Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: Motor neurons strongly activate CXCL13 in the spinal cord of fast mSOD1 mice, and the chemokine is released in the CSF during the disease progression. (a) Cxcl13 mRNA fold change levels from laser captured MNs of C57-mSOD1 and 129Sv-mSOD1 mice. mRNA levels are expressed as mean fold change ratio (± SEM) between C57-mSOD1 versus C57Ntg ( n = 4) mice and between 129Sv-mSOD1 ( n = 4) versus 129SvNTg ( n = 4) mice. ( b, c ) Real-time PCR for CXCL13 transcript in the lumbar spinal cord of (b) 129Sv-mSOD1 and (c) C57-mSOD1 mice. Data are normalised to β-actin and expressed as the mean ± SEM fold-change ratio between C57-mSOD1 and 129Sv-mSOD1 mice compared to respective age-matched Ntg littermates. ⁎⁎ P < 0.01; ⁎⁎⁎⁎ P < 0.0001 (Vs. Ntg) by unpaired t -test. ( d-g ) Fast progressing mice showed a striking activation of CXCL13 in the spinal cord at the disease onset, although the chemokine is upregulated in both (e) 129Sv-mSOD1 and (g) C57-mSOD1 mice compared to respective (d, f) Ntg littermates; scale bar, 50 μm. ( h ) Representative co-localization of CXCL13 (green) with the neuronal marker (Neurotrace; NT; red) in the ventral horn of the spinal cord of fast-progressing mice; scale bar: 20 μm. ( i ) Confocal representative image showing CXCL13 staining in correspondence of efferent motor axons (white arrowheads); scale bar: 20 μm. ( j ) Representative co-localization of CXCL13 (green) with the microglia marker (CD68) in the ventral horn of the spinal cord of fast-progressing mice. ( k ) Confocal image of the ventral portion of the spinal cord showing the lack of co-localisation of CXCL13 (green) with the astrocyte marker GFAP (red). ( l - o ) CXCL13 levels (pg/mL) in the CSF and serum of (l, n) 129Sv-mSOD1 and (m, o) C57-mSOD1 mice during the disease progression. Data are expressed as the mean ± SEM. * P < 0.05; ** P < 0.001; **** P < 0.0001 (Vs. Ntg); ° P < 0.05; °° P < 0.01; (between groups) by one-way ANOVA with Tukey's post-analysis.

Article Snippet: Primary antibodies were: goat anti-CXCL13 (1:200, R&D); mouse anti-nonphosphorylated neurofilament H (Smi32, 1:1000; Covance); mouse anti-GFAP (1:1000; Millipore) mouse anti-Iba1 (1:200; Wako).

Techniques: Real-time Polymerase Chain Reaction, Activation Assay, Marker, Staining

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: CXCR5 is upregulated in the motor neurons of mSOD1 mice during the disease progression. (a) Real-time PCR for CXCL13 transcript in the lumbar spinal cord of 129Sv-mSOD1 mice. Data are normalised to β-actin and expressed as the mean ± SEM fold-change ratio between 129Sv-mSOD1 mice compared to respective age-matched Ntg littermates. * P < 0.05; ⁎⁎ P < 0.01 (Vs. Ntg) by one-way ANOVA with Tukey's post-analysis. ( b ) Representative co-localization of CXCR5 (green) with NT (purple) but not with GFAP (red) in the ventral horn of the spinal cord of fast-progressing mice; scale bar: 20 μm. ( c ) Confocal representative images showing the progressive increase in CXCR5 staining in the lumbar spinal cord of 129Sv-mSOD1 mice.

Article Snippet: Primary antibodies were: goat anti-CXCL13 (1:200, R&D); mouse anti-nonphosphorylated neurofilament H (Smi32, 1:1000; Covance); mouse anti-GFAP (1:1000; Millipore) mouse anti-Iba1 (1:200; Wako).

Techniques: Real-time Polymerase Chain Reaction, Staining

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: The intra-cerebroventricular neutralisation of CXCL13 accelerates the symptom onset and reduced the survival of mSOD1 mice. (a) Body weight loss and (b) Paw Grip Endurance (PaGE) test for mAb-αCXCL13- ( n = 14) and IgG- treated ( n = 17) 129Sv-mSOD1 mice. Data are reported as mean ± SEM for each time point. * P < 0.05; ⁎⁎⁎⁎ P < 0.0001 by repeated-measures ANOVA with Sidak's post-analysis. ( c ) mAb-αCXCL13-treated mice show an earlier impairment in the extension reflex of hindlimbs * P < 0.05; ⁎⁎ P < 0.01; ⁎⁎⁎ P < 0.001 ⁎⁎⁎⁎ P < 0.0001 by repeated-measures ANOVA with Sidak's post-analysis. ( d ) mAb-αCXCL13-treated mice have an earlier onset of motor impairment than IgG- treated mice. P < 0.0055 by Mantel-Cox log-rank test. ( e ) mAb-αCXCL13-treated 129Sv-mSOD1 mice display a reduced survival in respect IgG- treated 129Sv-mSOD1 mice. P < 0.0303 by Mantel-Cox log-rank test. ( f ) Percentage of surviving mAb α-CXCL13 treated mice and controls at different time points (days) after the end of the treatment (16 weeks).

Article Snippet: Primary antibodies were: goat anti-CXCL13 (1:200, R&D); mouse anti-nonphosphorylated neurofilament H (Smi32, 1:1000; Covance); mouse anti-GFAP (1:1000; Millipore) mouse anti-Iba1 (1:200; Wako).

Techniques:

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: Intra-cerebroventricular neutralisation of CXCL13 exacerbated motor neuron impairment, and astrocytosis in the lumbar spinal cord of mSOD1 mice. (a, b) Real-time PCR for (a) Cxcl13 and (b) Cxcr5 transcripts in the lumbar spinal cord of mAb-αCXCL13- and IgG- treated 129Sv-mSOD1 mice compared to Ntg littermates at 16 weeks of age. Data are normalised to β-actin and expressed as the mean ± SEM fold-change ratio between mAb-αCXCL13- and IgG- treated 129Sv-mSOD1 mice and controls. ⁎⁎⁎⁎ P < 0.0001 (Vs Ntg); ° P <0.05 by one-way ANOVA with Tukey's post-analysis. ( c - f ) Representative Immunoblot images and densitometric analysis of (c, d) ChAT (c, e) GFAP (c, f) P-AKT expression in spinal cord extracts from mAb-αCXCL13- and IgG- treated 129Sv-mSOD1 mice compared to Ntg littermates at 16 weeks of age. Data are reported as percentages of the relative Ntg (mean ± SEM). * P < 0.05; ⁎⁎ P < 0.01: ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001 (Vs. Ntg); ° P < 0.05 (between groups) by one-way ANOVA with Tukey's post-analysis.

Article Snippet: Primary antibodies were: goat anti-CXCL13 (1:200, R&D); mouse anti-nonphosphorylated neurofilament H (Smi32, 1:1000; Covance); mouse anti-GFAP (1:1000; Millipore) mouse anti-Iba1 (1:200; Wako).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: Intra-cerebroventricular neutralisation of CXCL13 exacerbated the denervation atrophy of hindlimb skeletal muscles of mSOD1 mice. (a, b) Confocal micrographs showing higher levels of CXCL13 (red) in the sciatic nerve of 129Sv-mSDO1 mice at disease onset and co-localisation with the axonal marker NF200 (green); scale bars 200 μm. ( c-e ) Confocal micrographs showing higher levels of CXCR5 (green) in the sciatic nerve of 129Sv-mSDO1 mice than Ntg mice at disease onset; scale bar: 100 μm. CXCR5 is present in the bundles of Schwann cells (S100β, red), as evidenced by ( e ) the volume view of e. ( f, g ) Confocal micrographs showing the colocalisation of CXCL13 (red), and CXCR5 (green) in correspondence of peripheral axons (Smi32; purple) in the sciatic nerves of 129Sv-mSDO1 mice at the disease onset Scale bar: (f) 50 μm; (g) 10 μm. ( k ) Percent muscle atrophy (muscle wasting) calculated by measuring of the tibialis anterior muscle weight of mAb-αCXCL13- and IgG-treated 129Sv-mice at 16 weeks and endstage compared to relative Ntg littermates. Data are presented as mean ± SEM. ° P < 0.05 by unpaired t -test. ( h-j ) Analysis of muscle denervation on tibialis anterior (TA) muscle of both mAb-αCXCL13-, IgG-treated 129Sv-mice and Ntg littermates at 16 weeks of age. α-Bungarotoxin (BTX, green) was used to identify the postsynaptic domain, synaptic vesicle glycoprotein 2A (SV2, red) + neurofilaments (2H3, red) were used to identify presynaptic terminals. Scale bar 20 μm. ( l ) For each mouse group, the percentage of occupied endplates was calculated. Data are reported as mean ± SEM. ⁎⁎⁎⁎ P < 0.0001 (Vs. Ntg) ° P < 0.05 (between groups) by one-way ANOVA with Tukey's post-analysis.

Article Snippet: Primary antibodies were: goat anti-CXCL13 (1:200, R&D); mouse anti-nonphosphorylated neurofilament H (Smi32, 1:1000; Covance); mouse anti-GFAP (1:1000; Millipore) mouse anti-Iba1 (1:200; Wako).

Techniques: Marker

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: Silencing of CXCL13 expression heightened MN loss and astrocytosis in non-transgenic and mSOD1 primary co-cultures. (a-c) Confocal micrographs showing the co-localisation of CXCL13 (red) with (a, b) the neuronal marker Smi32 (green) and with (c) the microglia marker Iba1; Scale bar, (a, b): 100 μm; (c): 200 μm. ( d ) CXCL13 concentration (pg/mL) in the conditional media of shRNA-CXCl13- and shRNA- scramble (SCR)-treated or untreated (UT) mSOD1 and Ntg co-cultured with mSOD1 and Ntg microglia/astrocytes, respectively. Data are expressed as the mean ± SEM from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.001; ⁎⁎⁎ P < 0.001 Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis. (e) Representative SMI-32 immunostaining images (white) of shRNA-CXCl13- and shRNA-SCR-treated mSOD1 and Ntg motor neurons co-cultured with mSOD1 and Ntg microglia/astrocytes, respectively; scale bar 200 μm. ( f ) Cell count analysis showing greater MN loss in both shRNA-CXCl13 treated mSOD1 and Ntg co-cultures than or shRNA-SCR- treated or untreated (UT) co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.001; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001 by Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis. ( g ) Representative GFAP immunostaining images (green) of shRNA-CXCl13- and shRNA-SCR-treated mSOD1 and Ntg astrocytes co-cultured with mSOD1and Ntg motor neurons/microglia, respectively; scale bar 100 μm. ( h ) Cell Area Fraction (percentage) analysis showing greater astrocytosis in shRNA-CXCl13 treated mSOD1 co-cultures than shRNA-SCR-treated or UT mSOD1 co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.01; ⁎⁎⁎ P < 0.001 ⁎⁎⁎⁎ P < 0.0001 by Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis. ( i ) Representative Iba1 immunostaining images (red) of shRNA-CXCl13- and shRNA-SCR-treated mSOD1 and Ntg microglia co-cultured respectively with mSOD1and Ntg motor neurons/astrocytes; scale bar 100 μm. ( j ) Morphometric parameter of Iba1-positive microglia showing higher activation (mean microglial cell area) of shRNA-CXCl13-treated mSOD1 microglia than shRNA-SCR- treated or UT mSOD1 co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.001; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001 by Kruskall-Wallis ANOVA with uncorrected Dunn's post analysis.

Article Snippet: Primary antibodies were: goat anti-CXCL13 (1:200, R&D); mouse anti-nonphosphorylated neurofilament H (Smi32, 1:1000; Covance); mouse anti-GFAP (1:1000; Millipore) mouse anti-Iba1 (1:200; Wako).

Techniques: Expressing, Transgenic Assay, Marker, Concentration Assay, shRNA, Cell Culture, Immunostaining, Cell Counting, Activation Assay

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: CXCL13 protects motor neurons under acute and chronic treatment. (a, g) Representative SMI-32 immunostaining images (white) of rCXCL13- and rCXCL13+ mAb-αCXCL13- treated co-cultures in the presence (a) or (g) absence of LPS stimulus; scale bar 200 μm. (d, j ) Cell count analysis showing the protective effect of rCXCL13 in the presence (d) or (j) absence of LPS stimulus compared to CXCL13+ mAb-αCXCL13-treated co-cultures. Data are reported as the percentage of UT co-cultures (mean ± SEM of the No. fields analysed from three independent experiments for each experimental group). * P < 0.05; ⁎⁎ P < 0.01 (Vs. UT); °°° P < 0.01 (between groups) by one-way ANOVA with Tukey's post-analysis. (b, h) Representative GFAP immunostaining images (blue) of rCXCL13- and rCXCL13+ mAb-αCXCL13- treated co-cultures in the presence (b) or (h) absence of LPS stimulus; scale bar 100 μm. ( e, k ) Fluorescence Integrated Density (Int_Den) analysis showing greater astrocytosis in rCXCL13 + mAb-αCXCL13-treated co-cultures in the presence (e) or (k) absence of LPS stimulus compared to rCXCL13-treated or UT co-cultures. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001 (Vs. UT); °°°° P < 0.01 (between groups) by one-way ANOVA with Tukey's post-analysis. ( c, i ) Representative Iba1 immunostaining images (green) of rCXCL13- and rCXCL13+ mAb-αCXCL13- treated co-cultures in the presence (c) or (i) absence of LPS stimulus; scale bar 100 μm. ( f, l ) Morphometric parameter of Iba1-positive microglia showing no difference in activation (mean microglial cell area) between the experimental groups. Data are reported as mean ± SEM of the No. fields analysed from three independent experiments for each experimental group. * P < 0.05; ⁎⁎ P < 0.01; ⁎⁎⁎⁎ P < 0.0001 (Vs. UT) by one-way ANOVA with Tukey's post-analysis.

Article Snippet: Primary antibodies were: goat anti-CXCL13 (1:200, R&D); mouse anti-nonphosphorylated neurofilament H (Smi32, 1:1000; Covance); mouse anti-GFAP (1:1000; Millipore) mouse anti-Iba1 (1:200; Wako).

Techniques: Immunostaining, Cell Counting, Fluorescence, Activation Assay

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: CXCL13 is upregulated in the motor neurons of the spinal cord of familial and sporadic ALS patients. (a-l) Representative confocal images of immunofluorescence staining for pTDP43, CXCL13, and Neurotrace (neuronal marker) in the post-mortem lumbar spinal cord of (e-l) n = 4 sporadic (sALS) and n = 4 familial (fALS) ALS patients showing the upregulation of the CXCL13 staining in motor neurons compared to (a-d) n = 4 controls; Scale bar: 100 μm; Inset: 50 μm.

Article Snippet: Primary antibodies were: goat anti-CXCL13 (1:200, R&D); mouse anti-nonphosphorylated neurofilament H (Smi32, 1:1000; Covance); mouse anti-GFAP (1:1000; Millipore) mouse anti-Iba1 (1:200; Wako).

Techniques: Immunofluorescence, Staining, Marker

Journal: EBioMedicine

Article Title: CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2020.103097

Figure Lengend Snippet: CXCL13 has a low concentration in the CSF of ALS patients (a, b) CXCL13 concentration (pg/mL) in the CSF of (a) total ALS patients and (b) sub-grouped by disease onset and normative controls. ( c ) CXCL13 concentration (pg/mL) in the CSF of ALS patients and MS patients. Data are reported as mean ± SEM. P < 0.05 by Mann-Whitney test. ( d ) ROC curve and analysis of the area under the curve (AUC) were used to find the discriminatory power of CXCL13 CSF levels between ALS and MS patients. A 95% Confidence Interval (CI) was used and results are reported as percentage.

Article Snippet: Primary antibodies were: goat anti-CXCL13 (1:200, R&D); mouse anti-nonphosphorylated neurofilament H (Smi32, 1:1000; Covance); mouse anti-GFAP (1:1000; Millipore) mouse anti-Iba1 (1:200; Wako).

Techniques: Concentration Assay, MANN-WHITNEY