = 4 mice per group). Columns, mean; Bars, ± S.D.; ***P < 0.001.

Fig. 7. Schematic representation of our findings showing that light irradiation targets the melanopsin/TRPC6/Jab1 phototransduction pathway and regulates NSC proliferation and differentiation. See the Discussion section for further details.

transformation of NSCs located in the SGZ [6,36]. Therefore, an understanding of monochromatic light-induced mechanisms may be necessary to safely and effectively stimulate neurogenesis for nervous system rescue and repair. In addition, further investigation of optical modulation might provide insights into the deregulated mechanism of GBM tumor stem cell survival and may improve anti-cancer strategies in patients with brain tumors. The Opn4 gene product melanopsin was first identified in the intrinsically photosensitive retinal ganglion cells [20]. In contrast to the classical photopigment rhodopsin, melanopsin is described as a nonvisual opsin involved mainly in circadian entrainment and mood regulation [32,37]. As an opsin subgroup of G proteins, melanopsin also has a different light transduction mechanism from the classical photosensory pigments [38,39]. With respect to the melanopsin/TRPC6/Jab1 signaling pathway potential, the activation of Gq-coupled melanopsin by light irradiation may signal through the DAG-mediated phospholipase C (PLC) and phosphokinase C (PKC) activation to trigger a calcium influx into the cytosol by activation of TRPC6 [38,39]. The calcium influx activates the calcium sensor calmodulin (CaM) linked to the

transcription factor nuclear factor activated T cells (NFAT), which leads to the translocation of NFAT from the cytoplasm into the nucleus. Within the nucleus, the NFAT binds to its cognate promoter and induces specific gene expression in the target cells [40]. However, some specific proteins are likely involved in the correlation between NFAT and Jab1. Thus, investigating the target genes of the NFAT transcription factor and their regulatory roles with regard to Jab1 abundance may provide further details about the melanopsin/TRPC6/Jab1 signaling pathways. As a whole, these findings identified Jab1 as a critical modulator of cellular phenotype and function upon melanopsin/TRPC6 stimuli, revealing a previously unknown mechanism for modulation of NSC behavior involving the photoreceptor and phototransduction complex, as induced by monochromatic irradiation (Fig. 7). Jab1, which is highly conserved across species, was originally identified as a c-Jun coactivator and was subsequently discovered to be the fifth member of the constitutive photomorphogenic-9 (COP9) signalosome (CSN) complex, a multifunctional protein complex first isolated from plants as an essential regulator for light development [41]. In mammals, Jab1 is responsible for the removal of Nedd8 from cullin13

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RING ubiquitin ligases [42] and plays a role in a broad range of biological functions by functionally inactivating several key negative regulatory proteins through their subcellular localization, degradation, and deadenylation, including Smad 4/7, p53, and p27 [23,43–45]. Jab1 promotes cell proliferation by interacting directly with p27 and induces nuclear export and subsequent p27 degradation [46]. The significant increase in NSC proliferation mediated by Jab1 might be due to acceleration of the G1 phase or induction of survival signals through downregulation of p27, which is a substrate of the Jab1-HECT Ubiquitin ligase [44]. Finally, it has been demonstrated that Smurf1/2, which are members of the HECT family, target BMP-specific Smads for degradation [47]. Inhibitory Smads, such as Smad7, negatively regulate BMP signaling by interfering with R-Smad phosphorylation [48]. Jab1, a component of Smurf1/2 E3 ligases, serves to negatively regulate BMP signaling by promoting Smad7 degradation [49]. Consistent with the known function of Jab1, the current findings show that an increase in Jab1 levels induced by light exposure also leads to enhanced BMP signaling. BMP signaling pathways play multiple roles in nervous system development and differentiation [50]. Stimulated BMP signaling gives NSCs the ability to differentiate into astrocytes [51].

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5. Conclusions In summary, the present study demonstrated that NSCs constitutionally express photoreceptors and are responsive to blue/red light. A nongenetic optical modulation system (based on optical fibers and nanoparticles) was used to control cellular behavior in vivo. Our results indicate the potential to promote NSC proliferation, self-renewal, and glial cell differentiation via the melanopsin/TRPC6/Jab1 signaling pathway. Thus, our findings provide new insight into optogenetic manipulation by photostimulation of NSC behavior for nervous rescue and repair. The melanopsin/TRPC6/Jab1 pathway in NSCs also represents a potential target for therapeutic applications in neurodegenerative medicine. Declaration of competing interest The authors have no conflicts of interest to disclose. Acknowledgements This work was supported by the National Key Research and Development Program of China (2017YFB0405400), National Natural Science Foundation of China (81873737, 81671114, 81741055, 81873878), the Science Foundation of Shandong Province (ZR2018MH011), the National Key Research and Development Program of Shandong Province (2017GSF218041) and the Key Research and Development Program of Shandong Province (2016ZDJS07A08, 2018CXGC1211). The authors would like to thank Dr. Jihui Jia (School of Basic Medical Sciences, Shandong University) for illuminating discussions. The authors gratefully acknowledge Dr. Xiao Yu and Dr. Ziying Wang (School of Basic Medical Sciences, Shandong University) for technical assistances during TRPC6-mediated electrophysiological analysis. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biomaterials.2019.119539. References [1] F.H. Gage, Mammalian neural stem cells, Science 287 (5457) (2000) 1433–1438. [2] C. Anacker, R. Hen, Adult hippocampal neurogenesis and cognitive flexibility linking memory and mood, Nat. Rev. Neurosci. 18 (6) (2017) 335–346.

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