Journal: bioRxiv

Article Title: Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation

doi: 10.1101/2021.04.30.442163

Figure Lengend Snippet: A Immunostainings showing nuclear localization of TDP-43 Wt, 12D and 12A in HeLa cells. Endogenous TDP-43 expression was silenced by siRNAs, followed by transient transfection of the indicated siRNA-resistant myc-TDP-43 constructs. After 24 h, localization of TDP-43 Wt, 12D and 12A variants was visualized by TDP-43 immunostaining (mouse anti-TDP-43 antibody, Proteintech). G3BP1 (rabbit anti-G3BP1 antibody, Proteintech) and DAPI signal is shown to visualize the cytoplasm and nuclei, respectively. In the merge (right column), DAPI is show in turquoise, TDP-43 in green, and G3BP1 in magenta. Bar, 30 μm. B Electrophoretic mobility shift assay (EMSA) of TDP-43-MBP-His 6 variants (Wt, 12D and 12A) in a complex with (UG) 12 RNA. C SDS-PAGE followed by TDP-43 Western blot (upper blot) showing efficient siRNA-mediated knockdown of endogenous TDP-43 (running at ~43 kDa) in comparison to control siRNA and re-expression of myc-tagged TDP-43 Wt, 12D and 12A in Hela cells. Equal loading is demonstrated by α-Tubulin Western blot (bottom). TDP-43 was detected using rabbit anti-TDP-43 C-term antibody (Proteintech), α-Tubulin using mouse anti-alpha Tubulin antibody (Proteintech) and Myc-tag using mouse-anti Myc antibody (9E10, Helmholtz Center Munich).

Article Snippet: Knockdown was analyzed 48 h post transfection by immunohistochemistry using mouse anti TDP-43 antibody (Proteintech, Cat.No: 60019-2-Ig) and immunoblotting using rabbit anti TDP-43 C-Term antibody (Proteintech, Cat.No: 12892-1-AP) to detect TDP-43 and mouse anti alpha-Tubulin antibody (Proteintech, Cat.No: 66031-1-Ig) for detection of α-Tubulin as a control.

Techniques: Expressing, Transfection, Construct, Immunostaining, Electrophoretic Mobility Shift Assay, SDS Page, Western Blot

Journal: Nature Communications

Article Title: Neural deficits in a mouse model of PACS1 syndrome are corrected with PACS1- or HDAC6-targeting therapy

doi: 10.1038/s41467-023-42176-8

Figure Lengend Snippet: a (Top) PACS1 R203W patient (159 and 650) and parent (160 and 651, respectively) fibroblasts were stained for Giantin (red), α-tubulin (green) and nuclei (DAPI). Cell perimeter (white). (Bottom) Quantification of Golgi fragmentation (Golgi objects/cell) and dispersal (percentage of total Golgi area within or beyond the juxtanuclear/peripheral border (J/P) set 10 μm from the nuclear envelope (NE)). Data are mean ± SEM (2-tailed t -test), n = 36 (160), 37 (159), 30 (650), or 51 (651) cells/group from three independent experiments. Scale bar, 20 μm. p -values shown in figure. b Hela +/R203W cells and their isogenic WT control were stained for Giantin (red), α-tubulin (green), and nuclei (DAPI). (Bottom) Quantification of Golgi fragmentation and dispersal were quantified as in (a) except J/P was 3 μm from the NE. Data are mean ± SEM (2-tailed t-test), n = 65 cells/group from three independent experiments. Scale bar, 20 μm. p -values shown in figure. c Western blot of total α-tubulin and Ac-Lys 40 -α-tubulin in patient (159 and 650) and parent (160 and 651) fibroblasts. Data are normalized mean ± SEM (2-tailed t -test), n = 3 independent experiments, normalized individually to minimize inter-experimental variability. d Patient (159) cells expressing GFP-tagged K 40 Q-α-tubulin or K 40 R-α-tubulin were stained for Giantin (red), GFP (green), and nuclei (DAPI). Golgi area in GFP-positive or adjacent control cells (white bars) was measured as in ( a ). Data are mean ± SEM (1-way ANOVA), n = 105 (control), 62 (K 40 Q), or 57 (K 40 R) cells/group from three independent experiments. Scale bar, 20 μm. p -values shown in figure. e Western blot of total α-tubulin and Ac-Lys 40 -α-tubulin in WT and Pacs1 KO MEFs. Data are mean ± SEM (2-tailed t -test), n = 5 independent experiments, analyzed as in ( c ). f WT and Pacs1 KO MEFs expressing human furin were stained for Giantin (red), furin (green), and nuclei (DAPI). Golgi area measured as in ( a ) except J/P was 4 μm from the NE. Furin + endosomes >0.25 μm were quantified. Data are mean ± SEM (2-tailed t -test), n = 27 (WT) or 36 (Pacs1 KO ) cells/group from three independent experiments. Scale bar, 20 μm. Source data are presented as Sources Data files.

Article Snippet: Antibodies- actin (Millipore, MAB1501, 1:3000), α-actinin (Cell Signaling Technology (CST) 3134 S, 1:1000), α-tubulin (DMA1 Cell Signaling 3873 S 1:1000 and Thermo Fisher 66031, 1:250), Ac-Lys 40 -α -tubulin (CST 5335 S, 1:1000), cortactin (4F11 Sigma 05-180, 1:1000), Ac-cortactin (Sigma 09-881, 1:1000), CTIP2 (25B6 Abcam 18465, 1:500), SATB2 (Abcam 51502, 1:50), EB1 (BD Biosciences 610534, 1:50), V5 (Invitrogen, R960-25, 1:2000), HDAC6 (Abcam 253033, 1:50, D2E5 CST 7558 S 1:1000, and Assay biotech C0226, 1:1000), p62 (Abcam 56416, 1:100), Flag (Sigma-Aldrich, F7425, 1:5000 and A2220, 50% slurry), HA (CST 3724 S, 1:4000 and Biolegend 901513, 1:1000), furin (MON-152, kindly provided by J. Creemers, Leuven, 1:100), GAPDH (14C10, CST 2118 S, 1:1000), Giantin (kindly provided by Dr. A. Linstedt, CMU, 1:750), GM130 (BD Biosciences 610534, 1:500), pericentrin (AbCam 4888, 1:500), MAP2 (Biolegend 801810, 1:5000), βIII-tubulin (Biolegend 801213, 1:500), Nestin (AbClonal A11861, 1:100), Pax6 (CST 60433, 1:200), Sox2 (D6D9, CST 3579, 1:400), PSD95 (NeuroMab 75-028-020, 1:250), GABA A Rα1 (NeuroMab 75-136-020, 1:1000), AMPAR1 (CST 13185, 1:1000), WDR37 (Sigma HPA037565, 1:1000), RFP (Rockland 600-401-379, 1:800), PACS1 (BD Biosciences 611371, 1:100, Invitrogen PA558589, 1:100 and Ab 703 , 1:1000), PACS2 (Ab 193 , 1:1000), Goat anti-Rabbit IgG Alexa Fluor 488 (Invitrogen A11008, 1:400), Goat anti-Mouse IgG1 Alexa Fluor 568 (Invitrogen A11004, 1:400) Goat anti-Mouse IgG Alexa Fluor 647 (Invitrogen A-21242, 1:400), Goat anti-Chicken IgY Alexa Fluor 633 (Invitrogen A-21103, 1:400), Goat anti-Rat IgG Alexa Fluor 488 (Invitrogen A-11006, 1:400), Goat anti-Rabbit IgG Alexa Fluor 594 (Invitrogen A-11012, 1:400).

Techniques: Staining, Western Blot, Expressing

Journal: Nature Communications

Article Title: Neural deficits in a mouse model of PACS1 syndrome are corrected with PACS1- or HDAC6-targeting therapy

doi: 10.1038/s41467-023-42176-8

Figure Lengend Snippet: a , b Western blots ( a ) and immunohistochemistry (IHC) of coronal brain sections ( b ) prepared from adult R26 P1 and R26 P1R203W mice induced or not with Emx1 Cre to induce expression of HA-tagged PACS1 or PACS1 R203W (red). Scale bar, 1000 μm. Hippocampal CA1 region from specimens in ( b ). SO, stratum oriens ; SP, stratum pyramidale ; SR, stratum radiatum . Scale bar, 50 μm. d Dissociated Emx1 Cre ;R26 P1 or Emx1 Cre ;R26 P1R203W hippocampal neurons (DIV5) were fixed and stained for β3-tubulin (white, insets), Giantin, and HA-tagged PACS1 or PACS1 R203W . High-resolution tiled images of entire cultured hippocampal neurons were captured using a confocal microscope (see Methods and Fig. S ). (top) 3D surface reconstructions (Imaris, see Methods) of the captured images depict Golgi elements (red), HA-tagged PACS1 or PACS1 R203W (green), and nuclei (blue). Scale bar = 5 μm. (Bottom, left and right) Higher magnification of R26 P1 and R26 P1R203W neurites (yellow arrowheads in insets in the top panels) containing HA-tagged PACS1 or PACS1 R203W and Golgi (R26 P1R203W neurons). Scale bar = 1 μm. (Bottom middle) Quantification of deployed Golgi elements based on their subcellular localization. Somatic Golgi, within cell body; Neurite Golgi, Golgi deployed into developing neurites. Data mean ± SEM (2-tailed Mann–Whitney U -test), n = 8 cells/group from two independent experiments. See also Supplementary Movie . Source data are presented as Sources Data files.

Article Snippet: Antibodies- actin (Millipore, MAB1501, 1:3000), α-actinin (Cell Signaling Technology (CST) 3134 S, 1:1000), α-tubulin (DMA1 Cell Signaling 3873 S 1:1000 and Thermo Fisher 66031, 1:250), Ac-Lys 40 -α -tubulin (CST 5335 S, 1:1000), cortactin (4F11 Sigma 05-180, 1:1000), Ac-cortactin (Sigma 09-881, 1:1000), CTIP2 (25B6 Abcam 18465, 1:500), SATB2 (Abcam 51502, 1:50), EB1 (BD Biosciences 610534, 1:50), V5 (Invitrogen, R960-25, 1:2000), HDAC6 (Abcam 253033, 1:50, D2E5 CST 7558 S 1:1000, and Assay biotech C0226, 1:1000), p62 (Abcam 56416, 1:100), Flag (Sigma-Aldrich, F7425, 1:5000 and A2220, 50% slurry), HA (CST 3724 S, 1:4000 and Biolegend 901513, 1:1000), furin (MON-152, kindly provided by J. Creemers, Leuven, 1:100), GAPDH (14C10, CST 2118 S, 1:1000), Giantin (kindly provided by Dr. A. Linstedt, CMU, 1:750), GM130 (BD Biosciences 610534, 1:500), pericentrin (AbCam 4888, 1:500), MAP2 (Biolegend 801810, 1:5000), βIII-tubulin (Biolegend 801213, 1:500), Nestin (AbClonal A11861, 1:100), Pax6 (CST 60433, 1:200), Sox2 (D6D9, CST 3579, 1:400), PSD95 (NeuroMab 75-028-020, 1:250), GABA A Rα1 (NeuroMab 75-136-020, 1:1000), AMPAR1 (CST 13185, 1:1000), WDR37 (Sigma HPA037565, 1:1000), RFP (Rockland 600-401-379, 1:800), PACS1 (BD Biosciences 611371, 1:100, Invitrogen PA558589, 1:100 and Ab 703 , 1:1000), PACS2 (Ab 193 , 1:1000), Goat anti-Rabbit IgG Alexa Fluor 488 (Invitrogen A11008, 1:400), Goat anti-Mouse IgG1 Alexa Fluor 568 (Invitrogen A11004, 1:400) Goat anti-Mouse IgG Alexa Fluor 647 (Invitrogen A-21242, 1:400), Goat anti-Chicken IgY Alexa Fluor 633 (Invitrogen A-21103, 1:400), Goat anti-Rat IgG Alexa Fluor 488 (Invitrogen A-11006, 1:400), Goat anti-Rabbit IgG Alexa Fluor 594 (Invitrogen A-11012, 1:400).

Techniques: Western Blot, Immunohistochemistry, Expressing, Staining, Cell Culture, Microscopy, MANN-WHITNEY

Journal: Nature Communications

Article Title: Neural deficits in a mouse model of PACS1 syndrome are corrected with PACS1- or HDAC6-targeting therapy

doi: 10.1038/s41467-023-42176-8

Figure Lengend Snippet: a P1 Emx1 Cre ;R26 P1 and Emx1 Cre ;R26 P1R203W pups were ICV-injected with nASO or H6ASO (40 μg) together or not with Cre-inducible AAV9-FLEX-tdTomato (1 × 10 8 vp) and then analyzed as depicted in the timeline (created with Biorender.com). b Cerebral cortices from postnatal Emx1 Cre ;R26 P1 and Emx1 Cre ;R26 P1R203W mice treated with nASO or H6ASO were dissected and processed for Western blot with the indicated antisera. Numerical values depict the normalized signal intensity of Ac-α-tub bands determined with AlphaView (see Methods). c (Top) Representative dendritic arbor reconstructions of hippocampal CA1 pyramidal neurons from P18 Emx1 Cre ;R26 P1 and Emx1 Cre ;R26 P1R203W mice treated with H6ASO or nASO. Scale bar, 50 μm. (Right) Dendrite length, number of branching points, and Sholl Analysis. Data are mean ± SEM (2-way ANOVA followed by Tukey post hoc test), n = 20 neurons/5 mice (R26 P1 /nASO), 15 neurons/3 mice (R26 P1R203W /nASO), 20 neurons/5 mice (R26 P1 /H6ASO), 15 neurons/3 mice (R26 P1R203W /H6ASO). p -values shown in figure. (Bottom) Brain sections from specimens in ( c ) were stained for Golgi (GM130), and Golgi positioning was assessed in tdTomato + CA1 pyramidal neurons. Z-stacks containing the whole tdTomato + dendritic arbor were trimmed to show focal planes containing the soma and proximal segments of the apical dendrite. Arrowheads disseminated Golgi elements in the primary dendrite. Scale bar, 10 μm. d (Left) Patient (159)- and parent (160)-derived NPCs were treated with vehicle (DMSO) or tubacin (5 μM, 24 h) and then fixed and stained for the Golgi-marker Giantin (red), α-tubulin (green) and nuclei (DAPI). Scale bar, 20 μm. (Right) Quantification of Golgi localized to the soma or disseminated down the neurite (Golgi objects/cell) and dispersal (J/P was 3 μm of the NE). Data are mean ± SEM (2-way ANOVA followed by Tukey post hoc test), n = 53 (160/veh), 49 (159/veh), 53 (160/tubacin), 43 (159/tubacin) cells/condition from three independent experiments. Scale bar, 20 μm. p -values shown in figure. Source data are presented as Sources Data files.

Article Snippet: Antibodies- actin (Millipore, MAB1501, 1:3000), α-actinin (Cell Signaling Technology (CST) 3134 S, 1:1000), α-tubulin (DMA1 Cell Signaling 3873 S 1:1000 and Thermo Fisher 66031, 1:250), Ac-Lys 40 -α -tubulin (CST 5335 S, 1:1000), cortactin (4F11 Sigma 05-180, 1:1000), Ac-cortactin (Sigma 09-881, 1:1000), CTIP2 (25B6 Abcam 18465, 1:500), SATB2 (Abcam 51502, 1:50), EB1 (BD Biosciences 610534, 1:50), V5 (Invitrogen, R960-25, 1:2000), HDAC6 (Abcam 253033, 1:50, D2E5 CST 7558 S 1:1000, and Assay biotech C0226, 1:1000), p62 (Abcam 56416, 1:100), Flag (Sigma-Aldrich, F7425, 1:5000 and A2220, 50% slurry), HA (CST 3724 S, 1:4000 and Biolegend 901513, 1:1000), furin (MON-152, kindly provided by J. Creemers, Leuven, 1:100), GAPDH (14C10, CST 2118 S, 1:1000), Giantin (kindly provided by Dr. A. Linstedt, CMU, 1:750), GM130 (BD Biosciences 610534, 1:500), pericentrin (AbCam 4888, 1:500), MAP2 (Biolegend 801810, 1:5000), βIII-tubulin (Biolegend 801213, 1:500), Nestin (AbClonal A11861, 1:100), Pax6 (CST 60433, 1:200), Sox2 (D6D9, CST 3579, 1:400), PSD95 (NeuroMab 75-028-020, 1:250), GABA A Rα1 (NeuroMab 75-136-020, 1:1000), AMPAR1 (CST 13185, 1:1000), WDR37 (Sigma HPA037565, 1:1000), RFP (Rockland 600-401-379, 1:800), PACS1 (BD Biosciences 611371, 1:100, Invitrogen PA558589, 1:100 and Ab 703 , 1:1000), PACS2 (Ab 193 , 1:1000), Goat anti-Rabbit IgG Alexa Fluor 488 (Invitrogen A11008, 1:400), Goat anti-Mouse IgG1 Alexa Fluor 568 (Invitrogen A11004, 1:400) Goat anti-Mouse IgG Alexa Fluor 647 (Invitrogen A-21242, 1:400), Goat anti-Chicken IgY Alexa Fluor 633 (Invitrogen A-21103, 1:400), Goat anti-Rat IgG Alexa Fluor 488 (Invitrogen A-11006, 1:400), Goat anti-Rabbit IgG Alexa Fluor 594 (Invitrogen A-11012, 1:400).

Techniques: Injection, Western Blot, Staining, Derivative Assay, Marker