Spatially guided visual behavior , measured as frequency threshold with a virtual optokinetic system 9 days after immunization , is significantly decreased in MOG / CFA - injected mice compared to CFA - injected control litters . Data are shown as median ; boxes and whiskers illustrate 25 - 75 and 5 - 95 percentiles of values , respectively . Points below and above whiskers are drawn as individual points . At each trial , mice were measured three times . Home - made and commercial MOG / CFA suspensions were compared as well as PLP - injected mice with control - injected mice . Statistical analysis was done by ANOVA ( 0 , 05 significance level ) , mean comparison probabilities were Bonferroni corrected . N = 32 , n = 96 ( MOG / CFA ) ; N = 24 , n = 72 ( CFA ) . * * * , p < 0 . 001 ( Mann - Whitney test ) . Results from mice injected with home - made and commercial MOG / CFA suspensions as well as results obtained from PLP / CFA - injected mice ( and respective control - injected mice ) are

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-ORGANISM O O O O B-ORGANISM O O O O O O 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-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O B-ORGANISM O O

Spatially guided visual behavior, measured as frequency threshold with a virtual optokinetic system 9 days after immunization, is significantly decreased in MOG/CFA-injected mice compared to CFA-injected control litters. Data are shown as median; boxes and whiskers illustrate 25-75 and 5-95 percentiles of values, respectively. Points below and above whiskers are drawn as individual points. At each trial, mice were measured three times. Home-made and commercial MOG/CFA suspensions were compared as well as PLP-injected mice with control-injected mice. Statistical analysis was done by ANOVA (0,05 significance level), mean comparison probabilities were Bonferroni corrected. N=32, n=96 (MOG/CFA); N=24, n=72 (CFA). ***, p<0.001 (Mann-Whitney test). Results from mice injected with home-made and commercial MOG/CFA suspensions as well as results obtained from PLP/CFA-injected mice (and respective control-injected mice) are displayed


Optic nerve parameters are unaltered in early EAE ( 9 days after injection ) . ( A , B ) Cryostat sections of the optic nerve from MOG / CFA - injected mice ( own lab - made suspension ; for comparison with commercial MOG / CFA suspensions , see Appendix Fig . S12 ) and CFA - injected control mice were stained with anti - Nav antibody ( 1 : 50 dilution ) to label the node of Ranvier and with anti - CASPR1 antibody ( 1 : 500 dilution ) to stain the paranodal region . Length of the node of Ranvier and length of the paranodal region were quantified in C ) D )

B-TISSUE I-TISSUE O O O O O B-DISEASE O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O

Optic nerve parameters are unaltered in early EAE (9 days after injection). (A,B) Cryostat sections of the optic nerve from MOG/CFA-injected mice (own lab-made suspension; for comparison with commercial MOG/CFA suspensions, see Appendix Fig. S12) and CFA-injected control mice were stained with anti-Nav antibody (1:50 dilution) to label the node of Ranvier and with anti-CASPR1 antibody (1:500 dilution) to stain the paranodal region. Length of the node of Ranvier and length of the paranodal region were quantified in C) D)


( E1 , E2 ) Richardson - Blue stained semi - thin sections from the indicated optic nerve samples . ( E2 , E3 , F2 , F3 ) Transmission electron micrographs of the optic nerves from MOG / CFA - and CFA - injected animals . Abbreviations : ax , axon ; my , myelin sheath . Axon number and myelin thickness were quantified in G ) and H ) . Scale bars : 1μm ( A , B , E , F )

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-TISSUE O B-TISSUE I-TISSUE O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

(E1, E2) Richardson-Blue stained semi-thin sections from the indicated optic nerve samples. (E2, E3, F2, F3) Transmission electron micrographs of the optic nerves from MOG/CFA- and CFA-injected animals. Abbreviations: ax, axon; my, myelin sheath. Axon number and myelin thickness were quantified in G) and H). Scale bars: 1μm (A,B,E,F)


Retinal slices from MOG / CFA - injected and CFA - injected SypHy mice were stimulated by applying a 25mM K + - containing depolarization solution for 1min ( Fig . 6A ) . In response to depolarization , we observed a strong increase in fluorescence ( Fig . 6A ) that can be best fitted by a double - exponential curve ( B ) . Amplitudes of fast and slow release ( together with the respective time constants ) of the first depolarization response are plotted in C )

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Retinal slices from MOG/CFA-injected and CFA-injected SypHy mice were stimulated by applying a 25mM K+-containing depolarization solution for 1min (Fig. 6A). In response to depolarization, we observed a strong increase in fluorescence (Fig. 6A) that can be best fitted by a double-exponential curve (B). Amplitudes of fast and slow release (together with the respective time constants) of the first depolarization response are plotted in C)


The amount of CASPR1 protein in the synapse is also altered in the PLP mouse model of multiple sclerosis . Semi - thin sections of PLP / CFA and CFA - injected mice were probed with the indicated antibodies ( A , B ; see also Appendix Fig . S13 )

O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O B-GENEPROD B-ORGANISM O O B-DISEASE I-DISEASE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O

The amount of CASPR1 protein in the synapse is also altered in the PLP mouse model of multiple sclerosis. Semi-thin sections of PLP/CFA and CFA-injected mice were probed with the indicated antibodies (A, B; see also Appendix Fig. S13)


The synaptic amount of CASPR1 is strongly reduced both in the OPL and IPL . RIBEYE is only reduced in the OPL but not significantly in the IPL . CNTN1 is strongly reduced in both OPL and IPL ( C , see also Appendix Fig . S13 ) . N = 4 PLP / CFA - injected mice ; N = 4 CFA - injected mice ; n = 140 slices ( CFA , RIBEYE ) ; n = 143 slices ( PLP / CFA , RIBEYE ) ; n = 47 slices ( CFA , CASPR1 ) ; n = 53 slices ( PLP / CFA , CASPR1 ) ; n = 49 slices ( CFA , CNTN1 ) ; n = 46 slices ( PLP / CFA , CNTN1 ) ; n = 44 slices ( CFA , v - arrestin ) ; n = 44 slices ( PLP / CFA , v - arrestin )

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The synaptic amount of CASPR1 is strongly reduced both in the OPL and IPL. RIBEYE is only reduced in the OPL but not significantly in the IPL. CNTN1 is strongly reduced in both OPL and IPL (C, see also Appendix Fig. S13). N=4 PLP/CFA-injected mice; N=4 CFA-injected mice; n=140 slices (CFA, RIBEYE); n=143 slices (PLP/CFA, RIBEYE); n=47 slices (CFA, CASPR1); n=53 slices (PLP/CFA, CASPR1); n=49 slices (CFA, CNTN1); n=46 slices (PLP/CFA, CNTN1); n=44 slices (CFA, v-arrestin); n=44 slices (PLP/CFA, v-arrestin)


( D , E ) Cryostat sections of the optic nerve from PLP and control mice were stained with anti - Nav - channel antibody to label the node of Ranvier and with anti - CASPR1 antibody to stain the paranodal region . Length of the node of Ranvier and length of the paranodal region were quantified in G ) H ) . ( F ) transmission electron micrographs of the optic nerves from PLP / CFA - and CFA - injected animals ( F1 - F4 ) . Axon numbers and myelin thickness were quantified in I ) J )

O O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O B-ORGANISM O B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O B-GENEPROD O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O

(D,E) Cryostat sections of the optic nerve from PLP and control mice were stained with anti-Nav-channel antibody to label the node of Ranvier and with anti-CASPR1 antibody to stain the paranodal region. Length of the node of Ranvier and length of the paranodal region were quantified in G) H). (F) transmission electron micrographs of the optic nerves from PLP/CFA- and CFA-injected animals (F1-F4). Axon numbers and myelin thickness were quantified in I) J)


( B ) qRT - PCR analysis using primers overlapping exons 15 and 16 permitted to quantify the non ‐ skipped α1S form : 18 ± 6 % ( * * P ⩽ 0 . 001 , n = 19 ) and 10 ± 7 % ( * * P ⩽ 0 . 001 n = 19 ) ( black bars ) of the total α1S mRNA ( grey bars ) at 2 and 6 months post injection in TA of AAV1 ‐ ( U7 ‐ ESE ) and AAV1 ‐ ( U7 ‐ SA ) : ΔDHPR or AAV1 ‐ ( U7 ‐ Ctrl ) : c as a control , respectively .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O B-TISSUE O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O

(B) qRT-PCR analysis using primers overlapping exons 15 and 16 permitted to quantify the non‐skipped α1S form: 18±6% (**P⩽0.001, n=19) and 10±7% (**P⩽0.001 n=19) (black bars) of the total α1S mRNA (grey bars) at 2 and 6 months post injection in TA of AAV1‐(U7‐ESE) and AAV1‐(U7‐SA):ΔDHPR or AAV1‐(U7‐Ctrl): c as a control, respectively.


( C ) Six months post injection , lysates from TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were immuno ‐ blotted for α1S or α - actin for four mice . Graph depicts mean ± s . e . m . of relative expression of α1S subunit determined by densitrometry and nomalized to the α - actin expression for each muscle . Results were expressed in protein levels of α1S subunit in TAΔDHPR normalized to TACtrl for each mice , * * P ⩽ 0 . 001 , n = 4 .

O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O

(C) Six months post injection, lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for α1S or α-actin for four mice. Graph depicts mean±s.e.m. of relative expression of α1S subunit determined by densitrometry and nomalized to the α-actin expression for each muscle. Results were expressed in protein levels of α1S subunit in TAΔDHPR normalized to TACtrl for each mice, **P⩽0.001, n=4.


( D ) Longitudinal cryo ‐ sections from TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with anti ‐ α1S subunit ( red ) and anti ‐ laminin ( green ) antibodies , nuclei were visualized by Dapi ( blue ) and imaged by confocal microscopy . Bars represent 20 μm .

O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(D) Longitudinal cryo‐sections from TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red) and anti‐laminin (green) antibodies, nuclei were visualized by Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.


( A ) Six months post injection , TAs from eight mice were dissected and weighed .

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(A) Six months post injection, TAs from eight mice were dissected and weighed.


( B ) The internal diameters ( shortest diameter ) from all fibres throughout the total muscle section were recorded and analysed . Muscles from five different animals were examined . The bar graph presents mean ± s . e . m . of the number of myofibres by fibre diameter class for TAΔDHPR ( black ) and TACtrl ( grey ) .

O O O O O O O O O O O O B-TISSUE O O O O O O O O O O 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-TISSUE O O O O O B-TISSUE O O O O B-TISSUE O O O O

(B) The internal diameters (shortest diameter) from all fibres throughout the total muscle section were recorded and analysed. Muscles from five different animals were examined. The bar graph presents mean±s.e.m. of the number of myofibres by fibre diameter class for TAΔDHPR (black) and TACtrl(grey).


( C ) Transversal sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with haematoxylin and eosin , bars represent 100 μm .

O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(C) Transversal sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with haematoxylin and eosin, bars represent 100 μm.


( D ) To quantify fibrosis , transversal sections of total muscle were stained with Red Sirius and quantified using Histolab Software ( marked in blue ) , data were normalized with total surface of each muscle ( orange line surrounding the sections ) , bars represent 500 μm . ( E ) Quantification is presented in bar graph and showed 4 . 1 ± 0 . 1 fold increase of fibrosis in TAΔDHPR ( ΔDHPR ) compared to TACtrl ( c ) ( * * P0 . 001 , n = 4 ) .

O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O

(D) To quantify fibrosis, transversal sections of total muscle were stained with Red Sirius and quantified using Histolab Software (marked in blue), data were normalized with total surface of each muscle (orange line surrounding the sections), bars represent 500 μm. (E) Quantification is presented in bar graph and showed 4.1±0.1 fold increase of fibrosis in TAΔDHPR (ΔDHPR) compared to TACtrl (c) (**P0.001, n=4).


( A ) mRNA from TAΔDHPR and TACtrl tissues were extracted and nNOS expression was quantified by qRT - PCR . Results are expressed as mean ± s . e . m . , * * P0 . 001 , n = 4 .

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(A) mRNA from TAΔDHPR and TACtrl tissues were extracted and nNOS expression was quantified by qRT-PCR. Results are expressed as mean±s.e.m., **P0.001, n=4.


( B ) Transversal cryo ‐ sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with anti ‐ nNOS ( red ) , anti ‐ laminin ( green ) antibodies , nuclei with Dapi ( blue ) and imaged by confocal microscopy . Bars represent 20 μm .

O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(B) Transversal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐nNOS (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.


( C ) The tissues from extracts were analysed by western blot with FoxO3a ‐ P antibody and normalized with α ‐ actin antibody . Graph depicts mean ± s . e . m . of relative expression of FoxO3a ‐ P determined by densitometry and nomalized to the α ‐ actin expression for each muscle , * P ⩽ 0 . 005 , n = 3 .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O

(C) The tissues from extracts were analysed by western blot with FoxO3a‐P antibody and normalized with α‐actin antibody. Graph depicts mean±s.e.m. of relative expression of FoxO3a‐P determined by densitometry and nomalized to the α‐actin expression for each muscle, *P⩽0.005, n=3.


( D ) Longitudinal cryo ‐ sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with anti ‐ FoxO3a ( red ) , anti ‐ laminin ( green ) antibodies , nuclei with Dapi ( blue ) and imaged by confocal microscopy . Bars represent 20 μm .

(D) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐FoxO3a (red), anti‐laminin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Bars represent 20 μm.


( E ) mRNA from TAΔDHPR and TACtrl tissues were extracted and regulation of autophagy genes expression was followed by qRT - PCR . Bnip3 , CathepsinL , LC3 and PI3KIII expression ( noted in red ) were significantly increased in TAΔDHPR compared with the controlateral TACtrl , * * P0 . 001 , n = 4 .

O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O

(E) mRNA from TAΔDHPR and TACtrl tissues were extracted and regulation of autophagy genes expression was followed by qRT-PCR. Bnip3, CathepsinL, LC3 and PI3KIII expression (noted in red) were significantly increased in TAΔDHPR compared with the controlateral TACtrl, **P0.001, n=4.


( A ) Ultrathin sections were imaged by electron microscopy , M , mitochondria ; C , collagen fibres ; T , T ‐ tubule . Arrows show double ‐ membrane vesicules called auphagosomes .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O

(A) Ultrathin sections were imaged by electron microscopy, M, mitochondria; C, collagen fibres; T, T‐tubule. Arrows show double‐membrane vesicules called auphagosomes.


( B ) Transversal cryo ‐ sections were stained with anti ‐ LC3b ( red ) , anti ‐ dystrophin ( green ) antibodies , nuclei with Dapi ( blue ) and imaged by confocal microscopy . Upper panel : TACtrl ( Ctrl ) and lower panel : TAΔDHPR ( ΔDHPR ) . Bars represent 20 μm .

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(B) Transversal cryo‐sections were stained with anti‐LC3b (red), anti‐dystrophin (green) antibodies, nuclei with Dapi (blue) and imaged by confocal microscopy. Upper panel: TACtrl (Ctrl) and lower panel: TAΔDHPR (ΔDHPR). Bars represent 20 μm.


( C ) Myofibres isolated from control ( Ctrl ) or 6 months post ‐ injected FDB ( ΔDHPR ) muscles were processed for immuno ‐ fluorescent labelling for P62 ( green ) and LC3b ( red ) and imaged by confocal microscopy . Scale bars , 10 μm .

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(C) Myofibres isolated from control (Ctrl) or 6 months post‐injected FDB (ΔDHPR) muscles were processed for immuno‐fluorescent labelling for P62 (green) and LC3b (red) and imaged by confocal microscopy. Scale bars, 10 μm.


( A ) Ultrathin sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were imaged by electron microscopy . Cis , terminal cisternae ; T , tubule ; SR , sarcoplasmic reticulum . Bars : 500 nm .

O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O

(A) Ultrathin sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were imaged by electron microscopy. Cis, terminal cisternae; T, tubule; SR, sarcoplasmic reticulum. Bars: 500 nm.


( B ) Myofibres isolated from Ctrl or ΔDHPR FDB muscles 6 months post injection were processed for immuno ‐ fluorescent labelling for RyR1 ( green ) , SERCA ( red ) , Dapi ( blue ) and imaged by confocal microscopy . Scale bars , 10 μm .

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(B) Myofibres isolated from Ctrl or ΔDHPR FDB muscles 6 months post injection were processed for immuno‐fluorescent labelling for RyR1 (green), SERCA (red), Dapi (blue) and imaged by confocal microscopy. Scale bars, 10 μm.


( A ) Lysates from TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were immuno ‐ blotted for RyR1 or α ‐ actin . ( n = 4 ) . Graph depicts mean ± s . e . m . of relative expression of RyR1 determined by densitometry and nomalized to the α ‐ actin expression for each muscle . Results were expressed in protein levels of ΔDHPR in TAΔDHPR normalized to TACtrl for each mice , * P ⩽ 0 . 005 , n = 4 .

O O O O O B-TISSUE O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O

(A) Lysates from TAΔDHPR (ΔDHPR) and TACtrl (c) were immuno‐blotted for RyR1 or α‐actin. (n=4). Graph depicts mean±s.e.m. of relative expression of RyR1 determined by densitometry and nomalized to the α‐actin expression for each muscle. Results were expressed in protein levels of ΔDHPR in TAΔDHPR normalized to TACtrl for each mice, *P⩽0.005, n=4.


( B ) Longitudinal cryo ‐ sections of TAΔDHPR ( ΔDHPR ) and TACtrl ( c ) were stained with anti ‐ α1S subunit ( red ) , anti ‐ RyR1 ( green ) antibodies and imaged by confocal microscopy . Bars = 20 μm .

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(B) Longitudinal cryo‐sections of TAΔDHPR (ΔDHPR) and TACtrl (c) were stained with anti‐α1S subunit (red), anti‐RyR1 (green) antibodies and imaged by confocal microscopy. Bars=20 μm.


Localization of α 1S subunit . Whole skeletal muscle fibres enzymatically isolated from Ctrl ( A - D ) or ΔDHPR ( E - H ) FDB muscles 6 months post injection were processed for immuno ‐ fluorescent labelling for α1 S subunit ( red ) and laminin ( green ) . Scale bars , 10 μm ; Arrows indicate α1 S subunit expression on sarcolemma ; antibody labelling was visualized by serial confocal microscopy and represented as movies of the optical sections ( Supplementary Movies S5A and S5B ) . ( D , H ) Present projections of confocal Z ‐ series ( step between each frame is 1 μm ) along XZ and YZ planes as indicated .

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Localization of α 1S subunit. Whole skeletal muscle fibres enzymatically isolated from Ctrl (A-D) or ΔDHPR (E-H) FDB muscles 6 months post injection were processed for immuno‐fluorescent labelling for α1 S subunit (red) and laminin (green). Scale bars, 10 μm; Arrows indicate α1 S subunit expression on sarcolemma; antibody labelling was visualized by serial confocal microscopy and represented as movies of the optical sections (Supplementary Movies S5A and S5B). (D, H) Present projections of confocal Z‐series (step between each frame is 1 μm) along XZ and YZ planes as indicated.


Immunostaining for CCN1 ( green ) in the adult brain coronal section . Scale bar : 50 μm . The insert refers to the same image with DAPI staining to show cell nuclei ( blue ) . LV , lateral ventricle .

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Immunostaining for CCN1 (green) in the adult brain coronal section. Scale bar: 50 μm. The insert refers to the same image with DAPI staining to show cell nuclei (blue). LV, lateral ventricle.


Adult V - SVZ whole - mounts stained for CCN1 ( white ) and S100β ( green ) . Scale bar : 10 μm .

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Adult V-SVZ whole-mounts stained for CCN1 (white) and S100β (green). Scale bar: 10 μm.


Adult V - SVZ whole - mounts stained for CCN1 ( white ) , GFAP ( green ) and VCAM1 ( magenta ) . Arrowhead points to a VCAM1 + B1 cell surrounded by CCN1 - expressing ependymal cells . Scale bar : 10 μm .

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Adult V-SVZ whole-mounts stained for CCN1 (white), GFAP (green) and VCAM1 (magenta). Arrowhead points to a VCAM1+ B1 cell surrounded by CCN1-expressing ependymal cells. Scale bar: 10 μm.


Confocal images tracing a VCAM1 + GFAP + cell reveal CCN1 expression is absent in B1 cells . Yellow arrowheads point to the apical side of a B1 cell in the center of a NSC unit . White arrowheads point to the process of the B1 cell . Scale bar : 10 μm .

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Confocal images tracing a VCAM1+GFAP+ cell reveal CCN1 expression is absent in B1 cells. Yellow arrowheads point to the apical side of a B1 cell in the center of a NSC unit. White arrowheads point to the process of the B1 cell. Scale bar: 10 μm.


V - SVZ whole - mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment . Scale bar : 10 μm .

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V-SVZ whole-mount staining showing reduced CCN1 expression in Ccn1cKO mice 1 week after TAM treatment. Scale bar: 10 μm.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) and GFAP ( red ) . Arrowheads point to B1 cells in the pinwheel center . Scale bars : 500 μm ( left ) , 50 μm ( middle ) , 10 μm ( right ) .

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V-SVZ whole-mounts stained for γ-tubulin (cyan) and GFAP (red). Arrowheads point to B1 cells in the pinwheel center. Scale bars: 500 μm (left), 50 μm (middle), 10 μm (right).


Densities ( cells / mm2 ) of B1 cells in the adult V - SVZ . n = 4 , * * * p = 0 . 0005 .

B-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O

Densities (cells/mm2) of B1 cells in the adult V-SVZ. n=4, ***p=0.0005.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) and VCAM1 ( white ) . Arrowheads point to VCAM1 + B1 cells in NSC units . Scale bars : 50 μm ( left ) , 10 μm ( right ) .

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V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).


Densities ( cells / mm2 ) of VCAM1 + B1 cells in the adult V - SVZ . n = 4 , * * p = 0 . 002 . Densities ( units / mm2 ) of NSC units in the adult V - SVZ . n = 4 , * * p = 0 . 0022 . Distribution of NSC units with different numbers of B1 cells . n = 4 , * * * * p < 0 . 0001 ( 1 B1 cell / unit ) , * * p = 0 . 0025 ( 2 B1 cells / unit ) , * * p = 0 . 0063 ( 3 B1 cells / unit ) , * p = 0 . 0385 ( 4 B1 cells / unit ) .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O

Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ. n=4, **p=0.002. Densities (units/mm2) of NSC units in the adult V-SVZ. n=4, **p=0.0022. Distribution of NSC units with different numbers of B1 cells. n=4, ****p<0.0001 (1 B1 cell/unit), **p=0.0025 (2 B1 cells/unit), **p=0.0063 (3 B1 cells/unit), *p=0.0385 (4 B1 cells/unit).


Densities ( cells / mm2 ) of B1 cells in the aged V - SVZ . n = 3 , * * p = 0 . 0099 .

B-EXP_ASSAY O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O

Densities (cells/mm2) of B1 cells in the aged V-SVZ. n=3, **p=0.0099.


Densities ( cells / mm2 ) of VCAM1 + B1 cells in the aged V - SVZ . n = 3 , * * p = 0 . 0041 .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O

Densities (cells/mm2) of VCAM1+ B1 cells in the aged V-SVZ. n=3, **p=0.0041.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) and VCAM1 ( white ) in aged mice . Arrowheads point to VCAM1 + B1 cells in NSC units . Scale bars : 50 μm ( left ) , 10 μm ( right ) .

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V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white) in aged mice. Arrowheads point to VCAM1+ B1 cells in NSC units. Scale bars: 50 μm (left), 10 μm (right).


Densities ( units / mm2 ) of NSC units in the aged V - SVZ . n = 3 , * * p = 0 . 0093 . Average numbers of B1 cells in a NSC unit . n = 3 , n . s . not significant .

B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O

Densities (units/mm2) of NSC units in the aged V-SVZ. n=3, **p=0.0093. Average numbers of B1 cells in a NSC unit. n=3, n.s. not significant.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 2 days after TAM treatment . n = 4 , * p = 0 . 0141 . V - SVZ whole - mounts stained for GFAP ( red ) and Ki67 ( cyan ) . Arrowheads point to dividing GFAP + cells . Scale bar : 10 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 2 days after TAM treatment. n=4, *p=0.0141. V-SVZ whole-mounts stained for GFAP (red) and Ki67 (cyan). Arrowheads point to dividing GFAP+ cells. Scale bar: 10 μm.


Numbers of ASCL1 + cells in V - SVZ coronal sections 2 days after TAM treatment . n = 3 , * * p = 0 . 0087 . V - SVZ coronal sections stained for ASCL1 ( red ) and nuclei ( blue ) . Scale bar : 50 μm .

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Numbers of ASCL1+ cells in V-SVZ coronal sections 2 days after TAM treatment. n=3, **p= 0.0087. V-SVZ coronal sections stained for ASCL1 (red) and nuclei (blue). Scale bar: 50 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB granule cell layer ( GCL ) 2 days after TAM treatment . n = 4 ( Ctrl ) and 3 ( Ccn1cKO ) , * * p = 0 . 0086 . OB sections stained for BrdU ( yellow ) and nuclei ( blue ) . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB granule cell layer (GCL) 2 days after TAM treatment. n=4 (Ctrl) and 3 (Ccn1cKO), **p=0.0086. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 4 weeks after TAM treatment . Arrowheads point to dividing GFAP + cells . n = 4 , n . s . not significant . Scale bar : 10 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 4 weeks after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.


Numbers of ASCL1 + cells in V - SVZ coronal sections 4 weeks after TAM treatment . n = 3 , n . s . not significant . Scale bar : 20 μm .

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Numbers of ASCL1+ cells in V-SVZ coronal sections 4 weeks after TAM treatment. n=3, n.s. not significant. Scale bar: 20 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL 4 weeks after TAM treatment . n = 4 , n . s . not significant . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB GCL 4 weeks after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 16 months after TAM treatment . Arrowheads point to dividing GFAP + cells . n = 4 , n . s . not significant . Scale bar : 10 μm .

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 16 months after TAM treatment. Arrowheads point to dividing GFAP+ cells. n=4, n.s. not significant. Scale bar: 10 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL 16 months after TAM treatment . n = 4 , n . s . not significant . Scale bar : 50 μm .

Densities (cells/mm2) of BrdU+ cells in the OB GCL 16 months after TAM treatment. n=4, n.s. not significant. Scale bar: 50 μm.


Numbers of GFAP + BrdU + label - retaining NSCs in the V - SVZ . Arrowheads point to label - retaining NSCs . n = 4 , * p = 0 . 039 . V - SVZ coronal sections stained for GFAP ( cyan ) and BrdU ( red ) . Scale bar : 10 μm .

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Numbers of GFAP+BrdU+ label-retaining NSCs in the V-SVZ. Arrowheads point to label-retaining NSCs. n=4, *p= 0.039. V-SVZ coronal sections stained for GFAP (cyan) and BrdU (red). Scale bar: 10 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts 24h after TMZ treatment . Arrowheads point to dividing NSCs under regenerative condition . n = 6 , * p = 0 . 0173 . Scale bar : 10 μm .

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Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=6, *p= 0.0173. Scale bar: 10 μm.


Densities ( cells / mm2 ) of DCX + neuroblasts in V - SVZ whole - mounts 3 days after TMZ treatment . n = 3 , * p = 0 . 0179 . V - SVZ whole - mounts stained for DCX . Scale bar : 500 μm .

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Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts 3 days after TMZ treatment. n=3, *p= 0.0179. V-SVZ whole-mounts stained for DCX. Scale bar: 500 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL labeled 24h after TMZ treatment . n = 5 ( Ctrl ) and 6 ( Ccn1cKO ) , * p = 0 . 0484 . OB sections stained for BrdU ( yellow ) and nuclei ( blue ) . Scale bar : 50 μm .

B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O

Densities (cells/mm2) of BrdU+ cells in the OB GCL labeled 24h after TMZ treatment. n=5 (Ctrl) and 6 (Ccn1cKO), *p= 0.0484. OB sections stained for BrdU (yellow) and nuclei (blue). Scale bar: 50 μm.


Densities ( cells / mm2 ) of GFAP + Ki67 + cells in V - SVZ whole - mounts of aged mice 24h after TMZ treatment . Arrowheads point to dividing NSCs under regenerative condition . n = 3 , * p = 0 . 0321 . Scale bar : 10 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of GFAP+Ki67+ cells in V-SVZ whole-mounts of aged mice 24h after TMZ treatment. Arrowheads point to dividing NSCs under regenerative condition. n=3, *p= 0.0321. Scale bar: 10 μm.


Densities ( cells / mm2 ) of DCX + neuroblasts in V - SVZ whole - mounts of aged mice 3 days after TMZ treatment . n = 3 , n . s . not significant . Scale bar : 500 μm .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

Densities (cells/mm2) of DCX+ neuroblasts in V-SVZ whole-mounts of aged mice 3 days after TMZ treatment. n=3, n.s. not significant. Scale bar: 500 μm.


Densities ( cells / mm2 ) of BrdU + cells in the OB GCL of aged mice labeled 3 days after TMZ treatment . n = 4 ( Ctrl ) and 3 ( Ccn1cKO ) , n . s . not significant . Scale bar : 50 μm .

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Densities (cells/mm2) of BrdU+ cells in the OB GCL of aged mice labeled 3 days after TMZ treatment. n=4 (Ctrl) and 3 (Ccn1cKO), n.s. not significant. Scale bar: 50 μm.


Representative FACS plots showing gating strategy for the analysis of cell types in the adult V - SVZ .

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Representative FACS plots showing gating strategy for the analysis of cell types in the adult V-SVZ.


Total number of V - SVZ cells isolated from one mouse . n = 3 , n . s . not significant .

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Total number of V-SVZ cells isolated from one mouse. n=3, n.s. not significant.


Representative FACS plots showing aNSCs ( GFP + EGFR + ) , qNSCs ( GFP + EGFR - ) and C cells ( GFP - EGFR + ) in the adult V - SVZ .

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Representative FACS plots showing aNSCs (GFP+EGFR+), qNSCs (GFP+EGFR-) and C cells (GFP-EGFR+) in the adult V-SVZ.


Percentage of each cell population in the adult V - SVZ . n = 3 , * * p = 0 . 0014 ( GFP + EGFR - ) , * * * p = 0 . 0002 ( GFP + EGFR + ) , * p = 0 . 0111 ( GFP - EGFR + ) , * * * p = 0 . 0006 ( GFP + ) , n . s . not significant .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

Percentage of each cell population in the adult V-SVZ. n=3, **p=0.0014 (GFP+EGFR-), ***p=0.0002 (GFP+EGFR+), *p=0.0111 (GFP-EGFR+), ***p=0.0006 (GFP+), n.s. not significant.


Densities ( cells / mm2 ) of VCAM1 + B1 cells in the adult V - SVZ whole - mounts . n = 4 ( Ctrl + vehicle and Ccn1cKO + vehicle ) and 3 ( Ccn1cKO + erlotinib ) , * * p = 0 . 0021 ( Ccn1cKO + vehicle vs Ccn1cKO + erlotinib ) , * * p = 0 . 0014 ( Ccn1cKO + erlotinib vs Ctrl + vehicle ) , * * * * p < 0 . 0001 .

B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

Densities (cells/mm2) of VCAM1+ B1 cells in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), **p=0.0021 (Ccn1cKO + vehicle vs Ccn1cKO + erlotinib), **p=0.0014 (Ccn1cKO + erlotinib vs Ctrl + vehicle), ****p<0.0001.


Densities ( units / mm2 ) of NSC units in the adult V - SVZ whole - mounts . n = 4 ( Ctrl + vehicle and Ccn1cKO + vehicle ) and 3 ( Ccn1cKO + erlotinib ) , * p = 0 . 017 , * * p = 0 . 0061 , * * * p = 0 . 0002 .

B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

Densities (units/mm2) of NSC units in the adult V-SVZ whole-mounts. n=4 (Ctrl + vehicle and Ccn1cKO + vehicle) and 3 (Ccn1cKO + erlotinib), *p=0.017, **p=0.0061, ***p=0.0002.


V - SVZ whole - mounts stained for γ - tubulin ( cyan ) , GFAP ( red ) and VCAM1 ( white ) . Scale bar : 50 μm .

B-TISSUE I-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O

V-SVZ whole-mounts stained for γ-tubulin (cyan), GFAP (red) and VCAM1 (white). Scale bar: 50 μm.


A Kinetics of intracellular Ca2 + after EDN stimulation . UACC257 and A2058 ( WT and ENDRB - KO ) were seeded on 96 well plates , loaded with Fluo - 4 dye and stimulated with 100 nM EDN or PBS . Fluo - 4 fluorescence was monitored over ten minutes ( n = 6 ) . Representative example of three independent experiments .

O B-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

A Kinetics of intracellular Ca2+ after EDN stimulation. UACC257 and A2058 (WT and ENDRB-KO) were seeded on 96 well plates, loaded with Fluo-4 dye and stimulated with 100 nM EDN or PBS. Fluo-4 fluorescence was monitored over ten minutes (n = 6). Representative example of three independent experiments.


B Time course of AKT and CREB phosphorylation . Melanoma cell lines were stimulated with 100 nM EDN for 2 - 90 min . AKT and CREB phosphorylation and expression was evaluated by Western blot . Representative example of three independent experiments .

O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

B Time course of AKT and CREB phosphorylation. Melanoma cell lines were stimulated with 100 nM EDN for 2-90 min. AKT and CREB phosphorylation and expression was evaluated by Western blot. Representative example of three independent experiments.


D High reproducibility in the UACC257 and A2058 datasets . All CV values were grouped according to cell line to represent dataset specific CV distributions . Numbers and lines indicate the median .

O O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O

D High reproducibility in the UACC257 and A2058 datasets. All CV values were grouped according to cell line to represent dataset specific CV distributions. Numbers and lines indicate the median.


A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA . Colours indicate experimental groups and shading is mapped to time .

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A Phosphopeptide MS1 intensities in UACC257 samples were analysed by PCA. Colours indicate experimental groups and shading is mapped to time.


C Comparison of the kinetics of the 242 shared target phosphopeptides . Log2 FC of EDN / Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red .

O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

C Comparison of the kinetics of the 242 shared target phosphopeptides. Log2 FC of EDN/Ctr is plotted from top to bottom for each phosphopeptide for both cell lines with increases shown in green and decreases shown in red.


A , B EDN target phosphopeptides for ( A ) UACC257 and ( B ) A2058 were segregated into six clusters each , using c - means clustering ( MFuzz ) . Each line represents one phosphopeptide and cluster membership is indicated by colour . Most likely kinases for each phosphosite were predicted using NetworKIN and enrichment of kinase substrates was calculated for each cluster . Enriched kinases are indicated above each cluster . Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster .

O O O B-GENEPROD O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

A,B EDN target phosphopeptides for (A) UACC257 and (B) A2058 were segregated into six clusters each, using c-means clustering (MFuzz). Each line represents one phosphopeptide and cluster membership is indicated by colour. Most likely kinases for each phosphosite were predicted using NetworKIN and enrichment of kinase substrates was calculated for each cluster. Enriched kinases are indicated above each cluster. Note that substrates of these kinases only account for a fraction of all phosphosites in a cluster.


C Average MS1 intensity z - score kinetics for 217 predicted CaMKII - α sites that are shared between both cell lines . For comparison , average ( n = 6 ) calcium abundance kinetics are shown .

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C Average MS1 intensity z-score kinetics for 217 predicted CaMKII-α sites that are shared between both cell lines. For comparison, average (n = 6) calcium abundance kinetics are shown.


Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed . UACC257 cells were serum starved and stimulated with PBS or EDN at 100 nM for 60 min ( 5 min for CaMKII ) . Ten μg protein from RIPA lysates were analysed by Western blot . Kinase marker substrate phosphorylations and corresponding loading controls are shown . For each kinase , two independent replicates were performed and are shown . Each gel also had control lanes with increasing concentration of kinase inhibitors to support phospho band

O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-CELL_LINE O O B-TISSUE O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed. UACC257 cells were serum starved and stimulated with PBS or EDN at 100 nM for 60 min (5 min for CaMKII). Ten μg protein from RIPA lysates were analysed by Western blot. Kinase marker substrate phosphorylations and corresponding loading controls are shown. For each kinase, two independent replicates were performed and are shown. Each gel also had control lanes with increasing concentration of kinase inhibitors to support phospho band identity


The effect of kinase inhibitors on EDN induced cell migration of UACC257 cells was analysed using a scratch assay . Scratches were made in UACC257 monolayers . Serum was removed and cells were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0 . 1 μM or 1 μM in triplicates . A Example pictures showing the effect of EDN on migration . Cell free areas are delimited by blue lines . Migration was quantified as the change in cell free area between 0 h and 48 h . B The four inhibitors that were effective at 1 µM are shown in detail , i . e . every point corresponds to one well . C Abundance of E - Cadherin ( normalized to GAPDH and Actin ) was assessed by LC - SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM . Only the single inhibitor which could reverse EDN induced E - Cadherin repression is shown . * p < 0 . 05 two - sided unpaired t - test .

O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O

The effect of kinase inhibitors on EDN induced cell migration of UACC257 cells was analysed using a scratch assay. Scratches were made in UACC257 monolayers. Serum was removed and cells were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0.1 μM or 1 μM in triplicates. A Example pictures showing the effect of EDN on migration. Cell free areas are delimited by blue lines. Migration was quantified as the change in cell free area between 0 h and 48 h. B The four inhibitors that were effective at 1 µM are shown in detail, i.e. every point corresponds to one well. C Abundance of E-Cadherin (normalized to GAPDH and Actin) was assessed by LC-SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM. Only the single inhibitor which could reverse EDN induced E-Cadherin repression is shown. * p < 0.05 two-sided unpaired t-test.


( A - B ) Coronal brain section , bregma : - 2 . 88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus ( A ) . 8 h post - injection , majority of the injected Fib - Tau - 1N3R - ATTO550 ( red ) is taken up by the corpus callosum of the injected side ( B ) . Images are shown at low ( left , 10X ) and high ( right , 63X ) magnification . Data information : Scale bar , 100µm in the left panel in ( B ) , 10µm everywhere else .

O O O O O O B-TISSUE O O B-TISSUE O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Coronal brain section, bregma: - 2.88 from The Mouse Brain Atlas shows the site of injection in the CA1 cell body layer of the right hippocampus (A). 8 h post-injection, majority of the injected Fib-Tau-1N3R-ATTO550 (red) is taken up by the corpus callosum of the injected side (B). Images are shown at low (left, 10X) and high (right, 63X) magnification. Data information: Scale bar, 100µm in the left panel in (B), 10µm everywhere else.


Representative images of the distribution of Fib - Tau ( red ) 8 h and 24 h after injection . Dendrites are immunolabeled using MAP2 antibody ( green ) . 8 h after injection , both diffused and clustered distribution of exogenous Fib - Tau is observed in stratum oriens and CA1 cell body region No exogenous Fib - Tau is detected in the adjoining cortex , stratum radiatum and entrorhinal cortex 24 h after injection , clustered distribution is primarily restricted to the pyramidal ( CA ) cell body layer ( D ) . Uptake of Fib - Tau is seen in some cells in both stratum oriens and pyramidal cell body layer ( D ) . Paired - t - test performed between ' 8 / 24h - right ' and ' 8 / 24h - left ' ; Unpaired t - test performed between ' 8h - left ' and ' 24h - left ' columns ( n is number of animals : 6 for 8h and 5 for 24h ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns = not significant . Note : The images presented in panels C and D were independently acquired images under non - identical exposure setting at low magnification for display purpose Data information : Scale bar ,

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of the distribution of Fib-Tau (red) 8 h and 24 h after injection. Dendrites are immunolabeled using MAP2 antibody (green). 8 h after injection, both diffused and clustered distribution of exogenous Fib-Tau is observed in stratum oriens and CA1 cell body region No exogenous Fib-Tau is detected in the adjoining cortex, stratum radiatum and entrorhinal cortex 24 h after injection, clustered distribution is primarily restricted to the pyramidal (CA) cell body layer (D). Uptake of Fib-Tau is seen in some cells in both stratum oriens and pyramidal cell body layer (D). Paired-t-test performed between '8/24h-right' and '8/24h-left'; Unpaired t-test performed between '8h-left' and '24h-left' columns (n is number of animals: 6 for 8h and 5 for 24h); ***p<0.001, **p<0.01, *p<0.05, ns= not significant. Note: The images presented in panels C and D were independently acquired images under non-identical exposure setting at low magnification for display purpose Data information: Scale bar, 10µm


Representative images of the distribution of Fib - Tau ( red ) 8 h and 24 h after injection . Dendrites are immunolabeled using MAP2 antibody ( green ) . 8 h after injection , both diffused and clustered distribution of exogenous Fib - Tau is observed in stratum oriens The number of Fib - Tau clusters per µm2 within stratum oriens decreases between 8 h and 24 h post - injection ( G ) . A similar decrease in the total fluorescence ( clusters + diffused ) is observed indicating the diffusion and / or clearance of exogenous Fib - Tau ( H ) . Paired - t - test performed between ' 8 / 24h - right ' and ' 8 / 24h - left ' ; Unpaired t - test performed between ' 8h - left ' and ' 24h - left ' columns ( n is number of animals : 6 for 8h and 5 for 24h ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , ns = not significant . Panels ( E and F ) are shown for same exposure and are used for quantification . Data information : Scale bar

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of the distribution of Fib-Tau (red) 8 h and 24 h after injection. Dendrites are immunolabeled using MAP2 antibody (green). 8 h after injection, both diffused and clustered distribution of exogenous Fib-Tau is observed in stratum oriens The number of Fib-Tau clusters per µm2 within stratum oriens decreases between 8 h and 24 h post-injection (G). A similar decrease in the total fluorescence (clusters + diffused) is observed indicating the diffusion and/or clearance of exogenous Fib-Tau (H). Paired-t-test performed between '8/24h-right' and '8/24h-left'; Unpaired t-test performed between '8h-left' and '24h-left' columns (n is number of animals: 6 for 8h and 5 for 24h); ***p<0.001, **p<0.01, *p<0.05, ns= not significant. Panels (E and F) are shown for same exposure and are used for quantification. Data information: Scale bar 10µm


( A - C ) Time and concentration dependent clustering of Fib - Tau in primary neurons . Representative images are shown for certain conditions to illustrate Fib - Tau clustering time dependence ( A , top - row ) and concentration dependence ( A , bottom - row ) . Quantification of the number of Fib - Tau clusters per µm2 ( B ) or fluorescence intensity of clusters At low concentrations ( up to 0 . 72nM ) the density of clusters increased with time ( between 10 and 60 min ) but the increase in intensity was small . At high concentrations of Fib - Tau ( ≥ 1 . 8nM ) , both density and size increased with increasing time . Box - plot represents : median , interquartile range and 10 - 90 % distribution ; One - way ANOVA with Dunnett post - hoc test , number of images analysed from 3 cultures ( from left to right : 25 , 25 , 25 , 25 , 70 , 45 , 45 , 45 , 45 , 45 , 30 , 40 , 40 , 40 , 40 and 40 images ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar

O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O 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-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Time and concentration dependent clustering of Fib-Tau in primary neurons. Representative images are shown for certain conditions to illustrate Fib-Tau clustering time dependence (A, top-row) and concentration dependence (A, bottom-row). Quantification of the number of Fib-Tau clusters per µm2 (B) or fluorescence intensity of clusters At low concentrations (up to 0.72nM) the density of clusters increased with time (between 10 and 60 min) but the increase in intensity was small. At high concentrations of Fib-Tau (≥1.8nM), both density and size increased with increasing time. Box-plot represents: median, interquartile range and 10-90% distribution; One-way ANOVA with Dunnett post-hoc test, number of images analysed from 3 cultures (from left to right: 25, 25, 25, 25, 70, 45, 45, 45, 45, 45, 30, 40, 40, 40, 40 and 40 images). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar 2µm


( D - F ) Single Particle Tracking using Quantum Dots ( SPT - QD ) of biotin tagged Fib - Tau . Representative single molecule trajectories of Fib - Tau following 10 or 60 min exposure are shown ( D ) . Note after 60min exposure ( 0 . 36nM ) , single molecules are more confined suggesting they are trapped and clustered . Quantification of diffusion coefficient ( E ) and explored area ( F , extracted from mean squared displacement ( MSD ) , see methods ) shows that both these parameters decrease after 60min exposure to Fib - Tau . Unpaired t - test , n is averaged value per cells imaged in 3 experiments ( 10 min : 22 , 60 min : 23 ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar ,

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O 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-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-F) Single Particle Tracking using Quantum Dots (SPT-QD) of biotin tagged Fib-Tau. Representative single molecule trajectories of Fib-Tau following 10 or 60 min exposure are shown (D). Note after 60min exposure (0.36nM), single molecules are more confined suggesting they are trapped and clustered. Quantification of diffusion coefficient (E) and explored area (F, extracted from mean squared displacement (MSD), see methods) shows that both these parameters decrease after 60min exposure to Fib-Tau. Unpaired t-test, n is averaged value per cells imaged in 3 experiments (10 min: 22, 60 min: 23). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar, 2µm


( G ) Neurons were exposed for 60 min to Fib - Tau ( 0 . 36nM ) labeled both with biotin and ATTO488 ( red ) . Cell surface exposed biotin was labelled using streptavidin - 550 ( green ) followed by live imaging . Note that most of the clusters of ATTO488 ( red ) are co - labelled with Streptavidin 550 ( green ) indicating that the clusters are at the cell surface . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar ,

O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Neurons were exposed for 60 min to Fib-Tau (0.36nM) labeled both with biotin and ATTO488 (red). Cell surface exposed biotin was labelled using streptavidin-550 (green) followed by live imaging. Note that most of the clusters of ATTO488 (red) are co-labelled with Streptavidin 550 (green) indicating that the clusters are at the cell surface. Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar, 5µm


( H - J ) Clearance of Tau clusters from neurons . Neurons were exposed ( 0 . 36nM ) to ATTO 550 labeled Fib - Tau for 10 min and the unbound fibrils were washed . Cells were fixed immediately ( 10min ) or allowed to recover in culture medium for 60 min . Two representative images ( H ) and quantifications ( I , J ) show that following 60 min recovery most of the Tau clusters disappear / dissociate as indicated by a decrease in their density . Box - plot represents : median , interquartile range and 10 - 90 % distribution , Unpaired t - test , n is number of images analyzed from 3 cultures ( 49 images ) . Data information : * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant . Scale bar

O O O O O B-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-GENEPROD B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-J) Clearance of Tau clusters from neurons. Neurons were exposed (0.36nM) to ATTO 550 labeled Fib-Tau for 10 min and the unbound fibrils were washed. Cells were fixed immediately (10min) or allowed to recover in culture medium for 60 min. Two representative images (H) and quantifications (I, J) show that following 60 min recovery most of the Tau clusters disappear/dissociate as indicated by a decrease in their density. Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test, n is number of images analyzed from 3 cultures (49 images). Data information: *p<0.05; **p<0.01; ***p<0.001; ns= not significant. Scale bar 2µm


( A ) Super - resolution STORM images of neuronal membrane after exposure to ATTO647N labeled Fib - Tau ( 0 . 36nM ) . Primary neurons were fixed 10 , 60 , 120 and 240 min after exposure to ATTO647N labeled Fib - Tau . Images rendered with a pixel size of 5nm show both clustered and diffused ( non - clustered ) distribution of exogenous Fib - Tau molecules . Scale bar , 1µm . ( B - F ) Quantification of various parameters from the super - resolution images . Averaged values per image is plotted . Increased " total detections per µm2 " ( B ) and " cluster - density " ( C ) of Fib - Tau bound to neurons with increasing exposure time is observed . No change in the " size of cluster " ( D ) or " number of molecules within a cluster " ( E ) is seen with increased exposure time . The proportion of single molecule events detected within clusters remained nearly constant ( ~ 50 % ) in all conditions ( F ) indicating an equilibrium between clustered and non - clustered Fib - Tau single molecules . Unpaired t - test to compare difference with 10 min , n is number of images ( 8 images ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; ns = not significant .

O O O O O O B-EXP_ASSAY O O B-CELL_TYPE B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Super-resolution STORM images of neuronal membrane after exposure to ATTO647N labeled Fib-Tau (0.36nM). Primary neurons were fixed 10, 60, 120 and 240 min after exposure to ATTO647N labeled Fib-Tau. Images rendered with a pixel size of 5nm show both clustered and diffused (non-clustered) distribution of exogenous Fib-Tau molecules. Scale bar, 1µm. (B-F) Quantification of various parameters from the super-resolution images. Averaged values per image is plotted. Increased "total detections per µm2" (B) and "cluster-density" (C) of Fib-Tau bound to neurons with increasing exposure time is observed. No change in the "size of cluster" (D) or "number of molecules within a cluster" (E) is seen with increased exposure time. The proportion of single molecule events detected within clusters remained nearly constant (~ 50%) in all conditions (F) indicating an equilibrium between clustered and non-clustered Fib-Tau single molecules. Unpaired t-test to compare difference with 10 min, n is number of images (8 images). *p<0.05; **p<0.01; ***p<0.001; ns= not significant.


( D ) List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull - downs from neurons exposed to Fib - Tau . For each identified protein , the name of the protein , the gene name , the p - value ( t - test with Benjamini - Hochberg correction ) , and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given . In an independent analysis , after 10 min exposure of neurons to biotinylated Fib - Tau , a cross - linking step was performed during 20 min using 1mM of DTSSP added in the culture medium , in order to cross - link the protein complexes formed at the cell surface using a membrane impermeable cross - linker . After cross - linking , proteins were analyzed and identified exactly as non - cross - linked samples . Proteins identified with at least 2 peptides are labeled " + " , the other are labeled " - " .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

(D) List of synaptic and plasma membrane proteins with extracellular domains significantly enriched in pull-downs from neurons exposed to Fib-Tau. For each identified protein, the name of the protein, the gene name, the p-value (t-test with Benjamini-Hochberg correction), and the fold change corresponding to the ratio of spectral counts between exposed neuron and control samples are given. In an independent analysis, after 10 min exposure of neurons to biotinylated Fib-Tau, a cross-linking step was performed during 20 min using 1mM of DTSSP added in the culture medium, in order to cross-link the protein complexes formed at the cell surface using a membrane impermeable cross-linker. After cross-linking, proteins were analyzed and identified exactly as non-cross-linked samples. Proteins identified with at least 2 peptides are labeled "+", the other are labeled "-".


( E ) . Co - immunoprecipitation of exogenous biotin - labelled Fib - Tau with α3 - NKA , GluA2 and GluN1 . α3 - NKA , GluA2 and GluN1 were immunoprecipitated using specific antibodies as described in the methods section . The presence of Fib - Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin - HRP . A 2 . 4 - , 2 . 3 - , and 1 . 8 - fold enrichment in Tau band - intensity is observed in α3 - NKA , GluA2 and GluN1 immunoprecipitates , respectively , compared to controls performed with pre - immune goat or rabbit IgGs . Co - immunoprecipitates of exogenous biotin - labelled Fib - Tau with anti α3 - NKA , GluA2 and GluN1 specific antibodies were also subjected to SDS - PAGE and Western blot analysis . The presence of Fib - Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin - HRP . Fib - Tau co - immunoprecipitates with α3 - NKA and GluA2 - AMPA receptor but not with GluN1 - NMDA receptor .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SMALL_MOLECULE O O

(E). Co-immunoprecipitation of exogenous biotin-labelled Fib-Tau with α3-NKA, GluA2 and GluN1. α3-NKA, GluA2 and GluN1 were immunoprecipitated using specific antibodies as described in the methods section. The presence of Fib-Tau in the immunoprecipitate was assessed using a slot blot apparatus and nitrocellulose membranes probed with streptavidin-HRP. A 2.4-, 2.3-, and 1.8- fold enrichment in Tau band-intensity is observed in α3-NKA, GluA2 and GluN1 immunoprecipitates, respectively, compared to controls performed with pre-immune goat or rabbit IgGs. Co-immunoprecipitates of exogenous biotin-labelled Fib-Tau with anti α3-NKA, GluA2 and GluN1 specific antibodies were also subjected to SDS-PAGE and Western blot analysis. The presence of Fib-Tau in the immunoprecipitates was assessed by probing the nitrocellulose membranes with streptavidin-HRP. Fib-Tau co-immunoprecipitates with α3-NKA and GluA2-AMPA receptor but not with GluN1-NMDA receptor.


( A , B ) Primary neurons were exposed to Fib - Tau - ATTO550 ( 0 . 36nM , red ) for 10 ( top row ) or 60 min ( bottom row ) and excitatory ( Homer , green ) and inhibitory synapses ( Gephyrin , blue ) were immuno - labelled . Fib - Tau clusters co - localized and / or apposed to Homer ( arrowheads ) but not Gephyrin ( A ) . Quantitative analysis shows that 10 to 20 % of Fib - Tau clusters are localized at Homer containing synapses ( B ) . Box - plot represents : median , interquartile range and 10 - 90 % distribution , Unpaired t - test to compare difference between 10 and 60 min , n is number of images analyzed from 3 - experiments ( 10 min : 49 ; 60 min : 50 ) . Scale bar , 5µm .

O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Primary neurons were exposed to Fib-Tau-ATTO550 (0.36nM, red) for 10 (top row) or 60 min (bottom row) and excitatory (Homer, green) and inhibitory synapses (Gephyrin, blue) were immuno-labelled. Fib-Tau clusters co-localized and/or apposed to Homer (arrowheads) but not Gephyrin (A). Quantitative analysis shows that 10 to 20% of Fib-Tau clusters are localized at Homer containing synapses (B). Box-plot represents: median, interquartile range and 10-90% distribution, Unpaired t-test to compare difference between 10 and 60 min, n is number of images analyzed from 3-experiments (10 min: 49; 60 min: 50). Scale bar, 5µm.


Exposure of neurons to Fib - Tau - ATTO550 ( 0 . 36nM , 60 min , red ) and immuno - labelling of excitatory synapse ( Anti - Rabbit - Homer or Anti - Mouse - PSD , blue ) and α3 - NKA or GluA1 - AMPA or GluA2 - AMPA or GluN1 - NMDA or GluN2B - NMDA subunits ( green , post - permeabilization ) . Arrows indicate excitatory synapses where Fib - Tau and α3 - NKA / AMPA / NMDA are co - localized ( C ) . Mann - Whitney - test , n is number of images analyzed from 3 - 4 experiments ( α3 - NKA : 75 ; GluA1 : 95 ; GluA2 : 50 ; GluN1 / N2B : 45 ) .

O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM O B-GENEPROD O O O B-ORGANISM O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O

Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Arrows indicate excitatory synapses where Fib-Tau and α3-NKA/AMPA/NMDA are co-localized (C). Mann-Whitney-test, n is number of images analyzed from 3-4 experiments (α3-NKA: 75; GluA1: 95; GluA2: 50; GluN1/N2B: 45).


Exposure of neurons to Fib - Tau - ATTO550 ( 0 . 36nM , 60 min , red ) and immuno - labelling of excitatory synapse ( Anti - Rabbit - Homer or Anti - Mouse - PSD , blue ) and α3 - NKA or GluA1 - AMPA or GluA2 - AMPA or GluN1 - NMDA or GluN2B - NMDA subunits ( green , post - permeabilization ) . Quantification of the fluorescence intensity ( indicating size of clusters of synaptic α3 - NKA / AMPA / NMDA spots ( obtained after thresholding ) following exposure to Fib - Tau showed a reduction in the size of α3 - NKA and increase in the size of GluA2 - subunit containing AMPA receptors ( D ) . Box - plot represents : median , interquartile range and 10 - 90 % distribution , * * p < 0 . 01 ; ns = not significant . Scale bar , 5µm .

O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-ORGANISM O B-GENEPROD O O O B-ORGANISM O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Exposure of neurons to Fib-Tau-ATTO550 (0.36nM, 60 min, red) and immuno-labelling of excitatory synapse (Anti-Rabbit-Homer or Anti-Mouse-PSD, blue) and α3-NKA or GluA1-AMPA or GluA2-AMPA or GluN1-NMDA or GluN2B-NMDA subunits (green, post-permeabilization). Quantification of the fluorescence intensity (indicating size of clusters of synaptic α3-NKA/AMPA/NMDA spots (obtained after thresholding) following exposure to Fib-Tau showed a reduction in the size of α3-NKA and increase in the size of GluA2-subunit containing AMPA receptors (D). Box-plot represents: median, interquartile range and 10-90% distribution, **p<0.01; ns= not significant. Scale bar, 5µm.


( A - C ) SPT - QD of pHluorin tagged AMPA ( GluA1 and GluA2 ) and NMDA ( GluN1 and GluN2B ) receptor subunits using GFP - antibody pre - coupled to QD655 . Primary neurons were exposed or not to Fib - Tau ( 0 . 36nM , 60 min ) . Cumulative frequency of diffusion coefficient of all trajectories ( extracted from MSD plot , inset , A ) show a slow - down in diffusion of GluA2 - subunit but not GluA1 , GluN1 and GluN2B subunits ( A ) . To compute statistical differences , the diffusion coefficient of all trajectories per movie ( from 3 independent experiments ) were averaged ( B ) . To measure statistical differences in MSD , the explored area ( area under curve during 600 - 900ms of MSD plot ) per movie ( from 3 independent experiments ) were averaged ( C ) . Decreased diffusion coefficient and area explored for GluA2 - AMPA was observed indicating increased confinement concomitant with increased clustering ( Fig 5C ) following exposure to Fib - Tau . Mann - Whitney - test , n is number of movies ( left to right ) : 14 , 15 , 14 , 13 , 15 , 15 , 15 , and 15 . * * p < 0 . 01 ; ns = not significant .

O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) SPT-QD of pHluorin tagged AMPA (GluA1 and GluA2) and NMDA (GluN1 and GluN2B) receptor subunits using GFP-antibody pre-coupled to QD655. Primary neurons were exposed or not to Fib-Tau (0.36nM, 60 min). Cumulative frequency of diffusion coefficient of all trajectories (extracted from MSD plot, inset, A) show a slow-down in diffusion of GluA2-subunit but not GluA1, GluN1 and GluN2B subunits (A). To compute statistical differences, the diffusion coefficient of all trajectories per movie (from 3 independent experiments) were averaged (B). To measure statistical differences in MSD, the explored area (area under curve during 600-900ms of MSD plot) per movie (from 3 independent experiments) were averaged (C). Decreased diffusion coefficient and area explored for GluA2-AMPA was observed indicating increased confinement concomitant with increased clustering (Fig 5C) following exposure to Fib-Tau. Mann-Whitney-test, n is number of movies (left to right): 14, 15, 14, 13, 15, 15, 15, and 15. **p<0.01; ns= not significant.


( A ) Single Particle Tracking using PALM ( SPT - PALM ) was performed on α3 - NKA - Dendra expressing primary neurons . A representative image shows an epifluorescence image and trajectories ( strands of different colors ) of minimum 5 points in length . Scale bars : 1 µm in panel ( A )

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Single Particle Tracking using PALM (SPT-PALM) was performed on α3-NKA-Dendra expressing primary neurons. A representative image shows an epifluorescence image and trajectories (strands of different colors) of minimum 5 points in length. Scale bars: 1 µm in panel (A)


( B , C ) Extraction of diffusion coefficient using mean squared displacement ( MSD ) approach using minimum 5 - point trajectory length . Spine and shaft were visually identified . An upward shift of MSD curve , indicating reduced confinement was observed following exposure to Fib - Tau ( 0 . 36nM , 60 min ) ( B ) . As expected , an increased diffusion coefficient ( i . e . increased mobility ) ( C ) of α3 - NKA is observed in dendritic shaft following exposure to Fib - Tau . Mann - Whitney - test , n = 21 cells , obtained from 4 independent experiments ; Each dot represents average diffusion coefficient of all trajectories value per cell . Data information : * p < 0 . 05 ; ns = not significant .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

(B, C) Extraction of diffusion coefficient using mean squared displacement (MSD) approach using minimum 5-point trajectory length. Spine and shaft were visually identified. An upward shift of MSD curve, indicating reduced confinement was observed following exposure to Fib-Tau (0.36nM, 60 min) (B). As expected, an increased diffusion coefficient (i.e. increased mobility) (C) of α3-NKA is observed in dendritic shaft following exposure to Fib-Tau. Mann-Whitney-test, n=21 cells, obtained from 4 independent experiments; Each dot represents average diffusion coefficient of all trajectories value per cell. Data information: *p<0.05; ns= not significant.


Extraction of the number of diffusive states of α3 - NKA and the transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model The occupancy time of α3 - NKA in bound - state also decreased whereas that in free - state increased upon neuron exposure to Fib - Tau ( E ) . Box - plot represents : median , interquartile range and entire distribution , Mann - Whitney - test on averaged value per movie from 4 experiments ( n = 21 cells ) . Data information : * p < 0 . 05 ; ns = not significant .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model The occupancy time of α3-NKA in bound-state also decreased whereas that in free-state increased upon neuron exposure to Fib-Tau (E). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


Extraction of the number of diffusive states of α3 - NKA and the transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model Dwell - time of α3 - NKA is also reduced in bound state indicating destabilization of native α3 - NKA - complexes following interaction with Fib - Tau ( F ) . Box - plot represents : median , interquartile range and entire distribution , Mann - Whitney - test on averaged value per movie from 4 experiments ( n = 21 cells ) . Data information : * p < 0 . 05 ; ns = not significant .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model Dwell-time of α3-NKA is also reduced in bound state indicating destabilization of native α3-NKA-complexes following interaction with Fib-Tau (F). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


( G - H ) Cell - surface biotinylation on primary neurons exposed or not to Fib - Tau ( 1 . 8nM , 60 min ) . The amount of α - NKA at cell surface decreased upon exposure of neurons to Fib - Tau ( G ) . Quantification was performed by normalizing the western blot band intensity with input . Paired - t - test , 3 independent experiments . Data information : * p < 0 . 05 ; ns = not significant .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-H) Cell-surface biotinylation on primary neurons exposed or not to Fib-Tau (1.8nM, 60 min). The amount of α-NKA at cell surface decreased upon exposure of neurons to Fib-Tau (G). Quantification was performed by normalizing the western blot band intensity with input. Paired-t-test, 3 independent experiments. Data information: *p<0.05; ns= not significant.


( I ) Paraformaldehyde fixation α3 - NKA - pHluorin ( green , top row ) reveals endocytic spots otherwise not visible in live neurons ( green , bottom row ) . Many Fib - Tau - ATTO647 ( red ) clusters overlapped ( arrow ) with α3 - NKA - pHluorin spots ( exposure , 0 . 36nM , 60 min ) . Scale bars

O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(I) Paraformaldehyde fixation α3-NKA-pHluorin (green, top row) reveals endocytic spots otherwise not visible in live neurons (green, bottom row). Many Fib-Tau-ATTO647 (red) clusters overlapped (arrow) with α3-NKA-pHluorin spots (exposure, 0.36nM, 60 min). Scale bars 2µm


( J ) The majority of the overlapping Fib - Tau - ATTO647 clusters ( red ) and α3 - NKA - pHluorin spots ( green ) localized on top of Clathrin - mRFP ( blue ) puncta . Scale bars :

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O

(J) The majority of the overlapping Fib-Tau-ATTO647 clusters (red) and α3-NKA-pHluorin spots (green) localized on top of Clathrin-mRFP (blue) puncta. Scale bars: 2µm


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn ( shown here in green ) or unlabeled α - Syn A concentration - dependent increase in the binding and clustering of α - Syn fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α -

O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn (shown here in green) or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of α-Syn fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing Aβ ( given in monomer - equivalent ) of Aβ ( B ) fibrils [ in total 5 - experiments were performed with either ATTO647 labeled Aβ ( shown here in green ) or unlabeled Aβ fibrils ] . concentration - dependent increase in the binding and clustering of Aβ fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α - Syn , but not Aβ

O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing Aβ (given in monomer-equivalent) of Aβ (B) fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ (shown here in green) or unlabeled Aβ fibrils]. concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn or unlabeled α - Syn A concentration - dependent increase in the binding and clustering of both α - Syn fibrils is seen . One - Way ANOVA with Dunnett ' s post - hoc test was performed to compare the different condition to the reference condition ( 100nM ) . The number of images analyzed is 27 , 25 , 25 , 26 and 28 in ( C ) , from left to right . The data originate from 3 to 4 independent experiments . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

O B-CELL_TYPE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of both α-Syn fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 27, 25, 25, 26 and 28 in (C) , from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.