Protein Kinase C- δ Mediates Neuronal Apoptosis in the Retinas of Diabetic Rats via the Akt Signaling Pathway Young-Hee Kim,1 Yoon-Sook Kim,1 Chang-Hwan Park,1 In-Yong Chung,2 Ji-Myong Yoo,2 Jae-Geun Kim,3 Byung-Ju Lee,3 Sang-Soo Kang,1 Gyeong-Jae Cho,1 and Wan-Sung Choi1 당뇨 쥐의 망막에서, Akt signal pathway를 통한 protein kinase C-델타의 nueronal apoptosis 조절에 관한 논문입니다. Pf.박찬기 / R3 김재련

DM… a risk factor of glaucoma? Prevelence of COAG appears to be higher in the diabetic population. However, findings from numerous clinical studies on the association of diabetes and glaucoma are inconsistent. - in Shield’s textbook 당뇨가 녹내장의 위험인자 인지에 대해서는 논란이 많은데ㅡ 당뇨환자에서 COAG의 유병율이 높은것으로 알려져 있지만.. 여러 연구들에서 당뇨와 녹내장의 관계에서 일치되지않는 결과들을 나타내었습니다 교수님께서 이 논문의 내용이나 결과 해석에 치중하지말고… 크게 봐서 당뇨가 녹내장의 위험인자인지에 대한 논란이 있는 것을 부각시키고 당뇨가 녹내장의 위험인자가 될 수 있는 이론적 background를 찾는 의미에서 고른 논문임을 부각시키라고 하시네. 이에 관련된 내용을 좀더 찾아서 보강하면 좋을듯…

DM… a risk factor of glaucoma? what about the mechanism? Vascular dysfunction Hyperglycemic-induced cell apoptosis vs 당뇨가 녹내장의 원인이라면 그 메커니즘은 무엇인지에대해, ㄱVSCULAR DYSFUNCTION이 원인이지, 또는 HYPERGLYCEMIC INDUCED CELL APOPTOSIS가 원인이지에대해 연구가 되고 있습니다. 이 논문은 HYPERGLYCEMIC INDUCED CELL APOPTOSIS에 관련된 논문으로, 당뇨와 녹내장의 관련성에 대해 생각해보는 계기가 될 수 있을것입니다.

INTRODUCTION protein kinase C (PKC)-δ mediate anti-apoptotic signaling cascade through the phosphatidylinositol 3-kinase (PI3-kinase)–mediated survival pathway promote apoptosis by interfering with Akt signal Akt downstream target of PI 3-kinase through the phosphorylation of two key regulation residue, Thr308 and Ser473 on Akt plays an integral role in cell survival protein kinase C (PKC)-δ 는 phosphatidylinositol 3-kinase 통한 survival pathway를 통해 anti-apoptotic signaling cascade 를 조절하며, 또한 AKT signal을 억제하여 apoptosis를 유발한다고 알려져있습니다. Akt는 PI 3 kinase 의 downstream target으로, Thr308 and Ser473 두 residue의 phosphorylation에 의해 직용하여 cell survival에 관여한다고 알려져 있습니다.

INTRODUCTION Protein phosphatase 2A (PP2A) regulation of cell proliferation and survival through its ability to dephosphorylate Akt Heat shock protein 90 (HSP90) counteracts the effect of PP2A direct binding to Akt, protecting Akt from PP2A-mediated dephosphorylation functioning as a positive regulator of Akt signaling Akt- or HSP-mediated cytoprotection is regulated by PKC Protein phosphatase 2A 는 Akt를 dephosphorylatet시켜 cell proliferation 과 survival을 조절하며, Heat shock protein 90 는 PP2A와 길항작용으로, Akt와 결합하여 pp2a에 의한 Dephosphorylation을 방지합니다. 결론적으로 PKC에 의해 A kt나 HSP을 통해 세포 보호작용이 중제된다고 알려져 있습니다

INTRODUCTION Otsuka Long-Evans Tokushima fatty (OLETF) rats genetic animal model of late onset of hyperglycemia spontaneously develop type 2 diabetes exhibit hyperglycemia and insulin resistance at 20–40 weeks of age OLETF rat은 genetic animal model로서 20-40 주 사이에 insulin resistance와 hyperglycemia를 보여 사람에서 type 2 diabetes환경을 조성해 줍니다

INTRODUCTION In previous study PKC- δ activation is involved in neuronal apoptosis in 35-week OLETF rat retinas however, a direct association between PKC- δ and Akt was not defined. Purpose of this study.. Effects of PKC- δ on Akt-mediated survival pathways and neuronal apoptosis in the retinas of diabetic OLETF 이전연구에서는 35주의 당뇨쥐 의 망막에서 PKC- δ 활성화가 neuronal apoptosis 와 연관이 있음을 발혔으나 , PKC- δ 와 Akt 의 직접적인 상관관계를 알지 못하였습니다 이연구에서의 목적은 당뇨쥐위 망막에서 PKC-델타 의 Akt-mediated survival pathways에 대한 영향을 알아보고 자 하였습니다

RESEARCH DESIGN AND METHODS 8 LETO and 8 OLETF rats at 24 weeks of age 28 LETO and 28 OLETF rats at 35 weeks of age measured their body weights and blood glucose levels. Intravitreal injection Rottlerin (Sigma, St Louis, MO) highly specific PKC- δ inhibitor dissolved in 0.5% dimethyl sulfoxide (DMSO) 30-gauge needle was inserted into the vitreous 2 mm posterior to the limbus through the pars plana 3μ l rottlerin (5 mol/ μ l) was used for intravitreal injection into the right eye of 35-week-old LETO and OLETF rats DMSO (3 μ l) was injected into the left vitreous as a control All rats were killed 1 day after the injection. 24주의 OLETF 8마리와 대조군으로 LETO 8마리, 35주의 OLETF 28마리와 대조군으로 LETO 28마리가 사용되었습니다. 몸무게와 blood glucose level을 측정하였습니다 실험내용에서 Intravitreal injection에서 highly specific PKC- δ inhibitor 인 Rottlerin이 35주의 OLETF rats과 대조군쥐의 우안에 inject되었고 왼쪽눈에는 각각 DMSO을 주입하였습니다.

RESEARCH DESIGN AND METHODS PKC- δ and HSP90 gene silencing commercially available- small interfering RNAs (siRNAs) from Dharmacon To assess the effects of PKC- δ and HSP90 siRNAs on retinas, 1 μ and 3 μ l siRNAs, each at a concentration of 500mol/l intravitreally injected into the right eye of OLETF and LETO rats at 35 weeks. Control rats received 1 μ and 3 μ l distilled water into the left eye. Rats were killed at 1, 2, and 5 days after the injection effects of siRNAs on PKC- δ and HSP90 were determined by immunoblotting PKC- δ 와 HSP90 gene silencing 으로 small interfering RNAs 가 사용되어 마찮가지고 35주 OLETF and LETO 의 우안에 inj하였고, 좌안에는 distilled water를 주입하였습니다 Silencing: interruption of expression of a gene at transcriptonal, translational level

RESEARCH DESIGN AND METHODS Antibodies PKC- δ, PI 3-kinase p85(regulatory subunit), HSP90, and Akt : Mouse monoclonal antibodies Thy-1: goat polyclonal antibody PP2A: rabbit polyclonal antibody…… Immunoblot analysis Total protein (30 g) from LETO and OLETF rat retinas was subjected to SDS-PAGE transferred to a nitrocellulose membrane Antibody incubations and washing were performed immunoreactive proteins were visualized using an enhanced chemiluminescent kit (Amersham Biosciences). 실험에 쓰인 Antibodies 로는 다음의 것들이 사용되었고.. Immunoblot analysis로 다음의 키트와 방법들이 사용돠었습니다.

RESEARCH DESIGN AND METHODS PKC- δ kinase assay performed PKC- kinase assay using the SignaTECT PKC Assay System (Promega, Madison, WI) PP2A phosphatase assay determined using a PP2A-IP phosphatase assay kit Akt kinase assay Akt activity was measured using a nonradioactive Akt kinase assay kit Immunohistochemistry retinal sections were incubated in a mixture of primary antibodies rinsed in PBS, incubated in a mixture of secondary antibodies. Images were obtained using a BH-2 Olympus microscope PKC- kinase assay, PP2A phosphatase assay, Akt kinase assay 등을 통해 각각의 protein의 level를 측정하였습니다.

RESEARCH DESIGN AND METHODS Cell death assay determined using a Terminal dUTP transferase nick end labeling (TUNEL) assay TUNEL assay was performed after Thy-1 immunofluorescent staining on retinal section TUNEL-positive images were observed using a confocal microscope Cell death assay 로 TUNEL assay 방법이 사용되었습니다.

RESULTS OLETF rats gained weight faster than the control LETO rats (P <0.05; n =5, respectively) Blood glucose levels OLETF rats exhibited a steady increase in glucose levels from week 10 LETO rats sustained normoglycemia throughout the period of study OLETF LETO Blood glucose levels 14.5 ± 0.5 6.2 ± 0.3mmol/l at 24 weeks 21.6 ± 1.12 6.6 ± 0.5 mmol/l at35 weeks. OLETF rats 이 대조군보다 몸무게가 더 빠르게 증가하였고, Blood glucose levels은 24주, 35주 모두 유의 있게 차이가 있었습니다

Ganglion cell apoptosis in retinas of LETO and OLETF rats at 24 and 35 weeks. 다음은 tunel assay를 통한 aptotic ganglion cell의 크기 보여주고 있는데, 35주 oletf에서 다른 군보다 유의하게 증가된 양상을 보였습니다 사진에서 화살표는 ganlioncell layer에 aptosis가 일어난 것을 나타내고 있습니다. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 24주와 35주의 당뇨쥐 와 대조군 망막에서의 Ganglion cell apoptosis 를 나타내고 있는데, Specific ganglion cell marker인 Thy-1 immunostaining을 시행후 tunel assay를 시행하였습니다. B에서 화살표가 가르키는것이 TUNEL positive ganglion cell 이고, C와 F에서 화살표 머리부분은 Thy-1 immunostaining와 TUNEL positive ganglion cell의 CODISTRIBUTION을 나타낸것입니다. 이 CO positive cell이 apoptotic ganglion cell 수를 나타내는데, 35주의 당뇨쥐에서 유의있게 증가된 수지를 보였습니다. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Ganglion cell apoptosis in retinas of LETO and OLETF rats at 24 and 35 weeks. The TUNEL assay was performed after Thy-1 immunostaining, a specific ganglion cell marker, and then sections were stained with the nuclear marker DAPI. A and B–F: Representative images of 35-week LETO and OLETF retinas. The arrows indicate TUNEL-positive ganglion cells in 35-week-old OLETF rats (B). The arrowheads in F show the codistribution of TUNEL-positive signals (small arrowheads in C) and Thy-1–positive ganglion cells in 35-week OLETF retinas. The number of co-positive cells was counted and the fold changes are presented as the means SE (n 4) (G). **P < 0.01 compared with 24-week LETO and the other groups. INL, inner nuclear layer; IPL, inner plexiform layer; L (24) and L (35), 24- and 35-week LETO retinas, respectively; O (24) and O (35), 24- and 35-week OLETF retinas, respectively; ONL, outer nuclear layer. Bars, 12.5 m. number of TUNEL-positive ganglion cells in 35- week-old OLETF rats was significantly higher than in 24-week-old LETO rats

PKC- δ activity in retinas of LETO and OLETF rats at 24 and 35 weeks. PKC activity assay was performed using PKC- δ immune complexes and the SignaTECT PKC assay system PKC- δ activity 나타낸 것으로. 35주의 oleft만이 24주의 대조군 보다 높은 수치를 보였습니다. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ PKC- activity in retinas of LETO and OLETF rats at 24 and 35 weeks. A PKC activity assay was performed using PKC- immune complexes and the SignaTECT PKC assay system. [-32P]ATP-labeled PKC- was measured by scintillation counter. Data are the means SE (n 4). **P < 0.01 compared with 24-week LETO and the other groups. L (24) and L (35), 24- and 35-week LETO retinas, respectively; O (24) and O (35), 24- and 35-week OLETF retinas, respectively. **P <0.01; n = 4 PKC- δ activity was significantly higher (4.9-fold) in 35-week OLETF retinas than 24-week LETO retinas

Protein levels of PI 3-kinase, HSP90, PP2A, PP2B, phospho-Akt (Thr308) and -Akt (Ser473), and phospho-GSK in retinas of LETO and OLETF rats at 24 and 35 weeks. PI3 kinase HSP90 PP2A 쥐의 망막에서의 각 종류의 protein level을 보여주고 있습니다. PI3 kinase와 hsp90는 24주의 당뇨쥐에서 증가양상을 보였으며, HSP90은 35주 당뇨주에서는 24주 대조군보다 낮은 수치를 보였습니다. PI3 kinase는 당뇨쥐끼리는 의미있는 변화를 보이지 않았습니다. PP2A는 35주의 당뇨쥐에서 의미있게 높은 수치를 보였으며, Thr308 akt와 ser473 akt와 그리고 phospho-GSK는 24주 대조군에 비교해볼때 24당뇨주에서 높게 35주 당뇨쥐에서는 낮게 측정되었습니다. PI 3-kinase p85 and HSP90 were increased in 24-week OLETF retinas compared with LETO retinas (Fig. 3A–C) HSP90 levels were lower in 35-week OLETF retinas than in LETO retinas, and there was no significant difference between PI 3-kinase levels in both age-groups of OLETF rats. The levels of PP2A (catalytic subunit) and cleaved PP2B (48 kDa) were not significantly different in 24-week-old LETO and OLETF rats but were greatly increased in retinas from 35-week-old OLETF rats (Fig. 3D) Phospho-Akt (Thr308) and -Akt (Ser473) levels were increased (1.4- and 2.3-fold; P 0.05 and 0.01, respectively; n 4) in 24-week OLETF retinas compared with LETO retinas and decreased significantly Akt activity, based on phospho-GSK–3/ level (30 kDa), was significantly lower (twofold; P 0.01; n 4) in 35-week-old OLETF rats than 24-weekold LETO rats (Fig. 3I) Protein levels of PI 3-kinase, HSP90, PP2A, PP2B, phospho-Akt (Thr308) and -Akt (Ser473), and phospho-GSK in retinas of LETO and OLETF rats at 24 and 35 weeks. A: Representative immunoblots of PI 3-kinase, HSP90, PP2A, and PP2B. B–D: Fold changes in these protein levels. E, F, and G–I: Representative immunoblots of phospho-Akt and -GSK and the corresponding fold changes. The immunoblots were stripped and reprobed with anti–-tubulin or -Akt antibodies. Data are means SE (n 4). *P < 0.05 and **P < 0.01 compared with 24-week LETO and the other groups. L (24) and L (35), 24- and 35-week LETO retinas, respectively; O (24) and O (35), 24- and 35-week OLETF retinas, respectively. Akt-Thr308 Akt-Ser473 phospho-GSK

The associations with Akt and HSP90, PP2A, and PP2B in retinas of LETO and OLETF rats at 24 and 35 weeks AKT와 hsp와 PP2A의 결합관계를 보는 것으로, AKT-HSP결합은 35주 당뇨쥐에서 낮게 측정되었고, AKT-PP2A 결합은 35주 당뇨쥐에서 높게 측정되었습니다. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ The associations with Akt and HSP90, PP2A, and PP2B in retinas of LETO and OLETF rats at 24 and 35 weeks. Akt, HSP90, and PP2A immune complexes were subjected to immunoblot analysis (A– C). Data are representative blots of four independent experiments. The blots were reprobed with the immunoprecipitating antibody to account for loading differences. The immunoblot data were quantified using densitometry and the fold changes in Akt-HSP90 and Akt-PP2A binding are presented in D and E. Data are means SE (n 4). **P < 0.01 compared with 24-week LETO and the other groups. IP, immunoprecipitation; L (24) and L (35), 24- and 35-week LETO retinas, respectively; O (24) and O (35), 24- and 35-week OLETF retinas, respectively. Akt immune complexes to immunoblot analysis using anti-HSP90, -PP2A, and -PP2B antibodies Akt-HSP90 Akt-PP2A

Distribution of HSP90, PP2A, and phospho-Akt (Ser473) in retinas of LETO and OLETF rats at 35 weeks 35주의 OLETF와 대조군의 망막에서의 HSP90, PP2A, Phospho AKT의 분표를 보여주고 있는데, 앞의 결과와 마찮가지로, HSP90은 대조군에, PP2A는 당뇨주에서, P-AKT는 대조군에 많이 분포함을 나타내고 있습니다. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Distribution of HSP90, PP2A, and phospho-Akt (Ser473) in retinas of LETO and OLETF rats at 35 weeks. Their positive signals in the GCL are indicated by arrows, arrowheads, and small arrows, respectively. Insets show enlarged images of ganglion cells co-labeled with these proteins and Thy-1, a specific ganglion cell marker. Data are representative of three independent experiments. INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Bars, 12.5 m. (Please see http://dx.doi.org/ 10.2337/db07-1431 for a high-quality digital representation of this figure.) By double-immunostaining with Thy-1 of HSP90, PP2A, and phospho-Akt (Ser473),

The effects of rottlerin treatment on ganglion cell death in retinas of LETO and OLETF rats at 35 weeks The effects of rottlerin were examined 24 h after an intravitreal injection into the right eye of rats. Rottlerin을 intravitreal inj후의 ganglion cell death의 수 비교를 나타내는데, Rottlerin 주입하지 않은 당뇨쥐에서 가장 높게 측정되었고 주입한 당뇨쥐와도 의미있는 차이를 보였습니다. ~~~~~~~~~~~~~~~~~~~~~~~~

The effects of rottlerin treatment on protein levels of PI 3-kinase, HSP90, PP2A, and Akt; Akt activity; and the associations with Akt and HSP90 or PP2A in retinas of LETO and OLETF rats at 35 weeks. PI3 kinase HSP90 PP2A P-GSK Akt-Thr308 Akt-Ser473 Rottlerin 주입후의 각 protein level의 변화로, PI3kinase와 thr308 akt에서만 OLETF에서 주입전후에 의미있는 변화를 보이지 않았고, 나머지 HSP90, PP2A 레벨은 차이를 보였으며, Akt-hsp90 binding 과 Akt-pp2a binding의 변화에도 의미있는 차이를 보였습니다. ~~~~~~~~~~~~~~~~~~~~~~~ Akt-HSP90 Akt-PP2A

Changes in Akt signaling and cell death in retinas of 35-week-old OLETF rats after PKC- δ knockdown using RNA interference. PKC- δ PP2A PKC- δ knockdown위한 siRNA 주입의 영향으로 주입된 당뇨주에서는 PKC- δ 와 PP2A ACTIVITY가 감소함을 나타내었고 APTOSIS되는 GANGLION CELL도 줄었습니다. Aptotic ganglion cell

Changes in Akt signaling and cell death in retinas of 35-week-old LETO rats after HSP90 knockdown using RNA interference. HSP90 knockdown위한 siRNA 주입으로 GANGLION CELL APTOSIS는 증가함을 나타내고 있습니다. Aptotic ganglion cell

DISCUSSION PKC- δ activation is responsible for neuro-retinal apoptosis in diabetic OLETF rats via the inactivation of Akt. apoptosis occurs only in ganglion cells of the 35-week OLETF retinas PKC- δ activity is greatly increased in 35-week OLETF retinas compared with 24- or 35-week LETO and 24-week OLETF retinas PKC- δ activation is involved in ganglion cell death in OLETF rat retinas. PKC- δ activation은 AKT을 INACTIVATION 시켜서 당뇨쥐에서 NEURORETINAL APOPTOSIS를 일으킵니다. 이 APOPTOSIS는 GANGLION CELL에서만 일어났고, PKC- δ activity는 35주 당뇨쥐에서 가장 높게 측정되었습니다. 이결과로 PKC-델타는 당뇨쥐에서 GANGLION CELL DEATH를 일으킨다고 할수 있습니다..

DISCUSSION phospho-Akt functional compensation for diabetes-induced cellular stress. 24-week OLETF retina Akt activity HSP90 PI 3-kinase 24주 당뇨쥐에서는 PHOSPHO-AKT, AKT ACTIVITY HSP90 PI3KINASE 가 높았고 35주 당뇨쥐에서는 pi3KINASE만 제외하고 낮은 수치를 보였는데, 24주에서는 functional compensation으로 높은 수치를 보였다고 추정되며, 35주에서는 RETINAL DAMAGE가 진행되면서 그 수치가 감소된것으로 보입니다. phospho-Akt retinal damage associated with the pathological progression of diabetes 35-week-OLETF retina Akt activity HSP90

DISCUSSION inhibition PKC- δ rottlerin or siRNA treatment PP2A activation PP2A association with Akt PKC- δ inhibition phospho-Akt (Ser473) in 35-week OELTF retinas HSP90 Pkc-델타가 inactivation 되었을때 pp2a 활성도는 감소하였고, Hsp90, ser473akt 활성도 증가, 그리고 ganglion cell death의 감소를 보였지만, Pi3kinase 나 thr308 akt활성도는 변화를 보이지 않았다. phospho-GSK levels association of Akt with HSP90 no significant effects PI 3-kinase ganglion cell death PKC- δ–PI 3-kinase binding phospho-Akt (Thr308)

DISCUSSION HSP90 knockdown Akt-HSP90 binding Akt activity HSP90 KNOK DOWN으로는 맛-hsp binding감소 맛 활성 감소 맛 pp2 biding증가 ganglion cell death 증가를 보였는데. Akt-PP2A binding ganglion cell death

DISCUSSION PKC- δ PP2A activation PP2A association with Akt directly acts on PP2A PP2A association with Akt PKC- δ in 35-week OELTF retinas Dephosphorylate phospho-Akt (Ser473) 지금까지 결과를 종합해보면, pi3kinase independent pathway로 Pkc-d 는 pp2a에 직접 작용을 하는것으로 보이며 활성된 pp2a는 phosho-akt를 dephosphorylate 시키고, akt-sp90을 dissociation시켜 Ganglioncell death를 유발하는것으로 보인다. dissociation of Akt with HSP90 ganglion cell death PI 3-kinase–independent Akt pathway

DISCUSSION PKC- δ mediates neuronal death in retinas of diabetic rats via PP2A activation and Akt signaling inhibition Ganglion cell death occurs early as an initial event in diabetic retinopathy mechanism of this cell death is unknown. specific PKC- δ inhibitors may have potential for therapeutic agents for the prevention of human diabetic retinopathy. 마지막으로, Specific PKC –D INHIBITOR는 인간 당뇨망막증에서 치료제로 가능성을 갖을 수 있겠다.