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The P2X7 receptor antagonist JNJ-47965567 administered thrice weekly from disease onset does not alter progression of amyotrophic lateral sclerosis in SOD1G93A mice

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Abstract

The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate disease in SOD1G93A mice, but recent data suggests that this antagonist displays poor penetration of the central nervous system (CNS). Therefore, the current study aimed to determine whether the CNS-penetrant P2X7 antagonist, JNJ-47965567, could ameliorate ALS progression in SOD1G93A mice. A flow cytometric assay revealed that JNJ-47965567 impaired ATP-induced cation dye uptake in a concentration-dependent manner in murine J774 macrophages. Female and male SOD1G93A mice were injected intraperitoneally with JNJ-47965567 (30 mg/kg) or 2-(hydroxypropyl)-beta-cyclodextrin (vehicle control) three times a week from disease onset until end stage, when tissues were collected and studied. JNJ-47965567 did not impact weight loss, clinical score, motor (rotarod) coordination or survival compared to control mice. NanoString analysis revealed altered spinal cord gene expression in JNJ-47965567 mice compared to control mice, but such differences were not confirmed by quantitative PCR. Flow cytometric analyses revealed no differences between treatments in the frequencies or activation status of T cell or dendritic cell subsets in lymphoid tissues or in the concentrations of serum cytokines. Notably, serum IL-27, IFNβ and IL-10 were present in relatively high concentrations compared to other cytokines in both groups. In conclusion, JNJ-47965567 administered thrice weekly from disease onset did not alter disease progression or molecular and cellular parameters in SOD1G93A mice.

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Acknowledgements

The authors kindly acknowledge Sarah Toole (University of Wollongong) for advice regarding adverse events in mice; Kara L. Vine (University of Wollongong) and Anindya Bhattacharya (Janssen Research and Development) for advice regarding drug formulation; and Sam R. Adhikary (University of Wollongong) for assistance with necropsy. The authors also kindly acknowledge the technical staff of the Illawarra Health and Medical Research Institute and the University of Wollongong Rodent Facility.

Funding

This study was supported by grants from the Motor Neurone Disease Research Institute of Australia (R.S.), from the Australian National Health and Medical Research Council (NHMRC) Career Development Fellowship (APP1084144) (J.J.Y) and from the NHMRC Project Grant (APP1104295) (B.J.T). D.L. was a recipient of an Illawarra Health and Medical Research Institute Careers Development Grant. P.C., N.G and R.A.S were recipients of Australian Government Research Training Program (AGRTP) Scholarships.

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  1. Anjila Dongol and Peter Cuthbertson contributed equally to this work.

Authors and Affiliations

  1. Illawarra Health and Medical Research Institute, Wollongong, NSW, 2522, Australia

    Diane Ly, Anjila Dongol, Peter Cuthbertson, Thomas V. Guy, Nicholas J. Geraghty, Reece A. Sophocleous, Lucia Sin, Debbie Watson, Justin J. Yerbury & Ronald Sluyter

  2. Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, 2522, Australia

    Diane Ly, Anjila Dongol, Peter Cuthbertson, Nicholas J. Geraghty, Reece A. Sophocleous, Lucia Sin, Debbie Watson, Justin J. Yerbury & Ronald Sluyter

  3. Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC, 3052, Australia

    Bradley J. Turner

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All procedures performed in studies involving animals were in accordance with the ethical standards of the University of Wollongong at which the studies were conducted (Animal Ethics Committee protocols AE12/09 and AE16/18). This study does not contain any work with human participants performed by any of the authors.

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Fig. S1

JNJ-47965567 does not alter clinical disease progression in female or male SOD1G93A mice. β-CD- and JNJ-47965567-treated SOD1G93A mice (Fig. 1) were stratified according to sex (female, left panels; male, right panels) to compare (A) ALS score, (B) body weight loss (percent of pre-disease maximum), (C) motor (rotarod) coordination and (D) survival. (A-D) Results are mean ± SEM (β-CD, n = 12 females or 11 males; JNJ-47965567, n = 12 females or 12 males) with differences between groups compared using (A-C) a two-way ANOVA or (D) the log-rank (Mantel-Cox) test. (PNG 36 kb)

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Fig. S2

Gating strategies used to determine proportions of leukocyte subsets in SOD1G93A mice. (A) For analysis of T cell subsets, sequential flow cytometric gates were selected left to right as shown (top dot plots) and used to determine the proportion of total CD4+ and CD8+ T cells, as well as conventional CD4+ T cells (Foxp3−) and regulatory T cells (Foxp3+). The proportion of activated T cells (CD44+) in each T cell subset was then determined (bottom dot plots), with the relative CD39 expression on CD44+ T cell subsets also determined (histogram). (B) For analysis of DC subsets, single live cells were gated as for T cells (not shown) and then the proportion of conventional DCs (CD11c+MHC class IIlow cells) and migratory DCs (CD11c+MHC class IIhi cells) determined (left dot plot). The proportion of CD11b+ and CD11b− DCs amongst conventional and migratory DCs was then determined (middle dot plots). Finally, the relative CD80 expression on DC subsets was determined (histograms). (PNG 1937 kb)

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Ly, D., Dongol, A., Cuthbertson, P. et al. The P2X7 receptor antagonist JNJ-47965567 administered thrice weekly from disease onset does not alter progression of amyotrophic lateral sclerosis in SOD1G93A mice. Purinergic Signalling 16, 109–122 (2020). https://doi.org/10.1007/s11302-020-09692-4

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  • DOI: https://doi.org/10.1007/s11302-020-09692-4

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