Stimulation of the insulin/mtor pathway delays cone death in a mouse model of retinitis pigmentosa

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1 Stimultion of the insulin/mtor pthwy delys one deth in mouse model of retinitis pigmentos Cludio Punzo 1, Krl Kornker 2 & Constne L Cepko 1,3 Retinitis pigmentos is n inurle retinl disese tht leds to lindness. One puzzling spet onerns the progression of the disese. Although most muttions tht use retinitis pigmentos re in rod photoreeptor speifi genes, one photoreeptors lso die s result of suh muttions. To understnd the mehnism of non-utonomous one deth, we nlyzed four mouse models hroring muttions in rod-speifi genes. We found hnges in the insulin/mmmlin trget of rpmyin pthwy tht oinided with the tivtion of utophgy during the period of one deth. We inresed or deresed the insulin level nd mesured the survivl of ones in one of the models. Mie tht were treted systemilly with insulin hd prolonged one survivl, wheres depletion of endogenous insulin hd the opposite effet. These dt suggest tht the non-utonomous one deth in retinitis pigmentos ould, t lest in prt, e result of the strvtion of ones. Retinitis pigmentos is type of inherited retinl degenertion. It is urrently untretle nd usully leds to lindness. With over 40 reintitis pigmentos genes identified, it is the most ommon type of retinl degenertion used y single disese llele (RetNet, The phenotype is hrterized y n initil loss of night vision s result of the mlfuntion nd deth of rod photoreeptors. This phse is followed y progressive loss of ones. Beuse ones re responsile for olor nd high-uity vision, it is their loss tht leds to redution in the qulity of life. In mny ses, the disese-using llele is expressed exlusively in rods; nonetheless, ones die s well. Indeed, to dte there is no known form of retinl degenertion in humns or mie where rods die nd ones survive. In ontrst, muttions in one-speifi genes result only in one deth. Severl theories hve een proposed to explin this finding. For exmple, one deth ould e result of the relese of toxin produed y dying rods or the loss of trophi ftor tht is produed y helthy rods 1 6. Alterntively, one deth ould e used y mirogli tht re moilized initilly during rod deth 7 or y oxidtive stress 8,9.Oxidtive stress might lso diretly hrm ones. The onstnt flow of oxygen through the retinl pigmented epithelium (RPE) to photoreeptors nd the loss of rods, whih re 95% of the photoreeptors in humn nd mouse, my result in n overlod of oxygen to the remining ones 10. Evidene for ll of these mehnisms exists in mie, yet none re le to fully explin why ones my survive for mny yers in the sene of rods in humns. Nonetheless, rodents re very good model for this type of retinl degenertion. Although the rodent retin lks mul, whih is the one-rih nd rod-free re tht is present in humns, the mul is not involved in the initil phse of the disese. In humns, retinitis pigmentos strts outside of the mul, where the distriution of rods nd ones is similr to tht in mie. To determine the ommon underlying mehnism for one deth in retinitis pigmentos, we ompred four mouse models hroring muttions in rod-speifi genes ( (ref. 11), Pde6g / (ref. 12), Rho / (ref. 13) nd P23H 14, whih rries Rho trnsgene tht hs n mino id susitution of histidine for proline t mino id 23, s ours in some humn ses of retinitis pigmentos, see Methods). Affymetrix rrys were used to identify ommon hnges in gene expression tht ompny one deth. Chnges in sustntil numer of genes involved in ellulr metolism oinided with the onset of one deth. These hnges were suggestive of ones suffering from shortge of nutrients. We then found tht ones showed signs of utophgy, ellulr self-digestion proess, whih is onsistent with prolonged strvtion. We lso found tht severl spets of the insulin/ mmmlin trget of rpmyin (mtor) pthwy, n importnt pthwy tht regultes ellulr metolism, were ffeted during the period of one degenertion. As result of this finding, we inresed nd deresed the insulin level nd mesured the survivl of ones in one of the models. Mie treted systemilly with insulin hd prolonged one survivl, wheres depletion of endogenous insulin hd the opposite effet. Therefore, one strvtion is likely ontriutor to the slow demise of ones in humns with retinitis pigmentos. Tretments imed t improving nutrition of ones re thus plusile therpeuti venue. RESULTS Photoreeptor deth kinetis nd mirorry nlysis To estlish frmework for ompring gene expression in four different models of retinitis pigmentos, we estlished the equivlent stges of disese pthology through exmintion of the kinetis of rod (Fig. 1 nd Supplementry Figs. 1 nd 2 online) nd one (Fig. 2 nd 1 Hrvrd Medil Shool, Deprtment of Genetis nd Howrd Hughes Medil Institute, 77 Avenue Louis Psteur, Boston, Msshusetts 02115, USA. 2 The Reserh Institute t Ntionwide Children s Hospitl nd the Ohio Stte University, 169 Westwood Rod, Columus, Ohio 43214, USA. 3 Deprtment of Ophthmology, Hrvrd Medil Shool, 77 Avenue Louis Psteur, Boston, Msshusetts 02115, USA. Correspondene should e ddressed to C.L.C. (epko@genetis.med.hrvrd.edu). Reeived 30 Septemer; epted 30 Otoer; pulished online 7 Deemer 2008; doi: /nn VOLUME 12 [ NUMBER 1 [ JANUARY 2009 NATURE NEUROSCIENCE

2 d ONL PW5 PW7 PW7 PW7 e f g h ONL Wild type PW10 PW8 PW11 PW17 i j k l ONL PW7 PW17 PW20 PW25 m n o PW20 PW17 PW µm 50 µm Supplementry Fig. 3 online) deth. Rod deth kinetis ws estlished y determining the onset, progression nd end phse of rod deth (Fig. 1). The time from the onset of rod deth to the time when the outer nuler lyer (ONL) ws redued to one row of ells is referred to s the mjor rod deth phse. The time therefter until rod deth ws omplete will e referred to s the end phse of rod deth. To determine the eginning of the mjor phse of rod deth, we exmined the levge of the nuler envelope protein LminA (Fig. 1) nd of the poptoti protese spse3 (Fig. 1), nd used TUNEL stining (Fig. 1,d). The ontinution of the mjor rod deth phse ws monitored y these ssys, s well s inspetion of histologil setions (Fig. 1e h), s rods ount for more thn 95% of ll photoreeptors. One the ONL rehed one row of ells, the mjor phse of rod deth ws over. The end phse of rod deth ws determined using rod-speifi mrkers to perform either in situ hyridiztion (Supplementry Fig. 1) or immunohistohemistry (Fig. 1i l) on retinl setions. However, it is diffiult to determine whether ny rods remin unless every setion of single retin is olleted. Thus, retinl flt mounts were lso used to llow omprehensive nlysis of the end phse of rod deth (Fig. 1m q). Notly, lthough the end phse of rod deth ws lerly defined in the two GMP phosphodiesterse (PDE) mutnts nd in the Rho / mutnt, rods died so slowly in the P23H mutnt tht some rods were still present even 50 weeks (ltest time point nlyzed) fter the end of the mjor phse of rod deth (Supplementry Fig. 2). We used two methods to determine the onset nd progression of one deth. First, the overll time frme of one demise ws determined y quntittive rel-time PCR (qrt-pcr; Fig. 2) for the ventrl 15 one speifi trnsript Opn1sw (opsin1 short-wve sensitive, lue p q PW µm Figure 1 Rod deth kinetis in the Rho / mouse. All pnels show Rho / mie with the exeption of e. ( d) Onset of rod deth visulized y stining for leved nuler envelope protein LminA (), leved spse3 (rrowheds, mgent nd red signl, ) nd TUNEL (rrows, rown signl,,d). Blue in nd shows nuler DAPI stining. A retinl flt mount with view onto the photoreeptor lyer is shown in d. (e h) Progression of rod deth determined y the redution of the ONL ws visulized y hemtoxylin nd eosin stining. (i q) End phse of rod deth ws ssessed y setion nlysis (i l) or y retinl flt mounts (m q). In the Rho / mouse, the onset of rod deth ws round PW5 () nd progressed up to PW25 (l). By PW17, the ONL ws redued to one row of ells (h,j) nd the remining rods died in the following 8 weeks (j q), s seen y immunofluoresene with n ntiody to Gnt1 on setions of progressively older nimls (j l). Retinl flt mounts showing rods visulized y immunofluoresene with n ntiody Gnt1 re shown in m q. The entire retin is shown in m, higher mgnifitions round the opti nerve hed re shown in n nd o, nd the peripherl region is shown in p. No signl ws seen t PW25 on flt mounts (q) or setions (l). Age (in PW) is indited in the pnels. Vertil r in nd e l indites thikness of the ONL. one opsin). This llowed for n initil quntittive omprison mong different strins, ut ws not dequte to determine the numer of ones, s trnsript levels ould vry PW25 efore ell deth. Next, we used whole-mount immunohistohemistry for red/green opsin (Opn1mw, opsin1 medium-wve sensitive) nd penut gglutinin letin (PNA) (Fig. 2 n). Both mrkers were expressed throughout the murine retin, llowing for the visuliztion of ones (Fig. 2 d). Notly, the onset of one deth lwys ourred t the equivlent stge of rod deth, nmely fter the mjor rod deth phse, when the thikness of the ONL ws redued to only single row of ells. We found tht one deth ourred from the enter to the periphery in ll four models, s seen y stining with PNA (Fig. 2e). It ws preeded y grdul redution of the outer segment length (Fig. 2f i) ndy opsin loliztion from the outer segment to the entire ell memrne (Fig. 2j l). In ddition, red/green opsin protein, whih is normlly deteted throughout the mouse retin (Fig. 2), ws deteted minly dorslly during one degenertion (Fig. 2m,n). However, PNA stining showed no ppreile differene ross the dorsl/ventrl xis (Fig. 2m,n). Similrly, lue opsin expression, whih is normlly deteted only ventrlly 15 (Fig. 2,d), ws not ffeted during this erly phse of one degenertion (Fig. 2o). Shortening of one outer segments nd loss of one-speifi mrkers hs lso een desried in humn ses of retinitis pigmentos 16. In summry, the kinetis nd histologil hnges tht ompnied rod nd one deth shred severl fetures ross the four models. First, one degenertion lwys strted fter the end of the mjor rod deth phse (Fig. 3,). This point ws rehed t very different ges in three of the four mutnts, s the overll kinetis of rod deth were quite different. Seond, one deth ws lwys entrl to peripherl nd ws preeded y redution in outer segment length. Third, in ll four mutnts, red/green opsin protein levels were detetle minly dorslly during one degenertion (Fig. 3). These ommon fetures suggested NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 1 [ JANUARY

3 Reltive onentrtion in (log) Wild type P23H Rho / Pde6g / Time (PW) Red/green Red/green dorsl Red/green ventrl Blue dorsl Blue ventrl tht there might e ommon mehnism(s) of one deth nd tht lues out this mehnism(s) might e suggested y gene expression hnges tht were ommon ross the four models t the onset of one deth. To determine ommon gene expression hnges, we olleted RNA smples from ll four models hlfwy through the mjor phse of rod deth, t the onset of one deth nd from two time points during the one deth phse (Fig. 4). The RNA ws hyridized to n Affymetrix mouse rry. Gene expression hnges were ompred in the sme strin ross the four time points. Two riteri hd to e fulfilled to selet gene for ross omprison mong the four strins. First, the hnge over time hd to e sttistilly signifint (see Methods, P o 0.01). Seond, gene hd to e upregulted t lest twofold t the onset of one deth ompred with the other three time points. This seond riterion removed rod-speifi hnges tht were still ourring t the onset of one deth nd t the sme time enrihed for hnges t the onset of one deth. A totl of 240 Affymetrix ID numers were found tht stisfied oth riteri in eh of the four strins. The 240 ID numers mthed to 230 genes (Supplementry Tle 1 online). Of the 195 genes tht ould e nnotted, 34.9% (68 genes) were genes tht re involved in ellulr metolism (Fig. 4,). The signling pthwy with the highest numer of hits (12 genes) ws the insulin/ mtor signling pthwy (Fig. 4), n importnt pthwy for regulting mny spets of ellulr metolism. Thus, the dt suggested tht d e Peripherl Centrl 75 mm 12 µm j m Red/green dorsl n Red/green ventrl f g h 10 mm 75 µm 75 µm i Outer segment length µm 50 µm Blue opsin Centrl Peripherl ONL INL events t the onset of one deth oinided with hnges in ellulr metolism tht might e regulted y the insulin/mtor pthwy. mtor in wild-type nd degenerting retine On the sis of our findings from the mirorry nlysis, we exmined the insulin/mtor signling pthwy during the period of one deth. The kinse mtor is n importnt regultor of protein synthesis nd riosome iogenesis 17. When ellulr energy levels re high, mtor llows energy-onsuming proesses suh s trnsltion, nd prevents utophgy, wheres mtor hs reverse effet in nutrient-poor onditions. Therefore, gluose, whih inreses ellulr ATP levels, nd mino id vilility, espeilly tht of leuine, positively ffets mtor tivity. To egin to investigte the tivity level of mtor during degenertion, we exmined levels of phosphorylted mtor (P-mTOR) y immunofluoresene. Phosphoryltion of mtor inreses kinse tivity nd P-mTOR levels n therefore serve s n inditor of its tivity level. Beuse every eukryoti ell expresses mtor, ertin level of P-mTOR is likely to e found in every ell. Notly, high levels of P-mTOR were deteted only in dorsl ones of wild-type retine (Fig. 5 ). Notly, this pttern of P-mTOR is reminisent of the red/green opsin pttern tht ws seen during one degenertion (Fig. 3). We investigted whether the ventrl red/green opsin downregultion tht ourred during one degenertion ould e mimiked y Outer segment length (µm) k o l Wild type Figure 2 Cone deth kinetis. () qrt-pcr nlysis for Opn1sw during one degenertion. ( o) All pnels show retinl flt mounts exept for i nd l. Green signl indites PNA expression nd red signl indites red/green or lue opsin. The wild-type retin is shown t P35 in d. Red/green opsin () nd PNA (,d) expression were deteted oth dorslly nd ventrlly, wheres lue opsin (,d) ws deteted only ventrlly. The mouse is nlyzed in e g nd j o. A entrl to peripherl grdient of PNA nd shortening of one outer segments is shown in e g. At P20, there were fewer elongted outer segments in the enter (e) s ompred with the periphery. Higher mgnifitions of entrl or peripherl outer segment from e re shown in f nd g, nd wild-type outer segment is shown in h (white line mrks the outer segment). A quntifition of outer segment length t 3 weeks is shown in i (error rs represent s.d. of 15 mesurements eh). With the shortening of outer segments during degenertion, red/green opsin ws lolized throughout the memrne of the ell ody, nd PNA, whih detets n extrellulr protein(s), ws redued to smll dot tthed to the residul outer segment (j) (yellow shows red/green nd PNA overlp, rrow). A higher mgnifition of one showing red/green t the memrne is displyed in k (rrow). A ross setion showing red/green in ell ody is shown in l (rrows) (P70; j l). During degenertion, red/ green opsin ws deteted minly dorslly (m), wheres PNA (m, n) or lue opsin(o) expression were not ltered (m nd n, P21; o, P49). GCL, gnglion ell lyer; INL, inner nuler lyer. GCL 46 VOLUME 12 [ NUMBER 1 [ JANUARY 2009 NATURE NEUROSCIENCE

4 Mjor rod nd one deth phse End phse of rod or one deth Pde6g / 4 22 Rho / 8 28 P23H Figure 3 Summry of rod nd one deth kinetis. () Shemti representtion of the rod nd one deth kinetis found in the four mouse models of retinitis pigmentos. The onset of one deth is set s time zero. The orresponding time windows on the x xis re given in weeks. The mjor one deth phse ws the time period from onset until roughly 85% of ones hd died. The end phse of one deth ws the time period therefter. () Summry of rod nd one deth kinetis. Time is indited in postntl dys or weeks. () Immunofluoresene on retinl flt mounts showing the ventrl redution of red/green opsin expression found in the four mutnts during one degenertion. Strin nd time, in postntl dys, re indited elow eh imge (for higher mgnifition of wild type, see Fig. 2). 25 Strin Pde6g / Rho / P23H Mjor rod deth phse P11 20 P11 20 PW5 17 PW10 35 End phse of rod deth PW3 7 PW3 7 PW17 25 present t PW80 Mjor one deth phse PW3 15 PW3 15 PW17 45 PW35 on Wild type P35 P35 Pde6g / P175 Rho / P175 P23H P175 redution in mtor tivity. To this end, we treted wild-type mie with rpmyin, n mtor inhiitor 17. This tretment resulted in ventrl downregultion of red/green opsin without ffeting lue opsin or the PNA stining or dorsl phosphoryltion of mtor (Fig. 5d g). Thus, inhiition of mtor in wild type repitulted the expression pttern of red/green opsin nd lue opsin, s well s the pttern of PNA stining, tht ws seen in the mutnts during degenertion, suggesting tht the ventrl downregultion of red/ green opsin seen during degenertion might e the result of redued mtor tivity. As expeted for mtor funtion, the downregultion of red/green opsin did not our t the RNA level, ut insted t the protein level, in untreted mutnt mie, s well s in wild-type mie treted with rpmyin (Supplementry Fig. 4 online). Finlly, nlysis of mutnt retine showed derese of P-mTOR levels in dorsl ones during one degenertion (Fig. 5h m). To test whether the high level of P-mTOR found in dorsl wild-type ones ws gluose-dependent, retinl explnts of wild-type mie were ultured in medium for 4 h in the presene or sene of gluose. Dorsl P-mTOR ws olished in the sene of gluose, even when leuine onentrtions were inresed in the medium (Supplementry Fig. 5 online). Thus, our dt estlish link etween mtor tivity, the expression hnges of red/green opsin seen during degenertion, nd the mirorry dt, whih suggested metoli hnges t the onset of one deth. The hnges might e used y ompromised gluose uptke in ones. Responses of ones to nutritionl imlne Our mtor phosphoryltion dt suggested tht nutritionl imlne ws ourring in ones during degenertion, whih ws possily 40 End phse of one deth present t PW45 present t PW45 present t PW80 present t PW80 Figure 4 Affymetrix mirorry nlysis. () Equivlent time points in the four different mutnts t whih the mirorry nlysis ws performed (R, pproximtely hlfwy through the mjor phse of rod deth; C0, onset of one deth; C1 nd C2, first nd seond time point during one deth, respetively). Time is indited in postntl dys weeks. Crtoons depiting the progression of one deth re shown elow the orresponding time points. () Distriution in perentges of the 195 genes tht were nnotted. () Distriution in perentges of the 68 genes (34.9%) tht re prt of the metolism tegory shown in. used y redued gluose levels in ones. To test this ide, we exmined the level of the heterodimeri trnsription ftor hypoxi induile ftor 1 (HIF-1/), whih improves glyolysis under stress onditions suh s low oxygen. HIF-1 nd mtor re tightly linked, s low oxygen results in low energy s result of redued oxidtive phosphoryltion, leding to redued mtor tivity An upregultion of the regulted suunit HIF-1 would proly reflet low gluose levels in ones nd not hypoxi onditions, s oxygen levels re inresed s result of the loss of rods 10. Immunofluoresene nlysis of HIF-1 during one degenertion reveled n upregultion of the protein in the ones of ll four mouse models (Fig. 6 f nd Supplementry Fig. 6 online). Consistent with the upregultion of HIF-1, gluose trnsporter 1 (GLUT1, Sl21), HIF-1 trget gene 23,24 lso ws found to e upregulted in ones, gin in ll four mouse models (Fig. 6g j nd Supplementry Fig. 6). Thus, upregultion of HIF-1 nd GLUT1 in ones re onsistent with response to shortge of gluose. This lso provides link to the deresed P-mTOR levels tht we oserved during degenertion nd to the sensitivity of P-mTOR to gluose. Strin 1.0% 2.1% 2.1% 2.6% 3.1% 3.6% 3.6% 5.1% 6.2% 11.8% 16.2% 7.7% 5.9% R C0 C1 C2 P12 P20 PW5 PW9 Pde6g / P12 P20 PW9 PW25 Rho / PW8 PW17 PW25 PW40 P23H PW25 PW35 PW40 PW75 7.4% 6.2% 10.8% 23.5% 11.3% 34.9% 35.3% Metolism Trnsport Trnsription/trnsltion Cell deth/survivl Insulin/Akt/mTOR Differentition/development DNA inding/replition/repir Signl trnsdution Clium inding Oxidoredutse/redox tivuty Cytoskeleton GABA signling Hypoxi Rest Amino ids nd protein Crohydrte Lipid Nuleotide N-glyn Rest NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 1 [ JANUARY

5 d h k e i l To sertin whether ones re nutritionlly deprived, we ssessed utophgy in ones. Two types of utophgy re induile y vrious degrees of nutrient deprivtion: mroutophgy nd hperone-medited utophgy (CMA) Mroutophgy is nonseletive, trgets proteins or entire orgnelles, nd is mrked y de novo formtion of memrnes tht form intermedite vesiles f 20 µm 150 µm j m g OS IS 50 µm 20 µm Figure 5 P-mTOR in wild-type nd degenerting retine. ( ) P-mTOR levels in wild-type retine. Dorsl (top) enrihment of P-mTOR (). Higher mgnifitions of the dorsl nd ventrl regions re shown to the right, with P-mTOR in red nd one segments in green, s deteted y PNA. Dorsl retinl setions stined for P-mTOR (red) nd PNA () or ntiody to -gltosidse (green, ). Higher mgnifition (insets) imges suggest tht the P-mTOR signl is loted in the lower prt of the outer segment (OS). IS, inner segment. (d g) Rpmyin tretment of wild-type mie led to downregultion of red/green opsin ventrlly (e) ut not dorslly (red, d). Ventrl lue opsin (red, f) remined unffeted, s did PNA (d g) (green) nd P-mTOR itself (red, g). (h m) Redued levels of dorsl P-mTOR during photoreeptor degenertion (red). The wild-type ontrol is shown in h nd the Pde6 mutnt is shown in i nd j. Redution strted during rod deth t P15 (i) s the outer segments (green, PNA) dethed from the retinl pigmented epithelium. By P30, only few ones (green signl: --gltosidse) showed high levels of P-mTOR (red) (j). (k m) Similr redution ws seen in dorsl ones of the other three mutnts (ones mrked in green y PNA; Pde6g / is shown t P35 in k; Rho / is shown t PW20 in l; P23H is shown t PW70 in m). All pnels show immunofluoresene on retinl flt mounts (photoreeptor side up) with the exeption of, nd g, whih show retinl setions. Blue indites DAPI. (utophgosomes) tht fuse with the lysosomes. The mhinery required for mro- 20 µm utophgy hs een shown to e present in photoreeptors 29. In ontrst, CMA is seletive nd trgets individul proteins for trnsport to the lysosomes. We ssessed the presene of mroutophgy y infetion with virl vetor enoding fusion protein of green fluoresent protein (GFP) nd light hin 3 (LC3), n utophgosoml memrne mrker No differene ws oserved in GFP distriution in ones of wild-type nd mutnt mie, suggesting tht formtion of utophgosomes ws d e f 100 µm 50 µm 100 µm 25 µm Figure 6 Upregultion of Hif-1 nd GLUT1 in ones. All pnels show immunofluoresent stining. Blue shows nuler DAPI stining nd green shows ones mrked with PNA. ( f) Stining for HIF-1 (red signl). () Wild type (PW10) stined for HIF-1 (inset shows higher mgnifition). (,) Cross setions in wild type stined for HIF-1 (PW10). DAPI overlp j is shown in. (d f) During one degenertion in (PW10) mie, we found inresed levels of HIF-1 in ones (inset in d shows higher mgnifition with DAPI overlp). Cross setions indited tht the inrese 25 µm 50 µm of Hif-1 ourred minly in ones (rrows point to ones tht re loted in the top lyer of the inner nuler lyer t this stge; e). DAPI overlp is shown in f. (g) GLUT1 expression in wild type (PW10) (red). Most of the signl in etween the ones reflets expression in rods. (h j) Inresed expression of GLUT1 in ones during degenertion seen in flt mounts (h) nd setions (i,j). PNA overlp of j is shown in i. Retinl flt mounts re shown in, d, g nd h. Retinl setions re shown in,, e, f, i nd j. g h i 48 VOLUME 12 [ NUMBER 1 [ JANUARY 2009 NATURE NEUROSCIENCE

6 d Reltive onentrtion LAMP-2A LAMP-2B LAMP-2C 20 µm Wild type Rtio sent during one deth (Supplementry Fig. 7 online). In ddition, high levels of phosphorylted riosoml protein S6 were found in ll or most ones (Supplementry Fig. 7), refleting n inresed tivity of riosoml S6 kinse 1 (S6K1), n inhiitor of mroutophgy 27. Consistent with these findings is the ft tht mroutophgy reflets n ute short-term response to nutrient deprivtion or ellulr stress onditions 25,26. Prolonged nonseletive degrdtion of newly synthesized proteins to overome the stress ondition would not e fvorle to ells nd would proly result in the reltively rpid deth of most ones, rther thn the slow deth tht is seen in retinitis pigmentos. CMA is normlly tivted during extended periods of strvtion nd results in inresed levels of lysosoml-ssoited memrne protein type 2A (LAMP-2A) t the lysosoml memrne 25,26,33. Both strvtion nd oxidtive stress n indue CMA 25. Strvtion inreses LAMP-2A expression y preventing its degrdtion while oxidtive stress results in de novo synthesis of LAMP-2A 34. A LAMP-2 ntiody tht reognizes +streptozotoin Figure 7 Inresed levels of LAMP-2 t the lysosoml memrne. ( ) Immunofluoresene on retinl flt mounts (LAMP-2 is shown in green, red/green opsin in red nd DAPI in lue). Insets show enlrged ells (rrow). A wild-type retine t PW5 showing lysosome (smll green dots) with norml LAMP-2 distriution is imged in. Wek red/green opsin signl ws deteted t the level of the photoreeptor nulei, s it ws minly found in the outer segments in the wild type. Enlrged lysosomes (dots) resulting from n umultion of LAMP-2 t the lysosoml memrne were seen speifilly in ones from mie PW5 (). Confol setion of sme field s in tken t the level of the inner nuler lyer showed LAMP-2 levels similr to those in wild type (). (d) qrt-pcr for the three different LAMP-2 splie forms showing the reltive onentrtion nd the rtios etween the mutnt nd wild type. Error rs represent s.d. of three mesurements. the proteins resulting from ll three splie isoforms 35 (A, B nd C) indited tht there were high levels of LAMP-2 t the lysosoml memrne in ll four mutnts during one degenertion (Fig. 7 ; only dt for is shown). The high levels were speifi to ones nd were not seen in ells of the inner nuler lyer (Fig. 7,), whih might suggest tht ones re the only strving ells in the retinitis pigmentos retin. qrt-pcr for the three splie isoforms showed only minor inrese in mrna levels of LAMP-2A (1.2-fold) nd derese in LAMP-2C expression (Fig. 7d), suggesting tht the inrese seen in protein t the memrne is minly the result of nutritionl deprivtion, nd only to lesser extent to oxidtive stress 8,9,34. Tken together, the dt suggest tht nutritionl imlne in ones leds to the tivtion of CMA, proess tht is onsistent with prolonged strvtion. Our findings in reltion to mtor, HIF-1, GLUT1 nd the indution of CMA suggested tht shortge of gluose in ones ws ourring, resulting in strvtion, nd suggested tht the insulin/ mtor pthwy is importnt during one deth. To determine whether the insulin/mtor pthwy n influene one survivl, we stimulted the pthwy y systemi tretment of mie with insulin. The Pde6 mutnt ws hosen over the other three mutnts euse of its fster one deth kinetis. Mie were treted with dily intrperitonel injetions of insulin over 4-week period, strting t the onset of one +insulin d Perentge of ones +streptozotoin +insulin P < 0.05 P < e Gluose (mg/dl) f Weight (gr) Figure 8 Insulin levels ffet one survivl. 0 ( ) Retinl flt mounts of mutnts t PW7 stined for LZ 47,48 to detet ones (see g h Methods nd Supplementry Fig. 9). An exmple of untreted ontrol is shown in, n exmple of mouse injeted with streptozotoin is shown in nd n exmple of mouse injeted dily with insulin is shown in. (d) Quntifition of one +insulin survivl fter four weeks of tretment. Dt represents n verge of t lest eight retine. The y xis represents the perentge of the one surfe re versus the surfe re of entire retin (see Supplementry Figs. 8 nd 10). (e,f) Mesurements of lood gluose levels (e) nd ody weight (f) performed weekly over the time spn of the experiment. (g,h) Immunofluoresent stining on retinl flt mounts for HIF-1 (red) nd PNA (green) in untreted ontrol (g) nd mie treted for 4 weeks with insulin (h). DAPI is shown in lue. Error rs in d f represent s.d. NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 1 [ JANUARY

7 deth. To redue insulin, we injeted mie with streptozotoin, drug tht kills the insulin-produing et ells of the pnres. Systemi dministrtion of insulin results in desensitized insulin reeptor s result of feedk loop in the pthwy, whih uses n inrese in lood gluose levels. Injetion of streptozotoin, whih lso results in inresed lood gluose levels, served s ontrol for the effet of elevted lood gluose, nd lso provided nimls with redued levels of insulin. mie tht were injeted with insulin showed improved one survivl ompred with non-injeted ontrol mie. mie tht were injeted with streptozotoin showed derese in one survivl (Fig. 8 d). Improved one survivl ws therefore result of insulin nd not of the inresed lood gluose levels (Fig. 8e). Also, insulin tretment did not lter the overll gin in ody weight (Fig. 8f). In ddition, ones in mutnt mie tht were treted with insulin did not show the upregultion of HIF-1 tht is normlly seen in ones during degenertion, onsistent with the notion tht ones were responding to insulin diretly (Fig. 8g,h). DISCUSSION Here, we found tht ones show signs of nutritionl imlne during the period of one degenertion in mie with retinitis pigmentos. Our mirorry nlysis suggested tht there re hnges in ellulr metolism tht involve the insulin/mtor pthwy t the onset of one deth. We found tht inhiition of mtor in wild-type mie resulted in the sme pttern of loss of red/green opsin s is seen during degenertion. Consistent with hnges in P-mTOR nd its sensitivity to gluose, we oserved n upregultion of HIF-1 nd GLUT1, suggesting tht gluose uptke nd/or the intrellulr levels of gluose my e ompromised in ones of mie with retinitis pigmentos. In ddition, systemi dministrtion of insulin prolonged one survivl, wheres depletion of endogenous insulin hd the reverse effet. Systemi tretment with insulin prevented the upregultion of HIF-1 in ones tht is normlly seen during one degenertion, suggesting tht insulin ws diretly ting on ones. We lso treted group of mutnt mie with insulin for 7 weeks, rther thn for only 4 weeks. This prolonged tretment with insulin did not show ny sustntil improvement reltive to untreted mutnt mie (Supplementry Fig. 8 online). The differene in one survivl etween the two lengths of tretment my reflet the feedk loop of the pthwy, in whih S6K1 ts diretly on the insulin-reeptor sustrte. Alterntively, or in ddition, signling through the insulin reeptor might slow down utophgy, ut would not ddress fundmentl prolem, suh s insuffiient gluose. Eventully, the ones died, perhps in n elerted fshion one their metoli demnds exeeded the supply. An elertion of one deth might e predited, if in ft insulin suppressed utophgy, nd therey reted more of n energy imlne. Although the ross tlk in ones etween the insulin reeptor, mtor, HIF-1, S6K1 nd insulin-reeptor sustrte remins to e investigted, the results strengthen the notion tht nutrient vilility in ones my e ltered during the period of one degenertion nd tht the insulin/mtor pthwy is involved. A reent report showed tht onstitutive expression of proinsulin in the rd10 mouse model of retinitis pigmentos delys photoreeptor deth, oth of rods nd ones 36. However, proinsulin does not seem to t through the insulin reeptor, s mie treted with proinsulin did not develop hyperglyemi. Proinsulin loks developmentl ell deth nd thus my interfere with the poptoti pthwy in the postntl retin. Although downstrem effetors, suh s PI3K/Akt, re regulted y insulin nd proinsulin signling, the reltionship etween the results of the studies reported here on the effets of insulin nd proinsulin remin to e determined. Mroutophgy, whih is ontrolled y mtor through its downstrem trget S6K1, ws not deteted during one degenertion, wheres CMA ppered to e tivted. Inresed LAMP-2A levels t the lysosoml memrne re suggestive of n tivtion of CMA. In ddition, the oservtions onerning mtor, HIF-1 nd GLUT1 re onsistent with strvtion nd CMA. However, dditionl experiments re needed to prove tht strvtion nd CMA re indeed ourring nd to prove tht they re n importnt ontriutor to one deth. The lk of detetle mroutophgy does not rule out the possiility tht mroutophgy might our for short period of time (for exmple, 24 h) efore the tivtion of CMA. The dt only show tht mroutophgy is not the min form of utophgy over n extended period of time, whih is onsistent with the notion tht mroutophgy is short-term response. The prolonged inhiition of mroutophgy is proly the result of inresed S6K1 tivity, s seen y inresed phosphorylted-s6 levels. S6K1 is positively regulted y mtor nd AMP-tivted protein kinse 27, whih reds out ellulr ATP levels. Therefore, lthough mtor my report metoli prolems with respet to gluose uptke nd redue energy-onsuming proesses nd improve glyolysis through HIF-1, AMP-tivted protein kinse my report norml ellulr ATP levels nd inhiit mroutophgy. This my represent speifi response to the energy requirements of ones. Notly, most of the gluose tken up y photoreeptors never enters the Kres yle 37. Thus, the shortge of gluose my not use shortge of ATP. Ltte, provided y Muller gli, n generte ATP vi the Kres yle 38. However, gluose is needed to generte NADPH in the pentose phosphte yle nd NADPH is required for synthesis of phospholipids, the uilding loks of ell memrnes. Photoreeptors shed portion of their memrnes t the tip of the outer segments on dily yle. Beuse redued levels of gluose would result in redution of memrne synthesis, the rte of outer segment shedding my e higher thn the rte of memrne synthesis y ones. Consistent with this, outer segment shortening preeded ell deth in these four models, s is lso oserved in humn ses of retinitis pigmentos 16. In ddition, hnges tht ffet lipid metolism were lso seen in the mirorry nlysis. Why does the loss of rods result in one deth in retinitis pigmentos? The previous hypotheses mentioned ove shre ommonlity in tht they n t fully explin the pthology tht is found in humns. The rod nd one deth kinetis shown here lerly rgue ginst toxin produed y dying rods s use for one deth. If rod toxin used one deth, then the onset of one deth should hve either oinided with the onset of rod deth or should hve strted shortly therefter, s this would e the period of pek toxin prodution. Notly, the lk of trophi ftor produed y helthy rods would gree with the onset of one deth seen in ll four models, s one would expet the onset of one deth during the end stges of rod deth. However, the progression of one deth nd the end phse of rod deth mke this unlikely to e the sole reson for one deth. In the, Pde6g / nd Rho / mutnts, some of the ones survived for mny weeks fter ompletion of the end phse of rod deth, demonstrting tht they were not dependent on rod trophi ftor. In the P23H model, rods died so slowly during the end phse of rod deth tht they were still present during the entire period of one deth. Although our dt do not provide strong support for trophi ftor hypothesis, neither do they rule it out. Interprettion of the forementioned lk of onsisteny of the kinetis of rod nd one deth, s might e predited y the trophi ftor model, re further ompromised y the ft tht the four mouse models tht we used re from four different geneti kgrounds. Bkground differenes ould result in differenes, 50 VOLUME 12 [ NUMBER 1 [ JANUARY 2009 NATURE NEUROSCIENCE

8 inluding different levels of suh ftor, tht might ount for the oserved differenes in the progression of one deth. How ould our oservtions of nutritionlly deprived ones explin the dependene of ones on rods? The outer segment RPE intertions re vitl, s the RPE shuttles nutrition nd oxygen from the horoidl vsulture to photoreeptors. Roughly 95% of ll photoreeptors in mouse nd humn re rods nd pproximtely outer segments ontt one RPE ell 39,40. Thus, only 1 2 of those RPE outer segment ontts re vi ones. During the ollpse of the ONL, the remining one-rpe intertions re proly pertured. If these intertions drop elow threshold required for the proper flow of nutrients, the loss of rods might result in redued flow of nutrients to ones. In ll four mouse models, the onset of one deth ourred when the ONL rehed one row of ells. This ell density ould therefore represent the ritil threshold. Then, while the remining rods die s result of muttion in rod-speifi gene, one deth my egin euse of nutrient deprivtion. Consistent with this notion, one deth progressed more slowly when the remining rods died slowly. This proposed mehnism would lso explin why the loss of ones does not led to rod deth 41,42. Beuse ones re less thn 5% of ll photoreeptors in humns nd mouse, the ritil threshold tht perturs outer segment RPE intertions would not e rehed. Further support for this ide is provided y studies in zerfish, where the overll rtio of rods to ones is reversed (1 to 8). Additionlly, the distriution of rods nd ones in zerfish is uneven, with ertin regions eing rih in ones nd other regions eing rih in rods. A reently isolted muttion in one-speifi gene resulted in rod deth, ut only in regions of high one density 43, leding the uthors to onlude tht ell density is the ruil determinnt. We therefore propose tht one ritil threshold of ell density is rehed, improper outer segment RPE intertions result in redued flow of nutrients (for exmple, gluose). This results in redued outer segment memrne synthesis, whih in turn further ontriutes to redued uptke of nutrients from the RPE. Ultimtely, prolonged strvtion, s suggested y the tivtion of CMA, leds to ell deth. Beuse strvtion n our slowly over extended periods of time nd euse the rte my hnge s result of flututions in nutrient uptke, the slow nd irregulr demise of ones tht is oserved in humns my e expeted. Therefore, this study not only revels previously unknown mehnism of one deth in retinitis pigmentos tht should diret future therpeuti pprohes, ut lso provides n explntion for oservtions reported in the literture with respet to the deth kinetis of rods nd ones in mie nd humns with different retinitis pigmentos muttions. METHODS Animls. Wild-type mie (C57Bl/6N) nd mie (normlly referred to s rd1 or FVB/N) were purhsed from Toni Frms. The mie hve muttioninthe suunit of GMP PDE 11. The PDE-g knokout (Pde6g / ) lks the g-suunit of PDE 12. The rhodopsin knokout (Rho / ) lks the rodspeifi opsin gene 12,13. The P23H mouse rries trnsgene tht hs sustitution muttion in the Rho gene (proline 23 is repled with histidine) 14. Beuse this mouse rries trnsgene, we rossed the strin k to C57Bl/6N to ensure tht none of the progeny would rry two lleles of the trnsgene. The trnsgene is speifilly expressed in rods The one-lz line, whih ws provided y J. Nthns 47 (Johns Hopkins Shool of Mediine), rries trnsgene tht expresses LZ under the ontrol of the humn red/green opsin promoter nd is expressed in ll ones in mouse. All proedures were in ompline with the Assoition for Reserh in Vision nd Ophthlmology Sttement for the Use of Animls. Affymetrix rry nlysis. RNA ws extrted s desried previously 48.Four retine were used per RNA smple. A minimum of two rrys were nlyzed per time point. The sttistil signifine of eh gene expression profile ws determined y Jonkheere-Terpstr test of the hypothesized one deth ptterned lterntive, using ext P vlues lulted y the Hrding lgorithm 49. qrt-pcr. qrt-pcr for Opn1sw nd Gpdh ws performed s desried previously 48. We used the following primers for the Lmp2 splie forms: Lmp2 forwrd primer (CTG AAG GAA GTG AAT GTC TAC ATG), Lmp2 reverse primer (GCT CAT ATC CAG TAT GAT GGC), Lmp2 reverse primer (CAG AGT CTG ATA TCC AGC ATA G) nd Lmp2 reverse primer (GAC AGA CTG ATA ACC AGT ACG). The onditions tht we used for ll three PCRs were 951 for 3 s, 521 for 15 s nd 721 for 25 s (Figs. 2 nd 7d represent n verge of three mesurements orreted for Gpdh). Retinl explnt ultures. The retin ws disseted free from other oulr tissues in DMEM medium nd then inuted (onditions listed in Supplementry Fig. 5). The gluose onentrtions were 4.5 g L 1 for regulr DMEM nd 1 g L 1 for low-gluose DMEM. Leuine ws dded t 200 mmndfetllf serum t 10% (vol/vol). Inution ws performed for 4 h nd the retine were fixed nd proessed for ntiody stining s desried elow. TUNEL, X-gl histohemistry nd in situ hyridiztions. TUNEL stining, X-gl histohemistry nd in situ hyridiztions were rried out s desried previously 48. To doule lel ones (Supplementry Fig. 9 online), we fixed retine in 2% prformldehyde (weight) for 15 min, proessed them for the X-gl retion nd then post-fixed them in 4% prformldehyde for 15 min. Biotin-PNA ws used in n ntiody stining proedure nd deteted with streptvidin-peroxidse (1:500, Rohe) y 3,3 -diminoenzidine tetrhydrohloride stin (Sigm) ording to the mnufturer s instrutions. We used the BE nd BI ESTs for the red/green opsin nd lue opsin proes, respetively. The proe for Rho ws generted y suloning the oding sequene (forwrd primer, 5 -AGC CAT GAA CGG CAC AGA GGG-3 ; reverse primer, 5 -CTT AGG CTG GAG CCA CCT GGC T-3 ) of the gene into pgem- T Esy (Promeg). Virl injetions. We rried out virl injetions s desried previously 32.Mie were injeted t emryoni dy 10 nd hrvested t postntl week 10 (PW10). The GFP-LC3 fusion protein tht we used ws generted y reominnt PCR with NotI site t the 5 of the fusion protein followed y GFP, then LC3, nd then n XhoI site t the 3 of the fusion protein nd loned into pqcxix s NotI-XhoI frgment (Cloneteh, t# ). To rete the fusion protein, we used primers for 5 NotI-GFP (5 -ATG CGG GCC GCC ACC ATG GTG AGC AAGGGCGAGGAGC-3 ), 3 GFP-LC3 (5 -AGG TCT TCT CGG ACG GCA TCT TGT ACA GCT CGT CCA TGC CGA G-3 ), 5 LC3 (5 -ATG CCG TCC GAG AAG ACC TTC AAG C-3 ) nd3 LC3-XhoI (5 -ATC TCG AGT TAC ACA GCC ATT GCT GTC CCG AAT G-3 ). Rpmyin, streptozotoin nd insulin tretments. Rpmyin ws diluted to 10 mg ml 1 in ethnol. The stok ws diluted to mg ml 1 in drinking wter over period of 2 weeks. We injeted 150 ml (12 mg ml 1 in 0.1 M itri id, ph 4.5) of streptozotoin intrperitonelly t postntl dy 21 (P21). Insulin ws injeted intrperitonelly dily strting t P21. The onentrtion ws inresed weekly suh tht 10 U per kg of ody weight were injeted the first week, 15 U per kg the seond, 20 U per kg the third nd 30 U per kg the fourth. In the tretment tht lsted for 7 weeks, 30 U per kg were injeted for the remining 3 weeks. Blood gluose levels were mesured y olleting drop of lood from the til diretly onto test strip from the TrueTrk smrt system (CVS Phrmy). Eye leeds were voided euse we were ssying ell survivl in the retin. Quntifition of one survivl. For proedures, see Supplementry Figure 10 online. Whole-mount nd setion ntiody stining. Antiody stining ws rried out s previously 32 desried, with few modifitions. Triton X-100 ws repled with 0.01% sponin for LAMP-2 stining nd phosphte-uffered sline ws repled y tris-uffered sline in every step of the proedure for P-mTOR nd P-S6 stining. For primry ntiodies, we used mouse ntiody to rhodopsin Rho4D2 (1:200) 50, got ntiody to -gltosidse (1:400, Serote), rit ntiody to lue opsin (1:1,000, J. Nthns), rit ntiody to gunine NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 1 [ JANUARY

9 nuleotide protein lph trnsduin (Gnt1, 1:200, Snt Cruz), rit ntiody to leved spse3 (1:100, Cell Signling), rit ntiody to leved LminA (1:100, Cell Signling), rit ntiody to GLUT-1 (1:100, Alph Dignostis), rit ntiody to P-mTOR (1:300, Ser2448, Cell Signling), rit ntiody to P-S6 (1:100, Ser235/236, Cell Signling), rit ntiody to HIF-1 (1:300, R&D Systems) nd rt ntiody to LAMP-2 (1:200, lone GL2A7, from the Developmentl Studies Hyridom Bnk). We nlyzed rod nd one deth kinetis in Pde6 / mie dily from P10 20, weekly from PW3 10 nd t PW 12, PW15, PW18 nd PW45, in Pde6g / mie dily from P10 20, weekly from PW3 10 nd t PW15, PW25, PW45, in Rho / mie weekly from PW4 8, nd t PW10, PW11, PW17, PW20, PW25, PW27, PW31, PW34, PW37, PW45, PW55 nd PW80, nd in P23H mutnt mie t PW5, PW10, PW16, PW25, PW30, PW35, PW40, PW65, PW70, PW75, PW80 nd PW85. Note: Supplementry informtion is ville on the Nture Neurosiene wesite. ACKNOWLEDGMENTS We thnk J. Nthns for the one-lz mouse line nd the lue-one opsin ntiody. We re grteful to S. Tsng, M. Nsh nd J. Lem for the, P23H nd Rho / mie, respetively. The LAMP-2 ntiody, developed y B. Grnger, ws otined from the Developmentl Studies Hyridom Bnk developed under the uspies of the US Ntionl Institute of Child Helth nd Humn Development nd mintined y the University of Iow. We thnk J. Trimrhi, R. Kndi, N. Perrimon, J. Zirin, M. Feny nd C. Tin for ritil reding of the mnusript. This work ws supported y the US Ntionl Institutes of Helth (RO1 EY014466), Mulr Vision Reserh Foundtion, Foundtion for Retinl Reserh, Howrd Hughes Medil Institute, Merk nd y n EMBO fellowship to C.P. AUTHOR CONTRIBUTIONS C.P. onduted the experiments nd wrote the mnusript. K.K. performed omputtionl mirorry nlysis. C.L.C. supervised the projet nd wrote the mnusript. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Steinerg, R.H. Survivl ftors in retinl degenertions. Curr. Opin. Neuroiol. 4, (1994). 2. Mohnd-Sid, S. et l. 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