Figure 2A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . A . Representative carmine - alum stained mammary gland whole mounts . Arrow indicates the inguinal lymph node . Scale bars represent 2 mm . B . Quantification of mammary ductal outgrowth area ( n = 4 - 15 glands ) . C . Quantification of the number of ductal branch points per mammary gland ( n = 7 - 9 glands , 7 weeks old mice ) . A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . D . Cryosections from 7 weeks old wt or Sharpincpdm mouse mammary glands stained with HE ( upper panel ) or immunolabelled with the indicated antibodies . Scale bars represent 20 μmA - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . E . Representative carmine - alum stained images highlighting terminal end buds ( TEBs ) in 7 weeks old wt and Sharpincpdm mouse mammary glands and F . quantification of the number of TEBs per gland ( n = 9 - 10 glands ) . Scale bar represents 500 μm . A - G . Mammary ductal outgrowth in 3 - 7 weeks old wt or Sharpincpdm female mice . G . TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE ( upper panel ) or immunolabelled with the indicated antibodies ( lower panel ) . Scale bars represent 50 µm . Mean ± SEM .

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 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 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

Figure 2A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. A. Representative carmine-alum stained mammary gland whole mounts. Arrow indicates the inguinal lymph node. Scale bars represent 2 mm. B. Quantification of mammary ductal outgrowth area (n = 4 - 15 glands). C. Quantification of the number of ductal branch points per mammary gland (n = 7 - 9 glands, 7 weeks old mice).A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. D. Cryosections from 7 weeks old wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies. Scale bars represent 20 μmA-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. E. Representative carmine-alum stained images highlighting terminal end buds (TEBs) in 7 weeks old wt and Sharpincpdm mouse mammary glands and F. quantification of the number of TEBs per gland (n = 9 - 10 glands). Scale bar represents 500 μm.A-G. Mammary ductal outgrowth in 3-7 weeks old wt or Sharpincpdm female mice. G. TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies (lower panel). Scale bars represent 50 µm. Mean ± SEM.


Figure 3A - C . Monitoring of mammary gland development following transplantation of wt or Sharpincpdm mouse mammary epithelium in the mammary fat pads ( epithelium - free ) of virgin wt mice . A . Representative carmine - alum stained images of mouse mammary glands generated from wt or Sharpincpdm epithelial tissue transplants . B . Quantification of transplant growth take - on - rate ( n = 17 ) . C . Quantification of fat pad filling rate of grown transplants ( n = 7 - 10 ) in wt hosts . D . Monitoring of mammary gland differentiation during pregnancy following transplantation of wt or Sharpincpdm mouse mammary epithelium into wt hosts . Host animals were subsequently mated and mammary glands isolated at P15 . Representative Carmine - alum stained mammary gland whole mounts generated from wt and cdpm mammary epithelial transplants ( upper panel ) , and magnifications of the branched ductal epithelium ( lower panel ) are shown ( n = 5 mice ) . Scale bars represent 1 mm . E . Representative images of carmine - alum stained mouse mammary gland whole mounts generated from 7 weeks old S100a4 - Cre ; Sharpinfl / fl conditional knockout mice and their littermate controls ( S100a4 - Cre ; Sharpin fl / + ) F . Quantification of the normalized Sharpincpdm and S100a4 - Cre ; Sharpinfl / fl mammary ductal outgrowth area relative to littermate control mice ( wt and Sharpincpdm n = 10 glands ; S100a4 - Cre ; Sharpin fl / + and S100a4 - Cre ; Sharpinfl / fl n = 8 glands ) . G . Quantification of the number of TEBs per gland ( n = 8 - 9 glands ) . Mean ± SEM .

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 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

Figure 3A-C. Monitoring of mammary gland development following transplantation of wt or Sharpincpdm mouse mammary epithelium in the mammary fat pads (epithelium-free) of virgin wt mice. A. Representative carmine-alum stained images of mouse mammary glands generated from wt or Sharpincpdm epithelial tissue transplants. B. Quantification of transplant growth take-on-rate (n = 17). C. Quantification of fat pad filling rate of grown transplants (n = 7 - 10) in wt hosts.D. Monitoring of mammary gland differentiation during pregnancy following transplantation of wt or Sharpincpdm mouse mammary epithelium into wt hosts. Host animals were subsequently mated and mammary glands isolated at P15. Representative Carmine-alum stained mammary gland whole mounts generated from wt and cdpm mammary epithelial transplants (upper panel), and magnifications of the branched ductal epithelium (lower panel) are shown (n = 5 mice). Scale bars represent 1 mm.E. Representative images of carmine-alum stained mouse mammary gland whole mounts generated from 7 weeks old S100a4-Cre; Sharpinfl/fl conditional knockout mice and their littermate controls (S100a4-Cre; Sharpin fl/+) F. Quantification of the normalized Sharpincpdm and S100a4-Cre; Sharpinfl/fl mammary ductal outgrowth area relative to littermate control mice (wt and Sharpincpdm n = 10 glands; S100a4-Cre; Sharpin fl/+ and S100a4-Cre; Sharpinfl/fl n = 8 glands). G. Quantification of the number of TEBs per gland (n = 8 - 9 glands). Mean ± SEM.


Figure 4A . Second harmonic generation ( SHG ) and unfiltered multiphoton ( MP ) imaging of collagen fibres surrounding TEBs ( dashed line ) in 6 - 7 weeks old wt and Sharpincpdm mouse mammary glands ( n = 6 - 9 mice , 5 - 13 TEBs analysed per mouse ; scale bars represent 100 µm ) . Magnified images of collagen fibres are also shown ( scale bars represent 50 µm ) . B . Frequency of clear collagen bundles around TEBs per mouse ( Mean ± SEM , n = 6 - 9 mice ) . C . AFM indentation analysis of stromal stiffness in 6 - 7 weeks old wt and Sharpincpdm mouse mammary gland tissue sections ( n = 3 mice ; 3 - 6 sample sections per mouse and 192 indentation curves per sample ) .

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 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

Figure 4A. Second harmonic generation (SHG) and unfiltered multiphoton (MP) imaging of collagen fibres surrounding TEBs (dashed line) in 6-7 weeks old wt and Sharpincpdm mouse mammary glands (n = 6 - 9 mice, 5 - 13 TEBs analysed per mouse; scale bars represent 100 µm). Magnified images of collagen fibres are also shown (scale bars represent 50 µm). B. Frequency of clear collagen bundles around TEBs per mouse (Mean ± SEM, n = 6 - 9 mice).C. AFM indentation analysis of stromal stiffness in 6-7 weeks old wt and Sharpincpdm mouse mammary gland tissue sections (n = 3 mice; 3 - 6 sample sections per mouse and 192 indentation curves per sample).


Figure 5A . Ingenuity Pathway Analysis of differential gene expression ( fold - change ( FC ) > 1 . 5 , p < 0 . 05 ) in wt and Sharpincpdm MSFs ( passage 0 ) . The differentially expressed genes implicated in the most significantly altered canonical pathways ( leukocyte adhesion and transmigration , and tissue fibrosis ) are shown . Three independent cell isolation replicates were compared pairwise . FC colour scale represents a range from 0 − 4 . B . Analysis of collagen degradation on cross - bow - shaped micropatterns coated with the DQ collagen substrate . Mean intensity projection images of the DQ collagen fluorescence ( top panel ; cell area is outlined by dashed line ; original integer range 0 − 255 ) and single confocal images of the fibrinogen - Alexa647 - marked micropatterns ( bottom panel ) are shown ( n ≥ 53 from four independent experiments ) . Scale bars represent 10 μm . C - D . Collagen plug contraction assay using wt and Sharpincpdm MSFs or wt MSFs with SHARPIN siRNA silencing C . Representative light microscopy images of the collagen plugs three days post cell culture . D . Quantification of the average collagen plug area 1 − 3 days after seeding ( n = 3 - 6 ) . Scale bars represent 2 mm . E . SHARPIN protein level after silencing with negative control siRNA or SHARPIN siRNA in wt MSFs . Tubulin was used as loading control . F - I . Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen - coated matrices . F . Representative images of traction forces observed in wt and Sharpincpdm cells ( colour scale units : Pa ; cell boundaries are indicated by dashed lines ) . G . Quantification of strain energy per cell ( n = 8 − 11 cells from two independent experiments ) . F - I . Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen - coated matrices . H . Representative images of the traction forces and mCherry fluorescence of mCherry - Ctrl transfected wt cells and mCherry - Ctrl or mCherry - SHARPIN transfected Sharpincpdm cells . Scale bars represent 20 µm . I . Quantification of strain energy per cell ( wt mCherry control n = 26 ; Sharpincpdm mCherry - Ctrl n = 21 ; Sharpincpdm mCherry - SHARPIN n = 24 cells from five independent experiments ) . J - K . Focal adhesion dynamics . J . Visualization of GFP - Paxillin dynamics in a wt and a Sharpincpdm cell over time . Colour scale represents a range from early to late occurring focal adhesions . K . Quantification of focal adhesion average size , assembly rate and disassembly rate ( wt n = 17527 , 5250 , 5311 FAs ; and Sharpincpdm n = 9052 , 2790 , 3264 FAs , respectively from two independent experiments ) . Mean ± SEM .

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 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 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 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 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 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

Figure 5A. Ingenuity Pathway Analysis of differential gene expression (fold-change (FC) > 1.5, p < 0.05) in wt and Sharpincpdm MSFs (passage 0). The differentially expressed genes implicated in the most significantly altered canonical pathways (leukocyte adhesion and transmigration, and tissue fibrosis) are shown. Three independent cell isolation replicates were compared pairwise. FC colour scale represents a range from 0−4.B. Analysis of collagen degradation on cross-bow-shaped micropatterns coated with the DQ collagen substrate. Mean intensity projection images of the DQ collagen fluorescence (top panel; cell area is outlined by dashed line; original integer range 0 − 255) and single confocal images of the fibrinogen-Alexa647-marked micropatterns (bottom panel) are shown (n ≥ 53 from four independent experiments). Scale bars represent 10 μm.C-D. Collagen plug contraction assay using wt and Sharpincpdm MSFs or wt MSFs with SHARPIN siRNA silencing C. Representative light microscopy images of the collagen plugs three days post cell culture. D. Quantification of the average collagen plug area 1−3 days after seeding (n = 3-6). Scale bars represent 2 mm.E. SHARPIN protein level after silencing with negative control siRNA or SHARPIN siRNA in wt MSFs. Tubulin was used as loading control.F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. F. Representative images of traction forces observed in wt and Sharpincpdm cells (colour scale units: Pa; cell boundaries are indicated by dashed lines). G. Quantification of strain energy per cell (n = 8 − 11 cells from two independent experiments).F-I. Traction force microscopy analysis of wt and Sharpincpdm MSFs on collagen-coated matrices. H. Representative images of the traction forces and mCherry fluorescence of mCherry-Ctrl transfected wt cells and mCherry-Ctrl or mCherry-SHARPIN transfected Sharpincpdm cells. Scale bars represent 20 µm. I. Quantification of strain energy per cell (wt mCherry control n = 26; Sharpincpdm mCherry-Ctrl n = 21; Sharpincpdm mCherry-SHARPIN n = 24 cells from five independent experiments).J-K. Focal adhesion dynamics. J. Visualization of GFP-Paxillin dynamics in a wt and a Sharpincpdm cell over time. Colour scale represents a range from early to late occurring focal adhesions. K. Quantification of focal adhesion average size, assembly rate and disassembly rate (wt n =17527, 5250, 5311 FAs; and Sharpincpdm n= 9052, 2790, 3264 FAs, respectively from two independent experiments). Mean ± SEM.


Figure 6A . Quantification of soluble collagen levels in wt and Sharpincpdm MSF cultures on days 2 , 5 and 7 post serum removal ( n = 6 from two independent experiments ) . B - C . Analysis of the collagen network in cell - derived matrices ( CDMs ) produced by wt and Sharpincpdm MSFs using COL1A1 immunolabelling . B . Maximum intensity projections of confocal images are shown . Scale bars represent 50 μm . C . Quantification of distinct collagen fibre bundles in wt and Sharpincpdm CDM samples ( n = 5 ) . Mean ± SEM .

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 O O 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 6A. Quantification of soluble collagen levels in wt and Sharpincpdm MSF cultures on days 2, 5 and 7 post serum removal (n = 6 from two independent experiments).B-C. Analysis of the collagen network in cell-derived matrices (CDMs) produced by wt and Sharpincpdm MSFs using COL1A1 immunolabelling. B. Maximum intensity projections of confocal images are shown. Scale bars represent 50 μm. C. Quantification of distinct collagen fibre bundles in wt and Sharpincpdm CDM samples (n = 5). Mean ± SEM.


Figure 1 ( A , B ) ( A ) Representative IHC images and ( B ) semi quantification of CRMP4 protein in human spinal cords ( SC ) cross sections from 2 control patients and 3 ALS patients . We analyzed total of 7 SC sections of controls and 14 SC sections of ALS patients , Data presented as mean ± SE . DAB : labeled CRMP4 . Scale bar : left images 20 µm , right insets 10 µm . Mann - Whitney test * * * p = 0 . 0003 . ( C ) Representative images of P90 SC cross sections of SOD1G93A and WT mice . Red : denotes NeuN , Green : denotes CRMP4 . Scale bar : 10 μm . ( D ) Quantification of the percentage of CRMP4 positive SC neurons in 3 WT VS . 3 SOD1G93A mice . We monitored CRMP4 expression in total of 108 cells in WT condition and 123 cells in SOD1G93A , an average of 36 or 41 cells in each repeat respectively . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0161 .

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 O O O O O O O O O O O O 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) (A) Representative IHC images and (B) semi quantification of CRMP4 protein in human spinal cords (SC) cross sections from 2 control patients and 3 ALS patients. We analyzed total of 7 SC sections of controls and 14 SC sections of ALS patients, Data presented as mean ±SE. DAB: labeled CRMP4. Scale bar: left images 20 µm, right insets 10 µm. Mann-Whitney test ***p = 0.0003.(C) Representative images of P90 SC cross sections of SOD1G93A and WT mice. Red: denotes NeuN, Green: denotes CRMP4. Scale bar: 10 μm. (D) Quantification of the percentage of CRMP4 positive SC neurons in 3 WT VS. 3 SOD1G93A mice. We monitored CRMP4 expression in total of 108 cells in WT condition and 123 cells in SOD1G93A, an average of 36 or 41 cells in each repeat respectively. Student's t-test, n = 3, Data presented as mean ±SE, *p = 0.0161.


Figure 2 ( A ) Representative images of ALS patient or non - ALS human control intra - muscular nerves . Red : denotes NFH , Green : denotes CRMP4 , White : denotes co - localization area using Imaris software . Scale bar : 20 μm . ( B ) Quantification of CRMP4 intensity levels in NFH positive intra - muscular distal nerves from 5 non - ALS controls and 4 sALS patients . We analyzed 40 terminal axons from the healthy samples ( ~ 8 axons per sample ) and 36 terminal axons from sALS samples ( ~ 8 axons per sample ) . Data presented as mean ± SE . Student ' s t - test , * p = 0 . 0475 . ( C ) Representative images of SOD1G93A / ChAT : : tdTomato or WTChAT : : tdTomato neuromuscular junctions at P90 . White : denotes BTX , Red : denotes direct ChAT , Green : denotes CRMP4 , Yellow : denotes Z projection of 3D Imaris co - localization of CRMP4 and ChAT . Scale bar : 10 µm . ( D ) Quantification of CRMP4 positive NMJs in gastrocnemius muscles from 3 WT or 3 SOD1G93A P90 mice . Total of 44 NMJ ' s in WT condition and 60 NMJ ' s in SOD1G93A condition . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0157 . ( E ) Quantification of the percent of partially denervated NMJ ' s in the presence or absence of CRMP4 immunostaining in 3 different SOD1G93A mice . We counted 24 NMJ ' s in SOD1G93A CRMP4 negatives and 67 NMJ ' s in SOD1G93A CRMP4 positives . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0352 . ( F ) Representative images of P90 SOD1G93A and WT sciatic nerves . Red : denotes NFH , Blue : denotes GFAP and green denotes CRMP4 , Yellow : denotes the Z projection of 3D Imaris co - localization of CRMP4 and NFH . Scale bar : 5 μm . ( G ) Quantification of the co - localization area of CRMP4 with NFH in the sciatic nerve in 3 SOD1G93A mice compared to 3 WT mice using Imaris analysis . 14 WT sciatic nerve sections and 11 SOD1G93A sections were monitored . Data presented as mean ± SE . Student ' s t - test , n = 3 , * * * * p < 0 . 0001 .

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 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 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

Figure 2(A) Representative images of ALS patient or non-ALS human control intra-muscular nerves. Red: denotes NFH, Green: denotes CRMP4, White: denotes co-localization area using Imaris software. Scale bar: 20 μm. (B) Quantification of CRMP4 intensity levels in NFH positive intra-muscular distal nerves from 5 non-ALS controls and 4 sALS patients. We analyzed 40 terminal axons from the healthy samples (~8 axons per sample) and 36 terminal axons from sALS samples (~8 axons per sample). Data presented as mean ±SE. Student's t-test, *p = 0.0475. (C) Representative images of SOD1G93A/ChAT::tdTomato or WTChAT::tdTomato neuromuscular junctions at P90. White: denotes BTX, Red: denotes direct ChAT, Green: denotes CRMP4, Yellow: denotes Z projection of 3D Imaris co-localization of CRMP4 and ChAT. Scale bar: 10 µm. (D) Quantification of CRMP4 positive NMJs in gastrocnemius muscles from 3 WT or 3 SOD1G93A P90 mice. Total of 44 NMJ's in WT condition and 60 NMJ's in SOD1G93A condition. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0157. (E) Quantification of the percent of partially denervated NMJ's in the presence or absence of CRMP4 immunostaining in 3 different SOD1G93A mice. We counted 24 NMJ's in SOD1G93A CRMP4 negatives and 67 NMJ's in SOD1G93A CRMP4 positives. Student's t-test, n=3, Data presented as mean ±SE, *p = 0.0352. (F) Representative images of P90 SOD1G93A and WT sciatic nerves. Red: denotes NFH, Blue: denotes GFAP and green denotes CRMP4, Yellow: denotes the Z projection of 3D Imaris co-localization of CRMP4 and NFH. Scale bar: 5 μm. (G) Quantification of the co-localization area of CRMP4 with NFH in the sciatic nerve in 3 SOD1G93A mice compared to 3 WT mice using Imaris analysis. 14 WT sciatic nerve sections and 11 SOD1G93A sections were monitored. Data presented as mean ±SE. Student's t-test, n=3, ****p<0.0001.


Figure 3 ( A ) Representative images of healthy or C9orf72 iPSC - derived MNs treated with Sema3A or untreated in the distal compartment , 6 hours post treatment . Red : denotes Tubulin , Green : denotes CRMP4 . Scale bar : 5 μm . ( B ) Quantification of CRMP4 intensity levels in healthy or C9orf72 iPSC - derived MNs with Sema3A treatment or untreated . 14 untreated healthy axons , 12 healthy axons with Sema3A treatment , 49 untreated C9orf72 axons and 31 C9orf72 axons with Sema3A treatment were monitored from 3 different chambers . One - way ANOVA , Tukey ' s multiple comparisons test , n = 3 , Data presented as mean ± SE , * p = 0 . 0338 ; * * * * p < 0 . 0001 . ( D ) Representative images of healthy or C9orf72 human - derived MN cell somata with Sema3A treatment , Sema3A + dynein inhibitor treatment , or untreated . Gray : denotes CTX , Green : denotes CRMP4 . Scale bar : 5μm . ( E ) Quantification of CRMP4 intensity ( normalized to GAPDH + mCherry / area ) levels at the somata of healthy or C9orf72 human - derived MN after Sema3A treatment , Sema3A + dynein inhibitor treatment , or untreated . Analysis performed in 3 independent chambers per condition . 19 healthy untreated cell somata , 26 healthy cell somata with Sema3A treatment , 20 healthy cell somata with Sema3A + dynein inhibitor treatment and 14 C9orf72 cell somata from each condition were monitored . One - way ANOVA , Newman - Keuls multiple comparisons test , n = 3 , Data presented as mean ± SE , * p = 0 . 0207 ; * * * p = 0 . 0004 . ( F ) Representative images of healthy or C9orf72 human - derived MN proximal axons with Sema3A treatment , Sema3A + dynein inhibitor treatment or untreated control . Green : denotes CRMP4 , Red : denotes GAPDH . Scale bar : 5μm . ( G ) Quantification of CRMP4 intensity levels ( normalized to GAPDH + mCherry / area ) at the proximal axons in healthy or C9orf72 human - derived MN after Sema3A treatment , Sema3A + dynein inhibitor treatment , or untreated control . Analysis performed from 3 independent chambers in each condition . 21 healthy untreated proximal axons , 24 healthy proximal axons with Sema3A treatment , 16 healthy proximal axons with Sema3A + dynein inhibitor treatment , 12 C9orf72 untreated proximal axons , 8 C9orf72 proximal axons with Sema3A treatment and 13 C9orf72 proximal axons with Sema3A + dynein inhibitor treatment were monitored . One - way ANOVA , Newman - Keuls multiple comparisons test , n = 3 , Data presented as mean ± SE , * p = 0 . 0334 , * * * * p < 0 . 0001 .

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 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 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

Figure 3(A) Representative images of healthy or C9orf72 iPSC-derived MNs treated with Sema3A or untreated in the distal compartment, 6 hours post treatment. Red: denotes Tubulin, Green: denotes CRMP4. Scale bar: 5 μm. (B) Quantification of CRMP4 intensity levels in healthy or C9orf72 iPSC-derived MNs with Sema3A treatment or untreated. 14 untreated healthy axons, 12 healthy axons with Sema3A treatment, 49 untreated C9orf72 axons and 31 C9orf72 axons with Sema3A treatment were monitored from 3 different chambers. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p = 0.0338; ****p<0.0001. (D) Representative images of healthy or C9orf72 human-derived MN cell somata with Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Gray: denotes CTX, Green: denotes CRMP4. Scale bar: 5μm. (E) Quantification of CRMP4 intensity (normalized to GAPDH+mCherry/area) levels at the somata of healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated. Analysis performed in 3 independent chambers per condition. 19 healthy untreated cell somata, 26 healthy cell somata with Sema3A treatment, 20 healthy cell somata with Sema3A + dynein inhibitor treatment and 14 C9orf72 cell somata from each condition were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n=3, Data presented as mean ±SE, *p=0.0207; ***p=0.0004. (F) Representative images of healthy or C9orf72 human-derived MN proximal axons with Sema3A treatment, Sema3A + dynein inhibitor treatment or untreated control. Green: denotes CRMP4, Red: denotes GAPDH. Scale bar: 5μm. (G) Quantification of CRMP4 intensity levels (normalized to GAPDH+mCherry/area) at the proximal axons in healthy or C9orf72 human-derived MN after Sema3A treatment, Sema3A + dynein inhibitor treatment, or untreated control. Analysis performed from 3 independent chambers in each condition. 21 healthy untreated proximal axons, 24 healthy proximal axons with Sema3A treatment, 16 healthy proximal axons with Sema3A + dynein inhibitor treatment, 12 C9orf72 untreated proximal axons, 8 C9orf72 proximal axons with Sema3A treatment and 13 C9orf72 proximal axons with Sema3A + dynein inhibitor treatment were monitored. One-way ANOVA, Newman-Keuls multiple comparisons test, n = 3, Data presented as mean ±SE, *p =0.0334, ****p<0.0001.


Figure 4 ( F ) Upper panel - Immunoprecipitation assay with anti ‐ Flag antibody followed by Western blot analysis of dynactin ( p150 ) in COS7 cells overexpressing Flag - CRMP4 ( size of ~ 65 KDa ) . Lower panel - Total input ( size of ~ 150 KDa ) . ( G ) Quantification of the blot in F . The dynactin intensity band was normalized to the Flag - CRMP4 intensity band in each repeat . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0299 . ( H ) Immunoprecipitation of DIC ( size of ~ 75 KDa ) followed by Western blot analysis of CRMP4 ( size of ~ 64 KDa ) in COS7 cells that were transfected with CRMP4 and AAV9 - 50aa or its control . IgG antibody was used as a control . ( I ) Quantification of the blot in H from 3 independent repeats . The CRMP4 intensity band was normalized to the DIC intensity band in each repeat . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p = 0 . 0479 .

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 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

Figure 4(F) Upper panel - Immunoprecipitation assay with anti‐Flag antibody followed by Western blot analysis of dynactin (p150) in COS7 cells overexpressing Flag-CRMP4 (size of ~65 KDa). Lower panel - Total input (size of ~150 KDa). (G) Quantification of the blot in F. The dynactin intensity band was normalized to the Flag-CRMP4 intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0299. (H) Immunoprecipitation of DIC (size of ~75 KDa) followed by Western blot analysis of CRMP4 (size of ~64 KDa) in COS7 cells that were transfected with CRMP4 and AAV9-50aa or its control. IgG antibody was used as a control. (I) Quantification of the blot in H from 3 independent repeats. The CRMP4 intensity band was normalized to the DIC intensity band in each repeat. Student's t-test, n=3, Data presented as mean ±SE, *p=0.0479.


Figure 5 ( E ) Representative images from the proximity ligation assay ( For explanation of PLA technique ; please refer to method section ) for CRMP4 and dynein in SODG93A and WT primary MNs axons that were exposed to either control or Sema3A 8h post treatment . Scale bar : 5µm . ( F ) Quantification of the CRMP4 - DIC puncta number per primary motor neuron axon in each condition . We analyzed ~ 20 axons per condition from 3 independent chambers per group ( One - way ANOVA , Tukey ' s multiple comparisons test , n = 3 , Data presented as mean ± SE , * * p = 0 . 01 * p = 0 . 04 ) ( G ) Representative images of proximity ligation assay for CRMP4 and dynein in healthy and C9orf72 human - derived proximal axons post peptides treatment , Sema3A treatment , Sema3A + peptides treatment or untreated controls . Scale bar : 5µm . ( H ) Quantification of the CRMP4 - DIC puncta number per axon in healthy or C9orf72 human - derived MN proximal axons after Sema3A treatment , Sema3A + peptides treatment or untreated controls . Data collected from 3 independent chambers in each condition . Total of 37 healthy untreated proximal axons , 61 healthy proximal axons with peptides treatment , 59 healthy proximal axons with Sema3A treatment and 52 healthy proximal axons with Sema3A + peptides treatment . 67 C9orf72 untreated proximal axons , 63 C9orf72 proximal axons with peptides treatment , 41 C9orf72 proximal axons with Sema3A treatment and 50 C9orf72 proximal axons with Sema3A + peptides treatment monitored . Data presented as mean ± SE . One - way ANOVA , Tukey ' s multiple comparisons test , n = 3 , * * * * p < 0 . 0001 .

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 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

Figure 5(E) Representative images from the proximity ligation assay (For explanation of PLA technique; please refer to method section) for CRMP4 and dynein in SODG93A and WT primary MNs axons that were exposed to either control or Sema3A 8h post treatment. Scale bar: 5µm. (F) Quantification of the CRMP4-DIC puncta number per primary motor neuron axon in each condition. We analyzed ~20 axons per condition from 3 independent chambers per group (One-way ANOVA, Tukey's multiple comparisons test, n=3, Data presented as mean ±SE, **p=0.01 *p=0.04) (G) Representative images of proximity ligation assay for CRMP4 and dynein in healthy and C9orf72 human-derived proximal axons post peptides treatment, Sema3A treatment, Sema3A + peptides treatment or untreated controls. Scale bar: 5µm. (H) Quantification of the CRMP4-DIC puncta number per axon in healthy or C9orf72 human-derived MN proximal axons after Sema3A treatment, Sema3A + peptides treatment or untreated controls. Data collected from 3 independent chambers in each condition. Total of 37 healthy untreated proximal axons, 61 healthy proximal axons with peptides treatment, 59 healthy proximal axons with Sema3A treatment and 52 healthy proximal axons with Sema3A + peptides treatment. 67 C9orf72 untreated proximal axons, 63 C9orf72 proximal axons with peptides treatment, 41 C9orf72 proximal axons with Sema3A treatment and 50 C9orf72 proximal axons with Sema3A + peptides treatment monitored. Data presented as mean ±SE. One-way ANOVA, Tukey's multiple comparisons test, n=3, ****p<0.0001.


Figure 6 ( A ) Representative images of CTX signal in healthy and C9orf72 human IPSC - derived MNs before and after Sema3A application . Green : denotes CTX - positive cells . Yellow circles are numbered CTX positive cells . Purple circles are cells that are missing post Sema3A treatment . Scale bar : 40 μm . ( B - C ) Quantification of CTX signal in healthy and C9orf72 IPSC - derived MNs before and 3 days after applying Sema3A to distal compartment , compared with untreated control . 3 independent chambers in each condition were analyzed . Average of ~ 150 neurons per condition monitored . Student ' s t - test , n = 3 , Data presented as mean ± SE , * p < 0 . 05 . ( D ) Representative images of CTX signal in WT or SOD1G93A primary MNs before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor + Sema3A , Dynasore + Sema3A or untreated . Green : denotes CTX - positive cells . Yellow circles are numbered CTX positive cells . Purple circles are cells that are missing post Sema3A treatment . Scale bar : 30 μm . ( E ) Quantification of CTX signal in a SOD1G93A explant before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor + Sema3A , Dynasore + Sema3A . 3 independent chambers in each condition were analyzed . ~ 200 neurons were monitored per each condition . One - way ANOVA , Tukey ' s multiple comparisons test , n = 3 , Data presented as mean ± SE , * p < 0 . 05 , * * p < 0 . 01 . Dynein inhibitor and Dynasore treatments were used as a negative control .

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 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

Figure 6(A) Representative images of CTX signal in healthy and C9orf72 human IPSC-derived MNs before and after Sema3A application. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 40 μm. (B-C) Quantification of CTX signal in healthy and C9orf72 IPSC-derived MNs before and 3 days after applying Sema3A to distal compartment, compared with untreated control. 3 independent chambers in each condition were analyzed. Average of ~150 neurons per condition monitored. Student's t-test, n=3, Data presented as mean ±SE, *p<0.05. (D) Representative images of CTX signal in WT or SOD1G93A primary MNs before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A or untreated. Green: denotes CTX-positive cells. Yellow circles are numbered CTX positive cells. Purple circles are cells that are missing post Sema3A treatment. Scale bar: 30 μm. (E) Quantification of CTX signal in a SOD1G93A explant before and 2 days after Sema3A application to the distal compartment in the presence of either Dynein inhibitor+Sema3A, Dynasore+Sema3A. 3 independent chambers in each condition were analyzed. ~200 neurons were monitored per each condition. One-way ANOVA, Tukey's multiple comparisons test, n = 3, Data presented as mean ±SE, *p<0.05, **p<0.01. Dynein inhibitor and Dynasore treatments were used as a negative control.


Figure 7 ( A ) Representative images and insets of P90 WT and SOD1G93A SC cross sections at P90 . Blue : denotes DAPI , Red : denoted NeuN , and White : denotes activated caspase 3 . Scale bar : 20 µm . ( C ) Representative images of P90 SOD1G93A mice SC cross sections that were injected with AAV9 - GFP / AAV9 - 50aa - GFP . Blue : denotes DAPI , Red : denoted NeuN , and White : denotes activated caspase 3 . Scale bar : 10 µm .

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

Figure 7(A) Representative images and insets of P90 WT and SOD1G93A SC cross sections at P90. Blue: denotes DAPI, Red: denoted NeuN, and White: denotes activated caspase 3. Scale bar: 20 µm.(C) Representative images of P90 SOD1G93A mice SC cross sections that were injected with AAV9-GFP/AAV9-50aa-GFP. Blue: denotes DAPI, Red: denoted NeuN, and White: denotes activated caspase 3. Scale bar: 10 µm.


Figure 5 ( A , B ) Net charge distribution ( black ) of αSyn ( A ) and Aß ( B ) , plotted together with the binding distribution of wild - type SERF2 ( blue ) and the SERF2 charge mutant ( red ) . Blue boxes represent the SERF2 interacting regions . ( C ) ThT - monitored amyloid kinetics of 50uM alpha - synuclein in the presence of either wild - type SERF2 or mutant SERF2 in a 1 : 1 ratio . The average of three biological replicates is represented and error bars indicate mean ± SD . ( D ) ThT - monitored amyloid kinetics of 1uM amyloid - beta in the presence of either wild - type SERF2 or mutant SERF2 in a 1 : 4 ratio . The average of four technical replicates is represented and error bars indicate mean ± SD . ( E ) Net charge distribution of hIAPP ( black ) , plotted together with the binding distribution of wild - type SERF2 ( blue ) and the SERF2 charge mutant ( red ) . ( F ) ThT - monitored amyloid kinetics of 2uM human islet amyloid polypeptide in the presence of either wild - type SERF2 or mutant SERF2 in a 1 : 4 ratio . The average of five technical replicates is represented and error bars indicate mean ± SD . ( G ) Model and charge distribution graph of HA - Q74 and muHA - Q74 constructs . ( H ) Mutations and charge distribution in the SERF2 control mutant and SERF2 charge mutant proteins . Filter Trap with 5 - fold serial dilution and quantification of crude protein extract from SERF2 CRISPR deletion mutant cells transiently transfected with HA - polyQ74 and either wild - type SERF2 , empty vector or SERF2 charge mutant ( I ) , Western blots for HA - Q74 , SERF2 and Tubulin expression were included as controls For all Filter Trap assays , the depicted blots are from one representative experiment of four biological replicates . The average of four biological replicates with each three or four technical replicates is represented in the graphs . Data are represented as mean ± SD , significance was calculated using a one - way ANOVA , followed by a post - hoc Bonferroni multiple comparisons test . ( I ) p < 0 . 0001 , * * p < 0 . 01 ; * * * p < 0 . 001 . Filter Trap with 5 - fold serial dilution and quantification of crude protein extract from SERF2 CRISPR deletion mutant cells transiently transfected with HA - polyQ74 and either wild - type SERF2 , empty vector or SERF2 control mutant ( J ) or SERF2 charge mutant . Western blots for HA - Q74 , muHA - Q74 , SERF2 and Tubulin expression were included as controls For all Filter Trap assays , the depicted blots are from one representative experiment of four biological replicates . The average of four biological replicates with each three or four technical replicates is represented in the graphs . Data are represented as mean ± SD , significance was calculated using a one - way ANOVA , followed by a post - hoc Bonferroni multiple comparisons test . ( J ) = 0 . 0003 , ( K ) p = 0 . 9130 * * p < 0 . 01 ; * * * p < 0 . 001 .

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 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 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 B-PANEL_START 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 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

Figure 5(A,B) Net charge distribution (black) of αSyn (A) and Aß (B), plotted together with the binding distribution of wild-type SERF2 (blue) and the SERF2 charge mutant (red). Blue boxes represent the SERF2 interacting regions.(C) ThT-monitored amyloid kinetics of 50uM alpha-synuclein in the presence of either wild-type SERF2 or mutant SERF2 in a 1:1 ratio. The average of three biological replicates is represented and error bars indicate mean ± SD.(D) ThT-monitored amyloid kinetics of 1uM amyloid-beta in the presence of either wild-type SERF2 or mutant SERF2 in a 1:4 ratio. The average of four technical replicates is represented and error bars indicate mean ± SD.(E) Net charge distribution of hIAPP (black), plotted together with the binding distribution of wild-type SERF2 (blue) and the SERF2 charge mutant (red).(F) ThT-monitored amyloid kinetics of 2uM human islet amyloid polypeptide in the presence of either wild-type SERF2 or mutant SERF2 in a 1:4 ratio. The average of five technical replicates is represented and error bars indicate mean ± SD.(G) Model and charge distribution graph of HA-Q74 and muHA-Q74 constructs.(H) Mutations and charge distribution in the SERF2 control mutant and SERF2 charge mutant proteins.Filter Trap with 5-fold serial dilution and quantification of crude protein extract from SERF2 CRISPR deletion mutant cells transiently transfected with HA-polyQ74 and either wild-type SERF2, empty vector or SERF2 charge mutant (I), Western blots for HA-Q74, SERF2 and Tubulin expression were included as controls For all Filter Trap assays, the depicted blots are from one representative experiment of four biological replicates. The average of four biological replicates with each three or four technical replicates is represented in the graphs. Data are represented as mean ± SD, significance was calculated using a one-way ANOVA, followed by a post-hoc Bonferroni multiple comparisons test. (I) p<0.0001, **p < 0.01; ***p < 0.001.Filter Trap with 5-fold serial dilution and quantification of crude protein extract from SERF2 CRISPR deletion mutant cells transiently transfected with HA-polyQ74 and either wild-type SERF2, empty vector or SERF2 control mutant (J) or SERF2 charge mutant. Western blots for HA-Q74, muHA-Q74, SERF2 and Tubulin expression were included as controls For all Filter Trap assays, the depicted blots are from one representative experiment of four biological replicates. The average of four biological replicates with each three or four technical replicates is represented in the graphs. Data are represented as mean ± SD, significance was calculated using a one-way ANOVA, followed by a post-hoc Bonferroni multiple comparisons test. (J)=0.0003, (K) p=0.9130 **p < 0.01; ***p < 0.001.


Figure 6 ( B ) Representative images of Q40 and Q40 ; moag - 4 charge mutant animals . Scale bar , 75 mm . ( C ) Representative quantification of the number of aggregates in Q40 worms with moag - 4 deletion or expression of either wild - type moag - 4 , moag - 4 charge mutant or moag - 4 ctrl mutant . The results shown are representative experiments of three biological replicates of n = 20 worms in L4 stage . Data are represented as mean ± SD and significance was calculated using a one - way ANOVA , followed by a post - hoc Bonferroni multiple comparisons test . * * * * p < 0 . 0001 . ( D ) Representative TCSPC - FLIM images from the indicated strains . The dashed lines mark the retro vesicular ganglion and arrowheads indicate aggregates . Scale bar is 50 µm . ( E ) Number of aggregates per retro vesicular ganglion from three independent cohorts of the indicated Aβ1 - 42 strains with moag - 4 deletion mutant ( del ( tm4909 ) ) n = 11 , moag - 4 control mutant ( ctrl mutant ) n = 12 , moag - 4 charge mutant ( charge mutant ) n = 17 , and wild - type n = 15 . Data are represented as mean ± SD . One - way ANOVA + Bonferroni post - hoc test for significance ( * p < 0 . 05 , * * * * p < 0 . 0001 ) .

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 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 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

Figure 6(B) Representative images of Q40 and Q40;moag-4 charge mutant animals. Scale bar, 75 mm. (C) Representative quantification of the number of aggregates in Q40 worms with moag-4 deletion or expression of either wild-type moag-4, moag-4 charge mutant or moag-4 ctrl mutant. The results shown are representative experiments of three biological replicates of n=20 worms in L4 stage. Data are represented as mean ± SD and significance was calculated using a one-way ANOVA, followed by a post-hoc Bonferroni multiple comparisons test. ****p < 0.0001.(D) Representative TCSPC-FLIM images from the indicated strains. The dashed lines mark the retro vesicular ganglion and arrowheads indicate aggregates. Scale bar is 50 µm.(E) Number of aggregates per retro vesicular ganglion from three independent cohorts of the indicated Aβ1-42 strains with moag-4 deletion mutant (del (tm4909)) n=11, moag-4 control mutant (ctrl mutant) n=12, moag-4 charge mutant (charge mutant) n=17, and wild-type n=15. Data are represented as mean ± SD. One-way ANOVA + Bonferroni post-hoc test for significance (* p < 0.05, **** p < 0.0001).


Figure 1B Mass spectrometry analysis of proteins co - purified with Ccq1HA . Shown is a volcano plot for proteins significantly enriched in Ccq1HA relative to untagged Ccq1 ( left panel : WT cells ; right panel : rik1 ∆ cells ) . Members of the CLRC complex and shelterin are highlighted in red and blue , respectively . Bottom panel displays proteins enriched ( log2 ≥ 5 or pval ≤ 0 . 01 ) ChIP - qPCR analysis of epitope - tagged shelterin components in WT and mutant strains as indicated ( negative control : untagged strain ) . Positions on x - axis denote distances relative to telomeric repeats ( telomere - proximal PCR primer ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ± SEMChIP - qPCR analysis of epitope - tagged shelterin components in WT and mutant strains as indicated ( negative control : untagged strain ) . Positions on x - axis denote distances relative to telomeric repeats ( telomere - proximal PCR primer ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ± SEMChIP - qPCR analysis of epitope - tagged shelterin components in WT and mutant strains as indicated ( negative control : untagged strain ) . Positions on x - axis denote distances relative to telomeric repeats ( telomere - proximal PCR primer ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ± SEMF ChIP analysis of Ccq1HA and FLAG - TetR - Clr4 * ( see scheme ) . Expression and tethering of FLAG - TetR - Clr4 * are controlled by thiamine / AHT addition ( red dots indicate induced / tethered FLAG - TetR - Clr4 * ; empty circles indicate non - induced / non - bound controls ) ( n = 3 independent experiments ) Data information data are normalized to input and represented as mean ±

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 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 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 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

Figure 1B Mass spectrometry analysis of proteins co-purified with Ccq1HA. Shown is a volcano plot for proteins significantly enriched in Ccq1HA relative to untagged Ccq1 (left panel: WT cells; right panel: rik1∆ cells). Members of the CLRC complex and shelterin are highlighted in red and blue, respectively. Bottom panel displays proteins enriched (log2 ≥ 5 or pval ≤ 0.01)ChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEMChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEMChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEMF ChIP analysis of Ccq1HA and FLAG-TetR-Clr4* (see scheme). Expression and tethering of FLAG-TetR-Clr4* are controlled by thiamine/AHT addition (red dots indicate induced/tethered FLAG-TetR-Clr4*; empty circles indicate non-induced/non-bound controls) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM


Figure 2A Left : Schemes of CLRC and the shelterin complex . Middle and right : Scheme depicting the position of the ura4 + reporter gene and heterochromatin islands relative to telomeric repeats and TAS regions ; note that the minichromosomesome Ch16 does not contain TAS . B - C ChIP - qPCR analyses of H3K9me2 ( B ) and H3 ( C ) in WT and ccq1Δ cells ( n = 3 independent experiments . D Fold change of WT over ccq1Δ for H3K9me2 and H3 level Data information : In ( B , C ) , data are normalized to input and to the average of three euchromatic loci ( EC ) as internal contro and represented as mean ±

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Figure 2A Left: Schemes of CLRC and the shelterin complex. Middle and right: Scheme depicting the position of the ura4+ reporter gene and heterochromatin islands relative to telomeric repeats and TAS regions; note that the minichromosomesome Ch16 does not contain TAS. B-C ChIP-qPCR analyses of H3K9me2 (B) and H3 (C) in WT and ccq1Δ cells (n = 3 independent experiments. D Fold change of WT over ccq1Δ for H3K9me2 and H3 level Data information: In (B, C), data are normalized to input and to the average of three euchromatic loci (EC) as internal contro and represented as mean ± SEM


Figure 3ChIP - qPCR analyses of H3 in WT and mutants as indicated ( n = 3 independent experiments ) . Data information data are represented as mean ± SEChIP - qPCR analyses of H3 in WT and mutants as indicated ( n = 3 independent experiments ) . Data information data are represented as mean ± SEChIP - qPCR analyses of H3K9me2 in WT and mutants as indicated ( n = 3 independent experiments ) . Data information data are represented as mean ±

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Figure 3ChIP-qPCR analyses of H3 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SEChIP-qPCR analyses of H3 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SEChIP-qPCR analyses of H3K9me2 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SE


Figure 4A ChIP - seq reads mapped to TEL2R ( shown in reverse orientation for consistency )

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Figure 4A ChIP-seq reads mapped to TEL2R (shown in reverse orientation for consistency)


Figure 5A ChIP - qPCR analysis of H3 at endogenous TAS and a fragment spanning the TAS region from 115 - 905 bp ( relative to telomeric repeat ) inserted into the leu1 + locus ( see scheme ) . The TAS fragment was inserted into a strain that lacks endogenous TA Shown are ChIP analyses for WT ( left ) and ccq1 ∆ ( right ; note different the scale of the y - axis ) ( n = 9 - 10 independent experiments except for ectopic TAS in ccq1 ∆ strain where n = 3 ) Data information data are represented as mean ± SEB ChIP - qPCR analysis of H3 at reporter genes ( ura4 + and his3 + ) at various chromosomal locations ( see schemes ) in WT cells . TAS1 , TAS2 , and TAS3 correspond to position 116 bp , 2851 bp and 6291 bp ( relative to telomeric repeats ) , respectively ( n = 3 independent experiments ) . Data information data are represented as mean ±

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 O O O O O O O O O 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 ChIP-qPCR analysis of H3 at endogenous TAS and a fragment spanning the TAS region from 115-905 bp (relative to telomeric repeat) inserted into the leu1+ locus (see scheme). The TAS fragment was inserted into a strain that lacks endogenous TA Shown are ChIP analyses for WT (left) and ccq1∆ (right; note different the scale of the y-axis) (n = 9-10 independent experiments except for ectopic TAS in ccq1∆ strain where n = 3) Data information data are represented as mean ± SEB ChIP-qPCR analysis of H3 at reporter genes (ura4+ and his3+) at various chromosomal locations (see schemes) in WT cells. TAS1, TAS2, and TAS3 correspond to position 116 bp, 2851 bp and 6291 bp (relative to telomeric repeats), respectively (n = 3 independent experiments). Data information data are represented as mean ± SE


Figure 6A , B RT - qPCR analysis of transcript levels of TERRA ( A ) and tlh1 + ( B ) in WT strain and mutants as indicated ( double mutants with ccq1Δ are indicated by blue dot ) . Data information : In ( A , B ) , data is represented as mean ± SEM from 3 independent experiments and shown relative to WT

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Figure 6A, B RT-qPCR analysis of transcript levels of TERRA (A) and tlh1+ (B) in WT strain and mutants as indicated (double mutants with ccq1Δ are indicated by blue dot). Data information: In (A, B), data is represented as mean ± SEM from 3 independent experiments and shown relative to WT leve


Figure 7A Genomic copy number of his3 + , ura4 + and TAS1 in indicated strains harboring the reporter genes tel1L : : his3 + and tel2L : : ura4 + . Cultures from individual WT and freshly generated knockout clones ( ccq1Δ , n = 16 ; clr4Δ , n = 5 ; ccq1Δ clr4Δ , n = 5 ) were pre - grown on selective media for several days and inoculated at day 0 to grow in liquid media with regular back - dilution ( every 24 hours , approximately 7 generations ) . Samples were taken at indicated harvest times , and relative copy numbers of genomic regions were assessed by qPCR ( normalization against intrachromosomal loci ) . Black and rainbow color lines indicate WT strains and individual ccq1 + deletion mutants ( clones ) , respectivelyB qPCR analysis as in ( A ) but with strains harboring the minichromosome Ch16 m23 : : ura4 + ( ccq1Δ , n = 14 ) . Since Ch16 is unstable in ccq1Δ cell Note that Ch16 does not harbor TAS

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Figure 7A Genomic copy number of his3+, ura4+ and TAS1 in indicated strains harboring the reporter genes tel1L::his3+ and tel2L::ura4+. Cultures from individual WT and freshly generated knockout clones (ccq1Δ, n = 16; clr4Δ, n = 5; ccq1Δ clr4Δ, n = 5) were pre-grown on selective media for several days and inoculated at day 0 to grow in liquid media with regular back-dilution (every 24 hours, approximately 7 generations). Samples were taken at indicated harvest times, and relative copy numbers of genomic regions were assessed by qPCR (normalization against intrachromosomal loci). Black and rainbow color lines indicate WT strains and individual ccq1+ deletion mutants (clones), respectivelyB qPCR analysis as in (A) but with strains harboring the minichromosome Ch16 m23::ura4+ (ccq1Δ, n = 14). Since Ch16 is unstable in ccq1Δ cell Note that Ch16 does not harbor TAS sequences


figf1 ( b ) Levels of spermidine in the heads of wild - type flies fed with food supplemented by 1 or 5 mM spermidine ( Spd1mM + or Spd5mM + , respectively ) or food with no spermidine ( Spd − ) . Data are shown normalized to spermidine levels of 1 - d - old ( 1 d ) Spd − flies ( n = 4 independent experiments ; F = 14 . 08 for Spd − flies , F = 5 . 30 for Spd1mM + flies , F = 2 . 62 for Spd5mM + flies ; one - way - ANOVA with Bonferroni correction ) . 15 d , 15 d old ; 30 d , 30 d old . ( c ) Levels of putrescine in the heads of aged Spd1mM + or Spd5mM + flies compared with Spd − flies . Data are shown normalized to putrescine levels of 1 - d - old Spd − flies ( n = 4 independent experiments ; F = 24 . 31 for Spd - flies , F = 0 . 22 for Spd1mM + flies , F = 6 . 67 for Spd5mM + flies ; one - way - ANOVA with Bonferroni correction ) . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; ns indicates not significant , P > 0 . 05 . Data are presented as mean ± s . e . m .

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figf1(b) Levels of spermidine in the heads of wild-type flies fed with food supplemented by 1 or 5 mM spermidine (Spd1mM+ or Spd5mM+, respectively) or food with no spermidine (Spd−). Data are shown normalized to spermidine levels of 1-d-old (1 d) Spd− flies (n = 4 independent experiments; F = 14.08 for Spd− flies, F = 5.30 for Spd1mM+ flies, F = 2.62 for Spd5mM+ flies; one-way-ANOVA with Bonferroni correction). 15 d, 15 d old; 30 d, 30 d old.(c) Levels of putrescine in the heads of aged Spd1mM+ or Spd5mM+ flies compared with Spd− flies. Data are shown normalized to putrescine levels of 1-d-old Spd− flies (n = 4 independent experiments; F = 24.31 for Spd- flies, F = 0.22 for Spd1mM+ flies, F = 6.67 for Spd5mM+ flies; one-way-ANOVA with Bonferroni correction). *P 0.05; **P 0.01; ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.


figf2 ( a ) Aversive associative memory performance at 3 min after training ( STM ) of 3 - d - old ( 3 d ) Spd1mM + and Spd5mM + flies in comparison to Spd − flies ( n = 8 - 9 independent experiments , F = 2 . 28 , one - way - ANOVA with Bonferroni correction ) . ( b ) Aversive associative memory performance at 3 h after training ( ITM ) , ARM and ASM of 3 - d - old Spd1mM + and Spd5mM + compared with Spd − flies ( n = 8 - 9 independent experiments ; F = 2 . 05 for ITM , F = 0 . 02 for ARM , F = 1 . 74 for ASM ; one - way ANOVA with Bonferroni correction ) . ( c ) STM of 30 - d - old ( 30 d ) Spd1mM + and Spd5mM + and Spd − flies ( n = 8 - 9 independent experiments , F = 14 . 48 , one - way ANOVA with Bonferroni correction ) . ( d ) ITM , ARM and ASM of 30 - d - old Spd1mM + and Spd5mM + compared with Spd − flies ( n = 7 - 9 independent experiments ; F = 43 . 26 for ITM , F = 1 . 74 for ARM , F = 26 . 12 for ASM ; one - way ANOVA with Bonferroni correction ) . ( e ) Spermidine feeding for 10 d before measuring memory was sufficient to suppress AMI in aged ( 30 d old ) flies ( n = 9 - 11 independent experiments , F = 12 . 32 , one - way ANOVA with Bonferroni correction ) . ( f , g ) Spermidine feeding on days 21 - 30 resulted in a significant increase in the levels of spermidine and putrescine in 30 - d - old flies ( n = 3 independent experiments , F = 32 . 96 ( f ) and 64 . 77 ( g ) , one - way ANOVA with Bonferroni correction ) . The feeding regime of the flies is presented beneath the graphs , with + and − indicating food with or without spermidine . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; ns indicates not significant , P > 0 . 05 . Data are presented as mean ± s . e . m .

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 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 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 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

figf2(a) Aversive associative memory performance at 3 min after training (STM) of 3-d-old (3 d) Spd1mM+ and Spd5mM+ flies in comparison to Spd− flies (n = 8-9 independent experiments, F = 2.28, one-way-ANOVA with Bonferroni correction).(b) Aversive associative memory performance at 3 h after training (ITM), ARM and ASM of 3-d-old Spd1mM+ and Spd5mM+ compared with Spd− flies (n = 8-9 independent experiments; F = 2.05 for ITM, F = 0.02 for ARM, F = 1.74 for ASM; one-way ANOVA with Bonferroni correction).(c) STM of 30-d-old (30 d) Spd1mM+ and Spd5mM+ and Spd− flies (n = 8-9 independent experiments, F = 14.48, one-way ANOVA with Bonferroni correction).(d) ITM, ARM and ASM of 30-d-old Spd1mM+ and Spd5mM+ compared with Spd− flies (n = 7-9 independent experiments; F = 43.26 for ITM, F = 1.74 for ARM, F = 26.12 for ASM; one-way ANOVA with Bonferroni correction).(e) Spermidine feeding for 10 d before measuring memory was sufficient to suppress AMI in aged (30 d old) flies (n = 9-11 independent experiments, F = 12.32, one-way ANOVA with Bonferroni correction).(f,g) Spermidine feeding on days 21-30 resulted in a significant increase in the levels of spermidine and putrescine in 30-d-old flies (n = 3 independent experiments, F = 32.96 (f) and 64.77 (g), one-way ANOVA with Bonferroni correction). The feeding regime of the flies is presented beneath the graphs, with + and − indicating food with or without spermidine. *P 0.05, **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.


figf3 ( a ) Expression of GCaMP3 . 0 in the mushroom body of an individual fly , focused on the horizontal lobes . The red line indicates a region of interest used to determine changes in fluorescence emission . Scale bar represents 50 μm . ( b ) False color - coded image of Ca2 + activity in the horizontal mushroom body lobes shown in a . Warm colors indicate high levels , cold colors indicate low levels or no Ca2 + activity . The numbers indicate changes in fluorescence ΔF ( % ) . ( c , d ) Time course of Ca2 + increase in horizontal mushroom body lobes of 3 - d - old and 30 - d - old Spd − flies evoked by the odors 4 - methyl - cyclohexanol ( MCH ) or 3 - octanol ( OCT ) in comparison with the diluent , mineral oil ( n = 5 flies , non - parametric Mann - Whitney U test found no substantial difference between the Ca2 + increase of 3 - d - old and 30 - d - old Spd − flies evoked by the odors ) . The gray bars indicate the duration of the odor stimuli . Data are presented as mean ± s . e . m .

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figf3(a) Expression of GCaMP3.0 in the mushroom body of an individual fly, focused on the horizontal lobes. The red line indicates a region of interest used to determine changes in fluorescence emission. Scale bar represents 50 μm. (b) False color-coded image of Ca2+ activity in the horizontal mushroom body lobes shown in a. Warm colors indicate high levels, cold colors indicate low levels or no Ca2+ activity. The numbers indicate changes in fluorescence ΔF (%).(c,d) Time course of Ca2+ increase in horizontal mushroom body lobes of 3-d-old and 30-d-old Spd− flies evoked by the odors 4-methyl-cyclohexanol (MCH) or 3-octanol (OCT) in comparison with the diluent, mineral oil (n = 5 flies, non-parametric Mann-Whitney U test found no substantial difference between the Ca2+ increase of 3-d-old and 30-d-old Spd− flies evoked by the odors). The gray bars indicate the duration of the odor stimuli. Data are presented as mean ± s.e.m.


figf4 ( a ) Quantification of Atg8a proteins levels from head extracts normalized to α - tubulin ( n = 17 or 8 - 9 technical replicates from two independent biological aging replicates for all data , F = 7 . 52 , one - way - ANOVA with Tukey post - test ) . The full - length blot is shown in Supplementary Figure 9a . ( b ) Western blot analysis of poly - ubiquitinated proteins ( SDS - soluble protein fraction ) from head extracts from Spd − ( 3 and 30 d old ) flies compared with Spd5mM + ( 3 and 30 d old ) flies . Right , quantification of poly - ubiquitinated proteins levels normalized to α - tubulin ( n = 10 or 5 technical replicates from two independent biological aging replicates for all data , F = 6 . 32 , one - way ANOVA with Tukey post - test ) . The full - length blot is shown in Supplementary Figure 9b . ( c - f ) Adult brains of 3 - d - old and 30 - d - oldSpd − flies , as well as 3 - d - old and 30 - d - oldSpd5mM + fliesimmunostained for ref ( 2 ) P . Scale bar represents 25 μm . ( g ) Quantification of ref ( 2 ) P intensity in the central brain region normalized to 3 - d - oldSpd − ( n = 12 - 13 independent brains for all data , F = 26 . 58 , one - way ANOVA with Bonferroni correction ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; P > 0 . 05 . Data are presented as mean ± s . e . m .

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figf4(a) Quantification of Atg8a proteins levels from head extracts normalized to α-tubulin (n = 17 or 8-9 technical replicates from two independent biological aging replicates for all data, F = 7.52, one-way-ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9a.(b) Western blot analysis of poly-ubiquitinated proteins (SDS-soluble protein fraction) from head extracts from Spd− (3 and 30 d old) flies compared with Spd5mM+ (3 and 30 d old) flies. Right, quantification of poly-ubiquitinated proteins levels normalized to α-tubulin (n = 10 or 5 technical replicates from two independent biological aging replicates for all data, F = 6.32, one-way ANOVA with Tukey post-test). The full-length blot is shown in Supplementary Figure 9b.(c-f) Adult brains of 3-d-old and 30-d-oldSpd− flies, as well as 3-d-old and 30-d-oldSpd5mM+ fliesimmunostained for ref(2)P. Scale bar represents 25 μm. (g) Quantification of ref(2)P intensity in the central brain region normalized to 3-d-oldSpd− (n = 12-13 independent brains for all data, F = 26.58, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; P > 0.05. Data are presented as mean ± s.e.m.


figf5 ( a ) Aversive associative memory performance 3 min after training ( STM ) was markedly reduced in 3 - d - old Atg7 − / − flies ( Atg7d14 / Atg7d77 ) compared with Atg7 − / + flies ( Atg7d14 / CG5335d30 ) and wild - type flies ( Atg7 + / + ) ( n = 8 - 11 independent experiments , F = 25 . 82 , one - way ANOVA with Bonferroni correction ) . The CG5335d30 line harbors an Atg7 + chromosome related to Atg7d14 and Atg7d77 that serves as a genetic background control . ( b ) Olfactory learning was disrupted in 3 - d - old female flies homozygous for Atg8a hypomorph ( Atg8a − / − or Atg8aEP362 / Atg8aEP362 ) ( n = 8 - 9 independent experiments , F = 22 . 8 , one - way ANOVA with Bonferroni correction ) . ( c ) STM is severely impaired in 20 - d - old Atg7 − / − ( Atg7d14 / Atg7d77 ) flies and 20 - d - old Atg7 − / + heterozygous ( Atg7d14 / CG5335d30 ) when compared to age - matched wild - type flies ( n = 8 - 9 independent experiments , F = 18 . 02 , one - way ANOVA with Bonferroni correction ) . ( d ) Similarly , STM was markedly decreased in 20 - d - old Atg8a − / − ( Atg8aEP362 / Atg8aEP362 ) mutant female flies compared with control flies ( Atg8a + / + ) without any effect by spermidine feeding ( n = 8 - 9 independent experiments , F = 68 . 43 , one - way ANOVA with Bonferroni correction ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ; ns indicates not significant , P > 0 . 05 . Data are presented as mean ± s . e . m . The aversive olfactory avoidance and shock reactivity of different mutant are shown in Supplementary Table 5 .

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figf5(a) Aversive associative memory performance 3 min after training (STM) was markedly reduced in 3-d-old Atg7−/− flies (Atg7d14/Atg7d77) compared with Atg7−/+ flies (Atg7d14/CG5335d30) and wild-type flies (Atg7+/+) (n = 8-11 independent experiments, F = 25.82, one-way ANOVA with Bonferroni correction). The CG5335d30 line harbors an Atg7+ chromosome related to Atg7d14 and Atg7d77 that serves as a genetic background control.(b) Olfactory learning was disrupted in 3-d-old female flies homozygous for Atg8a hypomorph (Atg8a−/− or Atg8aEP362/Atg8aEP362) (n = 8-9 independent experiments, F = 22.8, one-way ANOVA with Bonferroni correction).(c) STM is severely impaired in 20-d-old Atg7−/− (Atg7d14/Atg7d77) flies and 20-d-old Atg7−/+ heterozygous (Atg7d14/CG5335d30) when compared to age-matched wild-type flies (n = 8-9 independent experiments, F = 18.02, one-way ANOVA with Bonferroni correction).(d) Similarly, STM was markedly decreased in 20-d-old Atg8a−/− (Atg8aEP362/Atg8aEP362) mutant female flies compared with control flies (Atg8a+/+) without any effect by spermidine feeding (n = 8-9 independent experiments, F = 68.43, one-way ANOVA with Bonferroni correction). *P 0.05, **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m. The aversive olfactory avoidance and shock reactivity of different mutant are shown in Supplementary Table 5.


figf7 ( a ) Aversive associative memory performance in 3 min after training ( STM ) in 3 - d - old female flies with pan - neuronal expression of UAS - Odc - 1 ( n = 7 independent experiments , F = 0 . 04 , one - way ANOVA with Bonferroni correction ) . ( b ) Aversive associative memory performance in 3 h after training ( ITM ) , ARM and ASM of 3 - d - old appl - driven UAS - Odc - 1 female flies compared with control flies ( n = 7 independent experiments ; F = 1 . 95 for ITM , F = 0 . 10 for ARM , F = 1 . 05 for ASM ; one - way ANOVA with Bonferroni correction ) . ( c ) Pan - neuronal expression of the UAS - Odc - 1 in a wild - type background suppressed AMI in 30 - d - old female flies ( n = 7 independent experiments , F = 21 . 35 , one - way - ANOVA with Bonferroni correction ) . ( d ) ITM , ARM and ASM of 30 - d - old female flies with pan - neuronal expression of UAS - Odc - 1 compared with their genetic controls ( n = 7 - 8 independent experiments ; F = 17 . 49 for ITM , F = 1 . 58 for ARM , F = 24 . 61 for ASM ; one - way ANOVA with Bonferroni correction ) . ( e ) Expressing Odc - 1 in just the mushroom body was sufficient to protect from STM decline in 30 - d - old flies ( n = 9 - 12 independent experiments , F = 9 . 25 , one - way ANOVA with Bonferroni correction ) . ( f ) ITM , ARM and ASM of 30 - d - old flies expressing mushroom body - specific Odc - 1 compared with their genetic controls ( n = 8 - 9 independent experiments ; F = 11 . 73 for ITM , F = 0 . 50 for ARM , F = 16 . 6 for ASM ; one - way ANOVA with Bonferroni correction ) . * * P 0 . 01 , * * * P 0 . 001 ; ns indicates not significant , P > 0 . 05 . Data are presented as mean ± s . e . m .

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figf7(a) Aversive associative memory performance in 3 min after training (STM) in 3-d-old female flies with pan-neuronal expression of UAS-Odc-1 (n = 7 independent experiments, F = 0.04, one-way ANOVA with Bonferroni correction).(b) Aversive associative memory performance in 3 h after training (ITM), ARM and ASM of 3-d-old appl-driven UAS-Odc-1 female flies compared with control flies (n = 7 independent experiments; F = 1.95 for ITM, F = 0.10 for ARM, F = 1.05 for ASM; one-way ANOVA with Bonferroni correction).(c) Pan-neuronal expression of the UAS-Odc-1 in a wild-type background suppressed AMI in 30-d-old female flies (n = 7 independent experiments, F = 21.35, one-way-ANOVA with Bonferroni correction).(d) ITM, ARM and ASM of 30-d-old female flies with pan-neuronal expression of UAS-Odc-1 compared with their genetic controls (n = 7-8 independent experiments; F = 17.49 for ITM, F = 1.58 for ARM, F = 24.61 for ASM; one-way ANOVA with Bonferroni correction).(e) Expressing Odc-1 in just the mushroom body was sufficient to protect from STM decline in 30-d-old flies (n = 9-12 independent experiments, F = 9.25, one-way ANOVA with Bonferroni correction).(f) ITM, ARM and ASM of 30-d-old flies expressing mushroom body-specific Odc-1 compared with their genetic controls (n = 8-9 independent experiments; F = 11.73 for ITM, F = 0.50 for ARM, F = 16.6 for ASM; one-way ANOVA with Bonferroni correction). **P 0.01, ***P 0.001; ns indicates not significant, P > 0.05. Data are presented as mean ± s.e.m.


Figure 1Ex vivo generation of CAR T cells . Activated human PBMC were left untransduced or incubated with CD8 - LVCD19CAR at an MOI of 2 . Five days later , expression of CD19 - CAR and CD8 were determined on CD3 + cells . Numbers indicate the percentage of cells in the respective gate . Detection of CAR T cells by vector copy numbers ( VCN ) . Genomic DNA was isolated from peritoneal cavity , spleen and blood cells . VCN were determined in technical duplicates by qPCR for two individual mice of each group . Presence of B cells in the transplanted PBMC is indicated below . Cells isolated from the peritoneal cavity ( peritoneum ) , spleen , or blood were evaluated by flow cytometry for the percentages of human CD8 + in CD3 + cells ( D ) , within the fraction of human CD45 + cells . Representative density plots are shown for the peritoneal cells . Cells isolated from the peritoneal cavity ( peritoneum ) , spleen , or blood were evaluated by flow cytometry for the percentages of CAR + or RFP + cells in the CD8 + and CD8 - fractions , respectively ( E ) within the fraction of human CD45 + cells . Representative density plots are shown for the peritoneal cells . Cells isolated from the peritoneal cavity ( peritoneum ) , spleen , or blood were evaluated by flow cytometry for the percentages of human CD19 + cells within the fraction of human CD45 + cells . Representative density plots are shown for the peritoneal cells . Mice were transplanted with B - cell depleted human PBMC and then received CD8 - LVCD19CAR ( filled circle ) or PBS ( open circle ) . As control , CD8 - LVCD19CAR or PBS were injected into mice transplanted with non - depleted PBMC . Data represent mean ± SD for all groups ( CD8 - LVCD19CAR : n = 6 in D , n = 4 in G ; CD8 - LVRFP : n = 4 ; PBS : n = 4 in G , all others n = 3 ) .

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Figure 1Ex vivo generation of CAR T cells. Activated human PBMC were left untransduced or incubated with CD8-LVCD19CAR at an MOI of 2. Five days later, expression of CD19-CAR and CD8 were determined on CD3+ cells. Numbers indicate the percentage of cells in the respective gate.Detection of CAR T cells by vector copy numbers (VCN). Genomic DNA was isolated from peritoneal cavity, spleen and blood cells. VCN were determined in technical duplicates by qPCR for two individual mice of each group. Presence of B cells in the transplanted PBMC is indicated below.Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD8+ in CD3+ cells (D), within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells.Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of CAR+ or RFP+ cells in the CD8+ and CD8- fractions, respectively (E) within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells.Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD19+ cells within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells.Mice were transplanted with B-cell depleted human PBMC and then received CD8-LVCD19CAR (filled circle) or PBS (open circle). As control, CD8-LVCD19CAR or PBS were injected into mice transplanted with non-depleted PBMC. Data represent mean ± SD for all groups (CD8-LVCD19CAR: n=6 in D, n=4 in G; CD8-LVRFP: n=4; PBS: n=4 in G, all others n=3).


Figure 2CAR T cell levels in the CD8 + and CD8 - T cells harvested from blood , spleen and bone marrow of PBS ( PBS ) or vector - injected ( CAR ) mice determined by flow cytometry . Detection of CAR T cells by determining vector copy numbers ( VCN ) in genomic DNA isolated from CD8 + enriched cells harvested from bone marrow . Detection of CAR T cells in bone marrow quantified by flow cytometry . CD19 + B cell levels in blood , spleen and bone marrow determined by flow cytometry . Relative human CD19 + B cell level in blood calculated by normalizing the levels at the day the animals were sacrificed to those before vector administration .

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Figure 2CAR T cell levels in the CD8+ and CD8- T cells harvested from blood, spleen and bone marrow of PBS (PBS) or vector-injected (CAR) mice determined by flow cytometry.Detection of CAR T cells by determining vector copy numbers (VCN) in genomic DNA isolated from CD8+ enriched cells harvested from bone marrow.Detection of CAR T cells in bone marrow quantified by flow cytometry.CD19+ B cell levels in blood, spleen and bone marrow determined by flow cytometry.Relative human CD19+ B cell level in blood calculated by normalizing the levels at the day the animals were sacrificed to those before vector administration.


Figure 3Cytokine levels in plasma of individual mice obtained from blood at 7 weeks after vector injection . The distinct symbols used for each individual mouse are identical to the ones used in Fig . 2 . Mean values ± SD of n = 2 technical replicates . Immunohistochemistry of paraffin embedded sections from the lungs , brain meninges , brain striatum , spleen and liver of the vector - injected mouse M16 ( CAR ) and a control mouse injected with PBS ( PBS ) stained against CD8 ( αCD8 ) , the CAR ( αCAR ) , CD4 ( αCD4 ) , or CD19 ( αCD19 ) . Black arrowheads point at infiltrated lymphocytes , red arrowheads at histiocytes , and the yellow line indicates B - lymphocyte rich zones .

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Figure 3Cytokine levels in plasma of individual mice obtained from blood at 7 weeks after vector injection. The distinct symbols used for each individual mouse are identical to the ones used in Fig. 2. Mean values ± SD of n=2 technical replicates.Immunohistochemistry of paraffin embedded sections from the lungs, brain meninges, brain striatum, spleen and liver of the vector-injected mouse M16 (CAR) and a control mouse injected with PBS (PBS) stained against CD8 (αCD8), the CAR (αCAR), CD4 (αCD4), or CD19 (αCD19). Black arrowheads point at infiltrated lymphocytes, red arrowheads at histiocytes, and the yellow line indicates B-lymphocyte rich zones.


Figure 1 ( C ) Blood and brain tissue levels of CNP520 in the rat after a 30 µmol / kg ( 15 . 4 mg / kg ) oral dose . Shown are unbound fraction ( main graph ) and total levels ( insert ) of CNP520 in the blood and brain , using values for rat plasma protein binding of 97 . 8 % and unspecific binding to brain homogenate of 99 % . Values are mean ± SD , with n = 5 per time point .

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Figure 1(C) Blood and brain tissue levels of CNP520 in the rat after a 30 µmol/kg (15.4 mg/kg) oral dose. Shown are unbound fraction (main graph) and total levels (insert) of CNP520 in the blood and brain, using values for rat plasma protein binding of 97.8% and unspecific binding to brain homogenate of 99%. Values are mean ± SD, with n = 5 per time point.


Figure 2 ( A ) Subjective assessment of hair depigmentation ( score 0 - 5 , with 0 = completely black and 5 = completely white ) at 2 doses of either CNP520 or the BACE - 1 / - 2 inhibitor NB - 360 ( mean ± SEM , n = 8 / group ) , statistical comparisons were made for every scoring day , versus vehicle using Kruskal - Wallis with Dunn ' s post - hoc test . ( B ) Summary of doses , steady - state effects on Aβ40 in brain ( % compared to vehicle - treated mice ) and the ratios between total skin concentrations of CNP520 and NB - 360 and their respective IC50 values for inhibition of BACE - 2 in vitro .

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Figure 2(A) Subjective assessment of hair depigmentation (score 0-5, with 0 = completely black and 5 = completely white) at 2 doses of either CNP520 or the BACE-1/-2 inhibitor NB-360 (mean ± SEM, n=8/group), statistical comparisons were made for every scoring day, versus vehicle using Kruskal-Wallis with Dunn's post-hoc test.(B) Summary of doses, steady-state effects on Aβ40 in brain (% compared to vehicle-treated mice) and the ratios between total skin concentrations of CNP520 and NB-360 and their respective IC50 values for inhibition of BACE-2 in vitro.


Figure 3 ( A ) Doses from 1 . 5 mg / kg ( 3 µmol / kg ) to 51 . 3 mg / kg ( 100 µmol / kg ) were given by oral gavage to rats ( n = 5 per dose ) ; animals were killed 4 hours later for determination of Aβ40 in brain tissue . Values are mean ± SEM as determined by ANOVA ( GraphPad Prism ) . ( B ) Time - dependent reduction of Aβ40 in rat brain and CSF after a single 15 . 4 mg / kg ( 30 µmol / kg ) oral dose of CNP520 . ( C ) CNP520 levels in dog blood and CSF , following a 3 . 1 mg / kg ( 6 µmol / kg ) oral dose ( mean ± SD = 4 ) . ( D ) Time - dependent reduction of Aβ40 and 42 in dog CSF , after a 3 . 1 mg / kg oral CNP520 dose ( mean ± s . d . ) .

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Figure 3(A) Doses from 1.5 mg/kg (3 µmol/kg) to 51.3 mg/kg (100 µmol/kg) were given by oral gavage to rats (n = 5 per dose); animals were killed 4 hours later for determination of Aβ40 in brain tissue. Values are mean ± SEM as determined by ANOVA (GraphPad Prism).(B) Time-dependent reduction of Aβ40 in rat brain and CSF after a single 15.4 mg/kg (30 µmol/kg) oral dose of CNP520.(C) CNP520 levels in dog blood and CSF, following a 3.1 mg/kg (6 µmol/kg) oral dose (mean ± SD = 4).(D) Time-dependent reduction of Aβ40 and 42 in dog CSF, after a 3.1 mg/kg oral CNP520 dose (mean ± s.d.).


Figure 4Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( A ) CNP520 levels in blood and cerebellum . Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( B ) Total cortical plaque area ( normalized to total sample area ( * 10 − 2 ) , stained with antibody NT - 12 . Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( C ) Formic acid - soluble Aβ40 in forebrain . ( D ) Formic acid - soluble Aβ42 in forebrain . Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( E ) Double - immunofluorescence stained brain sections with NT12 ( against amyloid - β , green ) and Iba1 ( against microglia , red ) or GFAP ( astrocytes , red ) for the different treatment groups as indicated . Scale bar : 500 µm . Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( F ) Quantification of total Iba1 positive microglia ( normalized to total area ( * 10 − 2 ) , only a subset of samples analyzed . Male APP23 mice ( age at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( G ) Quantification of total GFAP positive astrocyte area ( normalized to total area ( * 10 − 2 ) . Data were analyzed with Dunnett ' s multiple comparison test .

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 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 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 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 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 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

Figure 4Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (A) CNP520 levels in blood and cerebellum.Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (B) Total cortical plaque area (normalized to total sample area (*10−2), stained with antibody NT-12.Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (C) Formic acid-soluble Aβ40 in forebrain. (D) Formic acid-soluble Aβ42 in forebrain.Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (E) Double-immunofluorescence stained brain sections with NT12 (against amyloid-β, green) and Iba1 (against microglia, red) or GFAP (astrocytes, red) for the different treatment groups as indicated. Scale bar: 500 µm.Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (F) Quantification of total Iba1 positive microglia (normalized to total area (*10−2), only a subset of samples analyzed.Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (G) Quantification of total GFAP positive astrocyte area (normalized to total area (*10−2). Data were analyzed with Dunnett's multiple comparison test.