Figure 8A . Reduced TLR3 - induced mTORC1 signaling in mTOR S2159A knock - in primary macrophages ( mTORA / A ) relative to wild type ( mTOR + / + ) : BMDMs were pre - treated with Torin 1 [ 100 nM ] ( 30 min ) and stimulated - / + poly ( I : C ) [ 30 μg / ml ] for 30 min . Whole cells lysate ( WCL ) was immunoblotted as indicated . The graph quantitates three independent experiments each with n = 1 ( n = 3 total ) . The level of P - S6K1 - T389 normalized to total protein in mTOR + / + macrophages stimulated with poly ( I : C ) was set at 100 % . * The confidence interval at 95 % ( 52 . 7 - 59 . 1 ) indicates statistical significance between mTOR + / + vs . mTORA / A BMDMs . B . Reduced TLR4 - induced mTORC1 signaling in mTORA / A primary macrophages : BMDMs were treated as in A except stimulated - / + LPS [ 100 ng / mL ] ( 30 min ) . The graph quantitates three independent experiments each with n = 1 ( n = 3 total ) . The level of P - S6K1 - T389 normalized to total protein in mTOR + / + macrophages stimulated with LPS was set at 100 % . * The confidence interval at 95 % ( 58 . 6 - 71 . 2 % ) indicates statistical significance between mTOR + / + vs . mTORA / A BMDMs . C . Reduced TLR3 - stimulated IFNβ protein production in mTORA / A primary macrophages : BMDMs were serum starved for 6 hr . and stimulated - / + poly ( I : C ) [ 30 μg / ml ] for 6 hr . The secretion of IFNβ was measured by ELISA . Results represent the mean + / - SEM from three independent experiments , n = 8 samples total . * p = . 0002 relative to mTOR + / + + Poly ( I : C ) by unpaired t - test ( equal variance ; two - tailed ) . * * p = . 0001 relative to mTOR + / + + Poly ( I : C ) by paired t - test ( two - tailed ) . D . Reduced TLR3 - stimulated IRF3 nuclear translocation in mTORA / A primary macrophages : BMDMs were treated - / + poly ( I : C ) for 3 hr as in A , fixed , and processed for confocal immunofluorescence microscopy using an anti - IRF3 - Alexa 488 antibody and Dapi stain . The graph represents the mean + / - SEM of at least 270 cells total from four independent experiments . * p = . 001 relative to mTOR + / + no Poly ( I : C ) by unpaired t - test ( equal variance ; two - tailed ) . * * p = . 02 relative to mTOR + / + + Poly ( I : C ) by unpaired t - test ( equal variance ; two - tailed ) . Scale bar = 10 μM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. Reduced TLR3-induced mTORC1 signaling in mTOR S2159A knock-in primary macrophages (mTORA/A) relative to wild type (mTOR+/+): BMDMs were pre-treated with Torin 1 [100 nM] (30 min) and stimulated -/+ poly (I:C) [30 μg/ml] for 30 min. Whole cells lysate (WCL) was immunoblotted as indicated. The graph quantitates three independent experiments each with n=1 (n=3 total). The level of P-S6K1-T389 normalized to total protein in mTOR+/+ macrophages stimulated with poly (I:C) was set at 100%. *The confidence interval at 95% (52.7-59.1) indicates statistical significance between mTOR+/+ vs. mTORA/A BMDMs.B. Reduced TLR4-induced mTORC1 signaling in mTORA/A primary macrophages: BMDMs were treated as in A except stimulated -/+ LPS [100 ng/mL] (30 min). The graph quantitates three independent experiments each with n=1 (n=3 total). The level of P-S6K1-T389 normalized to total protein in mTOR+/+ macrophages stimulated with LPS was set at 100%. *The confidence interval at 95% (58.6-71.2%) indicates statistical significance between mTOR+/+ vs. mTORA/A BMDMs.C. Reduced TLR3-stimulated IFNβ protein production in mTORA/A primary macrophages: BMDMs were serum starved for 6 hr. and stimulated -/+ poly (I:C) [30 μg/ml] for 6 hr. The secretion of IFNβ was measured by ELISA. Results represent the mean +/- SEM from three independent experiments, n=8 samples total. *p=.0002 relative to mTOR+/+ + Poly(I:C) by unpaired t-test (equal variance; two-tailed). **p=.0001 relative to mTOR+/+ + Poly(I:C) by paired t-test (two-tailed).D. Reduced TLR3-stimulated IRF3 nuclear translocation in mTORA/A primary macrophages: BMDMs were treated -/+ poly (I:C) for 3 hr as in A, fixed, and processed for confocal immunofluorescence microscopy using an anti-IRF3-Alexa 488 antibody and Dapi stain. The graph represents the mean +/- SEM of at least 270 cells total from four independent experiments. *p=.001 relative to mTOR+/+ no Poly(I:C) by unpaired t-test (equal variance; two-tailed). **p=.02 relative to mTOR+/+ + Poly(I:C) by unpaired t-test (equal variance; two-tailed). Scale bar = 10 μM.


Figure 1Representative dual - channel kymographs showing that Rheb is recruited to mitochondria in axons and co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential ( ∆ ψm ) dissipation . The motility of mitochondria ( Mito ) and Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to the right ( negative slope ) represent anterograde movement ; those to the left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) . Time - lapse images ( B ) showing colocalization and comigration ( white arrows ) of Rheb with mitochondria in a retrograde direction along axons during a 148 - s recording period . Cortical neurons were transfected with GFP - Rheb and DsRed - Mito at 5 - 7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10 - 12 DIV . quantitative analysis showing that Rheb is recruited to mitochondria in axons and co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential ( ∆ ψm ) dissipation . The motility of mitochondria ( Mito ) and Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to the right ( negative slope ) represent anterograde movement ; those to the left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) . Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . The percentages of mitochondria targeted by Rheb or Rheb - SSVM in the presence or absence of CCCP were quantified , respectivelyrepresentative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . Rheb - SSVM , a farnesylation deficient mutant of Rheb , fails to be recruited to mitochondria upon dissipating ∆ ψm . Mitochondria associated with Rheb were marked by white arrows . F - H Representative dual - channel kymographs ( F ) and quantitative analysis ( G , H ) showing that Rheb - mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation . Note that Rheb , but not Rheb - SSVM , is associated with autophagic vacuoles ( AVs ) within axons upon ∆ ψm dissipation . Rheb - associated AVs move in a predominant retrograde direction along axons . The number of Rheb - associated AVs ( Rheb - AVs ) with or without CCCP treatment and the relative motilities of AVs and Rheb - AVs in CCCP - treated axons were quantified ,

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative dual-channel kymographs showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).Time-lapse images (B) showing colocalization and comigration (white arrows) of Rheb with mitochondria in a retrograde direction along axons during a 148-s recording period. Cortical neurons were transfected with GFP-Rheb and DsRed-Mito at 5-7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10-12 DIV.quantitative analysis showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. The percentages of mitochondria targeted by Rheb or Rheb-SSVM in the presence or absence of CCCP were quantified, respectivelyrepresentative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. Rheb-SSVM, a farnesylation deficient mutant of Rheb, fails to be recruited to mitochondria upon dissipating ∆ψm. Mitochondria associated with Rheb were marked by white arrows.F-H Representative dual-channel kymographs (F) and quantitative analysis (G, H) showing that Rheb-mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation. Note that Rheb, but not Rheb-SSVM, is associated with autophagic vacuoles (AVs) within axons upon ∆ψm dissipation. Rheb-associated AVs move in a predominant retrograde direction along axons. The number of Rheb-associated AVs (Rheb-AVs) with or without CCCP treatment and the relative motilities of AVs and Rheb-AVs in CCCP-treated axons were quantified, respectively


Figure 2Representative dual - channel kymographs showing that Rheb enhances mitophagy in CCCP - treated axons . quantitative analysis showing that Rheb enhances mitophagy in CCCP - treated axons . The numbers of mitochondria ( Mito ) , AVs , and Mito - AVs / mitophagosomes , the percentage of Mito within AVs , were quantifiedquantitative analysis the percentage of Mito within AVs , and the motilities of AVs and Mito - AVs along axons with and without elevated Rheb expression were quantifiedD , E Representative images ( D ) and quantitative analysis ( E ) showing that mitophagy in axons requires Rheb and Nix , but not Parkin . The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP - treated axons expressing scrambled shRNA , Rheb shRNA , Nix shRNA , or Parkin shRNA were quantified , respectively ( E ) . F , G Quantitative analysis ( F ) and representative blots ( G ) showing increased mitochondrial association with Rheb and LC3 - II in mature cortical neurons upon ∆ ψm depolarization . Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post - nuclear supernatant ( P ) , mitochondria - enriched membrane fraction ( M ) , and cytosolic supernatant ( S ) . Equal amounts of protein ( 20 μg ) were sequentially immunoblotted with antibodies against mitophagic / autophagic proteins Rheb , LC3 , and Rab7 , mitochondrial protein VDAC , and cytosolic protein GAPDH on the same membranes after stripping between each antibody application . The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH , compared to post - nuclear supernatant . Protein levels in mitochondrial fractions of CCCP - treated neurons were normalized to those in neurons incubated with DMSO . Data were quantified from four independent repeats . Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ ψm depolarization . The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm , respectively . Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP - treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA ( H ) . representative images showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ ψm depolarization . The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm , respectively . Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light . The ratio has been false colored with the indicated heat map , with high intensity indicative of ROGFP fluorescence in a more oxidative

O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative dual-channel kymographs showing that Rheb enhances mitophagy in CCCP-treated axons.quantitative analysis showing that Rheb enhances mitophagy in CCCP-treated axons. The numbers of mitochondria (Mito), AVs, and Mito-AVs/mitophagosomes, the percentage of Mito within AVs, were quantifiedquantitative analysis the percentage of Mito within AVs, and the motilities of AVs and Mito-AVs along axons with and without elevated Rheb expression were quantifiedD,E Representative images (D) and quantitative analysis (E) showing that mitophagy in axons requires Rheb and Nix, but not Parkin. The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP-treated axons expressing scrambled shRNA, Rheb shRNA, Nix shRNA, or Parkin shRNA were quantified, respectively (E).F,G Quantitative analysis (F) and representative blots (G) showing increased mitochondrial association with Rheb and LC3-II in mature cortical neurons upon ∆ψm depolarization. Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post-nuclear supernatant (P), mitochondria-enriched membrane fraction (M), and cytosolic supernatant (S). Equal amounts of protein (20 μg) were sequentially immunoblotted with antibodies against mitophagic/autophagic proteins Rheb, LC3, and Rab7, mitochondrial protein VDAC, and cytosolic protein GAPDH on the same membranes after stripping between each antibody application. The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH, compared to post-nuclear supernatant. Protein levels in mitochondrial fractions of CCCP-treated neurons were normalized to those in neurons incubated with DMSO. Data were quantified from four independent repeats.Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP-treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA (H).representative images showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light. The ratio has been false colored with the indicated heat map, with high intensity indicative of ROGFP fluorescence in a more oxidative level


Figure 3Aberrant accumulation of mitophagosomes co - labeled by antibodies against LC3 and cytochrome c ( Cyto c ) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains . Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse brainsThe average number of mitophagosomes per section ( 320 μm × 320 μm ) was quantified from the total number of brain slice sections ( n ) indicated on the top of the barsC Clustering of mitophagosomes ( white arrows ) co - labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen / dystrophic presynaptic terminals indicated by synaptophysin ( SYP ) surrounding amyloid plaques in AD mouse brains . Quantitative analysis showing presynaptic retention of mitophagosome - like structures in the hippocampal regions of mutant hAPP mouse brains . Data were quantified from the total number of the imaging fields ( n ) indicated in parenthesesrepresentative TEM images showing presynaptic retention of mitophagosome - like structures in the hippocampal regions of mutant hAPP mouse brains . White arrow - labeled mitophagosomes are not readily detected in WT mouse brains . Black arrows marked amphisomes or degradative autophagic vacuoles ( AVd ) . F , G Mitophagic accumulation at Aβ - enriched synapses in AD mouse brains . Note that the levels of mitophagic / autophagic proteins ( red box ) along with Aβ ( green box ) are increased in synaptic fractions isolated from AD mouse brains . Equal amounts ( 10 μg ) of synaptosomal preparations ( Syn ) and post - nuclear supernatants ( PNS ) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP / Aβ , Rheb , LC3 , p62 , and Rab7 , as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application . The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH ( F ) . Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls . Data were quantified from five repeats ( G ) . H , I Representative blots ( H ) and quantitative analysis ( I ) showing increased levels of Rheb along with LC3 - II , p62 , and Rab7 ( red box ) in Mito fractions purified from AD mouse brains . The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115 , relative to PNS fractions . Data were quantified from four repeats .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Aberrant accumulation of mitophagosomes co-labeled by antibodies against LC3 and cytochrome c (Cyto c) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains. Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse brainsThe average number of mitophagosomes per section (320 μm × 320 μm) was quantified from the total number of brain slice sections (n) indicated on the top of the barsC Clustering of mitophagosomes (white arrows) co-labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen/dystrophic presynaptic terminals indicated by synaptophysin (SYP) surrounding amyloid plaques in AD mouse brains.Quantitative analysis showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parenthesesrepresentative TEM images showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. White arrow-labeled mitophagosomes are not readily detected in WT mouse brains. Black arrows marked amphisomes or degradative autophagic vacuoles (AVd).F,G Mitophagic accumulation at Aβ-enriched synapses in AD mouse brains. Note that the levels of mitophagic/autophagic proteins (red box) along with Aβ (green box) are increased in synaptic fractions isolated from AD mouse brains. Equal amounts (10 μg) of synaptosomal preparations (Syn) and post-nuclear supernatants (PNS) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP/Aβ, Rheb, LC3, p62, and Rab7, as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH (F). Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls. Data were quantified from five repeats (G).H,I Representative blots (H) and quantitative analysis (I) showing increased levels of Rheb along with LC3-II, p62, and Rab7 (red box) in Mito fractions purified from AD mouse brains. The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115, relative to PNS fractions. Data were quantified from four repeats.


Figure 4Representative images showing increased mitochondrial association with Rheb in AD axons , relative to that of WT littermate controls . Note that CCCP treatment enhances Rheb localization to mitochondria . The percentages of white arrow - marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCPquantitative analysis The percentages of white arrow - marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified , respectivelyC - Representative dual - channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons . mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantifiedquantitative analysis , the numbers of Mito , AVs , and Mito - AVs , and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified , respectivelyquantitative analysis Relative motility of AVs and Mito - AVs / mitophagosomes in WT or mutant hAPP axons with and without CCCP were quantifiedF , G Quantitative analysis ( F ) and representative blots ( G ) showing that ∆ ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains . Note increased localization of Rheb along with LC3 - II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment ( red box ) . Data were quantified from three independent repeats . P : post - nuclear supernatants ; M : mitochondria - enriched membrane fractions ; S : cytosolic supernatants . Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths ( 405 nm and 488 nm ) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controlsrepresentative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons , which is exacerbated after dissipating ∆ ψm . Mean fluorescence intensity ratios evoked by the two excitation wavelengths ( 405 nm and 488 nm ) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT

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Figure 4Representative images showing increased mitochondrial association with Rheb in AD axons, relative to that of WT littermate controls. Note that CCCP treatment enhances Rheb localization to mitochondria. The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCPquantitative analysis The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified, respectivelyC- Representative dual-channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons. mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantifiedquantitative analysis , the numbers of Mito, AVs, and Mito-AVs, and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified, respectivelyquantitative analysis Relative motility of AVs and Mito-AVs/mitophagosomes in WT or mutant hAPP axons with and without CCCP were quantifiedF,G Quantitative analysis (F) and representative blots (G) showing that ∆ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains. Note increased localization of Rheb along with LC3-II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment (red box). Data were quantified from three independent repeats. P: post-nuclear supernatants; M: mitochondria-enriched membrane fractions; S: cytosolic supernatants.Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controlsrepresentative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons, which is exacerbated after dissipating ∆ψm. Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls


Figure 5A , B Representative images ( A ) and quantitative analysis ( B ) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin - cKO mouse brains . Note that white arrow - labeled mitophagosomes are not readily observed in WT mouse brains . The average number of mitophagosomes per section ( 320 μm × 320 μm ) was quantified . Representative blots showing aberrant mitophagic retention at synaptic terminals in snapin - mutant mouse brains . The levels of Rheb along with LC3 - II , p62 , and Rab7 are significantly elevated in the synaptosomal fractions ( Syn ) purified from snapin - deficient mouse brains , relative to those of WT littermate controls ( red box ) . Equal amounts ( 10 μg ) of Syn and post - nuclear supernatants ( PNSquantitative analysis The levels of Rheb along with LC3 - II , p62 , and Rab7 are significantly elevated in the synaptosomal fractions ( Syn ) purified from snapin - deficient mouse brains , relative to those of WT littermate controls WT and snapin - mutant mouse brains were sequentially immunoblotted with antibodies against TOM20 on the same membrane after stripping between each antibody application . The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH . Data were quantified from five independent repeats . E , F Representative blots ( E ) and quantitative analysis ( F ) showing mitophagic accumulation in the brains of snapin conditional KO ( cKO ) mice by crossing with Thy1 - cre Tg mice . Note increased levels of Rheb along with LC3 - II , p62 , and Rab7 in mitochondrial ( Mito ) fractions isolated from snapin - deficient mouse brains , relative to those of WT littermate controls ( red box ) . Data were quantified from five independent repeats . PNS : post - nuclear supernatants . G , H Representative TEM images ( G ) and quantitative analysis ( H ) showing abnormal presynaptic retention of mitophagosome - like structures in the hippocampal regions of snapin - deficient mouse brains . Mitophagosomes , indicated by black arrows , are not readily detected in WT mouse brains . Data were quantified from the total number of the imaging fields ( n ) indicated in parentheses ( H ) . I , J Representative images ( I ) and quantitative analysis ( J ) showing that deletion of snapin in mouse brains induces synaptic damage . Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin - mutant mice crossed with CamKII - cre Tg mice at the age of 8 months , relative to that of WT littermate controls .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A,B Representative images (A) and quantitative analysis (B) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin-cKO mouse brains. Note that white arrow-labeled mitophagosomes are not readily observed in WT mouse brains. The average number of mitophagosomes per section (320 μm × 320 μm) was quantified.Representative blots showing aberrant mitophagic retention at synaptic terminals in snapin-mutant mouse brains. The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Equal amounts (10 μg) of Syn and post-nuclear supernatants (PNSquantitative analysis The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls WT and snapin-mutant mouse brains were sequentially immunoblotted with antibodies against TOM20 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH. Data were quantified from five independent repeats.E,F Representative blots (E) and quantitative analysis (F) showing mitophagic accumulation in the brains of snapin conditional KO (cKO) mice by crossing with Thy1-cre Tg mice. Note increased levels of Rheb along with LC3-II, p62, and Rab7 in mitochondrial (Mito) fractions isolated from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Data were quantified from five independent repeats. PNS: post-nuclear supernatants.G,H Representative TEM images (G) and quantitative analysis (H) showing abnormal presynaptic retention of mitophagosome-like structures in the hippocampal regions of snapin-deficient mouse brains. Mitophagosomes, indicated by black arrows, are not readily detected in WT mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses (H).I,J Representative images (I) and quantitative analysis (J) showing that deletion of snapin in mouse brains induces synaptic damage. Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin-mutant mice crossed with CamKII-cre Tg mice at the age of 8 months, relative to that of WT littermate controls.


Figure 6Representative dual - channel kymographs ( A ) and quantitative analysis ( B showing that overexpression of Snapin , but not Snapin - L99K , a Snapin mutant deficient in dynein motor binding , reduces mitophagic accumulation within AD axons incubated with CCCP . Representative quantitative analysis showing that overexpression of Snapin , but not Snapin - L99K , a SnapinD Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin - L99K were quantified and normalized to those of mutant hAPP controls . Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Representative dual-channel kymographs (A) and quantitative analysis (B showing that overexpression of Snapin, but not Snapin-L99K, a Snapin mutant deficient in dynein motor binding, reduces mitophagic accumulation within AD axons incubated with CCCP.Representative quantitative analysis showing that overexpression of Snapin, but not Snapin-L99K, a SnapinD Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin-L99K were quantified and normalized to those of mutant hAPP controls. Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment.


Figure 7A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV - mCherry or AAV - mCherry - Snapin . mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus ( DG ) of mutant hAPP mouse brains . NeuN , a neuronal nuclear marker , indicates neurons . Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( white arrows ) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c * : color is converted from blue to red for better contrastAttenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( white arrows ) in mutant hAPP Tg mouse brains with elevated Snapin expressionD , E Quantitative analysis ( D ) and representative images ( E ) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV - mCherry - Snapin . Blue indicates the signal of DAPI staining . The percentage area of SYP - labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV - mCherry ( D ) . Data information : Data were quantified from a total number of 40 - 44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV-mCherry or AAV-mCherry-Snapin. mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus (DG) of mutant hAPP mouse brains. NeuN, a neuronal nuclear marker, indicates neurons.Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c*: color is converted from blue to red for better contrastAttenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expressionD,E Quantitative analysis (D) and representative images (E) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV-mCherry-Snapin. Blue indicates the signal of DAPI staining. The percentage area of SYP-labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV-mCherry (D). Data information: Data were quantified from a total number of 40-44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection.


Figure 1Primary rat cortical neurons transfected with eGFP - GA50 were examined to determine at which time points preceding cell death aggregates are found in neurites . Two days post - transfection , aggregates formed by eGFP - GA50 ( green ) are detectable in neurites ( SMI - 32 staining in red ) . These aggregates remain localized to neurites at 96 hours ( 4 days ) and 288 hours ( 8 days ) . Colocalization is indicated by yellow overlay of colors ( right panels ) . Inset below each image shows enlargement of neurite regions containing aggregates , Representative fields from 60x magnification z - stack confocal images , scale bar indicates 20μm . Inset scale bars indicate 5μm .

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Figure 1Primary rat cortical neurons transfected with eGFP-GA50 were examined to determine at which time points preceding cell death aggregates are found in neurites. Two days post-transfection, aggregates formed by eGFP-GA50 (green) are detectable in neurites (SMI-32 staining in red). These aggregates remain localized to neurites at 96 hours (4 days) and 288 hours (8 days). Colocalization is indicated by yellow overlay of colors (right panels). Inset below each image shows enlargement of neurite regions containing aggregates, Representative fields from 60x magnification z-stack confocal images, scale bar indicates 20μm. Inset scale bars indicate 5μm.


Figure 2Primary rat cortical neurons underwent live - cell imaging after 48 hours of eGFP - GAn expression to determine aggregate mobility within neurites . A ) Example of a cortical neuron expressing eGFP - GA100 ( green ) with brightfield overlay , 60x magnification , scale bar indicates 10μm . Inset in upper right shows enlargement of boxed area , to better visualize aggregates within neuronal processes . Inset scale bar indicates 5μm . Primary rat motor neurons underwent live - cell imaging after 48 hours of eGFP - GAn expression to determine aggregate mobility within neurites . B ) Example of a motor neuron expressing eGFP - GA25 ( green ) with brightfield overlay , 60x magnification , scale bar indicates 10μm . Inset in upper right shows enlargement of boxed area , to better visualize aggregates within neuronal processes . Inset scale bar indicates 5μm . C ) Quantification of eGFP - GAn aggregate velocity within cortical neurons . D ) Quantification of eGFP - GAn aggregate velocity within motor neuron neurites .

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Figure 2Primary rat cortical neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. A) Example of a cortical neuron expressing eGFP-GA100 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.Primary rat motor neurons underwent live-cell imaging after 48 hours of eGFP-GAn expression to determine aggregate mobility within neurites. B) Example of a motor neuron expressing eGFP-GA25 (green) with brightfield overlay, 60x magnification, scale bar indicates 10μm. Inset in upper right shows enlargement of boxed area, to better visualize aggregates within neuronal processes. Inset scale bar indicates 5μm.C) Quantification of eGFP-GAn aggregate velocity within cortical neurons.D) Quantification of eGFP-GAn aggregate velocity within motor neuron neurites.


Figure 3Primary rat neurons were co - transfected with Td - tomato and eGFP - GAn plasmids or eGFP - alone . The same neurons were imaged at 24 - hour intervals to assess viability . A ) Representative fields ( right ) of eGFP and eGFP - GA50 cortical neurons at days 1 , 7 , and 13 post - transfection follow the survival of individual cells over time ( white arrows ) . 20x magnification , scale bar indicates 25μm . B ) Kaplan - Meier survival analysis of cortical neurons followed for 13 days post - transfection . C ) Kaplan - Meier survival analysis ( left ) of motor neurons followed for 5 days post - transfection .

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Figure 3Primary rat neurons were co-transfected with Td-tomato and eGFP-GAn plasmids or eGFP-alone. The same neurons were imaged at 24-hour intervals to assess viability. A) Representative fields (right) of eGFP and eGFP-GA50 cortical neurons at days 1, 7, and 13 post-transfection follow the survival of individual cells over time (white arrows). 20x magnification, scale bar indicates 25μm. B) Kaplan-Meier survival analysis of cortical neurons followed for 13 days post-transfection.C) Kaplan-Meier survival analysis (left) of motor neurons followed for 5 days post-transfection.


Figure 4Cortical neurons were loaded with FM4 - 64 dye 48 hours after transfection . Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . A ) Representative dye loading showing defined puncta regions along neurites 40x magnification , scale bar indicates 10μm . B ) Representative intensities of FM4 - 64 puncta regions pre - and post - stimulation demonstrate loss of synaptic puncta fluorescence intensity following stimulation in GFP but not GA50 containing neurons . These images visually reflect the process performed in an automated manner through our imaging software to determine puncta fluorescence intensity . An unbiased constant threshold is placed on each image per experiment , then fluorescence intensity is measured over time . The threshold applied isolates puncta from background , giving distinct regions . 8 - bit images were thresholded and pseudo - color ( red ) was applied to enlarged puncta regions of interest , scale bar indicates 10μm . C ) Graphical representation is the change in fluorescence ( ΔF ) normalized to basal fluorescence ( F ) , ΔF / F . The final 10 seconds of baseline recording and 10 seconds of the depolarization phase are shown , arrow indicates start of depolarization . Non - linear regression was performed in GraphPad Prism7 using the plateau followed by one phase decay model . The decay constant ( k ) for GFP was 0 . 4971 s - 1 , while for GA50 it was 219 . 5 s - 1 . Similarly the tau ( τ ) values were 2 . 012 seconds for GFP and 0 . 00455 seconds for GA50 , respectively . Data presented as mean ± SEM for each imaged timepoint . Statistical significance was determined using the Sidak - Bonferroni method , * * * * p < 0 . 0001 , n = 20 puncta regions from 4 biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Cortical neurons were loaded with FM4-64 dye 48 hours after transfection. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. A) Representative dye loading showing defined puncta regions along neurites 40x magnification, scale bar indicates 10μm. B) Representative intensities of FM4-64 puncta regions pre- and post- stimulation demonstrate loss of synaptic puncta fluorescence intensity following stimulation in GFP but not GA50 containing neurons. These images visually reflect the process performed in an automated manner through our imaging software to determine puncta fluorescence intensity. An unbiased constant threshold is placed on each image per experiment, then fluorescence intensity is measured over time. The threshold applied isolates puncta from background, giving distinct regions. 8-bit images were thresholded and pseudo-color (red) was applied to enlarged puncta regions of interest, scale bar indicates 10μm.C) Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 10 seconds of baseline recording and 10 seconds of the depolarization phase are shown, arrow indicates start of depolarization. Non-linear regression was performed in GraphPad Prism7 using the plateau followed by one phase decay model. The decay constant (k) for GFP was 0.4971 s-1, while for GA50 it was 219.5 s-1. Similarly the tau (τ) values were 2.012 seconds for GFP and 0.00455 seconds for GA50, respectively. Data presented as mean ± SEM for each imaged timepoint. Statistical significance was determined using the Sidak-Bonferroni method, ****p < 0.0001, n=20 puncta regions from 4 biological replicates.


Figure 5Primary cortical or motor neurons were co - transfected with mCherry or mCherry - GA50 and GCaMP6f . After 48 hours , mCherry positive cells were determined . Green fluorescence intensity was then recorded from identified neurons . Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . A ) Representative images of a co - transfected cortical neuron expressing mCherry - GA50 ( left panel ) and GCaMP6f ( right two panels ) . This image shows a representative demonstration of the increase in detectable green fluorescence from the GCaMP6f indicator pre - and post - stimulation ( center and right respectively ) . 40x magnification , scale bar indicates 25μm . B - C ) Graphical representation of the peak change in fluorescence ( ΔF ) following ACSF perfusion normalized to basal fluorescence ( F ) , ΔF / F . The contribution of Ca2 + influx was determined by experiments in which stimulation was performed in ACSF containing high KCl but no Ca2 + . Quantification of the peak change in fluorescence in cortical neurons ( B ) and motor neurons ( C ) .

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Figure 5Primary cortical or motor neurons were co-transfected with mCherry or mCherry-GA50 and GCaMP6f. After 48 hours, mCherry positive cells were determined. Green fluorescence intensity was then recorded from identified neurons. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. A) Representative images of a co-transfected cortical neuron expressing mCherry-GA50 (left panel) and GCaMP6f (right two panels). This image shows a representative demonstration of the increase in detectable green fluorescence from the GCaMP6f indicator pre- and post- stimulation (center and right respectively). 40x magnification, scale bar indicates 25μm.B-C) Graphical representation of the peak change in fluorescence (ΔF) following ACSF perfusion normalized to basal fluorescence (F), ΔF/F. The contribution of Ca2+ influx was determined by experiments in which stimulation was performed in ACSF containing high KCl but no Ca2+. Quantification of the peak change in fluorescence in cortical neurons (B) and motor neurons (C).


Figure 6eGFP - GAn dipeptides were expressed in primary cortical or motor neurons for 48 hours , then immunostained for neurofilament ( gray ) and SV2 ( red ) . A - B ) Representative z - stack confocal images of neurites from eGFP , eGFP - GA50 , and eGFP - GA100 expressing cortical ( A ) or motor ( B ) neurons . 60x magnification , scale bar indicates 5μm . C - D ) Quantification of SV2 puncta along neurites in cortical neurons ( C ) or motor neurons ( D ) by ImageJ manual counting , normalized to eGFP - only expressing cells . ( C ) Cortical neurons demonstrated a significant GA - length dependent reduction , as well as a reduction in all cells containing GA - repeats greater than 50 ( left panel ) . ( D ) Motor neurons demonstrated a significant GA - length dependent reduction , as well as a reduction in all cells containing GA - repeats greater than 100 ( left panel ) . Evaluation of neurite length by NeuronJ software revealed no differences in total neurite length per cell in cortical neurons ( C ) or motor neurons ( D ) expressing any GA - repeat length compared with eGFP alone ( right panels ) .

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Figure 6eGFP-GAn dipeptides were expressed in primary cortical or motor neurons for 48 hours, then immunostained for neurofilament (gray) and SV2 (red). A-B) Representative z-stack confocal images of neurites from eGFP, eGFP-GA50, and eGFP-GA100 expressing cortical (A) or motor (B) neurons. 60x magnification, scale bar indicates 5μm. C-D) Quantification of SV2 puncta along neurites in cortical neurons (C) or motor neurons (D) by ImageJ manual counting, normalized to eGFP-only expressing cells. (C) Cortical neurons demonstrated a significant GA-length dependent reduction, as well as a reduction in all cells containing GA-repeats greater than 50 (left panel). (D) Motor neurons demonstrated a significant GA-length dependent reduction, as well as a reduction in all cells containing GA-repeats greater than 100 (left panel). Evaluation of neurite length by NeuronJ software revealed no differences in total neurite length per cell in cortical neurons (C) or motor neurons (D) expressing any GA-repeat length compared with eGFP alone (right panels).


Figure 7i3 cortical neurons from a C9orf72 - ALS patient and the paired isogenic control were differentiated for 21 days , then were immunostained for neurofilament ( red ) , and SV2 ( green ) ( A ) Shown are representative z - stack confocal images of neurites . B ) i3 cortical neurons from a C9orf72 - ALS patient and the paired isogenic control were differentiated for 21 days , then were immunostained for synaptophysin ( green ) ( B ) . Shown are representative z - stack confocal images of neurites . Quantification of SV2 ( C ) colocalized with neurofilament normalized to neurofilament area . This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control , Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . 15 non - overlapping regions from 3 differentiated wells for each genotype were evaluated . Quantification of Synaptophysin ( D ) colocalized with neurofilament normalized to neurofilament area . This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control , while synaptophysin remained unaltered . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . 15 non - overlapping regions from 3 differentiated wells for each genotype were evaluated . E ) mRNA was Trizol extracted from 3 independent wells of C9 - ALS and isogenic i3 cortical neurons . RNA was converted to cDNA using the Superscript First - strand kit , and q - PCR was performed using PowerUp SYBR Green . Measurements were normalized to the housekeeping gene GAPDH and then to isogenic levels . Analysis was performed using the ΔΔCT method . 2ΔΔCT ± SE , is presented . Unpaired t - test , * * p < 0 . 01 . F ) Whole cell lysates were generated from 3 independent wells of C9 - ALS and isogenic i3 cortical neurons differentiated for 21 days . Lysates were immunoblotted for SV2 , and normalized to total protein loading . Quantification band intensities revealed a significant decrease in SV2 protein levels in the C9orf72 lines compared with the isogenic . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . Whole cell lysates were generated from iMNs differentiated for 32 days . Lysates were immunoblotted for SV2 ( G ) Loading controls were β - actin Quantification band intensities normalized to loading controls revealed a trend towards reduction in SV2 protein levels in the C9orf72 cell lines compared with controls Data presented as mean ± SEM . Unpaired t - test . Samples from 5 independent control and 4 independent C9orf72 patient lines were used . Whole cell lysates were generated from iMNs differentiated for 32 days . Lysates were immunoblotted for synaptophysin ( H ) . Loading controls were total protein loading . synaptophysin levels remained unaltered . Data presented as mean ± SEM . Unpaired t - test . Samples from 5 independent control and 4 independent C9orf72 patient lines were used .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for neurofilament (red), and SV2 (green) (A) Shown are representative z-stack confocal images of neurites.B) i3 cortical neurons from a C9orf72-ALS patient and the paired isogenic control were differentiated for 21 days, then were immunostained for synaptophysin (green) (B). Shown are representative z-stack confocal images of neurites.Quantification of SV2 (C) colocalized with neurofilament normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control, Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. 15 non-overlapping regions from 3 differentiated wells for each genotype were evaluated.Quantification of Synaptophysin (D) colocalized with neurofilament normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in the C9orf72 line compared with the isogenic control, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05. 15 non-overlapping regions from 3 differentiated wells for each genotype were evaluated.E) mRNA was Trizol extracted from 3 independent wells of C9-ALS and isogenic i3 cortical neurons. RNA was converted to cDNA using the Superscript First-strand kit, and q-PCR was performed using PowerUp SYBR Green. Measurements were normalized to the housekeeping gene GAPDH and then to isogenic levels. Analysis was performed using the ΔΔCT method. 2ΔΔCT ± SE, is presented. Unpaired t-test, **p < 0.01.F) Whole cell lysates were generated from 3 independent wells of C9-ALS and isogenic i3 cortical neurons differentiated for 21 days. Lysates were immunoblotted for SV2, and normalized to total protein loading. Quantification band intensities revealed a significant decrease in SV2 protein levels in the C9orf72 lines compared with the isogenic. Data presented as mean ± SEM. Unpaired t-test, *p < 0.05.Whole cell lysates were generated from iMNs differentiated for 32 days. Lysates were immunoblotted for SV2 (G) Loading controls were β-actin Quantification band intensities normalized to loading controls revealed a trend towards reduction in SV2 protein levels in the C9orf72 cell lines compared with controls Data presented as mean ± SEM. Unpaired t-test. Samples from 5 independent control and 4 independent C9orf72 patient lines were used.Whole cell lysates were generated from iMNs differentiated for 32 days. Lysates were immunoblotted for synaptophysin (H). Loading controls were total protein loading. synaptophysin levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test. Samples from 5 independent control and 4 independent C9orf72 patient lines were used.


Figure 8A ) Cortical neurons were loaded with FM4 - 64 dye 48 hours after transfection with eGFP - GA50 and transduction with rSV2a - eGFP - pRRL lentivirus . Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . Graphical representation is the change in fluorescence ( ΔF ) normalized to basal fluorescence ( F ) , ΔF / F . The final 60 seconds of baseline recording and 60 seconds of the depolarization phase are shown , arrow indicates start of depolarization . The lack of synaptic release in the case of GA50 expression ( red line ) was rescued back to levels comparable to eGFP controls ( pink line ) in the case of coexpression of rSV2a - eGFP - pRRL ( orange line ) . Statistical significance was determined using the Sidak - Bonferroni method , * * * p < 0 . 001 , n = 10 puncta regions from each of 4 biological replicates . B ) Cortical neurons were co - transfected with mCherry or mCherry - GA50 and GCaMP6f , then transduced with rSV2a - eGFP - pRRL . Green fluorescence intensity was recorded from identified mCherry positive neurons . Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl . Quantification of the peak change in fluorescence ( ΔF ) following ACSF perfusion normalized to basal fluorescence ( F ) , ΔF / F . The significant increase in Ca2 + influx observed following GA50 expression ( * p < 0 . 05 ) is resolved when rSV2a - eGFP - pRRL is coexpressed . Data presented as mean ± SEM . One - way ANOVA with Sidak ' s multiple comparisons test , at least 10 cells per condition , pooled from n = 3 independent biological experiments . No significant differences were observed between mCherry or mCherry + SV2 conditions , groups were merged for this analysis . Neurons were co - transfected with mCherry - GAn plasmids or mCherry - alone and transduced with rSV2a - eGFP - pRRL , then imaged at 24 - hour intervals . ( C ) Kaplan - Meier survival analysis of cortical neurons expressing GA50 or GA50 + SV2 followed for 13 days . The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment . While the hazard ratio of GA50 was 1 . 473 , this was decreased to 0 . 7227 for GA50 + SV2 . At least 190 cells per condition were evaluated from a pooling of n = 3 independent experiments , log - rank Mantel - Cox test , * * * * p < 0 . 0001 . No significant differences were observed between eGFP or eGFP + SV2 conditions , and groups were merged for this analysis as Control . ( D ) A Kaplan - Meier survival analysis was performed for PR50 and PR50 + SV2 containing cells . Under these conditions significant toxicity is observed regardless of increased SV2 levels , with the hazard ratio of PR50 containing cells ( 1 . 772 ) remaining high for those expressing PR50 + SV2 ( 1 . 784 ) . At least 150 cells per condition were evaluated from a pooling of n = 3 independent experiments , log - rank Mantel - Cox test , * * * * p < 0 . 0001 . No significant differences were observed between eGFP or eGFP + SV2 conditions , and groups were merged for this analysis as Control . ( E ) Kaplan - Meier survival analysis of motor neurons expressing GA50 or GA50 + SV2 followed for 10 days . The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment . While the hazard ratio of GA50 was 1 . 345 , this was decreased to 0 . 7769 for GA50 + SV2 . At least 200 cells per condition were evaluated from a pooling of n = 3 independent experiments , log - rank Mantel - Cox test , * * * * p < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A) Cortical neurons were loaded with FM4-64 dye 48 hours after transfection with eGFP-GA50 and transduction with rSV2a-eGFP-pRRL lentivirus. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Graphical representation is the change in fluorescence (ΔF) normalized to basal fluorescence (F), ΔF/F. The final 60 seconds of baseline recording and 60 seconds of the depolarization phase are shown, arrow indicates start of depolarization. The lack of synaptic release in the case of GA50 expression (red line) was rescued back to levels comparable to eGFP controls (pink line) in the case of coexpression of rSV2a-eGFP-pRRL (orange line). Statistical significance was determined using the Sidak-Bonferroni method, ***p < 0.001, n=10 puncta regions from each of 4 biological replicates.B) Cortical neurons were co-transfected with mCherry or mCherry-GA50 and GCaMP6f, then transduced with rSV2a-eGFP-pRRL. Green fluorescence intensity was recorded from identified mCherry positive neurons. Basal fluorescence was monitored prior to induced depolarization via perfusion with ACSF containing 50mM KCl. Quantification of the peak change in fluorescence (ΔF) following ACSF perfusion normalized to basal fluorescence (F), ΔF/F. The significant increase in Ca2+ influx observed following GA50 expression (*p < 0.05) is resolved when rSV2a-eGFP-pRRL is coexpressed. Data presented as mean ± SEM. One-way ANOVA with Sidak's multiple comparisons test, at least 10 cells per condition, pooled from n= 3 independent biological experiments. No significant differences were observed between mCherry or mCherry+SV2 conditions, groups were merged for this analysis.Neurons were co-transfected with mCherry-GAn plasmids or mCherry-alone and transduced with rSV2a-eGFP-pRRL, then imaged at 24-hour intervals. (C) Kaplan-Meier survival analysis of cortical neurons expressing GA50 or GA50+SV2 followed for 13 days. The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment. While the hazard ratio of GA50 was 1.473, this was decreased to 0.7227 for GA50+SV2. At least 190 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001. No significant differences were observed between eGFP or eGFP+SV2 conditions, and groups were merged for this analysis as Control. (D) A Kaplan-Meier survival analysis was performed for PR50 and PR50+SV2 containing cells. Under these conditions significant toxicity is observed regardless of increased SV2 levels, with the hazard ratio of PR50 containing cells (1.772) remaining high for those expressing PR50+SV2 (1.784). At least 150 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001. No significant differences were observed between eGFP or eGFP+SV2 conditions, and groups were merged for this analysis as Control.(E) Kaplan-Meier survival analysis of motor neurons expressing GA50 or GA50+SV2 followed for 10 days. The significant cellular toxicity induced by GA50 is rescued by SV2 replenishment. While the hazard ratio of GA50 was 1.345, this was decreased to 0.7769 for GA50+SV2. At least 200 cells per condition were evaluated from a pooling of n=3 independent experiments, log-rank Mantel-Cox test, ****p < 0.0001.


Figure 9Spinal cord lysates were generated from fresh frozen tissue of 20 - month old mice either expressing GA149 - CFP under the Thy1 promoter ( Tgx GA149 ) or wild type controls . Lysates were immunoblotted for SV2 ( A ) Total protein levels were determined by imaging the Mini - PROTEAN TGX stain - free gel prior to transfer . Quantification of band intensities normalized to total protein loading revealed a significant reduction in SV2 protein levels in the GA - expressing cohort compared with wild type animals , Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . A total of 4 animals per group were examined . Spinal cord lysates were generated from fresh frozen tissue of 20 - month old mice either expressing GA149 - CFP under the Thy1 promoter ( Tgx GA149 ) or wild type controls . Lysates were immunoblotted for synaptophysin ( B ) Total protein levels were determined by imaging the Mini - PROTEAN TGX stain - free gel prior to transfer . synaptophysin levels remained unaltered . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . A total of 4 animals per group were examined . Spinal cord lysates were generated from fresh frozen tissue of 20 - month old mice either expressing GA149 - CFP under the Thy1 promoter ( Tgx GA149 ) or wild type controls . Lysates were immunoblotted for PSD - 95 ( C ) . Total protein levels were determined by imaging the Mini - PROTEAN TGX stain - free gel prior to transfer . PSD - 95 levels remained unaltered . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . A total of 4 animals per group were examined .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for SV2 (A) Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. Quantification of band intensities normalized to total protein loading revealed a significant reduction in SV2 protein levels in the GA-expressing cohort compared with wild type animals, Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for synaptophysin (B) Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. synaptophysin levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.Spinal cord lysates were generated from fresh frozen tissue of 20-month old mice either expressing GA149-CFP under the Thy1 promoter (Tgx GA149) or wild type controls. Lysates were immunoblotted for PSD-95 (C). Total protein levels were determined by imaging the Mini-PROTEAN TGX stain-free gel prior to transfer. PSD-95 levels remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 4 animals per group were examined.


Figure 10Muscle sections ( 30μm ) were cut from snap frozen tissue of 20 - month old GA149 mice ( Tgx GA149 ) or wild type controls . Immunohistochemical staining was performed to assess synaptic protein levels in proximity to NMJs . Sections were immunostained for SV2 or Synaptophysin , and counterstained with neurofilament . A ) Left Panels : Representative images show SV2 levels ( green ) in proximity to α - bungarotoxin positive NMJs ( red ) in wild type animals ( top ) versus GA149 animals ( bottom ) . Right Panels : Representative images show SV2 levels ( green ) overlapping with neurofilament ( blue ) at synaptic terminals approaching muscle in wild type animals ( top ) versus GA149 animals ( bottom ) . B - C ) Quantification of SV2 ( B ) or Synaptophysin ( C ) colocalized with neurofilament and normalized to neurofilament area . This analysis revealed a significant reduction in SV2 levels in GA149 animals compared with wild types , while synaptophysin remained unaltered . Data presented as mean ± SEM . Unpaired t - test , * p < 0 . 05 . A total of 10 non - overlapping regions of muscle from each of 5 animals per group were evaluated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 10Muscle sections (30μm) were cut from snap frozen tissue of 20-month old GA149 mice (Tgx GA149) or wild type controls. Immunohistochemical staining was performed to assess synaptic protein levels in proximity to NMJs. Sections were immunostained for SV2 or Synaptophysin, and counterstained with neurofilament. A) Left Panels: Representative images show SV2 levels (green) in proximity to α-bungarotoxin positive NMJs (red) in wild type animals (top) versus GA149 animals (bottom). Right Panels: Representative images show SV2 levels (green) overlapping with neurofilament (blue) at synaptic terminals approaching muscle in wild type animals (top) versus GA149 animals (bottom). B-C) Quantification of SV2 (B) or Synaptophysin (C) colocalized with neurofilament and normalized to neurofilament area. This analysis revealed a significant reduction in SV2 levels in GA149 animals compared with wild types, while synaptophysin remained unaltered. Data presented as mean ± SEM. Unpaired t-test, *p< 0.05. A total of 10 non-overlapping regions of muscle from each of 5 animals per group were evaluated.


Figure 1 ( A _ B ) In situs on adult brain sections reveal that ( A ) opn4 . 1 and ( B ) opn4xb are coexpressed in many brain domains including the epiphysis cerebri ( indicated with E ) or pineal . A horizontal line through a representation of the brain above the sagittal section indicates the location within the brain . Scale bar : 1 . 0 mm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A_B) In situs on adult brain sections reveal that (A) opn4.1 and (B) opn4xb are coexpressed in many brain domains including the epiphysis cerebri (indicated with E) or pineal. A horizontal line through a representation of the brain above the sagittal section indicates the location within the brain. Scale bar: 1.0 mm.


Figure 4 ( J ) ish with asmt probe on larvae sampled on the 6th dpf at ZT3 shows that asmt mRNA is confined to the pineal and indicates that the asmt gene is not ectopically expressed in opn4 dko larvae . The higher asmt mRNA level measured by qPCR in the opn4 dko therefore points at a defect in the pineal . Note that the eyes were removed after the ish for optimal visibility of expression in the brain . Scale bar : 0 . 5 mm . ( N ) ish with ddc probe on wild - type and opn4 dko larvae sampled on the 6th dpf represents ddc expression in pineals . Biological replicates indicated with BR . Scale bar : 0 . 05 mm ( O ) Quantification of the adjusted intensity in the pineals from larvae presented in N , implies a significant higher ddc transcript level in the opn4 dko pineal . Data information : boxplot divides the data in quartiles : the box indicates the interquartile range , with the horizontal line in the box denoting the median of the data set , the whiskers extend to the minimum and maximum , and meet the box at the median of the lower ( quartile 1 ) and median of the upper ( quartile 3 ) half of the dataset . Black dots indicate biological replicates O : n = 8 ) , the red dot indicates the mean , red error bars indicate the confidence interval ( 95 % ) , asterisks indicate significance ( 0 . 01 < p ( ✱ ) < 0 . 05 , 0 . 001 < p ( ✱ ✱ ) < 0 . 01 , p ( ✱ ✱ ✱ ) < 0 . 001 , ns = not significant ) .

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Figure 4(J) ish with asmt probe on larvae sampled on the 6th dpf at ZT3 shows that asmt mRNA is confined to the pineal and indicates that the asmt gene is not ectopically expressed in opn4 dko larvae. The higher asmt mRNA level measured by qPCR in the opn4 dko therefore points at a defect in the pineal. Note that the eyes were removed after the ish for optimal visibility of expression in the brain. Scale bar: 0.5 mm.(N) ish with ddc probe on wild-type and opn4 dko larvae sampled on the 6th dpf represents ddc expression in pineals. Biological replicates indicated with BR. Scale bar: 0.05 mm (O) Quantification of the adjusted intensity in the pineals from larvae presented in N, implies a significant higher ddc transcript level in the opn4 dko pineal. Data information: boxplot divides the data in quartiles: the box indicates the interquartile range, with the horizontal line in the box denoting the median of the data set, the whiskers extend to the minimum and maximum, and meet the box at the median of the lower (quartile 1) and median of the upper (quartile 3) half of the dataset. Black dots indicate biological replicates O: n=8), the red dot indicates the mean, red error bars indicate the confidence interval (95%), asterisks indicate significance (0.01<p(✱)<0.05, 0.001<p(✱✱)<0.01, p(✱✱✱)<0.001, ns = not significant).


Figure 1 ( A qRT - PCR for different ISGs relative to Rps29 using epidermal RNA from 8 week - old ( A ) K5 - R1 / R2 and control ( CTRL ) mice . qRT - PCR for different ISGs relative to Rps29 using epidermal RNA from 5 day - or 9 day - old ( B ) K5 - R1 / R2 and control ( CTRL ) mice . ( C ) Confocal microscopy images of epidermal sheets from back skin of K5 - R1 / R2 and CTRL mice stained for IRF7 and K14 , counterstained with DAPI . Arrows denote nuclear IRF7 ( red ) in basal and suprabasal keratinocytes of K5 - R1 / R2 mice . The strong red staining of the stratum corneum ( sc ) is unspecific background and is more pronounced in K5 - R1 / R2 mice due to the increased thickness of this layer . ( D ) qRT - PCR for different ISGs , Ifnb , and Ifnl3 relative to Rps29 using RNA from freshly isolated , non - cultured keratinocytes of K5 - R1 / R2 and control ( CTRL ) mice at P7 . ( E ) qRT - PCR for ISGs relative to Rps29 using RNA from primary keratinocytes of neonatal CTRL and K5 - R1 / R2 mice . ( F ) qRT - PCR for ISGs and Cre relative to Rps29 using RNA from primary keratinocytes of neonatal K5 - Cre and wild - type ( WT ) mice . ( G ) Western blot analysis of total ( L ) and nuclear ( N ) lysates from immortalized ( imm . ) keratinocytes of CTRL and K5 - R1 / R2 mice for IRF1 , IRF7 , IRF9 , Lamin A / C ( nuclear marker , loading control ) and GAPDH ( cytosolic marker , loading control ) . ( H ) qRT - PCR for RSAD2 and ISG15 relative to RPLP0 using RNA from HaCaT keratinocytes treated for 48 h with the FGFR inhibitor BGJ398 ( 3 . 5 μM ) or vehicle in the presence of serum .

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Figure 1(A qRT-PCR for different ISGs relative to Rps29 using epidermal RNA from 8 week-old (A) K5-R1/R2 and control (CTRL) mice.qRT-PCR for different ISGs relative to Rps29 using epidermal RNA from 5 day- or 9 day-old (B) K5-R1/R2 and control (CTRL) mice.(C) Confocal microscopy images of epidermal sheets from back skin of K5-R1/R2 and CTRL mice stained for IRF7 and K14, counterstained with DAPI. Arrows denote nuclear IRF7 (red) in basal and suprabasal keratinocytes of K5-R1/R2 mice. The strong red staining of the stratum corneum (sc) is unspecific background and is more pronounced in K5-R1/R2 mice due to the increased thickness of this layer.(D) qRT-PCR for different ISGs, Ifnb, and Ifnl3 relative to Rps29 using RNA from freshly isolated, non-cultured keratinocytes of K5-R1/R2 and control (CTRL) mice at P7.(E) qRT-PCR for ISGs relative to Rps29 using RNA from primary keratinocytes of neonatal CTRL and K5-R1/R2 mice.(F) qRT-PCR for ISGs and Cre relative to Rps29 using RNA from primary keratinocytes of neonatal K5-Cre and wild-type (WT) mice.(G) Western blot analysis of total (L) and nuclear (N) lysates from immortalized (imm.) keratinocytes of CTRL and K5-R1/R2 mice for IRF1, IRF7, IRF9, Lamin A/C (nuclear marker, loading control) and GAPDH (cytosolic marker, loading control).(H) qRT-PCR for RSAD2 and ISG15 relative to RPLP0 using RNA from HaCaT keratinocytes treated for 48 h with the FGFR inhibitor BGJ398 (3.5 μM) or vehicle in the presence of serum.


Figure 2 ( A ) Growth factor - starved primary keratinocytes from WT mice were treated for 3 or 6 h with FGF7 ( 10 ng / ml ) or vehicle ( CTRL ) . RNA samples were analyzed by qRT - PCR for the indicated ISGs relative to Rps29 . ( B ) Serum - starved HaCaT keratinocytes were treated for 6 h with FGF7 or FGF10 ( each 10 ng / ml ) . RNA samples were analyzed by qRT - PCR for IRF7 and RSAD2 relative to RPLP0 . ( C ) Serum - starved HaCaT keratinocytes were treated for 16 h with FGF7 ( 10 ng / ml ) . Protein lysates were analyzed by Western blot for IRF1 , IRF3 , IRF9 and GAPDH . ( D ) RNA samples from untreated , serum - starved primary keratinocytes from WT and Ifnar1 - KO mice were analyzed by qRT - PCR for Irf7 , Rsad2 , Oasl2 and Ifnar1 relative to Rps29 . ( E ) Growth factor - starved primary keratinocytes from neonatal WT , Ifnar1 - KO or Ifnar1 / Ifnlr1 - KO mice were treated for 24 h with FGF7 ( 10 ng / ml ) . RNA samples were analyzed by qRT - PCR for Irf7 relative to Rps29 . ( F ) HaCaT keratinocytes were transiently transfected with an ISRE - Luciferase reporter construct and starved 24 h after transfection for 9 h . They were then incubated in the presence or absence of FGF7 ( 10 ng / ml ) for 20 h . Lysates were analyzed using a DualGlo Luciferase Assay . HaCaT keratinocytes were pre - treated for 2 h with the proteasome inhibitors MG132 ( 10 μm ( G ) followed by a 20h treatment with FGF7 ( 10 ng / ml ) or vehicle . RNA samples were analyzed by qRT - PCR for IRF7 and IRF1 relative to RPLP0 . HaCaT keratinocytes were pre - treated for 2 h with epoxomicin ( 100 nM ) ( H ) , followed by a 20h treatment with FGF7 ( 10 ng / ml ) or vehicle . RNA samples were analyzed by qRT - PCR for IRF7 and IRF1 relative to RPLP0 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Growth factor-starved primary keratinocytes from WT mice were treated for 3 or 6 h with FGF7 (10 ng/ml) or vehicle (CTRL). RNA samples were analyzed by qRT-PCR for the indicated ISGs relative to Rps29.(B) Serum-starved HaCaT keratinocytes were treated for 6 h with FGF7 or FGF10 (each 10 ng/ml). RNA samples were analyzed by qRT-PCR for IRF7 and RSAD2 relative to RPLP0.(C) Serum-starved HaCaT keratinocytes were treated for 16 h with FGF7 (10 ng/ml). Protein lysates were analyzed by Western blot for IRF1, IRF3, IRF9 and GAPDH.(D) RNA samples from untreated, serum-starved primary keratinocytes from WT and Ifnar1-KO mice were analyzed by qRT-PCR for Irf7, Rsad2, Oasl2 and Ifnar1 relative to Rps29.(E) Growth factor-starved primary keratinocytes from neonatal WT, Ifnar1-KO or Ifnar1/Ifnlr1-KO mice were treated for 24 h with FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for Irf7 relative to Rps29.(F) HaCaT keratinocytes were transiently transfected with an ISRE-Luciferase reporter construct and starved 24 h after transfection for 9 h. They were then incubated in the presence or absence of FGF7 (10 ng/ml) for 20 h. Lysates were analyzed using a DualGlo Luciferase Assay.HaCaT keratinocytes were pre-treated for 2 h with the proteasome inhibitors MG132 (10 μm (G) followed by a 20h treatment with FGF7 (10 ng/ml) or vehicle. RNA samples were analyzed by qRT-PCR for IRF7 and IRF1 relative to RPLP0.HaCaT keratinocytes were pre-treated for 2 h with epoxomicin (100 nM) (H), followed by a 20h treatment with FGF7 (10 ng/ml) or vehicle. RNA samples were analyzed by qRT-PCR for IRF7 and IRF1 relative to RPLP0.


Figure 3Serum - starved HaCaT keratinocytes were treated with FGF7 ( 10 ng / ml ( A and / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . RNA was analyzed by qRT - PCR for ISGs relative to RPLP0 ( ASerum - starved HaCaT keratinocytes were treated with FGF7 different concentrations as indicated ( B ) ) and / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . RNA was analyzed by qRT - PCR for ISGs relative to RPLP0Serum - starved HaCaT keratinocytes were treated with FGF7 ( 10 ng / ml and / or IFN - α ( 500 - 1000 U / ml ) for 12 - 16 h . protein samples by Western blot for total and pSTAT1 ( Y701 and S727 ) , IRF1 , IRF9 and RSAD2 and GAPDH ( C ) . The vertical line in the RSAD2 blot indicates a place where the gel was broken . Densitometric quantification of the different blots ( N = 3 ) is shown on the right . ( D ) Human primary keratinocytes were starved and treated for 12 h with FGF7 ( 10 ng / ml ) and / or IFN - α ( 1000 U / ml ) . RNA samples were analyzed by qRT - PCR for ISGs relative to RPLP0 .

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Figure 3Serum-starved HaCaT keratinocytes were treated with FGF7 (10 ng/ml (A and/or IFN-α (500-1000 U/ml) for 12-16 h. RNA was analyzed by qRT-PCR for ISGs relative to RPLP0 (ASerum-starved HaCaT keratinocytes were treated with FGF7 different concentrations as indicated (B)) and/or IFN-α (500-1000 U/ml) for 12-16 h. RNA was analyzed by qRT-PCR for ISGs relative to RPLP0Serum-starved HaCaT keratinocytes were treated with FGF7 (10 ng/ml and/or IFN-α (500-1000 U/ml) for 12-16 h. protein samples by Western blot for total and pSTAT1 (Y701 and S727), IRF1, IRF9 and RSAD2 and GAPDH (C). The vertical line in the RSAD2 blot indicates a place where the gel was broken. Densitometric quantification of the different blots (N=3) is shown on the right.(D) Human primary keratinocytes were starved and treated for 12 h with FGF7 (10 ng/ml) and/or IFN-α (1000 U/ml). RNA samples were analyzed by qRT-PCR for ISGs relative to RPLP0.


Figure 4 ( A ) Serum - starved HaCaT cells were treated for 24 h with poly ( I : C ) ( 5 μg / ml ) in the presence or absence of FGF7 ( 10 ng / ml ) . RNA samples were analyzed by qRT - PCR for IFNL1 , IFNB , RSAD2 , IRF1 , IRF7 , CGAS , TLR3 or DDX58 , relative to RPLP0 . ( B ) Serum - starved HaCaT cells were treated for 18 h with poly ( I : C ) ( 5 μg / ml ) in the presence or absence of FGF7 ( 10 ng / ml ) . Protein lysates were analyzed by Western blot for total and pSTAT1 ( Y701 and S727 ) , total and pSTAT2 ( Y690 ) , IRF1 , IRF3 , pIRF3 ( S396 ) , IRF9 , RIG - 1 , RSAD2 and GAPDH .

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Figure 4(A) Serum-starved HaCaT cells were treated for 24 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). RNA samples were analyzed by qRT-PCR for IFNL1, IFNB, RSAD2, IRF1, IRF7, CGAS, TLR3 or DDX58, relative to RPLP0.(B) Serum-starved HaCaT cells were treated for 18 h with poly(I:C) (5 μg/ml) in the presence or absence of FGF7 (10 ng/ml). Protein lysates were analyzed by Western blot for total and pSTAT1 (Y701 and S727), total and pSTAT2 (Y690), IRF1, IRF3, pIRF3 (S396), IRF9, RIG-1, RSAD2 and GAPDH.