( H - I ) EPG - 7 : : GFP is expressed in pharyngeal muscles ( H , arrow ) and cells in the tail region ( I ) at larval stages .

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(H-I) EPG-7::GFP is expressed in pharyngeal muscles (H, arrow) and cells in the tail region (I) at larval stages.


( J ) Compared with wild - type animals , levels of EPG - 7 : : GFP are dramatically elevated in epg - 4 mutants , but remain unchanged in sqst - 1 mutants .

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(J) Compared with wild-type animals, levels of EPG-7::GFP are dramatically elevated in epg-4 mutants, but remain unchanged in sqst-1 mutants.


( K ) The expression level of EPG - 7 : : GFP is dramatically elevated and many EPG - 7 : : GFP aggregates are formed in lgg - 1 mutant embryos .

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(K) The expression level of EPG-7::GFP is dramatically elevated and many EPG-7::GFP aggregates are formed in lgg-1 mutant embryos.


( L and M ) No EPG - 7 aggregates , detected by anti - EPG - 7 antibody , are formed in wild - type embryos . ( L ) DAPI image of the embryo shown in M . ( N ) Levels of EPG - 7 are dramatically elevated and a large number of EPG - 7 aggregates are formed in atg - 3 mutants . ( O ) No EPG - 7 aggregates are formed in sqst - 1 mutant embryos .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O

(L and M) No EPG-7 aggregates, detected by anti-EPG-7 antibody, are formed in wild-type embryos. (L) DAPI image of the embryo shown in M. (N) Levels of EPG-7 are dramatically elevated and a large number of EPG-7 aggregates are formed in atg-3 mutants. (O) No EPG-7 aggregates are formed in sqst-1 mutant embryos.


( A ) EPG - 7 colocalizes with SQST - 1 aggregates in epg - 8 mutant embryos . Colocalization was defined as two aggregates showing > 70 % overlap in their fluorescence signals . Insets show a magnified view .

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(A) EPG-7 colocalizes with SQST-1 aggregates in epg-8 mutant embryos. Colocalization was defined as two aggregates showing >70% overlap in their fluorescence signals. Insets show a magnified view.


( B ) Percentage of EPG - 7 aggregates colocalized with SQST - 1 in indicated autophagy mutants .

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(B) Percentage of EPG-7 aggregates colocalized with SQST-1 in indicated autophagy mutants.


( C ) A coimmunoprecipitation assay reveals that EPG - 7 associates with SQST - 1 .

(C) A coimmunoprecipitation assay reveals that EPG-7 associates with SQST-1.


( D ) EPG - 7 interacts with SQST - 1 in a pull - down assay .

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(D) EPG-7 interacts with SQST-1 in a pull-down assay.


( E ) EPG - 7 self - associates in a pull - down assay .

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(E) EPG-7 self-associates in a pull-down assay.


( F ) SDS - PAGE and blue - native PAGE of purified EPG - 7 ( 520 - 714 ) proteins . Lane M shows molecular weight marker . Lanes 1 , 2 , 3 , and 4 contain 10 , 5 , 2 . 5 , and 1 . 25 µg of proteins , respectively .

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(F) SDS-PAGE and blue-native PAGE of purified EPG-7(520-714) proteins. Lane M shows molecular weight marker. Lanes 1, 2, 3, and 4 contain 10, 5, 2.5, and 1.25 µg of proteins, respectively.


( G ) An EPG - 7 fragment containing amino acids 531 - 631 self - interacts .

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(G) An EPG-7 fragment containing amino acids 531-631 self-interacts.


( H and I ) SQST - 1 aggregates accumulate in epg - 7 mutant embryos carrying an epg - 7 ( del 531 - 631 ) : : gfp transgene . ( H ) DAPI image of the embryo shown in I .

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(H and I) SQST-1 aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del 531-631)::gfp transgene. (H) DAPI image of the embryo shown in I.


( J and K ) EPG - 7 ( del 531 - 631 ) : : GFP is weakly expressed and diffusely localized in epg - 7 ( tm2508 ) embryos . Its expression is not elevated in lgg - 1 ( RNAi ) animals . H - K show embryos carrying the same transgene . Images in J and K were taken using the same exposure time .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J and K) EPG-7(del 531-631)::GFP is weakly expressed and diffusely localized in epg-7(tm2508) embryos. Its expression is not elevated in lgg-1(RNAi) animals. H-K show embryos carrying the same transgene. Images in J and K were taken using the same exposure time.


( L and M ) SQST - 1 self - interacts ( L ) and the self - interaction is mediated by a fragment containing amino acids 26 - 181 ( M ) .

(L and M) SQST-1 self-interacts (L) and the self-interaction is mediated by a fragment containing amino acids 26-181 (M).


( N ) Levels of SQST - 1 ( del 26 - 181 ) : : GFP are dramatically elevated in epg - 7 and atg - 3 mutants .

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(N) Levels of SQST-1(del 26-181)::GFP are dramatically elevated in epg-7 and atg-3 mutants.


( O and P ) SQST - 1 ( del 26 - 181 ) : : GFP is weakly expressed and diffusely localized in sqst - 1 ( ok2892 ) mutant embryos . ( O ) DIC image of the embryo shown in P .

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(O and P) SQST-1(del 26-181)::GFP is weakly expressed and diffusely localized in sqst-1(ok2892) mutant embryos. (O) DIC image of the embryo shown in P.


( Q and R ) SQST - 1 ( del 26 - 181 ) : : GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg - 1 ( Q ) and epg - 7 ( R ) mutants .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

(Q and R) SQST-1(del 26-181)::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg-1 (Q) and epg-7 (R) mutants.


( A - D ) Expression of LGG - 1 in wild - type embryos at the ∼ 100 - cell ( A and B ) , the ∼ 200 - cell ( C ) , and the fourfold ( D ) stages . ( A ) DAPI image of the embryo shown in B .

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(A-D) Expression of LGG-1 in wild-type embryos at the ∼100-cell (A and B), the ∼200-cell (C), and the fourfold (D) stages. (A) DAPI image of the embryo shown in B.


( E ) Loss of function of epg - 7 does not increase levels of LGG - 1 and also does not suppress accumulation of LGG - 1 in epg - 3 mutant embryos .

(E) Loss of function of epg-7 does not increase levels of LGG-1 and also does not suppress accumulation of LGG-1 in epg-3 mutant embryos.


( F - H ) Expression of LGG - 1 in epg - 7 mutant embryos before the ∼ 100 - cell stage ( F ) , at the ∼ 200 - cell stage ( G ) , and at the fourfold stage ( H ) . Images in B - D and F - H were taken using the same exposure time .

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(F-H) Expression of LGG-1 in epg-7 mutant embryos before the ∼100-cell stage (F), at the ∼200-cell stage (G), and at the fourfold stage (H). Images in B-D and F-H were taken using the same exposure time.


( I ) SQST - 1 aggregates are separable from LGG - 1 puncta in epg - 7 mutant embryos . Insets show a magnified view . ( J ) In epg - 6 mutant embryos , LGG - 1 puncta and SQST - 1 aggregates are enlarged and colocalized . ( K ) LGG - 1 and SQST - 1 aggregates are smaller in size and largely separable in epg - 6 ; epg - 7 double mutants . ( I - K ) DAPI images of the embryos shown in the same row .

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(I) SQST-1 aggregates are separable from LGG-1 puncta in epg-7 mutant embryos. Insets show a magnified view. (J) In epg-6 mutant embryos, LGG-1 puncta and SQST-1 aggregates are enlarged and colocalized. (K) LGG-1 and SQST-1 aggregates are smaller in size and largely separable in epg-6;epg-7 double mutants. (I-K) DAPI images of the embryos shown in the same row.


( L - O ) Enlarged LGG - 1 and SQST - 1 aggregates are colocalized in epg - 4 mutants ( L and M ) . Simultaneously depleting epg - 7 reduces the size of LGG - 1 puncta and SQST - 1 aggregates , which become largely separable ( N and O ) . ( L and N ) DAPI images of the embryos shown in M and O , respectively .

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(L-O) Enlarged LGG-1 and SQST-1 aggregates are colocalized in epg-4 mutants (L and M). Simultaneously depleting epg-7 reduces the size of LGG-1 puncta and SQST-1 aggregates, which become largely separable (N and O). (L and N) DAPI images of the embryos shown in M and O, respectively.


( P ) In atg - 9 mutant embryos , LGG - 1 puncta largely colocalize with SQST - 1 aggregates at the comma stage . Notably , there are more SQST - 1 aggregates than LGG - 1 puncta . ( Q ) SQST - 1 aggregates are separable from LGG - 1 puncta in atg - 9 ; epg - 7 mutants at the comma stage .

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(P) In atg-9 mutant embryos, LGG-1 puncta largely colocalize with SQST-1 aggregates at the comma stage. Notably, there are more SQST-1 aggregates than LGG-1 puncta. (Q) SQST-1 aggregates are separable from LGG-1 puncta in atg-9;epg-7 mutants at the comma stage.


( R ) Percentage of LGG - 1 puncta colocalized with SQST - 1 aggregates in indicated autophagy mutants . Comma - stage embryos were examined for atg - 9 and atg - 9 ; epg - 7 mutants . ∼ 200 - cell stage embryos were examined for other autophagy mutants .

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(R) Percentage of LGG-1 puncta colocalized with SQST-1 aggregates in indicated autophagy mutants. Comma-stage embryos were examined for atg-9 and atg-9;epg-7 mutants. ∼200-cell stage embryos were examined for other autophagy mutants.


( A ) EPG - 7 coimmunoprecipitates with ATG - 9 : : HA .

(A) EPG-7 coimmunoprecipitates with ATG-9::HA.


( B ) EPG - 7 directly interacts with ATG - 9 in a GST pull - down assay .

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(B) EPG-7 directly interacts with ATG-9 in a GST pull-down assay.


( C and D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 3 ( C )

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(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-3 (C)


( C and D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 1 ( D ) .

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(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-1 (D).


( E ) EPG - 7 directly interacts with LGG - 3 .

(E) EPG-7 directly interacts with LGG-3.


( F ) EPG - 7 fragments containing amino acids 852 - 1003 or 1004 - 1142 , but not 762 - 851 , interact with LGG - 1 .

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(F) EPG-7 fragments containing amino acids 852-1003 or 1004-1142, but not 762-851, interact with LGG-1.


( G ) Fragments of EPG - 7 containing amino acids 762 - 851 or 852 - 1003 directly interact with ATG - 18 .

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(G) Fragments of EPG-7 containing amino acids 762-851 or 852-1003 directly interact with ATG-18.


( H ) Fragments of EPG - 7 containing amino acids 1 - 704 or 1143 - 1330 directly interact with UNC - 51 .

(H) Fragments of EPG-7 containing amino acids 1-704 or 1143-1330 directly interact with UNC-51.


( I ) SQST - 1 associates with LGG - 1 in vivo . Embryonic extracts from epg - 7 mutants show that interaction of SQST - 1 with LGG - 1 is EPG - 7 independent .

(I) SQST-1 associates with LGG-1 in vivo. Embryonic extracts from epg-7 mutants show that interaction of SQST-1 with LGG-1 is EPG-7 independent.


( J ) Fragments of SQST - 1 containing amino acids 351 - 499 and 604 - 693 , but not 500 - 603 , interact with LGG - 1 .

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(J) Fragments of SQST-1 containing amino acids 351-499 and 604-693, but not 500-603, interact with LGG-1.


( K ) Fragments of SQST - 1 - containing amino acids 418 - 499 and 604 - 630 bind to LGG - 1 . Mutating the putative LIR in the 604 - 630 fragment ( 605YPDL608 to APDA ) does not affect the binding .

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(K) Fragments of SQST-1-containing amino acids 418-499 and 604-630 bind to LGG-1. Mutating the putative LIR in the 604-630 fragment (605YPDL608 to APDA) does not affect the binding.


( L and M ) SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP , in which the two LGG - 1 - binding fragments ( amino acids 418 - 499 and 604 - 630 ) are deleted , is weakly expressed and diffusely localized in the cytoplasm in sqst - 1 ( ok2892 ) mutants . ( N and O ) Loss of function of lgg - 1 dramatically elevates the expression level of SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP , which forms a large number of aggregates . ( L and N ) DIC images of the embryos shown in M and O ,

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(L and M) SQST-1(del LGG-1-1+2)::GFP, in which the two LGG-1-binding fragments (amino acids 418-499 and 604-630) are deleted, is weakly expressed and diffusely localized in the cytoplasm in sqst-1(ok2892) mutants. (N and O) Loss of function of lgg-1 dramatically elevates the expression level of SQST-1(del LGG-1-1+2)::GFP, which forms a large number of aggregates. (L and N) DIC images of the embryos shown in M and O, respectively


( P ) SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP aggregates colocalize with EPG - 7 in lgg - 1 ; sqst - 1 mutants . Insets show a magnified view .

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(P) SQST-1(del LGG-1-1+2)::GFP aggregates colocalize with EPG-7 in lgg-1;sqst-1 mutants. Insets show a magnified view.


Formation of ATG - 9 puncta in various autophagy mutants . ( A and B ) In wild - type embryos , ATG - 9 : : GFP is diffusely localized in the cytoplasm . ( A ) DIC image of the embryo shown in B . Insets show a magnified view . ( C ) ATG - 9 : : GFP forms a large number of small , intense puncta in epg - 1 mutants . ( D and E ) Accumulation of ATG - 9 : : GFP puncta in unc - 51 ( D ) and epg - 1 ( E ) mutants is independent of lgg - 1 . ( F ) The ATG - 9 : : GFP puncta largely disappear in epg - 1 ; epg - 7 mutants . ( G ) ATG - 9 : : GFP accumulates into large punctate structures in epg - 6 mutants . ( H ) Loss of function of lgg - 1 suppresses the accumulation of ATG - 9 : : GFP puncta in epg - 6 mutants . ( I ) Loss of function of epg - 7 has no effect on the formation of ATG - 9 : : GFP puncta in epg - 6 mutants . The ATG - 9 : : GFP puncta are smaller in epg - 6 ; epg - 7 mutants than those in epg - 6 single mutants . ( J - L ) ATG - 9 : : GFP accumulates into large punctate structures in epg - 4 mutants ( J ) and the accumulation is suppressed by simultaneously depleting the activity of lgg - 1 ( K ) or epg - 7 ( L ) . ( M and N ) In epg - 8 mutants , ATG - 9 accumulates into aggregates ( M ) that are suppressed by loss of lgg - 1 activity ( N ) . ( O ) epg - 7 mutants exhibit the same distribution pattern of ATG - 9 : : GFP as wild - type embryos . ( P ) Percentage of ATG - 9 : : GFP puncta colocalized with EPG - 7 and SQST - 1 aggregates in indicated autophagy mutants .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O

Formation of ATG-9 puncta in various autophagy mutants. (A and B) In wild-type embryos, ATG-9::GFP is diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. Insets show a magnified view. (C) ATG-9::GFP forms a large number of small, intense puncta in epg-1 mutants. (D and E) Accumulation of ATG-9::GFP puncta in unc-51(D) and epg-1 (E) mutants is independent of lgg-1. (F) The ATG-9::GFP puncta largely disappear in epg-1;epg-7 mutants. (G) ATG-9::GFP accumulates into large punctate structures in epg-6 mutants. (H) Loss of function of lgg-1 suppresses the accumulation of ATG-9::GFP puncta in epg-6 mutants. (I) Loss of function of epg-7 has no effect on the formation of ATG-9::GFP puncta in epg-6 mutants. The ATG-9::GFP puncta are smaller in epg-6;epg-7 mutants than those in epg-6 single mutants. (J-L) ATG-9::GFP accumulates into large punctate structures in epg-4 mutants (J) and the accumulation is suppressed by simultaneously depleting the activity of lgg-1 (K) or epg-7 (L). (M and N) In epg-8 mutants, ATG-9 accumulates into aggregates (M) that are suppressed by loss of lgg-1 activity (N). (O) epg-7 mutants exhibit the same distribution pattern of ATG-9::GFP as wild-type embryos. (P) Percentage of ATG-9::GFP puncta colocalized with EPG-7 and SQST-1 aggregates in indicated autophagy mutants.


( Q - T ) The ATG - 9 : : GFP puncta in epg - 6 ( Q ) , epg - 8 ( R ) , unc - 51 ( S ) , and atg - 18 ( T ) mutants colocalize with EPG - 7 aggregates .

(Q-T) The ATG-9::GFP puncta in epg-6 (Q), epg-8 (R), unc-51 (S), and atg-18 (T) mutants colocalize with EPG-7 aggregates.


( U and V ) ATG - 9 : : GFP puncta in epg - 6 ( U ) and epg - 8 ( V ) mutants colocalize with SQST - 1 aggregates . ( W and X ) In unc - 51 ( W ) and atg - 18 ( X ) mutants , ATG - 9 : : GFP partially colocalizes with SQST - 1 aggregates .

(U and V) ATG-9::GFP puncta in epg-6 (U) and epg-8 (V) mutants colocalize with SQST-1 aggregates. (W and X) In unc-51 (W) and atg-18 (X) mutants, ATG-9::GFP partially colocalizes with SQST-1 aggregates.


( A2 ) Levels of W07G4 . 5 : : GFP in wild type , epg - 7 mutants , and atg - 3 L1 larvae before and after 6 h starvation treatment . ( B2 ) Model for the role of epg - 7 in degradation of SQST - 1 aggregates . SQST - 1 directly interacts with EPG - 7 and is recruited into EPG - 7 aggregates . EPG - 7 also associates with multiple Atg proteins , which may be recruited to the SQST - 1 - EPG - 7 complex in a hierarchical order . IM , isolation membrane . ( C2 ) Hierarchical relationship of the scaffold proteins EPG - 2 and EPG - 7 and other essential ATG and EPG proteins in the aggrephagy

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A2) Levels of W07G4.5::GFP in wild type, epg-7 mutants, and atg-3 L1 larvae before and after 6 h starvation treatment. (B2) Model for the role of epg-7 in degradation of SQST-1 aggregates. SQST-1 directly interacts with EPG-7 and is recruited into EPG-7 aggregates. EPG-7 also associates with multiple Atg proteins, which may be recruited to the SQST-1-EPG-7 complex in a hierarchical order. IM, isolation membrane. (C2) Hierarchical relationship of the scaffold proteins EPG-2 and EPG-7 and other essential ATG and EPG proteins in the aggrephagy pathway


( A and B ) C17E4 . 2 : : GFP is weakly expressed and diffusely localized in a wild - type embryo . ( A ) DIC image of the embryo shown in B . ( C and D ) C17E4 . 2 : : GFP is expressed at dramatically elevated levels and accumulates into many aggregates in lgg - 1 ( C ) and epg - 7 ( D ) mutant embryos .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O

(A and B) C17E4.2::GFP is weakly expressed and diffusely localized in a wild-type embryo. (A) DIC image of the embryo shown in B. (C and D) C17E4.2::GFP is expressed at dramatically elevated levels and accumulates into many aggregates in lgg-1 (C) and epg-7 (D) mutant embryos.


( E and F ) W07G4 . 5 : : GFP is expressed in the intestine and a few aggregates are formed in a wild - type embryo at the fourfold stage . ( E ) DIC image of the embryo shown in F . ( G and H ) W07G4 . 5 : : GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg - 3 ( G ) and epg - 7 ( H ) mutants .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

(E and F) W07G4.5::GFP is expressed in the intestine and a few aggregates are formed in a wild-type embryo at the fourfold stage. (E) DIC image of the embryo shown in F. (G and H) W07G4.5::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg-3 (G) and epg-7 (H) mutants.


( I ) Levels of C33D9 . 6 : : GFP and W07G4 . 5 : : GFP are dramatically elevated in atg - 3 and epg - 7 mutant embryos .

(I) Levels of C33D9.6::GFP and W07G4.5::GFP are dramatically elevated in atg-3 and epg-7 mutant embryos.


( J and K ) C17E4 . 2 aggregates in atg - 3 mutants overlap with EPG - 7 aggregates ( J ) , but are separable from SQST - 1 aggregates ( K ) . Insets show a magnified view . ( L ) Colocalization frequency of C17E4 . 2 , C33D9 . 6 , and W07G4 . 5 aggregates with EPG - 7 and SQST - 1 aggregates in atg - 3 mutants .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

(J and K) C17E4.2 aggregates in atg-3 mutants overlap with EPG-7 aggregates (J), but are separable from SQST-1 aggregates (K). Insets show a magnified view. (L) Colocalization frequency of C17E4.2, C33D9.6, and W07G4.5 aggregates with EPG-7 and SQST-1 aggregates in atg-3 mutants.


( M ) SQST - 1 : : GFP accumulates in hypodermal cells in epg - 7 mutants at the larval stage . ( N ) SQST - 1 : : GFP aggregates disappear in rheb - 1 ( RNAi ) ; epg - 7 mutant larvae . ( O ) Dramatic accumulation of SQST - 1 : : GFP aggregates in hypodermal cells in atg - 3 mutant larvae . ( P ) SQST - 1 : : GFP aggregates persist in rheb - 1 ( RNAi ) ; atg - 3 mutant larvae .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O

(M) SQST-1::GFP accumulates in hypodermal cells in epg-7 mutants at the larval stage. (N) SQST-1::GFP aggregates disappear in rheb-1(RNAi);epg-7 mutant larvae. (O) Dramatic accumulation of SQST-1::GFP aggregates in hypodermal cells in atg-3 mutant larvae. (P) SQST-1::GFP aggregates persist in rheb-1(RNAi);atg-3 mutant larvae.


( Q - T ) At larval stages , W07G4 . 5 : : GFP forms aggregates in intestinal cells ( Q and R ) . These aggregates disappear upon RNAi knockdown of let - 363 ( S and T ) . ( U - X ) W07G4 . 5 : : GFP aggregates accumulate in intestinal cells in epg - 7 mutant larvae ( U and V ) . These aggregates disappear when let - 363 is simultaneously depleted ( W and X ) . ( Y and Z ) In atg - 3 mutants , W07G4 . 5 : : GFP aggregates accumulate in intestinal cells ( Y ) and persist after RNAi knockdown of let - 363 ( Z ) .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

(Q-T) At larval stages, W07G4.5::GFP forms aggregates in intestinal cells (Q and R). These aggregates disappear upon RNAi knockdown of let-363 (S and T). (U-X) W07G4.5::GFP aggregates accumulate in intestinal cells in epg-7 mutant larvae (U and V). These aggregates disappear when let-363 is simultaneously depleted (W and X). (Y and Z) In atg-3 mutants, W07G4.5::GFP aggregates accumulate in intestinal cells (Y) and persist after RNAi knockdown of let-363 (Z).


( a ) Plasma ornithine concentrations in Oatrhg mice following intravenous injections of AAV - OAT at the doses of 1x1013 ( n = 9 ) and 3x1013 genome copies ( gc ) / Kg ( n = 6 ) , or AAV - GFP at the dose of 1x1013 gc / Kg ( n = 9 ) . Normal range was established on wild - type mice ( n = 7 ) and average ± 2SD was calculated . Data are shown as averages ± SEM . * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Two - way ANOVA test ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Plasma ornithine concentrations in Oatrhg mice following intravenous injections of AAV-OAT at the doses of 1x1013 (n=9) and 3x1013 genome copies (gc)/Kg (n=6), or AAV-GFP at the dose of 1x1013 gc/Kg (n=9). Normal range was established on wild-type mice (n=7) and average ± 2SD was calculated. Data are shown as averages ± SEM. ***p<0.001, ****p<0.0001 (Two-way ANOVA test).


( c ) Plasma ornithine and lysine concentrations at 12 months of age ( 10 - months post - injection ) in Oatrhg mice injected with AAV - OAT ( n = 3 ) or AAV - GFP ( n = 3 ) ; WT mouse controls ( n = 5 ) are also shown ; each sample was analyzed as triplicates , data are shown as averages ± SEM . One - way ANOVA test with Tukey correction ; * p < 0 . 05 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Plasma ornithine and lysine concentrations at 12 months of age (10-months post-injection) in Oatrhg mice injected with AAV-OAT (n=3) or AAV-GFP (n=3); WT mouse controls (n=5) are also shown; each sample was analyzed as triplicates, data are shown as averages ± SEM. One-way ANOVA test with Tukey correction; *p <0.05; ****p<0.0001.


( e ) Western blot for OAT and GFP in livers of Oatrhg mice injected with AAV - GFP or AAV - OAT . A WT mouse liver was included as control . Vinculin was used as loading control .

O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(e) Western blot for OAT and GFP in livers of Oatrhg mice injected with AAV-GFP or AAV-OAT. A WT mouse liver was included as control. Vinculin was used as loading control.


( g ) Liver immunohistochemistry showing approximately 10 % of OAT - positive cells in Oatrhg mice injected with AAV - OAT . Livers of WT and Oatrhg mice injected with AAV - GFP are shown as controls . 5x ( images on the left side ) and 20x ( images on the right side ) fields of view are shown .

O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(g) Liver immunohistochemistry showing approximately 10% of OAT-positive cells in Oatrhg mice injected with AAV-OAT. Livers of WT and Oatrhg mice injected with AAV-GFP are shown as controls. 5x (images on the left side) and 20x (images on the right side) fields of view are shown.


( h ) Representative H & E staining of the retinas of 11 - month - old Oatrhg mice injected with AAV - GFP or AVV - OAT at the doses of 1x1013 or 3x1013 gc / Kg ; age - matched WT mice and uninjected Oatrhg mice were included as controls ( 40X ) . Abbreviations : OS , outer segment ; IS , inner segment ; RPE , retinal pigment epithelium ; ONL , outer nuclear layer ; INL , inner nuclear layer .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Representative H&E staining of the retinas of 11-month-old Oatrhg mice injected with AAV-GFP or AVV-OAT at the doses of 1x1013 or 3x1013 gc/Kg; age-matched WT mice and uninjected Oatrhg mice were included as controls (40X). Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.


( a ) Representative electron micrographs of the mouse retina of an AAV - GFP injected Oatrhg mouse showing retinal pigmented epithelium ( RPE ) and adjacent abnormal outer segment ( OS ) disk membranes ( black arrows ) . Phagolysosomes containing undigested OS disc membranes are present ( asterisks ) . ( b ) Retina of AAV - OAT injected Oatrhg mice showing photoreceptor disk membranes ( white arrows ) and RPE ultrastructure comparable to age matched WT mice ( c , white arrows ) . ( c ) Control retina of WT mouse is shown as comparison .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Representative electron micrographs of the mouse retina of an AAV-GFP injected Oatrhg mouse showing retinal pigmented epithelium (RPE) and adjacent abnormal outer segment (OS) disk membranes (black arrows). Phagolysosomes containing undigested OS disc membranes are present (asterisks). (b) Retina of AAV-OAT injected Oatrhg mice showing photoreceptor disk membranes (white arrows) and RPE ultrastructure comparable to age matched WT mice (c, white arrows). (c) Control retina of WT mouse is shown as comparison.


( a ) Plasma ornithine concentrations in AAV - GFP - ( n = 3 ) and AAV - OAT - injected ( n = 3 ) OatΔ mice . Normal range of wild - type mice is also shown . Each value plotted represents mean ± SEM ; Two - way ANOVA test , * p < 0 . 05 , * * p < 0 . 01 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Plasma ornithine concentrations in AAV-GFP- (n=3) and AAV-OAT-injected (n=3) OatΔ mice. Normal range of wild-type mice is also shown. Each value plotted represents mean ± SEM; Two-way ANOVA test, *p <0.05, **p<0.01.


( c ) Retinal H & E ( 40X ) of OatΔ mice injected with either AAV - GFP or AAV - OAT . Representative of three mice per groups are shown ; WT mice were included as control . Abbreviations : OS , outer segment ; IS , inner segment ; RPE , retinal pigment epithelium ; ONL , outer nuclear layer ; INL , inner nuclear layer .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Retinal H&E (40X) of OatΔ mice injected with either AAV-GFP or AAV-OAT. Representative of three mice per groups are shown; WT mice were included as control. Abbreviations: OS, outer segment; IS, inner segment; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer.


A - D Progression of microglial infiltration in the ONL . Early in the course of rod degeneration at P18 , few isolated apoptotic TUNEL - positive nuclei ( red , inset ) were present in the ONL , with Iba1 + microglialsomata and processes ( green ) confined to the inner retina ( A ) . At P21 , microglia in the outer plexiform layer ( OPL ) extended radial processes into the ONL ( inset ) , with a concurrent increase in the number of TUNEL + nuclei ( B ) . At P30 , retinalmicroglia infiltrated the entire depth of the markedly thinned ONL and the subretinal space ( SRS ) ( inset ) ( C ) . At P46 , microglia became less dense in the ONL and reverted to a more ramified morphology ( D ) . Scale bar , 50 μm . E - G Quantitative analyses demonstrate that ONL atrophy progression , characterized by ONL thickness ( empty symbols ) and number of rows of ONLnuclei ( filled symbols ) ( E ) , occurred concurrently with the progression of ONL apoptosis , followed as the density of TUNEL + nuclei in the ONL ( F ) , and microglia infiltration , followed as microglial density in the ONL ( G ) ( n = 3 animals at each time point ) . Data points and error bars indicate mean ± SEM . H - K Higher magnification images showing microglial process infiltration ( H ) , phagosome formation in infiltrating processes ( I ) , and microglial transformation into amoeboid morphologies containing multiple phagosomes ( J , K ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-D Progression of microglial infiltration in the ONL. Early in the course of rod degeneration at P18, few isolated apoptotic TUNEL-positive nuclei (red, inset) were present in the ONL, with Iba1+microglialsomata and processes (green) confined to the inner retina (A). At P21, microglia in the outer plexiform layer (OPL) extended radial processes into the ONL (inset), with a concurrent increase in the number of TUNEL+nuclei (B). At P30, retinalmicroglia infiltrated the entire depth of the markedly thinned ONL and the subretinal space (SRS) (inset) (C). At P46, microglia became less dense in the ONL and reverted to a more ramified morphology (D). Scale bar, 50 μm.E-G Quantitative analyses demonstrate that ONL atrophy progression, characterized by ONL thickness (empty symbols) and number of rows of ONLnuclei (filled symbols) (E), occurred concurrently with the progression of ONL apoptosis, followed as the density of TUNEL+nuclei in the ONL (F), and microglia infiltration, followed as microglial density in the ONL (G) (n = 3 animals at each time point). Data points and error bars indicate mean ± SEM.H-K Higher magnification images showing microglial process infiltration (H), phagosome formation in infiltrating processes (I), and microglial transformation into amoeboid morphologies containing multiple phagosomes (J,K).


A - C Microglia infiltrating the ONL demonstrate upregulation of the phagocytic molecule , CD68 . Top images in ( A ) show composites of CD68 ( red ) , Iba1 ( green ) , and DAPI ( blue ) staining ; bottom images show the same field with CD68 staining only . At P18 , non - infiltrating microglia expressed low or undetectable levels of CD68 , a lysosome - associated membrane protein . At P21 - 23 , microglia infiltrating the ONL strongly upregulated CD68 ( arrowheads ) . At P30 , CD68 immunopositivity in ONL microglia decreased and was mostly confined to amoeboid cells in the subretinal space . ( B ) Magnified view of inset in ( A ) demonstrates localization of CD68 to microglia phagosomes . ( C ) CD68 expression ( area of immunopositivity as a fraction of the ONL ) demonstrated a peaked at P22 and decreased by P30 . Scale bar , 20 μm .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C Microglia infiltrating the ONL demonstrate upregulation of the phagocytic molecule, CD68. Top images in (A) show composites of CD68 (red), Iba1 (green), and DAPI (blue) staining; bottom images show the same field with CD68 staining only. At P18, non-infiltrating microglia expressed low or undetectable levels of CD68, a lysosome-associated membrane protein. At P21-23, microglia infiltrating the ONL strongly upregulated CD68 (arrowheads). At P30, CD68 immunopositivity in ONL microglia decreased and was mostly confined to amoeboid cells in the subretinal space. (B) Magnified view of inset in (A) demonstrates localization of CD68 to microglia phagosomes. (C) CD68 expression (area of immunopositivity as a fraction of the ONL) demonstrated a peaked at P22 and decreased by P30. Scale bar, 20 μm.


D - F Upregulation of MFG - E8 , a secreted glycoprotein that serves as a bridging molecule for phagocytosis receptors , in the ONL . ( D ) MFG - E8 was low or absent in the ONL at P18 , but emerged at P21 - 23 , localizing to photoreceptor cell bodies in a column - like pattern ( arrow ) and as a punctate signal within infiltrating microglia ( arrowhead ) , before decreasing throughout the ONL at P30 . ( E ) A magnified orthogonal view of the inset from ( D ) demonstrating punctate MFG - E8 immunopositivity within ONL microglia . ( F ) Quantification of MFG - E8 expression demonstrated a prominent emergence at P22 and a subsequent decrement by P30 . Scale bar , 20 μm .

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D-F Upregulation of MFG-E8, a secreted glycoprotein that serves as a bridging molecule for phagocytosis receptors, in the ONL. (D) MFG-E8 was low or absent in the ONL at P18, but emerged at P21-23, localizing to photoreceptor cell bodies in a column-like pattern (arrow) and as a punctate signal within infiltrating microglia (arrowhead), before decreasing throughout the ONL at P30. (E) A magnified orthogonal view of the inset from (D) demonstrating punctate MFG-E8 immunopositivity within ONL microglia. (F) Quantification of MFG-E8 expression demonstrated a prominent emergence at P22 and a subsequent decrement by P30. Scale bar, 20 μm.


G - K Increase in phosphatidylserine ( PS ) exposure in the ONL during photoreceptor degeneration . ( G ) At P18 , PS immunopositivity is near absent in the ONL , but increased significantly in ONLsomata at P21 - 23 , before decreasing at P30 . ( H ) Quantitation of PS exposure by image analysis ( by fractional area of PS immunopositivity within the ONL ( top ) , and the mean intensity of PS staining in the ONL ( bottom ) ) demonstrated a transient increase at P22 . ( I ) Co - immunolabeling of rods with rhodopsin at P22 demonstrates that PS exposure was present in a majority of rods ( inset shows at high magnification the co - labeling of PS and rhodopsin in multiple rodsomata ) . ( J ) Conversely , immunolabeling of cones with conearrestin demonstrates the sparse co - localization of PS in cones ( inset shows close juxtaposition but no colocalization of PS and arrestin labeling ) . ( K ) Scoring of rhodopsin + rods and arrestin + cones for PS co - labeling demonstrates that a large majority of rods , but only a small minority of cones , showed PS exposure ( two - sided unpaired t - test , n = 3 animals at P22 ) . Scale bars = 20 μm .

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G-K Increase in phosphatidylserine (PS) exposure in the ONL during photoreceptor degeneration. (G) At P18, PS immunopositivity is near absent in the ONL, but increased significantly in ONLsomata at P21-23, before decreasing at P30. (H) Quantitation of PS exposure by image analysis (by fractional area of PS immunopositivity within the ONL (top), and the mean intensity of PS staining in the ONL (bottom)) demonstrated a transient increase at P22. (I) Co-immunolabeling of rods with rhodopsin at P22 demonstrates that PS exposure was present in a majority of rods (inset shows at high magnification the co-labeling of PS and rhodopsin in multiple rodsomata). (J) Conversely, immunolabeling of cones with conearrestin demonstrates the sparse co-localization of PS in cones (inset shows close juxtaposition but no colocalization of PS and arrestin labeling). (K) Scoring of rhodopsin+rods and arrestin+cones for PS co-labeling demonstrates that a large majority of rods, but only a small minority of cones, showed PS exposure (two-sided unpaired t-test, n = 3 animals at P22). Scale bars = 20 μm.


A - C Phagocytosis of rods by infiltrating microglia . ( A ) Representative example of a Iba1 + microglial process extending into the ONL with a phagosome at its terminal end . Each phagosome contained a photoreceptornucleus ( labeled with DAPI , arrow ) that was identified as a rodphotoreceptor by rhodopsin immunopositivity ( superposition of Iba1 + phagosome with rhodopsin + soma in orthogonal views ) . ( B ) Example of an amoeboid microglia in the ONL with multiple phagosomes containing both rhodopsin - positive ( arrowhead ) and rhodopsin - negative ( arrow ) nuclei . ( C ) Rhodopsin + nuclei can be localized within CD68 - positive phagosomes in infiltrating microglia , indicating phagocytic engulfment of rods . Scale bar , 10 μm . D , E The peak of microglial phagocytic activity occurred around P21 and declined subsequently , as measured by the number of rods phagocytosed ( D ) and the number of photoreceptors phagocytosed per microglial cell ( E ) ( n = 3 animals at each time point ) .

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A-C Phagocytosis of rods by infiltrating microglia. (A) Representative example of a Iba1+microglial process extending into the ONL with a phagosome at its terminal end. Each phagosome contained a photoreceptornucleus (labeled with DAPI, arrow) that was identified as a rodphotoreceptor by rhodopsin immunopositivity (superposition of Iba1+phagosome with rhodopsin+soma in orthogonal views). (B) Example of an amoeboid microglia in the ONL with multiple phagosomes containing both rhodopsin-positive (arrowhead) and rhodopsin-negative (arrow) nuclei. (C) Rhodopsin+nuclei can be localized within CD68-positive phagosomes in infiltrating microglia, indicating phagocytic engulfment of rods. Scale bar, 10 μm.D, E The peak of microglial phagocytic activity occurred around P21 and declined subsequently, as measured by the number of rods phagocytosed (D) and the number of photoreceptors phagocytosed per microglial cell (E) (n = 3 animals at each time point).


F - J Infiltrating microglia phagocytose TUNEL - negative rods in the ONL . ( F ) Although the peaks of TUNEL positivity and microglial phagocytosis occurred concurrently , the majority of rodphotoreceptors undergoing microglial phagocytosis were non - apoptotic ( arrows ) , while many apoptotic photoreceptors were not phagocytosed by microglia ( arrowheads ) ; representative example of a P21 retina demonstrates non - overlapping patterns of TUNEL positivity ( red ) and microglial phagocytosis ( Iba1 , green ) . ( G ) Confocal analysis of a representative amoeboid microglial cell demonstrates that while the photoreceptornuclei in phagosomes were rhodopsin - positive ( white ) , they were mostly TUNEL - negative . Only a small fraction of microglialphagosomes contained TUNEL + , rhodopsin + rods ( example shown in H ) . Scale bars , 20 μm . ( I ) Quantitative analysis of photoreceptornuclei within microglialphagosomes according to rhodopsin ( black symbols ) , and TUNEL labeling ( red symbols ) at different times during rod degeneration . ( J ) Quantitative analysis of all photoreceptors undergoing either apoptosis or phagocytosis at P21 - 23 demonstrates that TUNEL + apoptotic photoreceptors and phagocytosed photoreceptors consist of two distinct populations , indicating separate but parallel mechanisms of rod degeneration . Quantitative analyses involved retinal sections from n = 3 animals at each age .

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F-J Infiltrating microglia phagocytose TUNEL-negative rods in the ONL. (F) Although the peaks of TUNEL positivity and microglial phagocytosis occurred concurrently, the majority of rodphotoreceptors undergoing microglial phagocytosis were non-apoptotic (arrows), while many apoptotic photoreceptors were not phagocytosed by microglia (arrowheads); representative example of a P21 retina demonstrates non-overlapping patterns of TUNEL positivity (red) and microglial phagocytosis (Iba1, green). (G) Confocal analysis of a representative amoeboid microglial cell demonstrates that while the photoreceptornuclei in phagosomes were rhodopsin-positive (white), they were mostly TUNEL-negative. Only a small fraction of microglialphagosomes contained TUNEL+, rhodopsin+rods (example shown in H). Scale bars, 20 μm. (I) Quantitative analysis of photoreceptornuclei within microglialphagosomes according to rhodopsin (black symbols), and TUNEL labeling (red symbols) at different times during rod degeneration. (J) Quantitative analysis of all photoreceptors undergoing either apoptosis or phagocytosis at P21-23 demonstrates that TUNEL+ apoptotic photoreceptors and phagocytosed photoreceptors consist of two distinct populations, indicating separate but parallel mechanisms of rod degeneration. Quantitative analyses involved retinal sections from n = 3 animals at each age.


A - C Evidence of microglial phagocytosis of photoreceptors in other mouse models of RP . Histological analysis of a rd1mouseretina ( A ) demonstrates microglial phagocytosis of photoreceptornuclei in vibratome sections ( left ) and in flat - mounted retina ( right ) . Phagocytosed nuclei were predominantly negative for TUNEL staining ( arrows ) . Similar findings were found in the retinas of the rd16mouse ( loss - of - function mutation in the photoreceptor - expressed CEP290 gene , 1 month old ) ( B ) and the RPGRIP - deficient mouse ( 6 months old ) ( C ) . Scale bar , 20 μm .

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A-C Evidence of microglial phagocytosis of photoreceptors in other mouse models of RP. Histological analysis of a rd1mouseretina (A) demonstrates microglial phagocytosis of photoreceptornuclei in vibratome sections (left) and in flat-mounted retina (right). Phagocytosed nuclei were predominantly negative for TUNEL staining (arrows). Similar findings were found in the retinas of the rd16mouse (loss-of-function mutation in the photoreceptor-expressed CEP290 gene, 1 month old) (B) and the RPGRIP-deficient mouse (6 months old) (C). Scale bar, 20 μm.


D - G Evidence of microglial phagocytosis of photoreceptors in other human histopathological specimens of RP . ( D ) Retinal section from a 30 - year - old male donor with autosomal recessive RP ( AR RP ) showing extensive microglial infiltration of the ONL ; expanded inset ( right ) shows multiple photoreceptor nuclei in phagosomes that were predominantly negative for TUNEL staining . ( E , F ) Retinal sections from two separate donors with autosomal dominant RP ( AD RP1 = 68 - year - old man , T17M rhodopsin mutation , AD RP1 = 50 - year - old woman , Q - 64 - ter rhodopsin mutation ) showing similar evidence of microglial phagocytosis . ( G ) Retinal section from a 46 - year - old male donor with X - linked RP ( XL RP ) . Arrowheads indicate phagocytosed photoreceptor nuclei . Scale bars , 20 μm .

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D-G Evidence of microglial phagocytosis of photoreceptors in other human histopathological specimens of RP. (D) Retinal section from a 30-year-old male donor with autosomal recessive RP (AR RP) showing extensive microglial infiltration of the ONL; expanded inset (right) shows multiple photoreceptor nuclei in phagosomes that were predominantly negative for TUNEL staining. (E, F) Retinal sections from two separate donors with autosomal dominant RP (AD RP1 = 68-year-old man, T17M rhodopsin mutation, AD RP1 = 50-year-old woman, Q-64-ter rhodopsin mutation) showing similar evidence of microglial phagocytosis. (G) Retinal section from a 46-year-old male donor with X-linked RP (XL RP). Arrowheads indicate phagocytosed photoreceptor nuclei. Scale bars, 20 μm.


Time - lapse confocal imaging ( at a rate of one image stack / min ) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21 - 24 . Infiltrating microglia established rapid and transient physical contacts with nearby photoreceptorsomata ( red and yellow circles ) via their processes ; these processes often terminated in a " cup " - like structure ( arrow ) that upon soma contact proceeded to extend across the entire soma ( arrowhead , and red circle and * ) to engulf it completely . The majority of microglia - photoreceptor contacts were transient with the microglial processes dynamically contacting and releasing photoreceptorsomata in repetitive cycles ( e . g . yellow circle and * ) ( period ≅ 10 - 15 min ) .

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Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21-24.Infiltrating microglia established rapid and transient physical contacts with nearby photoreceptorsomata (red and yellow circles) via their processes; these processes often terminated in a "cup"-like structure (arrow) that upon soma contact proceeded to extend across the entire soma (arrowhead, and red circle and *) to engulf it completely. The majority of microglia-photoreceptor contacts were transient with the microglial processes dynamically contacting and releasing photoreceptorsomata in repetitive cycles (e.g. yellow circle and *) (period ≅10-15 min).


Time - lapse confocal imaging ( at a rate of one image stack / min ) was used to document dynamic behavior of ONL microglia in rd10 retinal explants at ages P21 - 24 . Example in which the engulfment of photoreceptorsomata by microglial phagocytic cup was followed up by actual phagocytosis in which the engulfed cell ( red * ) is translocated intracellularly within the microglial cell toward the cell body . Phagocytosis of soma occurred simultaneously with " probing " of other somata by processes of the same microglial cell ( yellow * ) .

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Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONL microglia in rd10 retinal explants at ages P21-24. Example in which the engulfment of photoreceptorsomata by microglial phagocytic cup was followed up by actual phagocytosis in which the engulfed cell (red *) is translocated intracellularly within the microglial cell toward the cell body. Phagocytosis of soma occurred simultaneously with "probing" of other somata by processes of the same microglial cell (yellow *).


Time - lapse confocal imaging ( at a rate of one image stack / min ) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21 - 24 . Phagocytosis of photoreceptorsomata occurred via flattened microgliallamellipodialmicroglial processes ( arrow ) that extended across somata ( yellow * ) to engulf them . Nuclei of phagocytosed photoreceptors within microglialphagosomes occasionally developed staining for propidium iodide ( red arrow ) ; these subsequently faded and disappeared over ≅ 10 min , possibly representing intraphagosomal breakdown .

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Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21-24. Phagocytosis of photoreceptorsomata occurred via flattened microgliallamellipodialmicroglial processes (arrow) that extended across somata (yellow *) to engulf them. Nuclei of phagocytosed photoreceptors within microglialphagosomes occasionally developed staining for propidium iodide (red arrow); these subsequently faded and disappeared over ≅10 min, possibly representing intraphagosomal breakdown.


Time - lapse confocal imaging ( at a rate of one image stack / min ) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21 - 24 . Amoeboid infiltrating microglia lacking extended processes were also observed to phagocytose photoreceptors via phagocytic cups formed at their cell body ( red * ) .

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Time-lapse confocal imaging (at a rate of one image stack/min) was used to document dynamic behavior of ONLmicroglia in rd10retinal explants at ages P21-24. Amoeboid infiltrating microglia lacking extended processes were also observed to phagocytose photoreceptors via phagocytic cups formed at their cell body (red *).


Retinalmicroglia were depleted in rd10 / CreDTAmice by the oral administration of tamoxifen ( in corn oil ) to activate microglia - specific Cre - mediated recombination and diphtheria toxin expression ; control CreDTA littermates were administered corn oil without tamoxifen . A - D Depletion of retinalmicroglia in the rd10 retina . Representative retinal section from a P28 control animal demonstrates Iba1 + microglia in the retina , including those infiltrating the ONL ( A ) , while a tamoxifen - administered littermate ( B ) was substantially depleted of retinalmicroglia . Scale bar , 40 μm . Microglial cell counts in the entire retina ( C ) and in the ONL only ( D ) confirmed efficient depletion of infiltrating microglia following tamoxifen administration ( n = 8 control and 11 depleted animals from four litters , two - sided unpaired t - test ) . E - J Effect of microglial depletion on retinal degeneration at P28 - 29 . ONL atrophy and thinning in control animals ( E ) was significantly more advanced relative to microglia - depleted littermates ( F ) . Scale bar , 40 μm . Quantification of mean ONL thickness ( G ) and mean number of layers of ONLnuclei ( H ) at P28 - 29 demonstrate significantly greater ONL preservation in depleted retinas ; the degree of ONL preservation correlated with the extent of microglia depletion ( I ) . ( J ) The mean density of TUNEL + nuclei in the ONL was not significantly decreased in depleted vs . control animals ( n = 8 control and 11 depleted animals from four litters , two - sided unpaired t - test ) . K - N Continuation of microglial depletion until P37 - 39 resulted in the persistence of morphological rescue ( K - M ) , with a significant reduction in TUNEL + nuclei density ( N ) ( n = 6 control and nine depleted animals from two litters , two - sided unpaired t - test ) . O - R Similar rescue effects as in ( K - N ) remained apparent when depletion was sustained until P50 ( O - R ) , a time when rod degeneration in the rd10 model is relatively complete ( n = 9 control and 10 depleted animals from three litters , two - sided unpaired t - test ) . S , T Functional rescue of photoreceptors was evident following microglial depletion until P50 in significantly increased dark - ( S ) and light - adapted ( T ) responses in ERG testing in depleted animals ( green lines ) relative to control animals ( black lines ) , in both a - and b - wave amplitudes across multiple flash intensities ( n = 9 control and nine depleted animals , * P < 0 . 05 in one - way ANOVA with Sidak ' s multiple comparison test ) .

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Retinalmicroglia were depleted in rd10/CreDTAmice by the oral administration of tamoxifen (in corn oil) to activate microglia-specific Cre-mediated recombination and diphtheria toxin expression; control CreDTA littermates were administered corn oil without tamoxifen.A-D Depletion of retinalmicroglia in the rd10 retina. Representative retinal section from a P28 control animal demonstrates Iba1+microglia in the retina, including those infiltrating the ONL (A), while a tamoxifen-administered littermate (B) was substantially depleted of retinalmicroglia. Scale bar, 40 μm. Microglial cell counts in the entire retina (C) and in the ONL only (D) confirmed efficient depletion of infiltrating microglia following tamoxifen administration (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).E-J Effect of microglial depletion on retinal degeneration at P28-29. ONL atrophy and thinning in control animals (E) was significantly more advanced relative to microglia-depleted littermates (F). Scale bar, 40 μm. Quantification of mean ONL thickness (G) and mean number of layers of ONLnuclei (H) at P28-29 demonstrate significantly greater ONL preservation in depleted retinas; the degree of ONL preservation correlated with the extent of microglia depletion (I). (J) The mean density of TUNEL+nuclei in the ONL was not significantly decreased in depleted vs. control animals (n = 8 control and 11 depleted animals from four litters, two-sided unpaired t-test).K-N Continuation of microglial depletion until P37-39 resulted in the persistence of morphological rescue (K-M), with a significant reduction in TUNEL+nuclei density (N) (n = 6 control and nine depleted animals from two litters, two-sided unpaired t-test).O-R Similar rescue effects as in (K-N) remained apparent when depletion was sustained until P50 (O-R), a time when rod degeneration in the rd10 model is relatively complete (n = 9 control and 10 depleted animals from three litters, two-sided unpaired t-test).S, T Functional rescue of photoreceptors was evident following microglial depletion until P50 in significantly increased dark- (S) and light-adapted (T) responses in ERG testing in depleted animals (green lines) relative to control animals (black lines), in both a- and b-wave amplitudes across multiple flash intensities (n = 9 control and nine depleted animals, *P < 0.05 in one-way ANOVA with Sidak's multiple comparison test).


A , B Effect of phagocytosis inhibition on retinal microglia ex vivo . Retinal explants acutely isolated from P21 - 23 rd10 / CX3CR1GFP / + mice were incubated in Ringer ' s solution ( control ) , or Ringer ' s solution containing either the vitronectin receptor inhibitor cRGD peptide or its inactive analog cRAD ( 400 μM ) for 1 h . GFP - labeled ( green ) ONL microglia in explants in cRGD transitioned from amoeboid morphologies containing multiple phagosomes to more ramified morphologies with fewer phagosomes . No morphological changes were detected in microglia incubated in cRAD ( insets show morphologies at higher magnification ) . Scale bar , 40 μm . Quantifications of total number of phagosomes per 40 × field ( left ) and the mean number of phagosomes per microglia ( right ) demonstrate significant reductions in phagocytic activity of cRGD , relative to cRAD , exposure ( n = 8 imaging fields from in each condition ) .

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A, B Effect of phagocytosis inhibition on retinal microglia ex vivo. Retinal explants acutely isolated from P21-23 rd10/CX3CR1GFP/+ mice were incubated in Ringer's solution (control), or Ringer's solution containing either the vitronectin receptor inhibitor cRGD peptide or its inactive analog cRAD (400 μM) for 1 h. GFP-labeled (green) ONL microglia in explants in cRGD transitioned from amoeboid morphologies containing multiple phagosomes to more ramified morphologies with fewer phagosomes. No morphological changes were detected in microglia incubated in cRAD (insets show morphologies at higher magnification). Scale bar, 40 μm. Quantifications of total number of phagosomes per 40× field (left) and the mean number of phagosomes per microglia (right) demonstrate significant reductions in phagocytic activity of cRGD, relative to cRAD, exposure (n = 8 imaging fields from in each condition).


C - N Structural effects of in vivo inhibition of microglial phagocytosis . P20 somatamice were injected intravitreally in one eye with the phagocytosis inhibitor cRGD , and in the control contralateral eye with the inactive analogue , cRAD . At P23 , prominent microglia infiltration in the ONL observed in control - injected eyes ( C ) was decreased in the contralateral cRGD - injected eyes ( D ) . ONLmicroglia in control eyes demonstrated more numerous phagosomes ( inset , arrows ) ( E ) compared with cRGD - injected eyes ( F ) . Pairwise comparisons of control - vs . cRGD - injected eyes demonstrated that phagocytosis inhibition significantly reduced the densities of infiltrating microglia ( G ) and microglialphagosomes ( H ) , and mean phagosome number per microglia ( I ) . ONL atrophy in control eyes ( J ) was more advanced compared to cRGD - injected eyes ( K ) , with significantly greater mean ONL thickness ( L ) , and ONLnuclear layers ( M ) in cRGD - injected eyes . Mean density of TUNEL + ONLnuclei was lower , with marginal significance ( N ) ( n = 13 animals ; measurements in cRGD - injected eyes normalized to contralateral control eyes , two - sided paired t - test ) . Scale bar , 40 μm .

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C-N Structural effects of in vivo inhibition of microglial phagocytosis. P20 somatamice were injected intravitreally in one eye with the phagocytosis inhibitor cRGD, and in the control contralateral eye with the inactive analogue, cRAD. At P23, prominent microglia infiltration in the ONL observed in control-injected eyes (C) was decreased in the contralateral cRGD-injected eyes (D). ONLmicroglia in control eyes demonstrated more numerous phagosomes (inset, arrows) (E) compared with cRGD-injected eyes (F). Pairwise comparisons of control- vs. cRGD-injected eyes demonstrated that phagocytosis inhibition significantly reduced the densities of infiltrating microglia (G) and microglialphagosomes (H), and mean phagosome number per microglia (I). ONL atrophy in control eyes (J) was more advanced compared to cRGD-injected eyes (K), with significantly greater mean ONL thickness (L), and ONLnuclear layers (M) in cRGD-injected eyes. Mean density of TUNEL+ONLnuclei was lower, with marginal significance (N) (n = 13 animals; measurements in cRGD-injected eyes normalized to contralateral control eyes, two-sided paired t-test). Scale bar, 40 μm.


O - T Functional effects of in vivo inhibition of microglial phagocytosis . Representative dark - adapted ERG recordings at P26 ( following intravitreal injections at P20 and P23 ) in a cRGD - injected eye ( red ) relative to the contralateral control eye ( black ) ( O ) showing that mean a - ( P ) and b - wave ( Q ) amplitudes were significantly greater in the cRGD - injected vs . control eyes at higher flash intensities . Representative light - adapted responses ( R ) illustrating that while mean light - adapted a - wave amplitudes ( S ) were slightly but not significantly greater in cRGD - injected eyes , mean b - wave amplitudes ( T ) were significantly greater at higher flash intensities ( n = 13 animals , two - sided paired t - test , * P < 0 . 05 , with exact values alongside ) .

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O-T Functional effects of in vivo inhibition of microglial phagocytosis. Representative dark-adapted ERG recordings at P26 (following intravitreal injections at P20 and P23) in a cRGD-injected eye (red) relative to the contralateral control eye (black) (O) showing that mean a- (P) and b-wave (Q) amplitudes were significantly greater in the cRGD-injected vs. control eyes at higher flash intensities. Representative light-adapted responses (R) illustrating that while mean light-adapted a-wave amplitudes (S) were slightly but not significantly greater in cRGD-injected eyes, mean b-wave amplitudes (T) were significantly greater at higher flash intensities (n = 13 animals, two-sided paired t-test, *P < 0.05, with exact values alongside).


Microglia infiltrating the rd10 retina upregulate IL - 1β expression during degeneration . Iba1 + microglia ( green ) were immunonegative for IL - 1β ( red ) at P18 but become prominently IL - 1β immunopositive at P22 ( arrows ) . Scale bars , 25 μm .

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Microglia infiltrating the rd10 retina upregulate IL-1β expression during degeneration. Iba1+ microglia (green) were immunonegative for IL-1β (red) at P18 but become prominently IL-1β immunopositive at P22 (arrows). Scale bars, 25 μm.


Depletion of infiltrating microglia in the rd10 retina decreases IL - 1β levels . Cytokine levels in rd10 / CreDTA mouse retinas were assayed following tamoxifen - induced microglial depletion ( from P21 to P37 - 50 , green bars ; n = 9 animals ) and compared with their untreated littermate controls ( white bars , n = 8 animals ; values normalized to control animals in the same litter ) . IL - 1β protein levels were significantly lowered following microglial depletion , but IL - 6 , CCL2 , or TNFα were not significantly changed .

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Depletion of infiltrating microglia in the rd10 retina decreases IL-1β levels. Cytokine levels in rd10/CreDTA mouse retinas were assayed following tamoxifen-induced microglial depletion (from P21 to P37-50, green bars; n = 9 animals) and compared with their untreated littermate controls (white bars, n = 8 animals; values normalized to control animals in the same litter). IL-1β protein levels were significantly lowered following microglial depletion, but IL-6, CCL2, or TNFα were not significantly changed.


Inhibition of microglial phagocytosis results in decreased microglial IL - 1β expression . Eyes of rd10 animals were treated with intravitreal injections of cRGD at P20 , and analyzed at P23 by immunohistochemistry for Iba1 ( green ) and IL - 1β ( red ) , and compared to uninjected eyes and with eyes injected with inactive analog , cRAD ( n = 3 eyes in each category ) . Immunopositivity was high in infiltrating microglia ( indicated by arrows ) for untreated and cRAD - injected control eyes and significantly reduced in microglia in cRGD - injected eyes ( upper panel ) , as demonstrated by the quantification of IL - 1β expression ( area of immunopositivity as a fraction of the ONL ) ( lower left panel ) . ONL thickness correlated negatively with the extent of IL - 1β immunopositivity ( lower right panel ) . Scale bar , 25 μm .

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Inhibition of microglial phagocytosis results in decreased microglial IL-1β expression. Eyes of rd10 animals were treated with intravitreal injections of cRGD at P20, and analyzed at P23 by immunohistochemistry for Iba1 (green) and IL-1β (red), and compared to uninjected eyes and with eyes injected with inactive analog, cRAD (n = 3 eyes in each category). Immunopositivity was high in infiltrating microglia (indicated by arrows) for untreated and cRAD-injected control eyes and significantly reduced in microglia in cRGD-injected eyes (upper panel), as demonstrated by the quantification of IL-1β expression (area of immunopositivity as a fraction of the ONL) (lower left panel). ONL thickness correlated negatively with the extent of IL-1β immunopositivity (lower right panel). Scale bar, 25 μm.


IL - 1R signaling during rod degeneration was inhibited by intravitreal injections of recombinant IL - 1 receptor antagonist , anakinra ( from P20 - 22 to P26 - 27 ) in one eye of each rd10 animal , while the contralateral eye was injected with PBS as a control . Upper panels show representative retinal sections from control PBS - injected and IL - 1RA - injected eyes from the same animal . ONL atrophy in control eyes was more advanced compared to IL - 1RA - injected eyes , with greater mean ONL thickness and lower mean density of TUNEL + ONL nuclei in IL - 1RA - injected eyes ( lower panels ) ( n = 15 animals ; measurements in IL - 1RA - injected eyes normalized to contralateral control eyes , paired t - test ) . Scale bar , 25 μm .

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IL-1R signaling during rod degeneration was inhibited by intravitreal injections of recombinant IL-1 receptor antagonist, anakinra (from P20-22 to P26-27) in one eye of each rd10 animal, while the contralateral eye was injected with PBS as a control. Upper panels show representative retinal sections from control PBS-injected and IL-1RA-injected eyes from the same animal. ONL atrophy in control eyes was more advanced compared to IL-1RA-injected eyes, with greater mean ONL thickness and lower mean density of TUNEL+ ONL nuclei in IL-1RA-injected eyes (lower panels) (n = 15 animals; measurements in IL-1RA-injected eyes normalized to contralateral control eyes, paired t-test). Scale bar, 25 μm.


( D ) Cells were treated with sodium arsenite ( 50 µM ) for 45 min , followed by recovery in drug - free medium . Kinetics of SG dissolution are reported . Images were taken over a time period of 4 hrs every 10 min . Dashed lines = 95 % confidence intervals . Number of cells counted : 159 ( siRNA control ) ; 166 ( siRNA Hsp90 α + β ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

(D) Cells were treated with sodium arsenite (50 µM) for 45 min, followed by recovery in drug-free medium. Kinetics of SG dissolution are reported. Images were taken over a time period of 4 hrs every 10 min. Dashed lines = 95% confidence intervals. Number of cells counted: 159 (siRNA control); 166 (siRNA Hsp90 α+β).


( E ) HeLa - Kyoto cells were treated with HS at 43 . 5 ˚ C for 1 hr . Cells were then allowed to recover at 37 ° C for 1 hr in drug - free medium ( control ) or in presence of VER ( 40 µM ) , GA ( 5 µM ) or 17AAG ( 5 µM ) . Cells were fixed , stained for the SG marker TIA - 1 and the percentage of cells with SGs was counted . Number of cells counted : 605 ( recovery control ) ; 510 ( recovery VER ) ; 1334 ( recovery GA ) ; 468 ( recovery 17AAG ) . n = 3 - 4 independent experiments , ± sem . p < 0 . 01 ( One - way ANOVA ) . ( F ) G3BP2 - GFP HeLa - Kyoto cells were treated with MG132 ( 20 µM ) for 3 hrs , followed by recovery for 2 hrs in drug - free medium ( control ) or in presence of VER ( 40 µM ) , GA ( 5 µM ) or 17AAG ( 5 µM ) . Cells were fixed and the percentage of cells with SGs was counted . Number of cells counted : 439 ( recovery control ) ; 649 ( recovery VER ) ; 637 ( recovery GA ) ; 649 ( recovery 17AAG ) . n = 3 independent experiments , ± sem . p < 0 . 0001 ( One - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) HeLa-Kyoto cells were treated with HS at 43.5˚C for 1 hr. Cells were then allowed to recover at 37 °C for 1 hr in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed, stained for the SG marker TIA-1 and the percentage of cells with SGs was counted. Number of cells counted: 605 (recovery control); 510 (recovery VER); 1334 (recovery GA); 468 (recovery 17AAG). n = 3-4 independent experiments, ± sem. p < 0.01 (One-way ANOVA). (F) G3BP2-GFP HeLa-Kyoto cells were treated with MG132 (20 µM) for 3 hrs, followed by recovery for 2 hrs in drug-free medium (control) or in presence of VER (40 µM), GA (5 µM) or 17AAG (5 µM). Cells were fixed and the percentage of cells with SGs was counted. Number of cells counted: 439 (recovery control); 649 (recovery VER); 637 (recovery GA); 649 (recovery 17AAG). n = 3 independent experiments, ± sem. p < 0.0001 (One-way ANOVA).


( C ) HeLa cells were subjected to proximity ligation assay ( PLA ) using antibodies specific for endogenous Hsp90 and DYRK3 . PLA foci and nuclei were segmented , and PLA foci / cell were automatically quantified . The mean of PLA / foci in cells incubated with no antibodies ( - ) , only Hsp90 antibody , only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown . Cells incubated with Hsp90 and DYRK3 antibodies and left untreated were used as control . n = 3 independent experiments , ± sem ; 368 - 504 cells counted / sample , p < 0 . 002 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies ; p = 10 - 6 between cells incubated with Hsp90 and DYRK3 antibodies and left untreated ( control ) or treated for 2 hrs with GA ( 5 µM ) ( One - way ANOVA ) . Scale bar is 10 µm .

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(C) HeLa cells were subjected to proximity ligation assay (PLA) using antibodies specific for endogenous Hsp90 and DYRK3. PLA foci and nuclei were segmented, and PLA foci/cell were automatically quantified. The mean of PLA/foci in cells incubated with no antibodies (-), only Hsp90 antibody, only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown. Cells incubated with Hsp90 and DYRK3 antibodies and left untreated were used as control. n = 3 independent experiments, ± sem; 368-504 cells counted/sample, p < 0.002 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies; p = 10-6 between cells incubated with Hsp90 and DYRK3 antibodies and left untreated (control) or treated for 2 hrs with GA (5 µM) (One-way ANOVA). Scale bar is 10 µm.


( F ) HeLa cells were lipofected with a cDNA encoding for GFP or GFP - DYRK3 . 6 hrs post - transfection , cells were incubated in drug - free medium ( - ) or in presence of GA , 17AAG or VER ( concentrations are shown ) . Total proteins were extracted 16 hrs later . GFP and TUBA4A protein levels were analyzed by immunoblotting . Representative immunoblotting of 3 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) HeLa cells were lipofected with a cDNA encoding for GFP or GFP-DYRK3. 6 hrs post-transfection, cells were incubated in drug-free medium (-) or in presence of GA, 17AAG or VER (concentrations are shown). Total proteins were extracted 16 hrs later. GFP and TUBA4A protein levels were analyzed by immunoblotting. Representative immunoblotting of 3 independent experiments.


( I ) HeLa cells were lipofected with a cDNA encoding for GFP - DYRK3 . 24 hrs post - transfection , cells were treated for 45 min with arsenite ( 50 µM ) alone ( control ) or with GA ( 5 µM ) or 17AAG ( 5 µM ) . Cell were then fixed and the percentage of cells expressing GFP - DYRK3 with SGs was counted . Total cells counted : 462 ( sodium arsenite ) ; 444 ( sodium arsenite and GA ) ; 561 ( sodium arsenite and 17AAG ) . n = 3 independent experiments , ± sem ; p < 0 . 001 ( One - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3. 24 hrs post-transfection, cells were treated for 45 min with arsenite (50 µM) alone (control) or with GA (5 µM) or 17AAG (5 µM). Cell were then fixed and the percentage of cells expressing GFP-DYRK3 with SGs was counted. Total cells counted: 462 (sodium arsenite); 444 (sodium arsenite and GA); 561 (sodium arsenite and 17AAG). n = 3 independent experiments, ± sem; p < 0.001 (One-way ANOVA).


( A , HeLa cells were left untreated ( control ) or treated with GA ( 5 µM ) , 17AAG ( 5 µM ) or VER ( 10 µM ) for 8 hrs . Cells were then fixed and immunostained for the nuclear speckle marker SC35 and DYRK3 . Automatic segmentation of nuclear speckles is based on SC35 . An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 ( using a fluorescent ratio 1 . 5 as threshold ) . Scale bar is 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(A, HeLa cells were left untreated (control) or treated with GA (5 µM), 17AAG (5 µM) or VER (10 µM) for 8 hrs. Cells were then fixed and immunostained for the nuclear speckle marker SC35 and DYRK3. Automatic segmentation of nuclear speckles is based on SC35. An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 (using a fluorescent ratio 1.5 as threshold). Scale bar is 10 µm.


D ) Representative confocal microscopy images of cells with SC35 - positive bodies ( D ) Scale bar is 10 µm .

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D) Representative confocal microscopy images of cells with SC35-positive bodies (D) Scale bar is 10 µm.


( F ) HeLa cells were lipofected with a cDNA encoding for GFP - DYRK3 - dN . 24 hrs post - transfection GFP - DYRK3 - dN cells were either left untreated or exposed to GA ( 5 µM ) for 4 hrs . In untreated cells ( control ) , GFP - DYRK3 - dN is diffusely distributed in the cytosol and in the nucleus . Upon GA treatment , GFP - DYRK3 - dN forms perinuclear ( PN ) aggregates . Representative confocal images are shown . Scale bar is 10 µm .

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(F) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3-dN. 24 hrs post-transfection GFP-DYRK3-dN cells were either left untreated or exposed to GA (5 µM) for 4 hrs. In untreated cells (control), GFP-DYRK3-dN is diffusely distributed in the cytosol and in the nucleus. Upon GA treatment, GFP-DYRK3-dN forms perinuclear (PN) aggregates. Representative confocal images are shown. Scale bar is 10 µm.


( I ) mCherry - G3BP1 expressing HeLa cells were lipofected with cDNAs encoding for GFP - DYRK3 - dN or Sup35 - NM - GFP - DYRK3 - dN . 24 hrs post - transfection cells were treated for 45 min with sodium arsenite to induce SGs , followed by recovery for 4 hrs in presence of GA ( 5 µM ; rec . GA ) . Representative confocal microscopy images are shown . Nucleic acid was stained with DAPI . Scale bar is 10 µm .

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(I) mCherry-G3BP1 expressing HeLa cells were lipofected with cDNAs encoding for GFP-DYRK3-dN or Sup35-NM-GFP-DYRK3-dN. 24 hrs post-transfection cells were treated for 45 min with sodium arsenite to induce SGs, followed by recovery for 4 hrs in presence of GA (5 µM; rec. GA). Representative confocal microscopy images are shown. Nucleic acid was stained with DAPI. Scale bar is 10 µm.


( C ) Quantitation of the fluorescence intensity recovery after bleach of GFP - DYRK3 in G3BP1 - mCherry HeLa - Kyoto cells 24 h after transfection . Cells were treated with sodium arsenite ( 50 µM ) for 45 min to induce SGs , which were visualized with G3BP1 - mCherry ; then , sodium arsenite was removed and the cells were incubated in drug - free medium or in presence of GA ( 5 µM ) or of GSK ( 5 µM ) . FRAP was performed during the stress recovery period . A representative image of cells treated with arsenite followed by recovery in drug - free medium is shown . Arrowheads and dotted circle indicate the ROI . Scale bar is 5 µm . The mean of 13 FRAP curves ( recovery control ) , 12 FRAP curves ( recovery GA ) and 14 FRAP curves ( recovery GSK ) is shown in red ; the sem is shown in gray .

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(C) Quantitation of the fluorescence intensity recovery after bleach of GFP-DYRK3 in G3BP1-mCherry HeLa-Kyoto cells 24 h after transfection. Cells were treated with sodium arsenite (50 µM) for 45 min to induce SGs, which were visualized with G3BP1-mCherry; then, sodium arsenite was removed and the cells were incubated in drug-free medium or in presence of GA (5 µM) or of GSK (5 µM). FRAP was performed during the stress recovery period. A representative image of cells treated with arsenite followed by recovery in drug-free medium is shown. Arrowheads and dotted circle indicate the ROI. Scale bar is 5 µm. The mean of 13 FRAP curves (recovery control), 12 FRAP curves (recovery GA) and 14 FRAP curves (recovery GSK) is shown in red; the sem is shown in gray.


( B ) Confocal microscopy images of P525L FUS eGFP MNs treated with sodium arsenite for 2 hrs and showing colocalization of FUS with TIAR - positive stress granules . Scale bar is 10 µm . ( C ) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs , followed by recovery in drug - free medium ( + rec . control ) or in presence of 17AAG ( 30 μM ; + rec . 17AAG ) or of GSK ( 5 μM ; + rec . GSK ) for 6 hrs . Quantitation of the number of SGs per cell in P525L FUS eGFP MNs . Number of P525L cells counted and n / condition : 2488 , n = 13 ( arsenite ) ; 1045 , n = 12 ( arsenite + rec . control ) ; 646 , n = 6 ( arsenite + rec . 17AAG ) ; 924 , n = 3 ( arsenite + rec . GSK ) , ± sem ( One - way ANOVA ) .

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(B) Confocal microscopy images of P525L FUS eGFP MNs treated with sodium arsenite for 2 hrs and showing colocalization of FUS with TIAR-positive stress granules. Scale bar is 10 µm. (C) P525L FUS eGFP MNs were treated with sodium arsenite for 2 hrs, followed by recovery in drug-free medium (+ rec. control) or in presence of 17AAG (30 μM; + rec. 17AAG) or of GSK (5 μM; +rec. GSK) for 6 hrs. Quantitation of the number of SGs per cell in P525L FUS eGFP MNs. Number of P525L cells counted and n/condition: 2488, n = 13 (arsenite); 1045, n = 12 (arsenite + rec. control); 646, n = 6 (arsenite + rec. 17AAG); 924, n = 3 (arsenite + rec. GSK), ± sem (One-way ANOVA).


( I ) Double immunofluorescence labeling using antibodies against DYRK3 and FUS in lumbar spinal cord α - MNs of healthy subjects ( control ) and fALS patients carrying the p . 521C mutation in the FUS gene . Control ( upper panel ) : α - MNs showing uniform cytoplasmic ( white arrows ) and speckled pattern of strong nuclear immunoreactivity ( white arrowhead ) of DYRK3 , as well as diffuse nuclear FUS immunoreactivity . fALS ( p . R521C - FUS ; lower panel ) : surviving α - MNs harboring large FUS aggregates ( yellow arrows ) showed markedly reduced DYRK3 immunoreactivity both in the cytoplasm as well as in the nucleus ( red arrowheads ) . Instead , α - MNs devoid of FUS aggregates ( white arrows ) showed normal DYRK3 immunoreactivity similar to the one of α - MNs from normal controls . Asterix ( * ) represents non - specific lipofuscin granules in one of the α - MN in FUS - ALS . Paraffin sections ; scale bars is 50 µm . ( J ) Quantification of α - MNs showing low or absent DYRK3 staining ( upper graphic ) and FUS aggregates ( lower graphic ) in lumbar spinal cord from five healthy subjects ( control ) and five fALS patients carrying the p . 521C mutation in the FUS gene . Total number of α - MNs analyzed : 403 ( control ) ; 132 ( fALS ) . n = 5 , ± sem ( Student ' s t - test ) .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Double immunofluorescence labeling using antibodies against DYRK3 and FUS in lumbar spinal cord α-MNs of healthy subjects (control) and fALS patients carrying the p.521C mutation in the FUS gene. Control (upper panel): α-MNs showing uniform cytoplasmic (white arrows) and speckled pattern of strong nuclear immunoreactivity (white arrowhead) of DYRK3, as well as diffuse nuclear FUS immunoreactivity. fALS (p.R521C-FUS; lower panel): surviving α-MNs harboring large FUS aggregates (yellow arrows) showed markedly reduced DYRK3 immunoreactivity both in the cytoplasm as well as in the nucleus (red arrowheads). Instead, α-MNs devoid of FUS aggregates (white arrows) showed normal DYRK3 immunoreactivity similar to the one of α-MNs from normal controls. Asterix (*) represents non-specific lipofuscin granules in one of the α-MN in FUS-ALS. Paraffin sections; scale bars is 50 µm. (J) Quantification of α-MNs showing low or absent DYRK3 staining (upper graphic) and FUS aggregates (lower graphic) in lumbar spinal cord from five healthy subjects (control) and five fALS patients carrying the p.521C mutation in the FUS gene. Total number of α-MNs analyzed: 403 (control); 132 (fALS). n = 5, ± sem (Student's t-test).