From transcriptome analysis to therapeutic anti-CD40L treatment in the SOD1 model of amyotrophic lateral sclerosis

Journal name:
Nature Genetics
Volume:
42,
Pages:
392–399
Year published:
DOI:
doi:10.1038/ng.557
Received
Accepted
Published online

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. Using unbiased transcript profiling in an ALS mouse model, we identified a role for the co-stimulatory pathway, a key regulator of immune responses. Furthermore, we observed that this pathway is upregulated in the blood of 56% of human patients with ALS. A therapy using a monoclonal antibody to CD40L was developed that slows weight loss, delays paralysis and extends survival in an ALS mouse model. This work demonstrates that unbiased transcript profiling can identify cellular pathways responsive to therapeutic intervention in a preclinical model of human disease.

At a glance

Figures

  1. Co-stimulatory pathway signaling in SOD1G93A skeletal muscle, spinal cord and sciatic nerve is upregulated during disease progression and is increased in a subset of blood samples from individuals with ALS.
    Figure 1: Co-stimulatory pathway signaling in SOD1G93A skeletal muscle, spinal cord and sciatic nerve is upregulated during disease progression and is increased in a subset of blood samples from individuals with ALS.

    We assessed the statistical differences in gene expression between SOD1G93A and wild-type medial gastrocnemius, spinal cord and sciatic nerve at a given time point using the nonlinear Bayes methodology in the Bioconductor LIMMA package and overlaid the resulting analysis onto the genMAPP pathway for co-stimulatory signaling (genMAPP: Mm-Std_20060628.gdb). Each colored box represents the estimated change in transcript abundance for a given gene after normalization and data summarization. Biological replicates for the SOD1G93A transgenic mice are n = 5. Age-matched, non-transgenic mice are biological replicates of n = 5. The key details the relative increase in transcript expression levels, with blue intensity representing decreasing levels and pink intensity representing increasing levels. (a) Gastrocnemius. (b) Spinal cord. (c) Sciatic nerve. (d) A heat-map visualization of the normalized expression data for genes of the human T cell co-stimulatory pathway from 27 non-ALS and 63 ALS blood samples. We normalized Affymetrix gene chip data using robust multi-array averaging and clustered samples using self-organizing maps. Gene expression intensity is represented colorimetrically, with low expression represented as blue rectangles and high expression as pink rectangles.

  2. Macrophages accumulate in peripheral nerves throughout the disease course.
    Figure 2: Macrophages accumulate in peripheral nerves throughout the disease course.

    Shown is a time course of immunofluorescence staining for CD68+ macrophage (green) and DAPI-counterstained nuclei (blue) in sciatic nerve. (a) Day 50. (b) Day 60. (c) Day 80. (d) Day 100. (e) Day 110. Scale bars in ae, 17 μm. (f) Quantification of accumulated CD68+ macrophages in the S100b+ nerves of the gastrocnemius. WT, wild type.

  3. Blocking CD40L with a monoclonal antibody to CD40L improves body-weight maintenance, delays disease onset and extends survival in SOD1 mice.
    Figure 3: Blocking CD40L with a monoclonal antibody to CD40L improves body-weight maintenance, delays disease onset and extends survival in SOD1 mice.

    SOD1G93A mice received a loading dose (5.22 mg per kg body weight for females, 6.75 mg per kg for males) followed by weekly maintenance doses (1 mg per kg for females, 1.34 mg per kg for males) given intraperitoneally beginning at 50 days of age and continuing until death. (a) Kaplan-Meier time-to-event analysis for time required to attain peak body weight. Time to peak was not significantly (P = 0.35) changed by anti-CD40L treatment. Ctrl., control; Drug, anti-CD40L. (b) Time-to-event analysis for the time from peak body weight until death. Body-weight maintenance was significantly (P = 0.0413) improved by anti-CD40L treatment. (c) Time-to-event analysis for disease onset, the age at which mice first showed signs of definitive neurological disease (neurological severity score of 2). Disease onset was significantly (P = 0.0038) delayed by anti-CD40L treatment. (d) Time-to-event analysis for survival, the age at which mice died. Survival was significantly (P = 0.0043) prolonged by anti-CD40L treatment. Results of statistical analyses are presented in Table 1.

  4. Meta-analysis of anti-CD40L treatment compared with riluzole, apocynin and historical controls.
    Figure 4: Meta-analysis of anti-CD40L treatment compared with riluzole, apocynin and historical controls.

    (a) Monte Carlo analysis of historical control SOD1 animals to assess the probability of randomly detecting a 9-d lengthening of survival. We randomly assigned 44 untreated SOD1G93A animals to either a mock control group or a mock treatment group and performed Kaplan-Meier survival analysis. We performed 1,000 iterations and plotted the frequency distribution as a function of percent change in survival. (b) The Kaplan-Meier survival data from historical control groups compared with a group treated with weekly intraperitoneal injection of 1 mg per kg (body weight) anti-CD40L. (c) Outlier analysis shows that anti-CD40L results do not fall within the distribution of results from other drugs tested in the SOD1G93A model. The jackknife outlier distance for each observation is calculated using estimates of the mean, s.d. and correlation matrix that do not include the observation itself. Extreme multivariate outliers are identified as points that exceed the upper distance value limit (dotted line). Results are tabulated by drug in Supplementary Table 4.

  5. MR1 treatment lowers the frequency of CD68+ cells in sciatic nerve and CD8+ T cells in sciatic lymph node.
    Figure 5: MR1 treatment lowers the frequency of CD68+ cells in sciatic nerve and CD8+ T cells in sciatic lymph node.

    (a) CD68+ macrophage (green) in S100b+ sciatic nerve (red), 100-day-old controls. (b) CD68+ macrophage (green) in S100b+ sciatic nerve (red), 100-day-old anti-CD40L–treated mice. (c) Quantification of reduction of CD68+ macrophage by anti-CD40L treatment, day 100. White bar, control; gray bar, anti-CD40L–treated; black bar, untreated age-matched non-transgenic mice. WT, wild type. (df) FACS analysis of CD4 and CD8 expression in CD3+ lymphocytes isolated from sciatic lymph node in age-matched (day 80) non-transgenic SOD1G93A (d), untreated control (e), and SOD1G93A anti-CD40L–treated mice (f). Full flow cytometry details and machine settings are described in the Online Methods.

  6. Anti-CD40L treatment decreases astrocytosis and microgliosis while reducing motor neuron loss in the spinal cord of SOD1G93A mice.
    Figure 6: Anti-CD40L treatment decreases astrocytosis and microgliosis while reducing motor neuron loss in the spinal cord of SOD1G93A mice.

    (a) Gfap staining (green; DAPI staining is blue) in reactive astrocytes in the lumbar spinal cord of untreated SOD1G93A mice. (b) Gfap staining (green; DAPI, blue) in the lumbar spinal cord of anti-CD40L–treated mice. (c) Mac-2 staining (red; DAPI, blue) in microglia in the lumbar spinal cord of untreated SOD1G93A mice. Inset, Mac-2+ cell from vehicle control. (d) Mac-2 staining (red; DAPI, blue) in the lumbar spinal cord of anti-CD40L–treated mice. Inset, Mac-2+ cell from treated mouse. (e,f) Representative field of Nissl-stained lumbar motor neurons in untreated SOD1G93A mice (e) and anti-CD40L SOD1G93A mice (f). (g) Quantitative comparison of lumbar spinal cord motor neuron counts per mm2 in control (n = 4) versus anti-CD40L–treated mice (n = 8).

  7. Treatment of SOD1G93A mice with anti-CD40L decreases the expression of genes in the co-stimulatory pathway in the spinal cord.
    Figure 7: Treatment of SOD1G93A mice with anti-CD40L decreases the expression of genes in the co-stimulatory pathway in the spinal cord.

    We used Affymetrix gene expression profiling to analyze spinal cords from non-transgenic animals (non Tg), SOD1G93A animals treated for 40 d with 1 mg per kg (body weight) per week of anti-CD40L (aCD40L) and SOD1G93A untreated animals (G93A). Plot shows fold differences in the expression of genes in the co-stimulatory pathway, comparing non-transgenic animals to untreated and treated SOD1G93A animals. Genes are noted on the x axis, sorted by difference in gene expression between treated and untreated groups.

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Author information

  1. These authors contributed equally to this work.

    • John M Lincecum &
    • Fernando G Vieira

Affiliations

  1. ALS Therapy Development Institute, Cambridge, Massachusetts, USA.

    • John M Lincecum,
    • Fernando G Vieira,
    • Monica Z Wang,
    • Kenneth Thompson,
    • Gerald S De Zutter,
    • Joshua Kidd,
    • Andrew Moreno,
    • Ricardo Sanchez,
    • Isarelis J Carrion,
    • Beth A Levine,
    • Bashar M Al-Nakhala,
    • Shawn M Sullivan,
    • Alan Gill &
    • Steven Perrin

Contributions

J.M.L., F.G.V., A.G. and S.P. designed the experiments. M.Z.W. performed the immunohistochemistry and FACS experiments. R.S. and I.J.C. performed the motor neuron histology. B.A.L. oversaw and consulted on the human blood sample study. K.T., J.K. and A.M. performed all the animal studies. G.S.D.Z. consulted on the interpretation of the results. B.M.A. and S.M.S. wrote the simulation and LIMS software. A.G. performed all pharmacological statistical analysis. S.P., J.M.L., A.G. and F.G.V. wrote the paper. All authors discussed the results and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (3.5M)

    Supplementary Figures 1–3 and Supplementary Table 4

Excel files

  1. Supplementary Table 1 (28K)

    Calculated Q scores of significantly different pathways in SOD1G93A mice compared to non-transgenic littermates.

  2. Supplementary Table 2 (736K)

    RMA normalized gene expression data of all genes in five significantly changing pathways in SOD1G93A mice compared to non-transgenic littermates.

  3. Supplementary Table 3 (108K)

    Calculated fold-change data of all genes in five significantly changing pathways in SOD1G93A mice compared to non-transgenic littermates derived from RMA normalized data.

  4. Supplementary Table 5 (212K)

    Relative normalized transcript expression of costimulatory genes in human clinical blood samples.

  5. Supplementary Note (72K)

    Clinical annotation of human clinical blood samples

Additional data