-methyl-n-nitrosourea-induced photoreceptor degeneration ...researcharticle...

Research ArticleN-Methyl-N-Nitrosourea-Induced Photoreceptor DegenerationIs Inhibited by Nicotinamide via the Blockade of UpstreamEvents before the Phosphorylation of Signalling Proteins

Eriko Sugano 1 Kitako Tabata1 Tsubasa Takezawa1

Raki Shiraiwa1 Hiroki Muraoka2 TomomiMetoki3 Asaka Kudo3 Yuki Iwama1

Mitsuru Nakazawa 3 and Hiroshi Tomita 14

1Laboratory of Visual Neuroscience Graduate Course in Biological Sciences Iwate University Division of Science and Engineering4-3-5 Ueda Morioka Iwate 020-8551 Japan

2Iwate University Division of Science and Engineering 4-3-5 Ueda Morioka Iwate 020-8551 Japan3Department of Ophthalmology Hirosaki University School of Medicine 5 Zaifu-cho Hirosaki Aomori 036-8562 Japan4Clinical Research Innovation and Education Center Tohoku University Hospital 1-1 Seiryo Aoba Sendai Miyagi 980-8574 Japan

Correspondence should be addressed to Hiroshi Tomita htomitaiwate-uacjp

Received 6 February 2019 Revised 9 April 2019 Accepted 15 April 2019 Published 23 April 2019

Academic Editor Arianna Scuteri

Copyright copy 2019 Eriko Sugano et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

N-methyl-N-nitrosourea (MNU) a known carcinogen is generally used in animal models to chemically induce photoreceptordegeneration It has been reported that nicotinamide (NAM) exerts a protective effect on MNU-induced photoreceptordegeneration We investigated the molecular mechanisms on MNU-induced photoreceptor degeneration Intraperitoneal MNUinjection (75 mgkg) in rats induced selective photoreceptor degeneration in 7 days NAM administration completely inhibitedphotoreceptor degeneration Photoreceptor layer abnormality was observed within 6 hours after MNU injection whereas it wasrestored in the NAM-treated retina as detected by optical coherence tomography One day following MNU administrationphosphorylation of the cell death-associated signalling proteins c-Jun N-terminal kinase (JNK) and p38 mitogen-activated proteinkinase (p38) increased while the apoptosis-related proteins full-length poly(ADP-ribose) polymerase (PARP) and apoptosis-inducing factor (AIF) were depletedThese changes were not observed in the NAM-treated retinas Cell survival signalling such asextracellular signal-regulated kinase (ERK) Akt and cAMP response element binding protein (CREB) phosphorylation increasedin the MNU- but not in the NAM-treated rat retinas Increased phosphorylated ERK (p-ERK) levels were observed within 6 hoursafterMNUadministration suggestive of cell survival signalling activationThis did not occur inNAM-treated retinasThese resultsindicate that NAM regulates upstream cellular events prior to the activation of cell death-related signalling events such as JNK andp38 phosphorylation

1 Introduction

Photoreceptor degenerative diseases such as retinitis pig-mentosa (RP) and age-related macular degeneration (AMD)are a major cause of blindness Although these two con-ditions present with similar pathological symptoms withrespect to photoreceptor degeneration they have differentcausative mechanisms RP is a genetic disorder with variousassociated genetic mutations having been identified mainlyin relation to phototransduction pathways (httpssphutheduRetnethomehtm) On the contrary the development

of age-related macular degeneration (AMD) has been mainlyassociated with various environmental factors such assmoking obesity and vitamin deficiencies with only a fewdisease-associated mutations having been identified to date[1]

There are various types of photoreceptor degenerationanimal models that simulate human pathology and representimportant tools for investigating the molecular mechanismsbehind RP and AMD and potential treatment targets Rodentmodels of photoreceptor degeneration can be classified intotwo types rat models such as P23H [2] S334ter [3] and the

HindawiBioMed Research InternationalVolume 2019 Article ID 3238719 10 pageshttpsdoiorg10115520193238719

2 BioMed Research International

one developed by the Royal College of Surgeons [4 5] thatharbour degenerative genetic mutations and the models ofphotoreceptor degeneration that are induced by light [6 7]or chemically [8ndash11] Although various genetic mutationsthat lead to RP have been identified they all eventuallylead to photoreceptor cell apoptosis [12 13] Photoreceptordegeneration induced by light or chemicals is also associ-ated with cellular apoptosis Apoptosis is the final commonpathway in the photoreceptor degeneration though some dif-ferences of molecular mechanisms involve in between gene-and light- or chemical-induced photoreceptor degenera-tions

Nicotinamide (NAM) a water-soluble B-group vitamin(vitamin B3) exerts protective effects against light- [14 15]andN-methyl-nitrosourea (MNU)-induced [16] photorecep-tor cell apoptosis A better understanding of the molecularmechanisms involved in the photoreceptor degeneration andNAM-mediated cellular protection will aid in the identifi-cation of successful therapeutic approaches against retinaldegenerative diseases

2 Materials and Methods

21 Animals All animal experiments were conducted inaccordance with the guidelines of the Animal ExperimentCommittee of Iwate University Japan Male Sprague-Dawleyrats were obtained fromCLEA Japan Inc (Tokyo Japan)Therats were housed in a 12 h12 h lightdark cycle with access towater ad libitum

22 Antibodies Antibodies against extracellular signal-reg-ulated kinase (ERK Cat No 4695S) phosphorylated ERK(pERK Cat No 9101S) Akt (Cat No 9272) pAkt (Cat No9271L) cAMP response element binding protein (CREBCat No 9197S) and pCREB (Cat No 9198S) were obtainedfromCell Signalling Technology (Tokyo)The anti-glutaminesynthetase (GS) antibody (Cat No MAB302) was obtainedfrom Millipore (Tokyo) The anti-c-Jun N-terminal kinase(JNK Cat No sc-571) and phosphorylated JNK (Cat Nosc-6254) were obtained from Santa Cruz BiotechnologySecondary antibodies against rabbit (Cat No S3731) andmouse immunoglobulin (Ig) G (IgG Cat No S3721) wereobtained from Promega (Tokyo)

23MNU andNAMAdministration Rats were administereda single intraperitoneal injection of MNU (Fujifilm WakoPure Chemical Corp Osaka Japan) at a dose of 75 mgkgbody weight The MNU solution was freshly prepared withsterile physiological saline containing 005 acetic acidimmediately prior to use and stored at 4∘C in the darkNAM (Nacalai Tesque Kyoto Japan) was freshly dissolved insterile physiological saline and subcutaneously injected at adose of 1000 mgkg 30 minutes prior to MNU administra-tion

24 Histology Seven days following MNU injection ratswere sacrificed using carbon dioxide and eyes were enu-cleated Eyes were immersed in 4 paraformaldehyde in

phosphate buffered saline (PBS) overnight at 4∘C and thenembedded in paraffin Serial sections (4 120583m) of the wholeeye were cut sagittally through the cornea and parallel tothe optic nerve stained against haematoxylin and eosin andvisualised by microscopy (AxioImager A1 Carl Zeiss TokyoJapan)

25 Optical Coherence Tomography (OCT) Rats were anaes-thetized by intramuscular injection of ketamine (75 mgkg)and medetomidine (05 mgkg) and their pupils were dilatedwith tropicamide (Midrin-P Santen Co Ltd Osaka Japan)The eye was placed under local anaesthesia using oxybup-rocaine (Santen Co Ltd Osaka Japan) and covered with acontact lens Image acquisition of 11 mm length of the ratretina including the optic disk was performed using the linescan mode on an OCT imaging device equipped with aspecial ordered lens (RS-3000 NIDEK Co Ltd AichiJapan)

26 Western Blot Analysis Western blot analysis was per-formed as previously described [17] Briefly the retinas wereisolated from the eyes 1 and 3 days afterMNUorMNUNAMadministration Proteins were extracted from the sampleswith a lysis buffer containing 10 mM Tris-HCl (pH 75) 1Triton X-100 05 NP-40 1 mM EDTA 150 mM NaCl 1xprotease inhibitor cocktail (Thermo Scientific Tokyo) and1x Halt phosphatase inhibitor cocktail (Thermo ScientificTokyo) The lysate was quantified using a bicinchoninic acidassay (BCA) protein assay kit (Pierce Rockford IL) Thirtymicrograms of proteinwas loaded for electrophoresis on 4ndash15 Mini-PROTEIN TGX gels (Bio-Rad Tokyo) and trans-ferred onto a polyvinylidene difluoride (PVDF) membraneAfter blocking with Block Ace (DS Pharma BiomedicalOsaka Japan) the membrane was incubated with the appro-priate primary antibody Following washing the membranewas incubated with the appropriate alkaline phosphatase-conjugated secondary antibody Chemiluminescence detec-tion (CDP-Star Detection Reagent GE Healthcare Tokyo)was performed according to the standard procedure Banddensities were measured using ImageQuant software (GEHealthcare Tokyo) and the density of each band was nor-malised to that of 120573-actin

27 Immunohistochemistry Immunohistochemistry wasperformed on the retina 6 hours and 1 day after MNU orMNUNAM administration Paraffin-embedded sectionswere deparaffinized according to the standard procedureThesections were treated in citrate buffer (pH60) in amicrowavefor antigen retrieval After blocking the sections wereincubated with anti-pERK (dilution 1200) and GS antibodiesas a marker of Muller cells or with a control solution of anti-rabbit and anti-mouse IgGs overnight at 4∘C The sectionswere then washed three times with PBS containing 001Tween-20 and incubated with Alexa 488- or Alexa 594-conjugated anti-rabbit and anti-mouse IgG solution at roomtemperature for 1 h Following washing the sections werecovered with mounting media including 410158406-diamidino-2-phenylindole (DAPI VECTASHIELD FunakoshiTokyo)

BioMed Research International 3

minus5 minus45 minus4 minus35 minus3 minus25 minus2 minus15 minus1 minus05 0 05 1 15 2 25 3 35 4 450

50

100

150

200

ControlMNUMNUNAM

Distance from the optic nerve (mm)

ILM

~RPE

(mm

)


10

20

30

40

50

ControlMNUMNUNAM


ON

L (

m)

ILM

ONL

RPE

(a)

(d) (e)

(b) (c)

Figure 1 Histological retinal examination seven days after MNU or MNUNAM administration All retinal layers were clearly observed incontrol retinas (a) Photoreceptor cells were completely degenerated in the retinas of MNU injected rats (b) whereas they were rescued fromthe degeneration by subcutaneous NAM injection (c) Scale bar is indicative of 50 micrometres The thickness of whole retina ((d) from ILMto RPE) and the ONL (e) weremeasured at 05 mm interval between the superior (+) and inferior (-) part Data are shown asmean plusmn standarderror (n = 4ndash8) MNUN-methyl-N-nitrosourea NAM nicotinamide OCT optical coherence tomography ONL outer nuclear layer PREretinal pigment epithelium and ILM internal limiting membrane

Statistical Analysis Statistical analysis was performedusing GraphPad Prism 4 (GraphPad software San DiegoCA) The statistical method used was Tukeyrsquos multiple com-parison test

3 Results31 Histology Seven days following the intraperitoneal injec-tion of MNU in rats the outer nuclear layer (ONL) of theretina was not detectable (Figure 1(b)) and photoreceptor


pre 6 hours 24 hours 72 hours

NAM

MNU

MNUNAM

(a)

pre 6 24 720

50

100

150

200

MNUMNUNAM

ON

L - R

PE (

m)

NAM

lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

(b)

Figure 2 Photoreceptor degeneration induced by intraperitoneal MNU injection OCT images were obtained from rats before and afteradministration of MNU NAM or MNUNAM (a) The thickness of the photoreceptor layer (arrow) was measured (b) The photoreceptorlayer (arrow) was present in the retina duringMNU preadministrationThe photoreceptor layer became thicker 6 hours afterMNU injectionand then gradually thinner at later time points Data are shown as mean plusmn standard error (n = 4ndash16 lowast lowastlowastlowast p lt 005 0001 Tukeyrsquos multiplecomparison test) MNU N-methyl-N-nitrosourea NAM nicotinamide and OCT optical coherence tomography

degeneration occurred in the whole retina (Figure 1(e)) TheMNU-induced degeneration was localised to the ONL anddid not affect any of the other retinal layers (Figure 1(d))On the contrary the retinal morphology was maintained inNAM-treated animals (Figures 1(c) and 1(e)) similar to thatin controls (Figure 1(a))

32 OCT The early phase of photoreceptor degenerationwas evaluated by OCT (Figure 2(a)) The ONL waspoorly marginated at 6 hours and 1 day after MNUadministration and its thickness significantly increasedat 6 hours but decreased 1 day and 3 days afterMNU administration (Figure 2(b)) This ONL thinning


MNU MNUNAM

pJNK(54kDa)panJNK

control 1 day 3 days 1 day 3 days

control control1 day 3 days 1 day 3 days

0

2

4

6lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n

lowastlowast

MNU MNUNAM

pJNK (54 kDa)pJNK (48 kDa)

panJNK

-actin

(a)

MNU MNUNAM

pp38p38

control 1 day 3 days 1 day 3 days0

2

4

6lowastlowast lowastlowast


relat

ive e

xpre

ssio

n

MNU MNUNAM

pp38

p38

-actin

(b)


relat

ive e

xpre

ssio

n

MNU

MNU

MNUNAM

MNUNAM

PARP-actin


2

4

6

lowast

full length PARP

-actin

(c)


relat

ive e

xpre

ssio

n

MNU MNUNAM

AIF-actin


2

4

6

lowast

lowastlowastlowast

lowast

lowastlowastlowast

MNU MNUNAM

AIF

-actin

(d)

Figure 3 Effect of NAM on cell death related signalling proteins Representative western blots of JNK (a) p38 (B) PARP (c) and AIF (d)are indicated in each densitometry analysis graph Increased phosphorylated forms of JNK and p38 were observed in retinas 1 and 3 daysand 1 day after MNU injection respectively The depletion of full-length PARP was observed in MNU-injected retinas The expression levelof AIF was decreased after MNU injection Data are shown as mean plusmn standard deviation (n = 4 lowastlowastlowast and lowast lowast lowast p lt 005 001 and 0001Tukeyrsquos multiple comparison test) AIF apoptosis-inducing factor JNK c-Jun N-terminal kinase MNU N-methyl-N-nitrosourea NAMnicotinamide PARP poly(ADP-ribose) polymerase and p38 p38 mitogen-activated protein kinase

induced by MNU was completely blocked by NAMcoadministration and the retinal architecture remainedunchanged at all time points following NAM treat-ment

33 Signalling Protein Expression We sought to evaluate pos-sible expression changes in cell death-related proteins Thephosphorylation of JNK (Figure 3(a) 54 kDa and 48 kDa)and p38 mitogen-activated protein kinase (p38 Figure 3(b))


pERK1pERK2

ERK1ERK2

MNU MNUNAM

MNU MNUNAM

MNU MNUNAM

pERK1ERK1


2

4

6

lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

nre

lativ

e exp

ress

ion

pERK2ERK2


2

4

6

lowastlowastlowast

lowastlowastlowast


-actin

(a)

MNU

MNU

MNUNAM

MNUNAM

pAkt

Akt

pAktAkt


2

4

6

lowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n


-actin

(b)

Figure 4 Continued


relat

ive e

xpre

ssio

n

pCREB

pATF1

CREB

MNU MNUNAM

MNU MNUNAM


2

4

6pCREBCREB

lowast


-actin

(c)

Figure 4 Effect of NAM on cellular stress-related proteins Representative western blots of ERK (a) Akt (b) and CREB (c) are indicatedin each densitometry analysis graph Increased phosphorylated forms of pERK1 pERK2 and pAKT were observed at Day 1 after MNUadministrationThereafter pCREB was also upregulated Data are shown as mean plusmn standard deviation (n = 4 lowast lowastlowast and lowastlowastlowast p lt 005 001and 0001 Tukeyrsquos multiple comparison test) CREB cAMP response element binding protein ERK extracellular signal-regulated kinaseMNU N-methyl-N-nitrosourea and NAM nicotinamide

was significantly increased 1 day after MNU administrationThe increase in phosphorylated JNKs (pJNKs) was main-tained for 3 days following MNU injection (Figure 3(a))The expression levels of both poly(ADP-ribose) polymerase(PARP Figure 3(c)) and the apoptosis-inducing factor (AIFFigure 3(d)) were decreased after MNU injection Thesechanges were not observed in the retinas of the animalscotreated with NAM Next we examined the expression ofvarious markers of cellular proliferation and survival Thelevels of pERK (Figure 4(a)) and pAkt (Figure 4(b)) increasedat 1 day and the pCREB (Figure 4(c)) significantly increasedafter MNU injection This MNU-induced protein phospho-rylation was completely abolished by NAM coadministration(Figures 4(a) 4(b) and 4(c))

34 Phospho-ERK and GS Immunohistochemistry Phospho-ERK is a well-known marker of retinal stress with protectiveeffects against retinal injuries [17ndash19] We evaluated the earlycellular responses following MNU administration Phospho-ERK immunoreactivity (IR) was mainly observed in theinner nuclear layer and partly in the ganglion cell layer incontrol retinas (Figure 5(a)) However six hours after MNUinjection pERK IR was mainly localised in the inner limitingmembrane and the cell bodies of the inner nuclear layer(Figure 5(b)) Phospho-ERK IR was subsequently detected inthe outer nuclear layer and the outer limiting membrane 1 day

after MNU administration (Figure 5(c)) with predominantnuclear localisation GS IRwas consistently detected from theinner limiting membrane to the outer limiting membrane incontrol retinas GS IR significantly increased 1 day afterMNUinjection in the outer nuclear layer (Figure 5(c))WhenNAMwas coadministered retinal GS IR was slightly increased ascompared to controls but no changes were observed in pERKlevels (Figures 5(d) and 5(e))

4 Discussion

MNU induced selective retinal photoreceptor degenerationin our rat model Photoreceptor cell death was completelyinduced 7 days after MNU injection OCT revealed pho-toreceptor layer degeneration as early as 6 hours after MNUinjection indicating the quick activation of the cell deathsignalling pathway (within 6 hours Figure 2(a)) HoweverNAM cotreated retinas were indistinguishable from controlsin OCT as early as 6 hours following administration Wehypothesized that NAM regulates the early phase of pho-toreceptor degeneration to induce such protective responsesagainst MNU-induced toxicity

Previously the JNKstress-activated protein kinase(SAPK) signalling pathway has been associated with MNU-induced photoreceptor cell death [20] and is known to induceneuronal apoptosis [21 22] It has also been reported that


MNU MNUNAM

(a) control (b) 6 hours (c) 1 day (d) 6 hours (e) 1 day

Nuclei

pERK

GS

Merge

50m

Figure 5 Retinal immunohistochemistry of pERKandGS after intraperitonealMNU injection Retinal cryosections from control (a) 6 hours(b d) and 1 day (c e) afterMNU (b c) orMNUNAM(d e) injectionwere immunostained against pERKandGSThe inner limitingmembraneand the cell bodies of the inner nuclear layer were colabelled with pERK and GS antibodies 6 hours after MNU injection (arrows in (b)) Oneday after MNU injection pERK immunoreactivity was observed in the outer nuclear layer and the outer limiting membrane (arrows in (c))In addition nuclear pERK staining was observed (arrowheads in (c)) The nuclei was stained with 410158406-diamidino-2-phenylindole (DAPI)shown as blue GS glutamine synthetase pERK phosphorylated extracellular signal-regulated kinase MNU N-methyl-N-nitrosourea andNAM nicotinamide

the cell death pathway induced by the phosphorylation ofp38 plays an important role in light-induced photoreceptordegeneration [23] We detected MNU-mediated increasesin pJNK and p-p38 NAM administration completelyabolished these changes (Figures 3(a) and 3(b)) and blockedthe MNU-induced photoreceptor degeneration (Figures1(c) 1(d) and 1(e)) Nevertheless there is little evidencesupporting the direct inhibition of pJNKs or p-p38 by NAMMNU is an alkylating agent that causes DNA damage [24]PARP is a known immediate cellular response activatorfollowing alkylating agent-induced DNA damage [25] Wedemonstrated that MNU leads to the depletion of full-lengthPARP within a day after its administration (Figure 3(c))and a subsequent decrease in AIF expression (Figure 3(d))Uehara N et al previously showed the increased PARP

in the photoreceptor cells with the same model [20] Ourresults seemed to be controversial However the MNUconcentration used in this study was higher than thatthey used indicating that more severe retinal damagewas induced in our model The PARP would be cleavedby the MNU administration and PARP and AIF werepossibly translocated into the nucleus Subsequently thefull-length PARP and AIF levels in the cellular componentwere decreased These results at least indicate that thePARPAIF-associated cell death pathway [26] is involvedin MNU-induced photoreceptor degeneration The nucleartranslocation of PARP leads to the depletion of nicotinamideadenine dinucleotide (NAD+) and a decrease in cellular ATPlevels which in turn induce cell death [27] Taking all theseinto consideration it is likely that NAM acts as a NAD+


supplier or a PARP inhibitor as NAM is a precursor forNAD+ a known PARP inhibitor by pretreatment of NAM[28]

To further explore the effects of NAM on cellular sig-nalling pERK [18] pAkt [29] and pCREB [30] were evalu-ated with respect to their potential protective effects againstretinal injuries NAM administration did not affect the phos-phorylation levels of these signalling proteins (Figures 4(a)4(b) and 4(c)) On the contrary MNU administration led toa significant increase in pERK and pAkt as well as pCREBat later time points Likewise we have previously reportedthat Akt also contributes in photoreceptor survival andmaintenance in light-induced photoreceptor degeneration[31] Increased pERK IR was mainly observed in Mullercells as early as 6 hours after MNU administration It hasbeen reported that the phosphorylation of signal transducerand activator of transcription 3 (STAT3) and ERK in Mullercells plays an important role in ciliary neurotrophic factor(CNTF)-mediated photoreceptor rescue in the retinal degen-eration [32] The observed MNU-mediated pERK increasein Muller cells might reflect an intrinsic retinal protectionmechanism against injuries

In this study we evaluated potential changes in theexpression patterns of various signalling proteins followingMNU administration The expression levels of all signallingproteins evaluated in this study were altered within one dayafter MNU injection The immunohistochemical evaluationof pERK revealed the activation of self-protective retinalresponses within 6 hours of MNU injection which was notthe case in the NAM cotreated retinas

5 Conclusions

NAM is likely to regulate upstream signalling pathways suchas NAD+ consumption or PARP inhibition to induce retinalprotection against photoreceptor degeneration prior to thephosphorylation of signalling proteins that occurs in laterstages The results of this study provide a better under-standing of the molecular mechanisms underlying retinaldegeneration and aid in the identification of novel therapeutictargets against photoreceptor degenerations

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Grants-in-Aid for ScientificResearch from the Ministry of Education Culture SportsScience and Technology Japan (Grant Nos 16H0548516K11314 18K09433 and 17H06330)We express our heartfeltappreciation to Ms Miho Sato of Laboratory of Visual

Neuroscience for preparing the histological sections used inthis study

References

[1] L S Lim P Mitchell J M Seddon F G Holz and T Y WongldquoAge-related macular degenerationrdquo e Lancet vol 379 no9827 pp 1728ndash1738 2012

[2] A S Lewin K A Drenser W W Hauswirth et al ldquoRibozymerescue of photoreceptor cells in a transgenic rat model ofautosomal dominant retinitis pigmentosardquo Nature Medicinevol 4 no 8 pp 967ndash971 1998

[3] C Liu Y Li M Peng A M Laties and R Wen ldquoActivationof caspase-3 in the retina of transgenic rats with the rhodopsinmutation S334ter during photoreceptor degenerationrdquo eJournal of Neuroscience vol 19 no 12 pp 4778ndash4785 1999

[4] M M LaVail R L Sidman and C O Gerhardt ldquoCongenicstrains of RCS rats with inherited retinal dystrophyrdquo Journal ofHeredity vol 66 no 4 pp 242ndash244 1975

[5] H Tomita E Sugano H Yawo et al ldquoRestoration of visualresponse in aged dystrophic RCS rats using AAV-mediatedchannelopsin-2 gene transferrdquo Investigative Ophthalmology ampVisual Science vol 48 no 8 pp 3821ndash3826 2007

[6] W K Noell and R Albrecht ldquoIrreversible effects of visible lighton the retina Role of vitamin Ardquo Science vol 172 no 3978 pp76ndash80 1971

[7] H Tomita Y Kotake and R E Anderson ldquoMechanism ofprotection from light-induced retinal degeneration by thesynthetic antioxidant phenyl-N-tert-butylnitronerdquo InvestigativeOphthalmology amp Visual Science vol 46 no 2 pp 427ndash4342005

[8] E L Pautler K T Wheeler R E Sheridan and B Hon ldquoTheeffect of iodoacetate on the retinal cell DNA in rabbitrdquo Experi-mental Eye Research vol 14 no 3 pp 183ndashIN1 1972

[9] A Hara M Niwa H Aoki et al ldquoA new model of retinal pho-toreceptor cell degeneration induced by a chemical hypoxia-mimicking agent cobalt chloriderdquo Brain Research vol 1109 no1 pp 192ndash200 2006

[10] K YugeHNambuH Senzaki et al ldquoN-methyl-n-nitrosourea-induced photoreceptor apoptosis in the Mouse retinardquo In Vivovol 10 no 5 pp 483ndash488 1996

[11] H Isago E Sugano N Murayama M Tamai and H TomitaldquoEstablishment of monocular-limited photoreceptor degenera-tionmodels in rabbitsrdquo BMCOphthalmology vol 13 no 1 2013

[12] F Doonan and T G Cotter ldquoApoptosis a potential thera-peutic target for retinal degenerationsrdquo Current NeurovascularResearch vol 1 no 1 pp 41ndash53 2004

[13] G-Q Chang Y Hao and F Wong ldquoApoptosis Final commonpathway of photoreceptor death in rd rds and mutant micerdquoNeuron vol 11 no 4 pp 595ndash605 1993

[14] C T Sheline Y Zhou and S Bai ldquoLight-induced photoreceptorand RPE degeneration involve zinc toxicity and are attenuatedby pyruvate nicotinamide or cyclic lightrdquoMolecularVision vol16 pp 2639ndash2652 2010

[15] S Bai and C T Sheline ldquoNAD+ maintenance attenuateslight induced photoreceptor degenerationrdquo Experimental EyeResearch vol 108 pp 76ndash83 2013

[16] K Kiuchi K Yoshizawa N Shikata M Matsumura and ATsubura ldquoNicotinamide prevents N-methyl-N-nitrosourea-induced photoreceptor cell apoptosis in Sprague-Dawley rats


and C57BL micerdquo Experimental Eye Research vol 74 no 3 pp383ndash392 2002

[17] Y Hayashi Y Kitaoka Y Munemasa et al ldquoNeuroprotectiveeffect of 17120573-estradiol against N-methyl-D-aspartate- inducedretinal neurotoxicity via p-ERK inductionrdquo Journal of Neuro-science Research vol 85 no 2 pp 386ndash394 2007

[18] H Akiyama T Nakazawa M Shimura H Tomita and MTamai ldquoPresence of mitogen-activated protein kinase in retinalMuller cells and its neuroprotective effect ischemia-reperfusioninjuryrdquo NeuroReport vol 13 no 16 pp 2103ndash2107 2002

[19] Y Munemasa R Ohtani-Kaneko Y Kitaoka et al ldquoContribu-tion of mitogen-activated protein kinases to NMDA-inducedneurotoxicity in the rat retinardquo Brain Research vol 1044 no 2pp 227ndash240 2005

[20] NUehara KMiki R Tsukamoto YMatsuoka andA TsuburaldquoNicotinamide blocks N-methyl-N-nitrosourea-inducedphotoreceptor cell apoptosis in rats through poly (ADP-ribose)polymerase activity and Jun N-terminal kinaseactivatorprotein-1 pathway inhibitionrdquo Experimental Eye Research vol82 no 3 pp 488ndash495 2006

[21] S Estus W J Zaks R S Freeman M Gruda R Bravo andE M Johnson Jr ldquoAltered gene expression in neurons duringprogrammed cell death Identification of c-jun as necessary forneuronal apoptosisrdquoe Journal of Cell Biology vol 127 no 6 Ipp 1717ndash1727 1994

[22] Z Xia M Dickens J Raingeaud R J Davis and M E Green-berg ldquoOpposing effects of ERK and JNK-p38 MAP kinases onapoptosisrdquo Science vol 270 no 5240 pp 1326ndash1331 1995

[23] L Yang X Zhu and M O Tso ldquoRole of NF-kappaB andMAPKs in light-induced photoreceptor apoptosisrdquo InvestigativeOpthalmology amp Visual Science vol 48 no 10 p 4766 2007

[24] Y Fujiwara ldquoPostreplication repair of alkylation damage toDNA of mammalian cells in culturerdquo Cancer Research vol 35no 10 pp 2780ndash2789 1975

[25] A Burkle ldquoPoly(APD-ribosyl)ation a DNA damage-drivenprotein modification and regulator of genomic instabilityrdquoCancer Letters vol 163 no 1 pp 1ndash5 2001

[26] S W Yu H Wang M F Poitras et al ldquoMediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factorrdquo Science vol 297 no 5579 pp 259ndash263 2002

[27] D A Carson S Seto D B Wasson and C J Carrera ldquoDNAstrand breaks NAD metabolism and programmed cell deathrdquoExperimental Cell Research vol 164 no 2 pp 273ndash281 1986

[28] M R Purnell and W J Whish ldquoNovel inhibitors of poly(ADP-ribose) synthetaserdquo Biochemical Journal vol 185 no 3 pp 775ndash777 1980

[29] T NakazawaM Shimura H Tomita et al ldquoIntrinsic activationof PI3KAkt signaling pathway and its neuroprotective effectagainst retinal injuryrdquo Current Eye Research vol 26 no 1 pp55ndash63 2003

[30] J Sancho-Pelluz B Arango-Gonzalez S Kustermann et alldquoPhotoreceptor cell death mechanisms in inherited retinaldegenerationrdquo Molecular Neurobiology vol 38 no 3 pp 253ndash269 2008

[31] G Li R E Anderson H Tomita et al ldquoNonredundant role ofAkt2 for neuroprotection of rod photoreceptor cells from light-induced cell deathrdquo e Journal of Neuroscience vol 27 no 1pp 203ndash211 2007

[32] KD Rhee S Nusinowitz K Chao F YuD Bok andX-J YangldquoCNTF-mediated protection of photoreceptors requires initialactivation of the cytokine receptor gp130 in Muller glial cellsrdquo

Proceedings of the National Acadamy of Sciences of the UnitedStates of America vol 110 no 47 pp E4520ndashE4529 2013

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one developed by the Royal College of Surgeons [4 5] thatharbour degenerative genetic mutations and the models ofphotoreceptor degeneration that are induced by light [6 7]or chemically [8ndash11] Although various genetic mutationsthat lead to RP have been identified they all eventuallylead to photoreceptor cell apoptosis [12 13] Photoreceptordegeneration induced by light or chemicals is also associ-ated with cellular apoptosis Apoptosis is the final commonpathway in the photoreceptor degeneration though some dif-ferences of molecular mechanisms involve in between gene-and light- or chemical-induced photoreceptor degenera-tions

Nicotinamide (NAM) a water-soluble B-group vitamin(vitamin B3) exerts protective effects against light- [14 15]andN-methyl-nitrosourea (MNU)-induced [16] photorecep-tor cell apoptosis A better understanding of the molecularmechanisms involved in the photoreceptor degeneration andNAM-mediated cellular protection will aid in the identifi-cation of successful therapeutic approaches against retinaldegenerative diseases

2 Materials and Methods

21 Animals All animal experiments were conducted inaccordance with the guidelines of the Animal ExperimentCommittee of Iwate University Japan Male Sprague-Dawleyrats were obtained fromCLEA Japan Inc (Tokyo Japan)Therats were housed in a 12 h12 h lightdark cycle with access towater ad libitum

22 Antibodies Antibodies against extracellular signal-reg-ulated kinase (ERK Cat No 4695S) phosphorylated ERK(pERK Cat No 9101S) Akt (Cat No 9272) pAkt (Cat No9271L) cAMP response element binding protein (CREBCat No 9197S) and pCREB (Cat No 9198S) were obtainedfromCell Signalling Technology (Tokyo)The anti-glutaminesynthetase (GS) antibody (Cat No MAB302) was obtainedfrom Millipore (Tokyo) The anti-c-Jun N-terminal kinase(JNK Cat No sc-571) and phosphorylated JNK (Cat Nosc-6254) were obtained from Santa Cruz BiotechnologySecondary antibodies against rabbit (Cat No S3731) andmouse immunoglobulin (Ig) G (IgG Cat No S3721) wereobtained from Promega (Tokyo)

23MNU andNAMAdministration Rats were administereda single intraperitoneal injection of MNU (Fujifilm WakoPure Chemical Corp Osaka Japan) at a dose of 75 mgkgbody weight The MNU solution was freshly prepared withsterile physiological saline containing 005 acetic acidimmediately prior to use and stored at 4∘C in the darkNAM (Nacalai Tesque Kyoto Japan) was freshly dissolved insterile physiological saline and subcutaneously injected at adose of 1000 mgkg 30 minutes prior to MNU administra-tion

24 Histology Seven days following MNU injection ratswere sacrificed using carbon dioxide and eyes were enu-cleated Eyes were immersed in 4 paraformaldehyde in

phosphate buffered saline (PBS) overnight at 4∘C and thenembedded in paraffin Serial sections (4 120583m) of the wholeeye were cut sagittally through the cornea and parallel tothe optic nerve stained against haematoxylin and eosin andvisualised by microscopy (AxioImager A1 Carl Zeiss TokyoJapan)

25 Optical Coherence Tomography (OCT) Rats were anaes-thetized by intramuscular injection of ketamine (75 mgkg)and medetomidine (05 mgkg) and their pupils were dilatedwith tropicamide (Midrin-P Santen Co Ltd Osaka Japan)The eye was placed under local anaesthesia using oxybup-rocaine (Santen Co Ltd Osaka Japan) and covered with acontact lens Image acquisition of 11 mm length of the ratretina including the optic disk was performed using the linescan mode on an OCT imaging device equipped with aspecial ordered lens (RS-3000 NIDEK Co Ltd AichiJapan)

26 Western Blot Analysis Western blot analysis was per-formed as previously described [17] Briefly the retinas wereisolated from the eyes 1 and 3 days afterMNUorMNUNAMadministration Proteins were extracted from the sampleswith a lysis buffer containing 10 mM Tris-HCl (pH 75) 1Triton X-100 05 NP-40 1 mM EDTA 150 mM NaCl 1xprotease inhibitor cocktail (Thermo Scientific Tokyo) and1x Halt phosphatase inhibitor cocktail (Thermo ScientificTokyo) The lysate was quantified using a bicinchoninic acidassay (BCA) protein assay kit (Pierce Rockford IL) Thirtymicrograms of proteinwas loaded for electrophoresis on 4ndash15 Mini-PROTEIN TGX gels (Bio-Rad Tokyo) and trans-ferred onto a polyvinylidene difluoride (PVDF) membraneAfter blocking with Block Ace (DS Pharma BiomedicalOsaka Japan) the membrane was incubated with the appro-priate primary antibody Following washing the membranewas incubated with the appropriate alkaline phosphatase-conjugated secondary antibody Chemiluminescence detec-tion (CDP-Star Detection Reagent GE Healthcare Tokyo)was performed according to the standard procedure Banddensities were measured using ImageQuant software (GEHealthcare Tokyo) and the density of each band was nor-malised to that of 120573-actin

27 Immunohistochemistry Immunohistochemistry wasperformed on the retina 6 hours and 1 day after MNU orMNUNAM administration Paraffin-embedded sectionswere deparaffinized according to the standard procedureThesections were treated in citrate buffer (pH60) in amicrowavefor antigen retrieval After blocking the sections wereincubated with anti-pERK (dilution 1200) and GS antibodiesas a marker of Muller cells or with a control solution of anti-rabbit and anti-mouse IgGs overnight at 4∘C The sectionswere then washed three times with PBS containing 001Tween-20 and incubated with Alexa 488- or Alexa 594-conjugated anti-rabbit and anti-mouse IgG solution at roomtemperature for 1 h Following washing the sections werecovered with mounting media including 410158406-diamidino-2-phenylindole (DAPI VECTASHIELD FunakoshiTokyo)



50

100

150

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ControlMNUMNUNAM


ILM

~RPE

(mm

)


10

20

30

40

50

ControlMNUMNUNAM


ON

L (

m)

ILM

ONL

RPE

(a)

(d) (e)

(b) (c)






NAM

MNU

MNUNAM

(a)

pre 6 24 720

50

100

150

200

MNUMNUNAM

ON

L - R

PE (

m)

NAM


lowastlowastlowast


(b)





MNU MNUNAM

pJNK(54kDa)panJNK



0

2

4

6lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n

lowastlowast

MNU MNUNAM


panJNK

-actin

(a)

MNU MNUNAM

pp38p38


2

4



relat

ive e

xpre

ssio

n

MNU MNUNAM

pp38

p38

-actin

(b)


relat

ive e

xpre

ssio

n

MNU

MNU

MNUNAM

MNUNAM

PARP-actin


2

4

6

lowast

full length PARP

-actin

(c)


relat

ive e

xpre

ssio

n

MNU MNUNAM

AIF-actin


2

4

6

lowast

lowastlowastlowast

lowast

lowastlowastlowast

MNU MNUNAM

AIF

-actin

(d)





pERK1pERK2

ERK1ERK2

MNU MNUNAM

MNU MNUNAM

MNU MNUNAM

pERK1ERK1


2

4

6

lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

nre

lativ

e exp

ress

ion

pERK2ERK2


2

4

6

lowastlowastlowast

lowastlowastlowast


-actin

(a)

MNU

MNU

MNUNAM

MNUNAM

pAkt

Akt

pAktAkt


2

4

6

lowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n


-actin

(b)

Figure 4 Continued


relat

ive e

xpre

ssio

n

pCREB

pATF1

CREB

MNU MNUNAM

MNU MNUNAM


2

4

6pCREBCREB

lowast


-actin

(c)





4 Discussion




MNU MNUNAM


Nuclei

pERK

GS

Merge

50m








5 Conclusions


Data Availability




Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















50

100

150

200

ControlMNUMNUNAM


ILM

~RPE

(mm

)


10

20

30

40

50

ControlMNUMNUNAM


ON

L (

m)

ILM

ONL

RPE

(a)

(d) (e)

(b) (c)






NAM

MNU

MNUNAM

(a)

pre 6 24 720

50

100

150

200

MNUMNUNAM

ON

L - R

PE (

m)

NAM


lowastlowastlowast


(b)





MNU MNUNAM

pJNK(54kDa)panJNK



0

2

4

6lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n

lowastlowast

MNU MNUNAM


panJNK

-actin

(a)

MNU MNUNAM

pp38p38


2

4



relat

ive e

xpre

ssio

n

MNU MNUNAM

pp38

p38

-actin

(b)


relat

ive e

xpre

ssio

n

MNU

MNU

MNUNAM

MNUNAM

PARP-actin


2

4

6

lowast

full length PARP

-actin

(c)


relat

ive e

xpre

ssio

n

MNU MNUNAM

AIF-actin


2

4

6

lowast

lowastlowastlowast

lowast

lowastlowastlowast

MNU MNUNAM

AIF

-actin

(d)





pERK1pERK2

ERK1ERK2

MNU MNUNAM

MNU MNUNAM

MNU MNUNAM

pERK1ERK1


2

4

6

lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

nre

lativ

e exp

ress

ion

pERK2ERK2


2

4

6

lowastlowastlowast

lowastlowastlowast


-actin

(a)

MNU

MNU

MNUNAM

MNUNAM

pAkt

Akt

pAktAkt


2

4

6

lowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n


-actin

(b)

Figure 4 Continued


relat

ive e

xpre

ssio

n

pCREB

pATF1

CREB

MNU MNUNAM

MNU MNUNAM


2

4

6pCREBCREB

lowast


-actin

(c)





4 Discussion




MNU MNUNAM


Nuclei

pERK

GS

Merge

50m








5 Conclusions


Data Availability




Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















NAM

MNU

MNUNAM

(a)

pre 6 24 720

50

100

150

200

MNUMNUNAM

ON

L - R

PE (

m)

NAM


lowastlowastlowast


(b)





MNU MNUNAM

pJNK(54kDa)panJNK



0

2

4

6lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

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lowastlowast

MNU MNUNAM


panJNK

-actin

(a)

MNU MNUNAM

pp38p38


2

4



relat

ive e

xpre

ssio

n

MNU MNUNAM

pp38

p38

-actin

(b)


relat

ive e

xpre

ssio

n

MNU

MNU

MNUNAM

MNUNAM

PARP-actin


2

4

6

lowast

full length PARP

-actin

(c)


relat

ive e

xpre

ssio

n

MNU MNUNAM

AIF-actin


2

4

6

lowast

lowastlowastlowast

lowast

lowastlowastlowast

MNU MNUNAM

AIF

-actin

(d)





pERK1pERK2

ERK1ERK2

MNU MNUNAM

MNU MNUNAM

MNU MNUNAM

pERK1ERK1


2

4

6

lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

nre

lativ

e exp

ress

ion

pERK2ERK2


2

4

6

lowastlowastlowast

lowastlowastlowast


-actin

(a)

MNU

MNU

MNUNAM

MNUNAM

pAkt

Akt

pAktAkt


2

4

6

lowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n


-actin

(b)

Figure 4 Continued


relat

ive e

xpre

ssio

n

pCREB

pATF1

CREB

MNU MNUNAM

MNU MNUNAM


2

4

6pCREBCREB

lowast


-actin

(c)





4 Discussion




MNU MNUNAM


Nuclei

pERK

GS

Merge

50m








5 Conclusions


Data Availability




Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















MNU MNUNAM

pJNK(54kDa)panJNK



0

2

4

6lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n

lowastlowast

MNU MNUNAM


panJNK

-actin

(a)

MNU MNUNAM

pp38p38


2

4



relat

ive e

xpre

ssio

n

MNU MNUNAM

pp38

p38

-actin

(b)


relat

ive e

xpre

ssio

n

MNU

MNU

MNUNAM

MNUNAM

PARP-actin


2

4

6

lowast

full length PARP

-actin

(c)


relat

ive e

xpre

ssio

n

MNU MNUNAM

AIF-actin


2

4

6

lowast

lowastlowastlowast

lowast

lowastlowastlowast

MNU MNUNAM

AIF

-actin

(d)





pERK1pERK2

ERK1ERK2

MNU MNUNAM

MNU MNUNAM

MNU MNUNAM

pERK1ERK1


2

4

6

lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

nre

lativ

e exp

ress

ion

pERK2ERK2


2

4

6

lowastlowastlowast

lowastlowastlowast


-actin

(a)

MNU

MNU

MNUNAM

MNUNAM

pAkt

Akt

pAktAkt


2

4

6

lowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n


-actin

(b)

Figure 4 Continued


relat

ive e

xpre

ssio

n

pCREB

pATF1

CREB

MNU MNUNAM

MNU MNUNAM


2

4

6pCREBCREB

lowast


-actin

(c)





4 Discussion




MNU MNUNAM


Nuclei

pERK

GS

Merge

50m








5 Conclusions


Data Availability




Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















pERK1pERK2

ERK1ERK2

MNU MNUNAM

MNU MNUNAM

MNU MNUNAM

pERK1ERK1


2

4

6

lowastlowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

nre

lativ

e exp

ress

ion

pERK2ERK2


2

4

6

lowastlowastlowast

lowastlowastlowast


-actin

(a)

MNU

MNU

MNUNAM

MNUNAM

pAkt

Akt

pAktAkt


2

4

6

lowastlowast

lowastlowastlowast

relat

ive e

xpre

ssio

n


-actin

(b)

Figure 4 Continued


relat

ive e

xpre

ssio

n

pCREB

pATF1

CREB

MNU MNUNAM

MNU MNUNAM


2

4

6pCREBCREB

lowast


-actin

(c)





4 Discussion




MNU MNUNAM


Nuclei

pERK

GS

Merge

50m








5 Conclusions


Data Availability




Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















relat

ive e

xpre

ssio

n

pCREB

pATF1

CREB

MNU MNUNAM

MNU MNUNAM


2

4

6pCREBCREB

lowast


-actin

(c)





4 Discussion




MNU MNUNAM


Nuclei

pERK

GS

Merge

50m








5 Conclusions


Data Availability




Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















MNU MNUNAM


Nuclei

pERK

GS

Merge

50m








5 Conclusions


Data Availability




Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























5 Conclusions


Data Availability




Acknowledgments



References








































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















-methyl-n-nitrosourea-induced photoreceptor degeneration ...researcharticle...

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