JP4925155B2 - Retinal nerve cell function recovery agent - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
  The present inventionRetinal nerve cell function recovery agentAbout.
[0002]
[Prior art]
Nerve survival and differentiation are affected by cellular redox conditions. TRX is a small 12 kDa multifunctional protein with a redox-active disulfide / dithiol in -Cys-Gly-Pro-Cys- and also acts as a protein disulfide reduction system for NADPH and thioredoxin reductase (Holmgren , 1985). Several reports have shown that TRX-dependent redox regulation is closely involved in signaling mediated by AP-1, NF-kB, p53, ASK1, and p38 MAP kinases (Hirota et al., 1997 Saitoh et al., 1998; Hashimoto et al., 1999; Ueno et al., 1999). TRX is widely distributed and induced by various stresses (Nakamura et al., 1997; Masutani, 1999). TRX Expression is also increased by hemin (Kim et al., 2001), which is a differentiation-inducing agent for K562 erythroleukemia cells, or cyclic AMP analogs of retinal pigment epithelial cells (Yamamoto et al., 1997). Within the regulatory region of the TRX gene are several SP-1 binding motifs, antioxidant response elements (ARE), and cyclic AMP response elements (CRE). In neural tissue, TRX is induced in astrocytes after ischemia (Tomimoto et al., 1993) and motor neurons after nerve injury (Mansur et al., 1998). TRX is known to have cytoprotective action (Nakamura et al., 1994) and neuroprotective activity (Hori et al., 1994) against oxidative stress. Furthermore, overexpression of TRX in transgenic mice reduces focal ischemic brain injury (Takagi et al., 1999). TRX has also been reported as a neurotrophic factor in central cholinergic neurons and has neurotrophic activity (Endoh et al., 1993), but the molecular basis of its effects has not been elucidated.
[0003]
Other members of the neurotrophin family, such as nerve growth factor (NGF) and brain-derived neurotrophic factor, have profound effects on neurons, including promoting survival and differentiation (Lo, 1992). NGF has been reported as a potential therapeutic agent in age-related neurodegenerative diseases such as Alzheimer's disease (Connor and Dragunow, 1998). A current understanding of these mechanisms relies heavily on studies of NGF action on the pheochromocytoma cell line PC12 (Greene and Tischler, 1976). When exposed to NGF, PC12 cells differentiate into sympathetic neuron-like cells. The signal is initiated by NGF binding to its highly active receptor, TrkA, on the cell membrane (Kaplan et al., 1991), ras and mitogen-activated protein kinase (MAPK) cascade (Thomas et al., 1992). ). NGF treatment of PC12 cells results in the activation of genes such as c-fos that are thought to be important for NGF action (Milbrandt, 1986). NGF activates the c-fos gene by several elements including serum response elements (Treisman, 1986) and CRE (Ginty et al., 1994; Ahn et al., 1998).
[0004]
[Problems to be solved by the invention]
  The purpose of the present invention is toretinaNeuronalFunctional recoveryIt relates to technology that promotes.
[0005]
[Means for Solving the Problems]
  The present inventor said that TRXretinaNeuronalFunctional recoveryPromote,It was found useful for the treatment of diseases in the ophthalmic field.
[0006]
  The present invention includes the following:Retinal nerve cell function recovery agentConcerning
Item 1.An agent for restoring retinal nerve cell function, which contains NGF and restores the function of photodamaged retinal neurons by inducing endogenous thioredoxin expression.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
  Functional recoveryAs a nerve to be doneSightGodSutraAnd theseEachFor seed stem cellsNerve growth factor (NGF)By actingInduces the expression of TRX and thereby the retinaNerveRestore cell functionIn addition, various nervous system diseases caused by nerve degeneration or neuronal cell death can be treated.
[0016]
Examples of various stem cells that can differentiate into nerves include neural stem cells, retinal stem cells, mesenchymal stem cells, and embryonic stem cells.
[0017]
  The present inventors have found that a large amount of TRX is expressed in neural stem cells. From the following examples, TRX is also expressed.Functional recovery of retinal neuronsIt is clear to promote.
[0019]
  The present inventionRetinal neuronal function recoveryThe agentRetinal neuronsBy applying toInduces TRX expression,as a resultRetinal neuronsofFunctional recoveryCan be promoted.
[0021]
As an administration route, any of oral (tablet, capsule, granule, powder, liquid, syrup, etc.) and parenteral (injection, inhalation, nasal drop, suppository, etc.) can be administered.
[0022]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
(1) Cell lines and culture
NGF, polyethyleneimine (PEI), PD98059 and NAC were purchased from Sigma. Rat pheochromocytoma tumor cell line PC12 has 10% heat-inactivated horse serum and 5% heat-inactivated fetal calf serum (FCS) at 37 ° C in a humid atmosphere containing 5% CO2, Substances were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 100 IU / ml penicillin and 100 mg / ml streptomycin.
Plasmid: pTrxCAT plasmid was constructed according to a known method (Taniguchi et al., 1996). The HindIII-BamH I insert from the pTrxCAT vector was subcloned into pBluescriptII KS (+) (pTRXblue vector. pTRX (-1148) -Luc, pTRX (-1062) -Luc, pTRX (-352) -Luc and pTRX ( The -263) -Luc vector was constructed by ligating the KpnI / BamHI fragment of the pTRXblue vector to the KpnI / BglII site of the pGL3 basic vector (Promega, Wis.) The ApaI / PvuII insert of the pTRX (-263) -Luc vector was PTRX (-217) -Luc vector obtained by cutting, filling and self-ligation, pGL3-c-fos (-40, +42) and pGL3-c-fos (-99, +42) luc vectors Was constructed by subcloning the MluI / HindIII fragment of Fos-40 luc (Masutani et al., 1997) and the pFDE-luc vector into the MluI / HindIII site of the pGL3 basic vector (Promega). The BamHI / HindIII fragment of FDE-CAT (Trouche et al., 1993) was replaced with BglII-H of pGL2 basic vector (Promega). The pCDSR a (alpha) -TRX and pCDSRa -TRX (C32S / C35S) vectors were constructed by the method described previously (Hirota et al., 1997) The pcDNA3TRX (32S / 35S) ) Vectors were constructed by the previously described method (Nishiyama et al., 1999) BamHI inserts from pCDSRa-TRX and pCDSRa-TRXm (Tagaya et al., 1989; Hirota et al., 1997) were each pBluescript. II Subcloned into the BamHI site of KS (pBS-wtTRX, pBS-dmTRX), pBI-EGFP-wtTRX and pBI-EGFP-dmTRX (32S / 35S) are EcoRV / XbaI fragments of pBS-wtTRX and pBS-dmTRX vectors, respectively. Was constructed by ligating to the PvuII / NheI site of the pBI-EGFP vector (Clontech). All constructs were controlled by direct nucleotide sequencing using a Thermo Sequenase II dye terminator cycles equencing kit (Amersham Pharmacia). pRL-TK vector was purchased from Promega. pcDNA3 was purchased from Invitrogen.
・ Western blot analysis
Cells were collected and washed twice with ice-cold phosphate buffered saline (PBS) and then lysed solution (10 mM Tris-HCI (pH 7.4), 150 mM NaCI, 1% NP-40, 1 mM EDTA). , 0.1 mM PMSF, 8 mg / ml aprotinin and 2 mg / ml leupeptin) for 30 minutes on ice. The extract was clarified by centrifugation. Cell lysates were maintained at 95 ° C. for 5 minutes and then separated by 15% SDS-polyacrylamide gel electrophoresis. The separated protein was transferred to a polyvinylidene difluoride membrane (Millipore Co., Bedford, Mass.). The membrane was treated overnight with 10% (w / v) skim milk in T-PBS (PBS containing 0.05% Tween20), incubated with anti-mouse TRX rabbit polyclonal antibody (Takagi et al., 1998c) for 1 hour, then peroxidase Incubated with conjugated anti-rabbit IgG (Amershan Pharmacia Biotech) for 1 hour. Epitopes were visualized with ECL Western blot detection kit (Amershan Pharmacia Biotech). The inventor has previously reported that this anti-mouse antibody cross-reacts with rat TRX (Takagi et al., 1998b).
・ Northern blot analysis
Total RNA was extracted using TRIzol reagent according to the manufacturer's specifications (Maruyama et al., 1997). 20 mg of total RNA was electrophoresed and transferred to Maximum strength Nytran nylon (Schleicher and Schrul, Knee, NH) using a Turbo-Blotter system (Schleicher and Schrul). Filters were hybridized with mouse TRX probes that cross-react with rat TRX mRNA as previously described (Takagi et al., 1998b).
・ Transfection and luciferase assays
PC12 cells were seeded at 70% confluence in 35-mm dishes before transfection. Cells in serum-free medium were transfected with PEI reagent as described by Boussif et al. (Boussif et al., 1995). After 24 hours, the transfected cells were treated with 50 ng / ml NGF (Sigma). Luciferase gene expression normalized by Renilla luciferase activity was analyzed 24 hours later using an assay kit (Promega, Madison, Wis.). The relative fold activation of luciferase was calculated. The same experiment was performed three times. PC12 cells were transfected with bidirectional expression vectors pBI-EGFP, pBI-EGFP-wtTRX or pBI-EGFP-dmTRX in which the active site of TRX was inactivated (Ueno et al., 1999). After transfection, NGF was added to the medium. After 24 hours, cell-expressed EGFP was examined with a laser confocal microscope.
・ Immunofluorescent cell staining
PC12 cells were seeded before staining with 70% confluence in culture slides coated with poly-L-lysine. Cells were then fixed with 3.7% paraformaldehyde in PBS containing 10% FCS for 20 minutes at room temperature. This was then membrane permeabilized with 0.2% (W / V) Triton X-100 in PBS for 10 minutes and blocked with PBS containing 5% bovine serum albumin and 10% FCS for 20 minutes. Slides were incubated with 2 mg / ml mouse TRX antibody (provided by Fujirebio) for 60 minutes and then washed with PBS. The slides were then incubated with 1 mg / ml fluorescein isothiocyanate labeled secondary antibody for 60 minutes and washed again with PBS. Stained cells were examined with a laser confocal microscope (Bio-Rad).
·result
NGF-induced TRX expression in PC12 cells
The inventor examined the effect of NGF on TRX expression in PC12 cells. The protein expression was increased in PC12 cells by NGF (FIG. 1A). TRX mRNA also increased 2 hours after NGF treatment (FIG. 1B). To further analyze the induction mechanism by NGF treatment, we transfected PC12 cells with TRX promoter luciferase reporter construct (TRX-Luc). Treatment with NGF significantly enhanced TRX promoter activity (FIG. 1C).
Identification of NGF-responsive region in TRX promoter
In order to understand the mechanism of TRX gene activation by NGF, a luciferase reporter construct containing various deletion mutants of the TRX promoter region was used. The gene region from −263 to −217 with respect to the translation initiation site was required for NGF response. This region contains a sequence resembling consensus CRE, indicating the involvement of CRE in activation (Figure 2). TRX expression was suppressed by PD98059 or NAC.
[0023]
ERK inhibitors PD98059 and NAC (Kamata et al., 1996) are known to suppress ERK-CRE-mediated activation by NGF. Treatment with NGF for 2 days induced neuronal differentiation of PC12 cells with the appearance of neurite outgrowth. As previously reported, PD98059 (50 mM) or NAC (20 mM) suppressed NGF-induced morphological changes in PC12 cells (FIG. 3A). The inventor then tested the effect of PD98059 or NAC on NGF-mediated TRX gene activation. PD98059 or NAC blocked NGF-induced activation of the TRX gene (FIG. 3B). PD98059 or NAC also caused a decrease in TRX protein expression (FIG. 3C).
NGF-induced nuclear transport of TRX is blocked by PD98059 or NAC.
[0024]
ERK is transported from the cytoplasm to the nucleus during NGF treatment (Chen et al., 1992) .TRX is also transported from the cytoplasm to the nucleus when exposed to H2O2 or UV radiation (Hirota et al. , 1997). In order to analyze the involvement of TRX in NGF-induced signaling, the present inventors studied the subcellular localization of TRX during NGF treatment. After treatment with NGF for 16 hours, TRX migrated to the nucleus. The migration was blocked by PD98059 or NAC (Figure 4). Overexpression of dominant TRX blocked NGF-induced differentiation of PC12.
[0025]
The inventor then examined whether TRX is required for NGF-dependent differentiation of PC12 cells. We have transiently transfected PC12 cells with pBI-EGFP, pBI-EGFP-wtTRX or pBI-EGFP-dmTRX (32S / 35S), where the redox active site of TRX is inactivated. It was done. Mutant TRX inhibited TRX-dependent activation of transcription factors (Ueno et al., 1999). After transfection, NGF was added to the medium. Cells were examined with a laser confocal microscope 24 hours after NGF treatment. Cells transfected with mutant TRX vector showed suppression of differentiation, whereas cells transfected with wild type TRX vector or control vector showed NGF-induced differentiation (FIG. 5).
[0026]
Overexpression of dominant negative mutant TRX blocked CRE-mediated c-fos induction by NGF. TRX has been reported to regulate various transcription factors by promoting DNA binding (Hirota et al., 1997) (Ueno et al., 1999) or co-activator interactions (Ema et al., 1999). It was done. Therefore, we analyzed whether TRX is involved in the regulation of CGF-mediated NGF-mediated activation. After 4 hours of treatment, NGF caused a 40-fold increase in luciferase activity in pGL3-c-fos (-99, +42), but increased pGL3-c-fos (-40, +42). There was no (FIG. 6A). The response of the reporter gene pGL3-c-fos (-99, +42) to NGF was remarkably suppressed by transfection of the dominant negative mutant of TRX. Transactivation was suppressed by 75% by mutant TRX (FIG. 6B).
・ Consideration
In this study, the inventor found that NGF induces TRX expression at the protein and mRNA levels in PC12 cells. The present inventor has identified an NGF response region located at −263 to −217 bp of the TRX gene by luciferase assay. This region contained a CGTCA sequence carrying analogs that consensus CRE (Montminy et al., 1986). In addition, the present inventors have shown that PD98059, an ERK inhibitor, suppresses NGF-induced TRX expression. NGF-induced CRE activation is mediated by extracellular signaling-regulated protein kinase (ERK) (Impey et al., 1998). These results indicate that the TRX gene is induced by NGF through the ERK and CRE cascades. Further research is ongoing on the analysis of the TRX gene induction mechanism by NGF.
[0027]
The inventor has also shown that NGF induces nuclear translocation of TRX as well as PMA (Hirota et al., 1997), UV (Ueno et al., 1999) and Hemin (Kim et al., 2001). Demonstrated. The inventor has shown that the ERK inhibitor PD98059 blocks this effect of NGF, and this result suggests that ERK is involved in the regulation of NGF-induced nuclear migration of TRX. The mechanism and physiological significance of the nuclear migration should be studied in more detail. NGF-induced expression and nuclear translocation of TRX appear to be related to NGF-induced differentiation of PC12 cells, because PD98059 and NAC repress nuclear translocation as well as NGF-induced differentiation and TRX expression, respectively (Fig. 3 and 4). More importantly, overexpression of the dominant negative mutant TRX almost completely inhibited differentiation (FIG. 5). These results indicate that TRX is required for PC12 differentiation induced by NGF.
[0028]
Genes such as c-fos were thought to be required for NGF action. In the upstream regulatory region of the c-fos gene, CRE is critical for the regulation of c-fos transcription in response to various extracellular stimuli that induce neural differentiation (Ahn et al., 1998) (Sheng et al ., 1988). The present inventors have shown that overexpression of the dominant negative mutant form of TRX blocks NGF-induced activation of the pGL3-c-fos (-99, +42) reporter gene containing CRE, but pGL3-c-fos (- 40, +42) showed no block. These results demonstrate that TRX is required for CGF-mediated NGF signaling leading to c-fos expression. TRX regulates the activity of DNA-binding proteins including Jun / Fos (AP-1) and interacts with redox factor 1 (Ref-1), a nuclear reducing molecule (Hirota et al., 1997). AP-1 and Ref-1 have been reported to be involved in differentiation (Sheng and Greenberg, 1990) (Chiarini et al., 2000). Recently, the inventor has reported that TRX and Ref-1 regulate the interaction between transcription factors and coactivators (Ema et al., 1999), and that CREB activation is regulated by TRX. (Hirota et al., 2000). Thus, TRX may increase the interaction of CREB with DNA or coactivators and promote NGF signaling. Further research is needed for the relevant mechanisms of TRX in the NGF signaling pathway.
[0029]
NGF has been shown to promote axonal regeneration (Hollowell et al., 1990). Endoh et al. Reported the neurotrophic activity of TRX on cholinergic neurons (Endoh et al., 1993). The results confirm and develop this finding and suggest that TRX is a neurotrophic cofactor that enhances the effects of NGF on neuronal differentiation and regeneration
NGF also acts as a neuronal survival factor. NGF has been shown to prevent the death of axotomized septal neurons (Pallage et al., 1986). Loss of NGF results in apoptosis of PC12 cells, which is mediated by p38 MAPK and apoptosis signaling kinase 1 (ASK1) (Xia et al., 1995; Kummer et al., 1997; Kanamoto et al., 2000). TRX has been reported to act as an endogenous inhibitor of ASK1 and p38 MAPK (Saitoh et al., 1998; Hashimoto et al., 1999), and loss of NGF also results in downregulation of TRX expression in PC 12 cells ( Bai, et al. Unpublished observations). These results demonstrate that maintaining TRX levels by NGF plays a role in preventing neuronal death. The protective role of TRX against nerve injury was shown. TRX is induced in astrocytes after ischemia (Tomimoto et al., 1993). Overexpression of TRX in transgenic mice reduces focal cerebral ischemic injury (Takagi et al., 1999) and excitotoxic hippocampal injury (Takagi et al., 2000). Decreased expression of TRX in the brains of Alzheimer's disease patients has been reported (Lovell et al., 2000). NGF administration has been proposed to maintain cholinergic neurons in Alzheimer's patients (Serrano Sanchez et al., 2001). Taken together, these results and the results of the present invention show that administration of TRX can enhance the effects of NGF in neurological diseases such as neurodegenerative diseases. Further research is underway to clarify the therapeutic potential of TRX for neurodegenerative diseases.
Example 2 (data on retinal photoreceptor cells)
animal
Four-week-old male BALB / c mice (albino) were obtained from Japan SLC (Shizuoka, Japan) and bred in the inventor's colony room for 2-5 days prior to the experiment. The light intensity in the Japanese SLC and the inventors' laboratory was 300 lux, and the light intensity in the laboratory cage was 20-40 lux. All mice were maintained in Japan SLC and the inventor's colony room in a light / dark cycle of 12 hours (8:00 A.M. to 8:00 P.M.).
Light irradiation
Four week old mice were placed in the dark for 24 hours before the experiment. The pupil was dilated with 1% cyclopentrate hydrochloride eye drops (Santen Pharmaceutical). Unanesthetized mice were exposed to 8,000 lux of divergent cold white fluorescent light (Matsushita Electric Industrial) for 2 hours in a cage equipped with a reflective interior. All light irradiation started at 10 am. The temperature during light irradiation was maintained at 25 ± 1.5 ° C. Special care was taken to ensure that both eyes received the same degree of irradiation during irradiation.
Preparation of retinal tissue sections
After induction of deep anesthesia by intraperitoneal injection with pentobarbital, mice were perfused with phosphate buffered saline (PBS) (pH, 7.4) to perfuse the left ventricle to wash out blood before fixation. It was then perfused with freshly prepared 4% paraformaldehyde containing 0.25% glutaraldehyde in PBS. The eyes were then removed. All tissues were fixed in paraffin for 12 hours at 4 ° C. with the same fixative as described above and cut into 1 μm sagittal sections with whole retina including the optic disc. A 7-0 silk suture was placed as a landmark on the temporal side of the eye. Tissue sections were collected on glass slides and treated with xylene and a series of varying amounts of alcohol for 30 minutes to remove paraffin from the sections.
Morphological measurement
Retinal paraffin sections (1 μm) containing the optic disc were stained with hematoxylin-eosin (H-E), and digitized color images of four positions of each section were obtained using the PDMC le digital imaging system (Olympus). Two images were obtained from the upper retina of the upper optic disc 100-800 μm and two from the lower retina of the lower optic disc 100-800 μm. The number of hematoxylin positive photoreceptor cell nuclei in each image was counted and compared by the one-way ANOVA followed by the Bonfferoni / Dunn post hoc test.
[0030]
TdT-mediated dUTP nick end labeling (TUNEL)
TUNEL was performed on 1-μm paraffin sections using an in situ apoptosis detection kit (Takara Shuzo). 3 ', 3'-diaminobenzene (Dako, Carpinteria, CA) was used as a color former. The number of TUNEL-positive nuclei was counted by the same method used for the hematoxylin positive cell count described above.
antibody
Rabbit anti-mouse TRX antibody (polyclonal) was prepared as previously reported¹⁹.
Immunohistochemistry of mouse and human TRX
For immunohistochemical analysis of mouse TRX, the inventor¹⁹It was used. Briefly, endogenous peroxidase activity was inactivated with 0.6% H2O2. Primary antibody or control normal rabbit serum was added and incubated overnight at 4 ° C. Biotinylated goat anti-rabbit immunoglobulin (Biomeda, Foster
City, CA) was used as the secondary antibody. Avidin-biotin amplification (Biomeda) was performed and then incubated with the substrate 0.1% 3 ′, 3′-diaminobenzidine (Dako).
Western blot of mouse TRX
Retina sample preparation and Western blotting were performed as previously reported.¹⁵. Briefly, after induction of deep anesthesia by intraperitoneal injection of pentobarbital, the mouse's left ventricle was perfused with ice-cold phosphate buffered saline (PBS) (pH, 7.4) before blood fixation. Was washed off and then the eyes were removed. The cornea and lens were removed from the eye and the inner layer of the retina (neural retina) was separated from the eyecup under the microscope. In the eyes after perfusion with ice-cold PBS, the adhesion between the photoreceptor cell layer and the retinal pigment epithelial cell layer was weakened and they were easily separated. The eyecup after removal of the retinal nerve was analyzed as a retinal pigment epithelial cell fraction. This fraction therefore contained the choroid and sclera. Equal amounts of retinal protein (5 μg protein / lane) were electrophoresed on a 12% sodium dodecyl sulfate (SDS) -polyacrylamide gel and then electrophoretically polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). After blocking, the membrane was incubated with the first antibody and then with the peroxidase-linked second antibody. Chemiluminescence was detected with an ECL Western blot detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Intravitreal injection of recombinant thioredoxin (rTRX)
5 μg of rTRX or mutant rTRX (TRXC32S / C35S) 20 or 3 μl of 0.9% NaCl was injected intravitreally 2 hours before light irradiation. rTRX was injected intravitreally from the temporal margin of the right eye using a 30-G fine disposable needle (Hamilton, Reno, NV) equipped with a 10-μl microinjection syringe.
Detection of tyrosine phosphorylated protein
Tyrosine phosphorylated protein was detected using an ECL tyrosine phosphorylation detection system (RPN 2220/1, Amersham Pharmacia Biotech). Following the manufacturer's recommendations, protein samples of neural retina were prepared and electrophoresed on a 12% SDS-polyacrylamide gel (10 μg protein / lane) and then electrophoretically transferred to a PVDF membrane. After blocking, the membrane was incubated with a peroxidase-conjugated anti-phosphotyrosine antibody (PY-20, Amersham Pharmacia Biotech) and then chemiluminescence was detected with an ECL Western blot detection kit.
Detection of oxidized protein
Oxidized protein was detected as previously reported using an oxidized protein detection kit (OxyBlot, Intergen, Purchase, NY)¹⁷. OxyBlot provides a sensitive immunodetection reagent for carbonyl groups. Prepare a 2,4-dinitrophenyl (DNP) -hydrazone derivatized protein sample of the retinal nerve according to the manufacturer's protocol, separate by 12% SDS-polyacrylamide gel electrophoresis (5 μg protein / lane), and then PVDF Transferred to the membrane. After blocking, the membrane was incubated with a primary antibody specific for the DNP portion of the protein. The protein band was detected in the same way as the Western blot for mouse TRX.
DNA ladder
Internucleosomal DNA breakage was detected using the Quick Apoptotic DNA ladder Detection Kit (MBL, Nagoya, Japan). Retinal DNA was extracted, loaded onto a 1% agarose gel and electrophoresed according to the manufacturer's protocol. The gel was stained with ethidium bromide and the DNA band was visualized with an ultraviolet transluminator.
Statistical analysis
All statistical analyzes were performed on a Macintosh personal computer using StatView software, version 5.0 (SAS, Cary, NC).
result
Expression of endogenous TRX in the retina
To determine the severity of retinal damage, total and TUNEL-positive photoreceptor nuclei (Fig. 7) in retinal sections were counted before, immediately after, 12, 24, 48 and 96 hours after light irradiation. did. Compared to the number of photoreceptor cells in mice not irradiated (mean ± SD; 248.5 ± 11.4 cells / 100 μm), the number is 24 hours after light irradiation (182.0 ± 10.7, P <0.05) and thereafter ( 178.3 ± 18.3 cells / 100 μm, P <0.01 and 50.0 ± 9.8 cells / 100 μm, P <0.01, 48 hours and 96 hours, respectively). TUNEL-positive nuclei were observed 12 hours after light irradiation (mean ± SD; 8.7 ± 2.2%) and maintained until 96 hours after light irradiation (44.2 ± 6.9%, 34.5 ± 2.4%, and 38.0 ± 11.7). % At 24, 48, and 96 hours respectively). .
Since TRX is up-regulated in response to various oxidative stresses, the present inventors have shown TRX expression during the retina response to photooxidative stress by immunohistochemistry (FIG. 8A) and Western blot (FIG. 8B, C). analyzed. Immediately after light irradiation, TRX nuclear labeling was observed in the inner and outer nuclear layers of the posterior pole of the retina; the labeling was not significant around the retina immediately after the iris. The TRX label in the nuclear inner layer disappeared 24 hours after light irradiation and thereafter. On the other hand, labeling of the outer nuclear layer was maintained up to 96 hours. Twenty-four hours after light irradiation, strong TRX labeling was observed in the posterior polar retinal pigment epithelium (RPE), which was maintained up to 96 hours after light irradiation. The labeling was not significant in the peripheral retina RPE throughout the analyzed time course. TRX Western blot results showed that TRX up-regulation was observed in the retinal nerve and RPE fractions at 12 and 24 hours after light irradiation (FIGS. 8B and C). There were no significant band changes in the Coomassie blue stained gel in both preretinal retinal nerve and RPE fractions at 12 and 24 hours after light irradiation (data not shown).
Detection of oxidized and tyrosine phosphorylated proteins in retinal samples of rTRX injected mice
The present inventor analyzed protein oxidation and tyrosine phosphorylation to evaluate oxidative stress after light irradiation in mice injected with rTRX, vehicle or mutant rTRX into the intravitreal cavity before light irradiation.
[0031]
In vehicle or mutant rTRX mice, the amount of oxidized protein in the retinal nerve increased immediately after light irradiation (FIG. 9A). Compared to vehicle or mutant rTRX treated mice, the amount of oxidized protein was reduced in rTRX-treated mice. In a retinal specimen derived from a mouse not irradiated with light, two strong bands and three weak bands phosphorylated at tyrosine were detected (FIG. 9B). Immediately after light irradiation, one of the two bands of strong intensity was enhanced and an additional band of weak intensity was detected in vehicle or mutant rTRX treated mice. These band enhancements were less pronounced in rTRX-treated mice compared to vehicle or mutant rTRX-treated mice.
Cytoprotective effect of rTRX against photooxidative stress
The inventor then examined the effect of rTRX administration on retinal damage. Either rTRX, vehicle or mutant rTRX was injected into the intravitreal cavity prior to light irradiation, and surviving photoreceptor cell nuclei were compared between these eyes (FIGS. 10A, B). After 96 hours of light irradiation, the number of photoreceptor cell nuclei was significantly higher in rTRX-treated eyes than in vehicle (P <0.001) or mutant rTRX-treated eyes (P <0.001). Internucleosomal DNA ladders were evaluated in retinal samples from rTRX-, vehicle- or mutant rTRX-treated eyes. After 36 hours of light irradiation, DNA ladders were detected in retinal nerve samples from vehicle- and mutant rTRX-treated mice but not in retinal nerve samples from rTRX-treated mice (data not shown).
[0032]
After 96 hours of light irradiation, DNA ladders were detected in retinal nerve samples from vehicle and mutant rTRX-treated mice. In contrast, it decreased in retinal nerve samples from rTRX-treated mice (FIG. 10C).
Consideration
Light irradiation causes significant loss of photoreceptor nuclei (FIG. 7). Since TUNEL-positive photoreceptor cell nuclei (FIG. 7) and DNA ladder formation (FIG. 10C) are observed in the retina after light irradiation, the present data of the present inventor are that apoptosis is the main cell death of photoreceptor cells. This is a difficult route and this is the previous literature^TenMatches. According to immunohistochemistry, TRX was upregulated after light irradiation in both retinal nerves and RPE, and was not upregulated in the peripheral retina in the immediate vicinity of the iris (FIG. 8A). In Western blotting, TRX was upregulated in both retinal nerve and RPE fractions (FIGS. 8B and 8C). Taken together, the results of the present invention indicate that TRX is a light-induced endogenous molecule and that TRX plays an important role in the regeneration of retinal nerves by light. Protein oxidation occurs by free radical production and tyrosine kinases, including src family kinases, phosphatidylinositol 3-kinases, and mitogen-activated protein kinases, are activated by oxidative stress^12,22,23. This study shows that the enhancement in retinal nerve after light irradiation of both oxidized and tyrosine phosphorylated proteins is decreased in rTRX-treated mice but not in mutant rTRX-treated mice (FIGS. 9A, B), This suggests that intravitreal administration of rTRX reduces retinal nerve damage due to photooxidative stress in the retina, and that cysteine residues in its conserved active site play an important role in reducing photooxidative stress.
[0033]
Compared to vehicle-treated mice, the decrease in photoreceptor cell nuclei and DNA ladder formation was significantly excluded in rTRX-treated mice, while the effect disappeared in mutant TRX-treated mice (FIG. 10). This result suggests that TRX has an anti-apoptotic effect in retinal photopathy and that cysteine residues in its conserved active site play an important role in this cytoprotection. Previous studies have shown that exogenous rTRX ischemia / reperfusion injury lungs¹⁶,retina¹⁸And vascular endothelial disorders^{twenty four}Suggests cytoprotective effect against The mechanism by which exogenous rTRX ameliorates retinal photoreceptor damage is unknown. Reactive oxygen species induced by photooxidation in extracellular space and plasma membrane are TRX-dependent peroxidase^{twenty five}Or direct scavenging action of TRX on singlet oxygen or hydroxy radicals²⁶May be reduced. Another possibility is that exogenous TRX binds to the cell membrane and is taken up into the intracellular space.
[0034]
Previously, ascorbic acid²⁷, Dimethylthiourea^28,29And WR-77913⁸Cytoprotective effects against antioxidants such as The results of the present invention further emphasize the role of antioxidants against retinal photopathy. In addition, thioredoxin may act as a redox regulator, transcription factor regulatory function and stress signaling kinase.^12,13These mechanisms of action may be important for the cytoprotective effect of thioredoxin against retinal light stress.
[0035]
Excessive light is related to human age-related macular degeneration and possibly some forms of retinitis pigmentosa^1,2Progression and severity. Broad spectrum light hazards resulting from manipulation of microscopes used in ophthalmic practices can cause photomacular disease^3,4. The present invention has demonstrated the possibility of protecting retinal photodamage by exogenous TRX administration. The present invention further suggests that the induction of endogenous TRX is associated with increased resistance to retinal photopathy. The inventor is prostaglandin E1^30,31And geranylgeranylacetone³²Have been shown to effectively induce endogenous TRX in cells or tissues. TRX augmentation with these TRX inducers can be a useful therapeutic strategy for the protection of human photooxidative stress-related retinal diseases
References for Example 2
1. Cruickshanks KJ, Klein R, Klein BE. Sunlight and age-related macular degeneration. The Beaver Dam Eye Study. Arch Ophthalmol. 1993; 111 (4): 514-518.
2. Cideciyan AV, Hood DC, Huang Y, et al. Disease sequence from mutant rhodopsin allele to rod and cone replica degeneration in man.Proc Natl Acad Sci U S A. 1998; 95 (12): 7103-7108.
3. Byrnes GA, Chang B, Loose I, Miller SA, Benson WE. Prospective incidence of photic maculopathy after cataract surgery. Am J Ophthalmol. 1995; 119 (2): 231-232.
4. Minckler D. Retinal light damage and eye surgery. Ophthalmology. 1995; 102 (12): 1741-2.
5. Wiegand RD, Giusto NM, Rapp LM, Anderson RE.Evidence for rod outer segment lipid peroxidation following constant illumination of the rat retina.Invest Ophthalmol Vis Sci. 1983; 24 (10): 1433-1435.
6. Penn JS, Naash MI, Anderson RE.Effect of light history on retinal antioxidants and light damage susceptibility in the rat.Exp Eye Res. 1987; 44 (6): 779-788.
7. Organisciak DT, Wang HM, Xie A, Reeves DS, Donoso LA.Intense-light mediated changes in rat rod outer segment lipids and proteins.Prog Clin Biol Res. 1989; 314: 493-512.
8. Reme CE, Braschler UF, Roberts J, Dillon J. Light damage in the rat retina: effect of a radioprotective agent (WR-77913) on acute rod outer segment disk disruptions. Photochem Photobiol. 1991; 54 (1): 137 -142.
9. De La Paz MA, Zhang J, Fridovich I. Antioxidant enzymes of the human retina: effect of age on enzyme activity of macula and periphery.Curr Eye Res. 1996; 15 (3): 273-278.
10. Hafezi F, Steinbach JP, Marti A, et al. The absence of c-fos prevents light-induced apoptotic cell death of inhibitors in retinal degeneration in vivo. Nat Med. 1997; 3 (3): 346-349.
11. Holmgren A. Thioredoxin. Annu Rev Biochem. 1985; 54: 237-271.
12. Saitoh M, Nishitoh H, Fujii M, et al. Mammalian thioredoxin is a direct inhibitor of apoptosis signal- regulating kinase (ASK) 1. Embo J. 1998; 17 (9): 2596-2606.
13. Hirota K, Murata M, Sachi Y, et al. Distinct roles of thioredoxin in the cytoplasm and in the nucleus.A two-step mechanism of redox regulation of transcription factor NF- kappaB.J Biol Chem. 1999; 274 (39 ): 27891-27897.
14. Nakamura H, Nakamura K, Yodoi J. Redox regulation of cellular activation. Annu Rev Immunol. 1997; 15: 351-369.
15. Ohira A, Honda O, Gauntt CD, et al. Oxidative stress induces adult T cell leukemia derived factor / thioredoxin in the rat retina.Lab Invest. 1994; 70 (2): 279-285.
16. Okubo K, Kosaka S, Isowa N, et al. Amelioration of ischemia-reperfusion injury by human thioredoxin in rabbit lung. J Thorac Cardiovasc Surg. 1997; 113 (1): 1-9.
17. Takagi Y, Mitsui A, Nishiyama A, et al. Overexpression of thioredoxin in transgenic mice attenuates focal ischemic brain damage.Proc Natl Acad Sci U S A. 1999; 96 (7): 4131-4136.
18. Shibuki H, Katai N, Kuroiwa S, Kurokawa T, Yodoi J, Yoshimura N. Protective effect of adult T-cell leukemia-derived factor on retinal ischemia-reperfusion injury in the rat.Invest Ophthalmol Vis Sci. 1998; 39 ( 8): 1470-1477.
19. Takagi Y, Gon Y, Todaka T, et al. Expression of thioredoxin is enhanced in atherosclerotic plaques and during neointima formation in rat arteries.Lab Invest. 1998; 78 (8): 957-966.
20. Hirota K, Matsui M, Iwata S, Nishiyama A, Mori K, Yodoi J. AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc Natl Acad Sci US A. 1997; 94 (8 ): 3633-3638.
21. Oliver CN, Starke-Reed PE, Stadtman ER, Liu GJ, Carney JM, Floyd RA.Oxidative damage to brain proteins, loss of glutamine synthetase activity, and production of free radicals during ischemia / reperfusion- induced injury to gerbil brain. Proc Natl Acad Sci US A. 1990; 87 (13): 5144-5147.
22. Nakamura K, Hori T, Sato N, Sugie K, Kawakami T, Yodoi J. Redox regulation of a src family protein tyrosine kinase p56lck in T cells. Oncogene. 1993; 8 (11): 3133-3139.
23. Nakamura K, Hori T, Yodoi J. Alternative binding of p56lck and phosphatidylinositol 3-kinase in T cells by sulfhydryl oxidation: implication of aberrant signaling due to oxidative stress in T lymphocytes. Mol Immunol. 1996; 33 (10): 855 -865.
24. Nakamura H, Matsuda M, Furuke K, et al. Adult T cell leukemia-derived factor / human thioredoxin protects endothelial F-2 cell injury caused by activated neutrophils or hydrogen peroxide [published erratum appears in Immunol Lett 1994 Oct; 42 ( 3): 213]. Immunol Lett. 1994; 42 (1-2): 75-80.
25. Chae HZ, Chung SJ, Rhee SG. Thioredoxin-dependent peroxide reductase from yeast. J Biol Chem. 1994; 269 (44): 27670-27678.
26. Das KC, Das CK. Thioredoxin, a singlet oxygenquencher and hydroxyl redical scavenger: redox independent functions. Biochem Biophys Res Commun. 2000; 277 (2): 443-447.
27. Organisciak DT, Wang HM, Li ZY, et al. The protective effect of ascorbate in retinal light damage of rats.Invest Ophthalmol Vis Sci 1985; 26 (11): 1580-1588.
28. Lam S, Tso MO, Gurne DH. Amelioration of retinal photic injury in albino rats by dimethylthiourea. Arch Ophthalmol 1990; 108 (12): 1751-1757.
29. Organisciak DT, Darrow RM, Jiang YI, et al. Protection by dimethylthiourea against retinal light damage in rats.Invest Ophthalmol Vis Sci 1992; 33 (5): 1599-1609.
30. Yamamoto M, Ohira A, Honda O, et al. Analysis of localization of adult T-cell leukemia-derived factor in the transient ischemic rat retina after treatment with OP-1206 alpha-CD, a prostaglandin E1 analogue. J Histochem Cytochem 1997; 45 (1): 63-70.
31. Yamamoto M, Sato N, Tajima H, et al. Induction of human thioredoxin in cultured human retinal pigment epithelial cells through cyclic AMP-dependent pathway; involvement in the cytoprotective activity of prostaglandin E1. Exp Eye Res. 1997; 65 (5 ): 645-652.
32. Hirota K, Nakamura H, Arai T, et al. Geranylgeranylacetone enhances expression of thioredoxin and suppresses ethanol-induced cytotoxicity in cultured hepatocytes. Biochem Biophys Res Commun. 2000; 275 (3): 825-830.
Example 3
PC12 cells were incubated in RPMI 1640 medium containing 10% bovine serum (HS) and 5% fetal calf serum (FCS), 100 μg / ml streptomycin and 100 U / ml penicillin. Cells are 5% CO₂Incubate at 37 ° C. in the lower humid atmosphere. 0, 0.3, 1 mM MPP⁺After incubation with (1-methyl-4-phenylpyridinium ion) for 3 hours, PC12 cells were recovered and a cell lysate was prepared. TRX expression was detected by Western blot in PC12 cells. Briefly, cell lysates are maintained at 95 ° C. for 5 minutes, then applied to a 12% dodecyl sulfate acrylamide gel, electrophoresed (5 μg / lane), and then transferred to a transfer membrane (Millipore, Bedford, Mass.). did. After blocking with 5% skim milk in phosphate buffered saline (PBS) containing 0.5% Tween 20, the membrane was cross-reacted with rat TRX for 1 hour (Y. Takagi et al. J. Cereb Incubation with Blood Flow Metab. 18 (1998) pp. 206-214). The membrane was then incubated with peroxidase-conjugated anti-rabbit immunoglobulin antibody. The conjugated peroxidase is chemiluminescent (ECL) according to the manufacturer's specifications.^TM(RPN2106, Amersham Pharmacia Biotech) Western blot detection kit). The amount of TRX in each sample was evaluated by analyzing the density of each band using a computerized densitometer, NIH image. MPP⁺Under treatment conditions, TRX expression was reduced in PC12 cells (FIG. 11). Similar experiments were performed three times.
[0036]
Next, the inventor confirmed that MPP for PC12 cell viability by lactate dehydrogenase (LDH) release assay.⁺The effect of was examined in detail. LDH released from damaged cells was measured in aliquots of cell culture. The remaining cellular LDH was obtained by lysing the cells with 0.2% Tween 20 in PBS. LDH in a 50 μl sample of cell culture or cell lysate was measured using an LDH assay kit (kyokuto, Tokyo) according to the manufacturer's method. The percent cell lysis was measured as the LDH ratio (LDH in medium / total LDH per well). Here, total LDH refers to LDH of medium + cell lysate. MPP⁺When cultured with, cell viability decreased in a dose-dependent manner (FIG. 12). Similar experiments were repeated three times.
[0037]
Overexpression of TRX is MPP⁺To determine whether to protect PC12 cells from induced damage, PC12 cells (1 × 10⁷/ Ml) Incubated overnight in RPMI 1640 medium supplemented with 10% HS and 5% FCS. Cells were mechanically detached from the tissue dish, washed 3 times with serum-free RPMI 1640 medium, and then the cells were mixed with 10 μg pBI-EGFP-wtTRX. pBI-EGFP-wtTRX was constructed by ligating a TRX cDNA fragment (Y. Tagaya et al. EMBO J. 8 (1989), pp. 757-764) to a pBI-EGFP control vector (Clontech). Cells were then incubated on ice for 5 minutes and exposed to the appropriate single electrical pulse. Transient TRX overexpression and administration of recombinant human TRX causes PC12 cells to MPP⁺And MPP when incubated for 24 hours⁺Induced damage was suppressed (FIGS. 13A, B).
[0038]
The above results show that TRX has a neuronal protective effect, thereby MPP⁺It shows that the damage-inducing action was suppressed.
Example 4
Thioredoxin transgenic (trx-tg) mice are C57BL / 6 mice (wild type) that carry human TRX transgene under the control of β-actin promoter and express human TRX throughout the body including the brain (Takagi Y et al., Proc. Natl. Acad. Sci. USA, 96 (1999) 4131-4136). The present inventor confirmed that human TRX is expressed throughout the trx-tg retina (FIGS. 14A and B). Retinal sample preparation, immunohistochemistry, and Western blotting were performed as previously described (Takagi Y. et al., Proc. Natl. Acad. Sci. USA, 96 (1999) 4131-4136; Ohira A et al., Lab. Invest., 70 (1994) 279-285). Therefore, the present inventor evaluated whether trx-tg mice are resistant to retinal damage induced by intense light. All methods in this study followed the ARVO statement on Use of Animals in Ophthalmic and Vision Research.
[0039]
First, we analyzed endogenous mouse TRX before and after light exposure to the retina of trx-tg and wild type mice by Western blotting with anti-mouse TRX antibody (Takagi Y. et al., Proc. Natl. Acad. Sci. USA, 96 (1999) 4131-4136; Ohira A. et al., Lab. Invest., 70 (1994) 279-285). Prior to light exposure, 3-4 week old male mice were acclimated for 48 hours in the dark and their pupils were dilated using 0.5% tropicamide and 0.5% phenylephrine hydrochloride eye drops (Santen Pharmaceutical). Unanesthetized mice were exposed to 8000 lux diffusive daylight white fluorescent light (Matsushita Electric Industrial) in a cage with a reflective interior for 2 hours (Wenzel A. et al., J. Neurosci., 20 (2000) 81-88). Care was taken to ensure that the eyes received the same level of light during illumination. Prior to light exposure, the expression of endogenous mouse TRX in the retina of trx-tg mice could be compared with that of wild type mice. After a total of 24 hours of light exposure, a two-fold upregulation of endogenous mouse TRX was observed in both trx-tg and wild type mice, where there was no significant difference in both mice (data not shown) . The amount of human TRX protein in each tissue of trx-tg mice has been reported to be 3-6 times greater than endogenous mouse TRX protein (Takagi Y. et al., Proc. Natl. Acad. Sci. USA, 96 (1999) 4131-4136). Furthermore, the activity of the β-actin promoter is considered to be sufficiently higher than that of the endogenous mouse TRX promoter. As a result, the amount of total TRX protein in the retina is believed to be considerably higher in trx-tg mice than in wild-type mice before and after light exposure.
[0040]
Next, we analyzed protein oxidation and tyrosine phosphorylation to assess oxidative stress after light exposure in trx-tg and wild type mice. Oxidized protein was analyzed as described above (Takagi Y. et al., Proc. Natl. Acad. Sci. USA, 96 (1999) 4131-4136) Oxidized protein detection kit (OxyBlot, Intergen, Purchase, NY) It detected using. The kit provides reagents for sensitive immunodetection of carbonyl groups. Prepare 2,4-dinitrophenyl (DNP) hydrazone derivatized retinal protein samples according to the manufacturer's protocol, separate by 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (10 μg protein / lane), and polyvinylidene Transferred to fluoride (PVDF) membrane. After blocking, the membrane was incubated with a primary antibody specific for the DNP portion of the protein. The protein band was then detected by the same Western blotting method as human TRX. Tyrosine phosphorylated protein was detected using the ECL tyrosine phosphorylation detection system (RPN 2220 / l, Amersham Biosciences). Retinal protein samples were prepared and electrophoresed on a 12% SDS polyacrylamide gel (10 μg protein / lane) and then electrophoretically transferred to a PVDF membrane according to the manufacturer's recommendations. After blocking, the membrane was incubated with a peroxidase-conjugated anti-phosphotyrosine antibody (PY-20, Amersham Biosciences) and chemiluminescence was detected with an ECL Western blot detection kit. In wild-type mice, the amount of oxidized protein in the retinal nerve increased immediately after light exposure compared to before light exposure (FIG. 15A, lane 2). Compared to wild-type mice, the amount of oxidized protein decreased in trx-tg mice (FIG. 15A, lane 4). In wild type mouse retinal specimens, three strong bands (arrows) of tyrosine phosphorylated protein were detected before light exposure (FIG. 15B, lane 1). Immediately after light exposure, these bands were enhanced and at least two additional bands (arrowheads) were detected (FIG. 15B, lane 2). Compared to wild type mice, the increase in these bands was less pronounced in trx-tg mice (FIG. 15B, lane 4). Protein oxidation is the result of free radical production (Oliver CN et al., Proc. Natl. Acad. Sci. USA, 87 (1990) 5144-5147), src family kinase, phosphatidylinositol 3-kinase and mitogen-activated protein Tyrosine kinases including kinases are activated by oxidative stress (Nakamura K. et al., Mol. Immunol., 33 (1996) 855-865; Saitoh, M. et al., EMBO J., 17 (1998) 2596-2606). Thus, this result indicates that TRX overexpression reduces photooxidative stress in the retina.
[0041]
Intense light causes photoreceptor DNA damage (Organisciak D.T. et al., Photochem Photobiol., 70 (1999) 261-268). In order to evaluate the cytoprotective effect of overexpressed TRX against light-induced DNA damage, the inventors used quantitative immunohistochemistry (8OhdG index) for 8-hydroxy-2-deoxyguanosine. Methods for preparing paraffin-treated retinal samples, immunohistochemistry for 8OhdG using the alkaline phosphatase method and quantification of 8OhdG immunostaining are known (Ohira A., et al., Lab. Invest., 70 (1994 279-285; Toyokuni S. et al., Lab. Invest., 76 (1997) 365-374). Anti-8OhdG monoclonal antibody was purchased from NOF Corporation. In order to calculate the 8OhdG index, digitized color images at two positions (upper and lower retina, approximately 100 μm from the optic disc) of each mouse were used in a PICT file using a digital imaging system (PDMC le, Olympus). And analyzed using the National Institutes of Health image version 1.61 software on a Macintosh personal computer. There was no significant difference in 8OhdG index in photoreceptor nuclei (outer granule layer) between mouse strains prior to light exposure (FIG. 16B, -2h). At 12 and 24 hours after light exposure, the 8OhdG index was significantly higher in wild-type mice than in trx-tg mice (P <0.01 and P <0.01, respectively) (FIGS. 16B, 12h and 24h). ). In wild type mice, strong staining was maintained 24 hours after light exposure (FIG. 16A, left panel). On the other hand, the staining of the outer granule layer of trx-tg mice decreased after 24 hours of light exposure (FIG. 16A, right panel). The major DNA base modification product, 8OhdG, is induced by either hydroxy radicals, singlet oxygen, or photodynamic action and is an established marker of oxidative stress-induced DNA damage (Toyokuni S. et al., Lab. Invest., 76 (1997) 365-374). Therefore, this result indicates that overexpressed TRX prevents photoreceptor DNA damage caused by photooxidative stress.
[0042]
Electroretinography (ERG) is a recording of action potentials (a-waves) generated by photoreceptor cells and action potentials (b-waves) generated by secondary neurons in the inner granule layer that interact with Muller glial cells. Thus, the amplitudes of the a- and b-waves reflect the functional state of these first two retinal neurons. To test the effect of overexpressed TRX on retinal function, we compared a-wave and b-wave between both mouse strains. Flash ERG was recorded using PE-3000 (Tomey, Nagoya). A gold contact lens electrode (3 mm diameter, 1.5-mm base curve, Kyoto contact lens) was placed on the left eye, the same reference electrode was placed in the mouth, and the outer electrode was placed on the left leg pad. Prior to light exposure, the amplitudes of a-wave and b-wave were not different between these lines (FIGS. 17, -2h). After light exposure, the amplitudes of a-wave and b-wave are 6 hours (P <0.01 and P <0.05, respectively) and 12 hours (P <0.01 and P <0.05, respectively). And significantly higher in trx-tg mice compared to wild type mice (FIGS. 17, 6h and 12h). Thus, this result indicates that overexpressed TRX regenerates photodamaged retinal nerves.
References
Ahn S, Olive M, Aggarwal S, Krylov D, Ginty DD, Vinson C (1998) A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus-dependent transcription of c-fos. Mol Cell Biol 18: 967 -977.
Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.Proc Natl Acad Sci USA 92: 7297-7301.
Chen RH, Sarnecki C, Blenis J (1992) Nuclear localization and regulation of erk- and rsk-encoded protein kinases. Mol Cell Biol 12: 915-927.
Chiarini LB, Freitas FG, Petrs-Silva H, Linden R (2000) Evidence that the bifunctional redox factor / AP endonuclease Ref-1 is an anti-apoptotic protein associated with differentiation in the developing retina.Cell Death Differ 7: 272-281 .
Connor B, Dragunow M (1998) The role of neuronal growth factors in neurodegenerative disorders of the human brain.Brain Res Rev 27: 1-39.
Ema M, Hirota K, Mimura J, Abe H, Yodoi J, Sogawa K, Poellinger L, Fujii-Kuriyama Y (1999) Molecular mechanisms of transcription activation by HLF and HIF1alpha in response to hypoxia: their stabilization and redox signal-induced interaction with CBP / p300. EMBO J 18: 1905-1914.
Endoh M, Kunishita T, Tabira T (1993) Thioredoxin from activated macrophages as a trophic factor for central cholinergic neurons in vitro.Biochem Biophys Res Commun 192: 760-765.
Ginty DD, Bonni A, Greenberg ME (1994) Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription viaphosphorylation of CREB.Cell 77: 713-725.
Greene LA, Tischler AS (1976) Establishment of a noradrenerergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.Proc Natl Acad Sci U S A 73: 2424-2428.
Hashimoto S, Matsumoto K, Gon Y, Furuichi S, Maruoka S, Takeshita I, Hirota K, Yodoi J, Horie T (1999) Thioredoxin negatively regulates p38 MAP kinase activation and IL-6 production by tumor necrosis factor-alpha.Biochem Biophys Res Commun 258: 443-447.
Hirota K, Matsui M, Iwata S, Nishiyama A, Mori K, Yodoi J (1997) AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1. Proc Natl Acad Sci U S A 94: 3633-3638.
Hirota K, Matsui M, Murata M, Takashima Y, Cheng FS, Itoh T, Fukuda K, Yodoi J (2000) Nucleoredoxin, glutaredoxin, and thioredoxin differentially regulate NF-kappaB, AP-1, and CREB activation in HEK293 cells.
Biophys Res Commun 274: 177-182.
Hollowell JP, Villadiego A, Rich KM (1990) Sciatic nerve regeneration across gaps within silicone chambers: long-term effects of NGF and consideration of
Axonal branching.Exp Neurol 110: 45-51.Holmgren A (1985) Thioredoxin.Annu Rev Biochem 54: 237-271.Hori K, Katayama M, Sato N, Ishii K, Waga S, Yodoi J (1994) Neuroprotection by glial cells through adult T cell leukemia-derived factor / human thioredoxin (ADF / TRX). Brain Res 652: 304-310.
Impey S, Obrietan K, Wong ST, Poser S, Yano S, Wayman G, Deloulme JC, Chan G, Storm DR (1998) Cross talk between ERK and PKA is required for Ca2 + stimulation of CREB-dependent transcription and ERK nuclear translocation. Neuron 21: 869-883.
Kamata H, Tanaka C, Yagisawa H, Matsuda S, Gotoh Y, Nishida E, Hirata H (1996) Suppression of nerve growth factor-induced neuronal differentiation of PC12 cells.N-acetylcysteine uncouples the signal transduction from ras to the mitogen-activated protein kinase cascade. J Biol Chem 271: 33018-33025.
Kanamoto T, Mota M, Takeda K, Rubin LL, Miyazono K, Ichijo H, Bazenet CE (2000) Role of apoptosis signal-regulating kinase in regulation of the c-Jun N-terminal kinase pathway and apoptosis in sympathetic neurons. Biol 20: 196-204.
Kane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS, Ord T, Bredesen DE (1993) Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species.Science 262: 1274-1277..Kaplan DR, Martin-Zanca D, Parada LF (1991) Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF.Nature 350: 158-160.
Kim YC, Masutani H, Yamaguchi Y, Itoh K, Yamamoto M, Yodoi J (2001) Hemin-induced activation of the thioredoxin gene by Nrf2. A differential regulation of the antioxidant responsive element by a switch of its binding factors.J Biol Chem 276: 18399-18406.
Kummer JL, Rao PK, Heidenreich KA (1997) Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase.
J Biol
Chem 272: 20490-20494.
Lo DC (1992) Signal transduction and regulation of neurotrophins. Curr Opin Neurobiol 2: 336-340.
Lovell MA, Xie C, Gabbita SP, Markesbery WR (2000) Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer's disease brain.Free Radic Biol Med 28: 418-427.
Mansur K, Iwahashi Y, Kiryu-Seo S, Su Q, Namikawa K, Yodoi J, Kiyama H (1998) Up-regulation of thioredoxin expression in motor neurons after nerve injury.Brain Res Mol Brain Res 62: 86-91.
Maruyama T, Kitaoka Y, Sachi Y, Nakanoin K, Hirota K, Shiozawa T, Yoshimura Y, Fujii S, Yodoi J (1997) Thioredoxin expression in the human endometrium during the menstrual cycle.Mol Hum Reprod 3: 989-993.
Masutani H, Magnaghi-Jaulin L, Ait-Si-Ali S, Groisman R, Robin P, Harel-Bellan A. (1997) Activation of the c-fos SRE through SAP-1a.Oncogene 15: 1661-1669.
Masutani H, Ueno M, Ueda S and Yodoi J (1999) in Antioxidant and redox regulation of genes (Sen, C.K., Sies, H., Baeuerle, P.A., eds), pp.297-311, Academic Press, San Diego
Milbrandt J (1986) Nerve growth factor rapidly induces c-fos mRNA in PC12 Rat pheochromocytoma cells. Proc Natl Acad Sci U S A 83: 4789-4793.
Montminy MR, Sevarino KA, Wagner JA, Mandel G, Goodman RH (1986) Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.Proc Natl Acad Sci U S 83: 6682-6686.
Nakamura H, Matsuda M, Furuke K, Kitaoka Y, Iwata S, Toda K, Inamoto T, Yamaoka Y, Ozawa K, Yodoi J (1994) Adult T cell leukemia-derived factor / human thioredoxin protects endothelial F-2 cell injury caused by activated neutrophils or hydrogen peroxide. Immunol Lett 42: 75-80.
Nakamura H, Nakamura K, Yodoi J (1997) Redox regulation of cellular activation. Annu Rev Immunol 15: 351-369.
Nishiyama A, Matsui M, Iwata S, Hirota K, Masutani H, Nakamura H, Takagi Y, Sono H, Gon Y, Yodoi J (1999) Identification of thioredoxin-binding protein-2 / vitamin D (3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression.J Biol Chem 274: 21645-21650.
Pallage V, Toniolo G, Will B, Hefti F (1986) Long-term effects of nerve growth factor and neural transplants on behavior of rats with medial septal lesions.Brain Res 386: 197-208.
Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, Kawabata M, Miyazono K, Ichijo H (1998) Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1.EMBO J 17: 2596-2606.
Serrano Sanchez T, Robinson Agramonte Md M, Lorigados Pedre L, Diaz Armesto I, Gonzalez Fraguela ME, Dorta-Contreras AJ (2001) Endogenous nerve growth factor in patients with Alzheimer s disease.Rev Neurol 32: 825-828.
Sheng M, Dougan ST, McFadden G, Greenberg ME (1988) Calcium and growth factor pathways of c-fos transcriptional activation require distinct upstream regulatory sequences. Mol Cell Biol 8: 2787-2796.
Sheng M, Greenberg ME (1990) The regulation and function of c-fos and other immediate early genes in the nervous system.Neuron 4: 477-485.
Tagaya Y, Maeda Y, Mitsui A, Kondo N, Matsui H, Hamuro J, Brown N, Arai K, Yokota T, Wakasugi H, Yodoi J. (1989) ATL-derived factor (ADF), an IL-2 receptor / Tac inducer homologous to thioredoxin; possible involvement of dithiol-reduction in the IL-2 receptor induction. EMBO J 8: 757-764.
Takagi Y, Horikawa F, Nozaki K, Sugino T, Hashimoto N, Yodoi J (1998a) Expression and distribution of redox regulatory protein, thioredoxin during transient focal brain ischemia in the rat.Neurosci Lett 251: 25-28..Takagi Y, Tokime T, Nozaki K, Gon Y, Kikuchi H, Yodoi J (1998b) Redox control of neuronal damage during brain ischemia after middle cerebral artery occlusion in the rat: immunohistochemical and hybridization studies of thioredoxin.J Cereb Blood Flow Metab 18: 206- 214.
Takagi Y, Gon Y, Todaka T, Nozaki K, Nishiyama A, Sono H, Hashimoto N, Kikuchi H, Yodoi J (1998c) Expression of thioredoxin is enhanced in atherosclerotic plaques and during neointima formation in rat arteries.Lab Invest 78: 957 -966.
Takagi Y, Mitsui A, Nishiyama A, Nozaki K, Sono H, Gon Y, Hashimoto N, Yodoi J (1999) Overexpression of thioredoxin in transgenic mice attenuates focal ischemic brain damage.Proc Natl Acad Sci USA 96: 4131-4136 Takagi Y, Hattori I, Nozaki K, Mitsui A, Ishikawa M, Hashimoto N, Yodoi J (2000) Excitotoxic hippocampal injury is attenuated in thioredoxin transgenic mice.J Cereb Blood Flow Metab 20: 829-833.
Taniguchi Y, Taniguchi-Ueda Y, Mori K, Yodoi J (1996) A novel promoter sequence is involved in the oxidative stress-induced expression of the adult T-cell leukemia-derived factor (ADF) / human thioredoxin (Trx) gene. Nucleic Acids Res 24: 2746-2752.
Thomas SM, DeMarco M, D'Arcangelo G, Halegoua S, Brugge JS (1992) Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphorylation of MAP kinases.Cell 68: 1031-1040..Tomimoto H, Akiguchi I, Wakita H, Kimura J, Hori K, Yodoi J (1993) Astroglial expression of ATL-derived factor, a human thioredoxin homologue, in the gerbil brain after transient global ischemia.Brain Res 625: 1-8.
Treisman R (1986) Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors.Cell 46: 567-574.
Trouche D, Grigoriev M, Lenormand JL, Robin P, Leibovitch SA, Sassone-Corsi P, Harel-Bellan A (1993) Repression of c-fos promoter by MyoD on muscle cell differentiation.Nature 363: 79-82.
Ueno M, Masutani H, Arai RJ, Yamauchi A, Hirota K, Sakai T, Inamoto T, Yamaoka Y, Yodoi J, Nikaido T (1999) Thioredoxin-dependent redox regulation of p53-mediated p21 activation.J Biol Chem 274: 35809 -35815.
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326-1331.
Yamamoto M, Sato N, Tajima H, Furuke K, Ohira A, Honda Y, Yodoi J (1997) Induction of human thioredoxin in cultured human retinal pigment.Exp Eye Res 65: 645-652.
[Brief description of the drawings]
FIG. 1A NGF-induced TRX expression.
FIG. 1A shows the increase in TRX protein caused by NGF. PC12 cells treated with NGF (50 ng / ml) for 24 and 48 hours were harvested and detected by Western blotting.
FIG. 1B NGF-induced TRX expression.
FIG. 1B shows increased expression of TRX mRNA induced by NGF. NGF-treated PC12 cells were harvested at each indicated time and then analyzed by Northern blotting.
FIG. 1C NGF-induced TRX expression.
FIG. 1C shows activation of the TRX gene by NGF. PC12 cells were transfected with the pTRX-Luc vector pTRX (-1148) and pRL-TK and then treated in the presence or absence of NGF.
[Figure 2] Identification of the region that responds to NGF in the TRX promoter
PC12 cells were transfected with pTRX-Luc vector and pRL-TK as shown in the left panel, and the values shown represent the ratio of luciferase activity of NGF (50 ng / ml) -treated cells to untreated cells . This result is representative of three independent experiments.
FIG. 3A. Repression of TRX expression by PD98059 or NAC.
FIG. 3A shows blockade of PC12 differentiation by PD98059 or NAC. PC12 cells were cultured for 2 days in the presence or absence of NGF (50 ng / ml) and then with NGF (50 ng / ml) in the presence of PD98059 (50 mM) or NAC (20 mM).
FIG. 3B. Repression of TRX expression by PD98059 or NAC.
FIG. 3B is inhibition of NGF-induced transactivation by addition of PD98059 or NAC. PC12 cells were transfected with pTRX (−263) -Luc vector and pRL-TK and then treated with NGF (50 ng / ml) and PD98059 (50 mM) or NAC (20 mM). The values shown are the ratio of luciferase activity to untreated cells in cells treated with NGF 50 (ng / ml) and PD98059 or NAC. The results are representative of three independent experiments.
FIG. 3C. Repression of TRX expression by PD98059 or NAC.
FIG. 3C shows suppression of TRX protein expression by PD98059 or NAC. PC12 cells were treated as in FIG. 3A and protein samples were fractionated and screened with anti-TRX antibodies.
Fig. 4 Suppression of NGF-induced TRX nuclear migration
FIG. 4 (A) shows PC12 cells cultured without NGF, FIG. 4 (B) shows PC12 cells treated with NGF, and FIGS. 4 (C) and 4 (D) show NGF and PD98059 (FIG. 4). 4C) or PC12 cells treated with NAC (FIG. 4D).
These cells were stained with anti-TRX mAb.
FIG. 5. Inhibition of NGF-induced differentiation by mutant TRX overexpression.
PC12 cells were transfected with pBI-EGFP, pBI-EGFP-wtTRX or pBI-EGFP-dmTRX (32S / 35S) vectors and then treated with NGF 50 (ng / ml). After 24 hours, these cells were examined with a laser confocal microscope.
FIG. 6A shows suppression of CRE-mediated c-fos induction by dominant negative mutant TRX.
FIG. 6A shows that NGF induces c-fos through CRE. PC12 cells were transfected with pGL3-c-fos (-40, +42) or pGL3-c-fos (-99, +42) as shown in the upper panel with pRL-TK and then 4 with NGF. Time processed. The value shown is the ratio of luciferase activity of NGF treated cells to untreated cells.
FIG. 6B shows suppression of CRE-mediated c-fos induction by dominant negative mutant type TRX.
FIG. 6B shows inhibition of c-fos activation by NGF by mutant TRX. PC12 cells were cotransfected with pGL-TK, pGL3-c-fos (-99, +42) and pcDNA3 (1g) or pcDNA3-TRX (32S / 35S) (1g) vector as shown in the upper panel. It was effected. Transfected PC12 cells were treated with NGF for 4 hours. The value shown is the ratio of luciferase activity of NGF treated cells to untreated cells.
FIG. 7 shows H-E and TUNEL staining of the irradiated retina. Representative H-E and TUNEL staining in retinal specimens is shown. A significant decrease in photoreceptor cell nuclei was observed after 24 hours and thereafter. TUNEL-positive cells were observed after 12 hours and thereafter (arrows). INL is the inner nuclear layer and ONL is the outer nuclear layer.
FIG. 8. Immunohistochemistry and Western blot for TRX expression in retinal samples. Representative immunohistochemistry for TRX is shown in (A). TRX nuclear labeling was observed in the inner nuclear layer (INL) immediately after light irradiation (short arrow), but disappeared after 24 hours. Nuclear labeling of the outer nuclear layer (ONL) was observed immediately after light irradiation (long arrow) and was maintained until 96 hours later. Immunity labeling is not prominent in the peripheral retina near the ciliary body immediately after light irradiation (0 hour). TRX labeling is observed with RPE after 24 hours or later (arrows) but not prominent around the retina (white arrows, 12 hours and 24 hours later). Shown is a representative Western blot (B) for TRX in the retinal nerve and RPE fractions, and semi-quantitative analysis of band intensity (C). The band intensity at 12 hours and 24 hours after light irradiation is represented by the induction ratio compared to that of mice not irradiated with light.
FIG. 9. Detection of oxidized (A) and tyrosine-phosphorylated (B) proteins in retinal samples from eyes pre-intravitreally treated with vehicle, rTRX (5 μg) or mutant rTRX (5 μg). (A) A representative Western blot for oxidized protein in the retinal nerve is shown. Eyes before light irradiation (first lane) and after vehicle, rTRX and mutant rTRX treatment (second lane, third lane and fourth lane, respectively). (B) A representative Western blot for tyrosine phosphorylated proteins in retinal nerves is shown. Eyes before light irradiation (first lane) and after vehicle, rTRX and mutant rTRX treatment (second lane, third lane and fourth lane, respectively). Two strong bands and three weak bands (arrows) were detected before light irradiation. Immediately after light irradiation, one of the two bands with strong intensity (upper arrow) was enhanced, and one additional band with lower intensity (lower arrow) was detected in vehicle or mutant rTRX-treated mice.
FIG. 10 shows cytoprotective effect of recombinant TRX. Shown are representative HE-E staining (A) and number of photoreceptor nuclei (B) from retinal samples from eyes pre-intravitreally treated with vehicle, rTRX (5 μg) or mutant rTRX (5 μg). 96 hours after light irradiation The number of photoreceptor nuclei in the eyes treated with rTRX (mean ± SE, 101.3 ± 5.1 cells / 100 μm; n = 6) is the vehicle (63.3 ± 2.7 cells / 100 μm; n = 6) and Mutant rTRX-treated (51.4 ± 3.3 cells / 100 μm; n = 4) significantly greater than in eyes. P values were calculated by one-way ANOVA followed by Bonferroni / Dunn post hoc test. Representative DNA ladder detection in retinal samples is shown in (C). Analysis of DNA ladder formation was detected 96 hours after light exposure in vehicle, rTRX or mutant rTRX treated eyes. ONL is the outer nuclear layer; n.s. is not statistically significant.
FIG. 11 MPP⁺Reduces TRX in PC12 cells. PC12 cells pre-cultured overnight in 35 mm dishes (2 × 10^Five/ Ml) is 0, 0.3 and 1 mM MPP⁺The cells were cultured for 3 hours in a medium containing and then cell lysates were collected. TRX is recognized as a 12 kDa band by Western blotting.
FIG. 12 MPP⁺Reduces the viability of PC12 cells. PC12 cells (2 × 10^Four/ Ml) is 0, 0.3 and 1 mM MPP⁺And incubated for 24 hours. The percent cell lysis was measured by LDH release assay.
FIG. 13: TRP overexpression and recombinant TRX administration is MPP⁺Suppresses induced damage. 1 mM MPP 24 hours after TRX overexpression⁺Was added to the medium and incubated for 24 hours. The percent cell lysis was measured by LDH release assay.
TRX overexpressing PC12 cells are treated with MPP as measured by LDH release assay.⁺Resistant to induced damage (FIG. 13A). Furthermore, administration of 100 μg / ml rTRX also allowed PC12 cells to MPP⁺0.3 and 1 mM MPP when incubated for 24 hours with⁺Induced damage was suppressed. Two asterisks (*) indicate statistical significance (^**P <0.001)
FIG. 14. Representative immunohistochemistry (A); and Western blot (B) for human TRX expression in retinal samples of wild type and trx-tg mouse eyes. GLC is the ganglion cell layer; INL is the inner granular layer; ONL is the outer granular layer; and RPE is the retinal pigment epithelial layer and the scale bar is 100 μm.
FIG. 15 Representative Western blots of oxidized (A) and tyrosine phosphorylated (B) proteins in retinal nerves of wild type and trx-tg mice. Before light exposure of wild type and trx-tg mice (-2h) (lanes 1 and 3, respectively) and immediately after light exposure of wild type and trx-tg mice (0h) (lanes 2 and 4 respectively).
FIG. 16 Quantitative immunohistochemical analysis of 8OhdG in photoreceptor granule layer.
(A) Representative immunohistochemical analysis of 8OhdG in specimens 24 hours after light exposure of wild type mice (left panel) and trx-tg mice (right panel). ONL is outer granule layer; scale bar is 20 μm.
(B) The 8OhdG index is summarized. Each column is expressed as mean ± SE (n = 5 for each group). P value was calculated by Mann-Whitney U-test.
FIG. 17 ERG. Average a- and b-wave amplitudes are summarized. Each column is expressed as mean ± SE (n = 5 for each group). P value was calculated by Mann-Whitney U-test.

Claims (1)

An agent for restoring retinal nerve cell function, which contains NGF and restores the function of photodamaged retinal neurons by inducing endogenous thioredoxin expression.

Images