Journal: Neurotherapeutics

Article Title: Neuroprotective effects of niclosamide on disease progression via inflammatory pathways modulation in SOD1-G93A and FUS-associated amyotrophic lateral sclerosis models

doi: 10.1016/j.neurot.2024.e00346

Figure Lengend Snippet: Niclosamide decreases microgliosis and increase P2Y12-positive microglia . (a) Representative confocal images of lumbar spinal cord sections of vehicle- and 20 ​mg/kg niclosamide-treated SOD1-G93A mice immunolabeled with Iba1 (green). Scale bar: 100 ​μm. Quantification of Iba1 staining in hemisections of both groups. Data are expressed as means ​± ​SEM (n ​= ​3/4 animals for group, at least four sections for animal). Statistical significance was calculated by student's t -test ∗∗∗∗p ​< ​0.0001. (b) Representative western blots and quantification of Iba1 in vehicle- (n ​= ​4) and niclosamide-treated (n ​= ​4) SOD1-G93A mice. GAPDH was used as a loading control. Data represent means ​± ​SEM. Statistical significance was calculated by student's t-test ∗p ​< ​0.05, (c) Microglia in vehicle- and niclosamide-treated SOD1-G93A mice were analyzed by ImageJ software for different size descriptors (Area, Perimeter). The transformation index was calculated as (perimeter) 2 /4πarea. Scale bar: 50 ​μm. Data represent mean ​± ​S.E.M. (n ​= ​4 mice/group, at least 20 ​cells/mice). Statistical significance was calculated by t -test. ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001. Representative confocal images of lumbar spinal cord sections cells labeled with anti-CD68 (d, in red) and anti-P2Y12 (e, in green) in vehicle- and 20 ​mg/kg niclosamide-treated SOD1-G93A mice. Scale bar: 100 ​μm. Quantification of CD68 (d) and P2Y12 (e) staining in hemisections of vehicle- and niclosamide-treated SOD1-G93A mice. Data represent means ​± ​S.E.M. Statistical significance was calculated by t -test, ∗∗p ​< ​0.01, ∗∗∗∗p ​< ​0.0001. (n ​= ​3/4 animals for group, at least four sections for animal).

Article Snippet: Immunofluorescences were performed with the following primary antibodies: rabbit anti-P2Y12 (1:500, Alomone), rabbit anti-HA-FUS (1:500, Cell Signaling), mouse anti-SMI32 (1:1000, Covance), rat anti-CD68 (1:500, AbdSerotec), rabbit anti-phospho-TDP43 (1:500, Proteintech), mouse anti-glial fibrillary acidic protein (GFAP) (1:500, Cell Signaling), rabbit anti-Iba1 (1:500 Wako).

Techniques: Confocal, Immunolabeling, Staining, Western Blot, Control, Software, Transformation Assay, Labeling, Immunofluorescence

Journal: Neurotherapeutics

Article Title: Neuroprotective effects of niclosamide on disease progression via inflammatory pathways modulation in SOD1-G93A and FUS-associated amyotrophic lateral sclerosis models

doi: 10.1016/j.neurot.2024.e00346

Figure Lengend Snippet: Niclosamide ameliorates spinal cord pathology in FUS mice . (a) Nissl-stained spinal cord sections of Ntg (∼40 days) and terminal stage FUS mice after vehicle or niclosamide 50 ​mg/kg treatment. Scale bar: 100 ​μm. Quantification of motor neuron (MNs) numbers/ventral horn is provided. (b) Representative confocal images of motoneurons labeled with SMI32 (red) and HA-FUS (green) in heterozygous FUS (FUS+/−), vehicle and niclosamide-treated FUS mice. Scale bar: 10 ​μm. (c) Quantification of FUS nuclear/cytoplasmatic ratio in FUS+/−, vehicle- and niclosamide-treated FUS mice. (d) Representative confocal images of lumbar spinal cord sections cells labeled with anti-CD68 (red) and anti-P2Y12 (green) in Ntg, vehicle- and niclosamide-treated FUS mice. Scale bar: 100 ​μm. (e) Quantification of CD68 and P2Y12 staining in hemisections of Ntg, vehicle- and niclosamide-treated FUS mice. Data represent means ​± ​S.E.M. Data represent means ​± ​S.E.M. Statistical significance was calculated by ANOVA, ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001. (n ​= ​3/4 animals for group, at least four sections for animal). (f) Representative western blots and quantification of p-mTOR and STAT3 in vehicle- (n ​= ​4) and niclosamide-treated (n ​= ​4) FUS mice. GAPDH was used as a loading control. Data represent means ​± ​SEM. Statistical significance was calculated by student's t -test or by Mann-Whitney test referred to vehicle-FUS group, ∗ p ​< ​0.05.

Article Snippet: Immunofluorescences were performed with the following primary antibodies: rabbit anti-P2Y12 (1:500, Alomone), rabbit anti-HA-FUS (1:500, Cell Signaling), mouse anti-SMI32 (1:1000, Covance), rat anti-CD68 (1:500, AbdSerotec), rabbit anti-phospho-TDP43 (1:500, Proteintech), mouse anti-glial fibrillary acidic protein (GFAP) (1:500, Cell Signaling), rabbit anti-Iba1 (1:500 Wako).

Techniques: Pathology, Staining, Confocal, Labeling, Western Blot, Control, MANN-WHITNEY, Transwell Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. α 1 +/- and α 1 +/+ platelets in PRP were pooled from five mice from the same strain, and platelet concentration was adjusted to 2.5E+08/mL with PPP. The PRP was divided into 4 equal parts, repleted with MgCl 2 /CaCl 2 to a final concentration of 1 mM, and then treated with 2.5 µM ADP for the indicated periods with rotation. The reaction was stopped by adding an EDTA/PGE1 cocktail containing 500 ng/mL PGE1 and 2 mM EDTA. Platelets were pelleted by a quick spin at 13,000 rpm for 15 seconds, lysed in RIPA buffer, and used for western blot assay of AKT activation. GAPDH was blotted as a loading control. The blot represents two repeats. B. WT platelets pooled from 6 mice were lysed and used for IP of NKA α1 (with ab2872 antibody, 2 µg antibody/1 mg total protein), and then IB for P2Y12 was conducted. IP was also conducted with #1 WT sample pool with normal mouse IgG (2 µg normal IgG/1 mg total protein) as control. C. Platelet lysates prepared from α1 +/- and α 1 +/+ mice were subjected to the Blue-Native PAGE assay. The membrane was blotted for α1 first, stripped, and re-blotted for P2Y12. The image represents two repeats. D. COS-7 cells were transiently transfected with P2Y12-tango plasmid (p-hP2Y12, addgene, #66471), or pEYFP-N1-A2BR (p-hA2BR, addgene, #37202) for 36 h and then cell lysates were used for IP α1 (with ab2872) and immunoblotted for P2Y12. The membrane was stripped and re-blotted for α1 and A2BR (no band was detected in the IP and data was not shown).

Article Snippet: Antibodies to P2Y12 (AGP-098) and A2BR (AAR-003) were purchased from Alomone Lab. Antibodies to NKAα1 subunit (ab2872, ab7671, and ab76020) and P2Y12 (ab184411, ab183066) were purchased from abcam (Waltham, MA).

Techniques: Concentration Assay, Western Blot, Activation Assay, Control, Blue Native PAGE, Membrane, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Sodium/Potassium ATPase Alpha 1 Subunit Fine-tunes Platelet GPCR Signaling Function and is Essential for Thrombosis

doi: 10.1101/2024.05.13.593923

Figure Lengend Snippet: A. COS-7 cells were transiently transfected with plasmids encoding mouse NKA α1 and WT or LGL>SFT mutated mouse P2Y12 for 36 hours and then cells were lysed and used for the IP-IB assay. B & C. Human platelet lysates prepared from two individuals were used for IP of α1 (with ab2872) and then IB for PAR4 and TP. The membranes were stripped and reblotted for α1 (ab76020). HRP-conjugated anti-rabbit IgG light chain antibody was used for the detection of PAR4, TP, and α1 (middle blot) signals. Due to the weak signal of α1 in the IP, the membranes were re-blotted using the HRP-conjugated anti-rabbit whole IgG, and then the α1 signal was visualized by DAB staining. D. COS-7 cells were transiently transfected with plasmids encoding human P2Y12 for 24 hours. The cells were then treated with LGL at the indicated concentrations for an additional 16 hours. Cells were lysed and used for the co-IP assay. GAPDH was blotted for loading control of input. E. COS-7 cells were transiently transfected with plasmids encoding mouse α1 and P2Y12, either WT or LGL(LGI)>SFT mutated genes, in different combinations and then cells were lysed and used for the IP-IB assay. Two dishes of cells transfected with mouse WT α1 and P2Y12 were treated with 100 µM Ouabain or 2 µM Digoxin, respectively, for 1 hour before being harvested for IP-IB assay. SB indicates sample buffer only was loaded in the corresponding lane.

Article Snippet: Antibodies to P2Y12 (AGP-098) and A2BR (AAR-003) were purchased from Alomone Lab. Antibodies to NKAα1 subunit (ab2872, ab7671, and ab76020) and P2Y12 (ab184411, ab183066) were purchased from abcam (Waltham, MA).

Techniques: Transfection, Staining, Co-Immunoprecipitation Assay, Control

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Figure Lengend Snippet: Primary antibodies employed in the study.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

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Figure Lengend Snippet: P2Y 12 antibodies validation and RT-PCR analysis. (a) Scheme of human P2RY 12 gene location, transcript variants [ , ], and protein structure, with amino acid epitopes recognized by the used antibodies and highlighted in color ( intra1 , intra2 , and intra fl , red circle; c-ter , green oval). Species conservation for each epitope was calculated by using BLAT tool of UCSC genome browser . (b) Total protein extracts from SH-SY5Y or HEK293 cells expressing Myc-tagged P2Y 12 receptor were subjected to Western blot analysis with the indicated antibodies. (c) Total protein extracted from human, rat and mouse brain, from primary mouse microglia (mMG) and rat oligodendrocyte (OL) cultures were subjected to Western blot analysis with the indicated antibodies. For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (d) RT-PCR using primers specific for P2Y 12 mRNA was performed on total RNA from rat microglia (rMG) and OL. Control lanes show RT-PCR performed without reverse transcriptase enzyme.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

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Figure Lengend Snippet: P2Y 12 receptor in dissociated and organotypic primary cultures. (a) Mouse primary cortical microglia were subjected to immunofluorescence and confocal analysis with phalloidin (green, merged field) and P2Y 12 receptor antibodies (red, insets and merged) and Hoechst (white, merged). Scale bars in insets: 20 μ m. (b) Double immunofluorescence and confocal analysis of primary rat mature (OL) and precursor (OPC) oligodendrocytes was performed with antibodies for P2Y 12 receptor, MBP, NG2 . For intra2 antibodies, lots AN01/02/04/0502/0602 were used. (c) Rat cerebellar organotypic cultures were analyzed by double immunofluorescence and confocal microscopy for intra1 (red) and c-ter (red), highlighting different structures (see also insets), and MBP (green).

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

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Figure Lengend Snippet: P2Y 12 receptor in rat brain tissue. Double immunofluorescence and confocal analysis was performed on sections from rat cerebellum (panels (a), (b), (c), (g), (h), and (i)) and striatum (panels (d), (e), (f), (j), (k), and (l)) with intra1 , intra2 -lots AN01/02/04, intra fl , c-ter (all red), and GFAP (green, inset b1), CD11b (green, insets c1, f1, h1; yellow merged, inset i1; green, panel (k); merged, panel (l)), MBP (yellow merged, inset c2; green, inset e1; green, panels (h) and (i); green, inset j1) antibodies.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence

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Figure Lengend Snippet: Temporal and regional pattern of P2Y 12 expression in SOD1-G93A ALS spinal microglia. (a) Double immunofluorescence and confocal analysis on lumbar spinal cord sections (L3–L5) of wild-type (WT) mice was performed with c-ter antibody (green and yellow, merged and insets), CD11b (left panel, yellow, merged and inset), and CD68 (right panel, red, merged and inset), in both dorsal (DH) and ventral (VH) horns of spinal cord. (b) Double immunofluorescence and confocal analysis on SOD1-G93A lumbar spinal cord sections (L3–L5) at two different stages of ALS disease, that is, 20 weeks, and end stage, was performed with c-ter (green) and CD68 (red) antibodies. (c) Equal amount of total lumbar spinal cord lysates (L3–L5) from WT and SOD1-G93A ( n = 4 for each group) were subjected to Western blotting and immunoreactions with c-ter and CD68 antibodies; anti- β -actin was used for protein normalization. Data represent means ± SEM. Statistical significance was calculated by Student's t -test, * P < 0.05.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Expressing, Immunofluorescence, Western Blot

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Figure Lengend Snippet: P2Y 12 receptor in human cortex. Sections from human healthy and SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for the immunoreactive markers c-ter (red, panels (a), (c); insets a1, a2, c1; yellow merged, inset c2), intra1 (red, panels (d), insets d1, d2, d3; yellow merged, panel f, insets f1, f2), MBP (green, panels (b), (c), (e), insets c1, e1; yellow merged, panel (f), inset f1), MHC II (green, inset d3), and integrin α II/ β 3 (green, insets b2, e2; yellow merged, insets c2, f2). The asterisks show decreased P2Y 12 immunoreactivity in proximity to MS lesion.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy

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Figure Lengend Snippet: Regional distribution of P2Y 12 in proximity to MS lesions. Sections from SPMS frontal cortex were analyzed by double immunofluorescence and confocal microscopy for c-ter (red) and MHC II (green) immunoreactivity. In proximity to the demyelinating active cortical lesion expressing augmented positivity for MHC II, microglia gradually lose immunoreactivity for c-ter antibody. Microglia express differential immunoreactivity in the four chosen areas which are found inside (circled b-c) and around (circled a–d) a lesion.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Immunofluorescence, Confocal Microscopy, Expressing

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Figure Lengend Snippet: Draw of microglial marker expression as a function of activation. Branched microglia are represented in blue and activated microglia in red. Iba1 , CD11b, and MHC II are mostly expressed in microglia throughout the different morphological states and their expression increases during activation (light blue to red color). P2Y 12 c-ter (light blue) and CD68 (red) are expressed, respectively, in branched or roundish/activated microglia.

Article Snippet: P2Y 12 receptor-ATTO-594 ( intra fl ) , Polyclonal , 125–142 , P2Y 12 receptor , 1 : 50 , Alomone.

Techniques: Marker, Expressing, Activation Assay