Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons, axon degeneration, and denervation of neuromuscular junctions (NMJ). Here we show that death receptor 6 (DR6) levels are elevated in spinal cords from post-mortem samples of human ALS and from SOD1(G93A) transgenic mice, and DR6 promotes motor neuron death through activation of the caspase 3 signaling pathway. Blocking DR6 with antagonist antibody 5D10 promotes motor neuron survival in vitro via activation of Akt phosphorylation and inhibition of the caspase 3 signaling pathway, after growth factor withdrawal, sodium arsenite treatment or co-culture with SOD1(G93A) astrocytes. Treatment of SOD1(G93A) mice at an asymptomatic stage starting on the age of 42 days with 5D10 protects NMJ from denervation, decreases gliosis, increases survival of motor neurons and CC1(+) oligodendrocytes in spinal cord, decreases phosphorylated neurofilament heavy chain (pNfH) levels in serum, and promotes motor functional improvement assessed by increased grip strength. The combined data provide clear evidence for neuroprotective effects of 5D10. Blocking DR6 function represents a new approach for the treatment of neurodegenerative disorders involving motor neuron death and axon degeneration, such as ALS.

Figure 1

DR6 mRNA and protein levels are upregulated in spinal cord of SOD1^G93A mice and ALS post-mortem samples. (a) In situ hybridization of DR6⁺ motor neurons at the age of 60 days SOD1^G93A and WT mice, scale bar=25 μm. (b) Quantification of DR6⁺ motor neuron number from a, n=4 fields/3 animals/group. (c) IHC analysis of DR6 expression in SOD1^G93A and WT mice lumbar spinal cord ventral horn region (age 60 days), DR6 (red), NeuN (green), scale bar=50 μm. (d) Quantification of DR6⁺/NeuN⁺ (>20 μm) neuron number from c, n=9 sections/3 animals/group. (e) Western blot analysis in SOD1^G93A and WT mice spinal cord (age 60 days) for DR6 expression, WT: n=7, SOD1^G93A: n=8. β-actin was used as an internal control. (f) IHC analysis of DR6 expression in human non-ALS or ALS post-mortem spinal cord tissue, adjacent frozen sections were used to stain DR6 (brown, top panel) and NeuN (blue, bottom panel), respectively, arrows denote DR6⁺ (top panel) or NeuN⁺ (bottom panel) neurons, respectively, scale bar=20 μm. (g) Quantification of DR6⁺/NeuN⁺ (>30 μm) neuron number from f, n=3 samples/group. (h) Western blot analysis showing DR6 expression in human post-mortem spinal cord samples run in duplicates. β-actin was used as an internal control. (i) Quantification of DR6 expression from h plus an additional nine ALS and five non-ALS spinal cords by densitometry. Total ALS samples: n=13, total non-ALS samples: n=9. Data presented as the ratio of DR6 over actin. Data in b, d, g, and i were shown as mean±S.E.M. P-values were determined by two-tailed unpaired t test

Figure 2

Anti-DR6 antibody promotes human motor neuron survival and preserves axon integrity in vitro. (a) ICC images of human motor neurons following growth factor removal, and treated with 10 μg/ml isotype control antibody or 5D10, NF (green), scale bar=95 μm. (b) Quantification of surviving motor neuron number from a, growth factor supplemented condition was used as a positive control (100%), n=12–48 fields/group. (c) Quantification of axon length from a, n=∼30 axons/15 fields/group. (d) ICC images of rat motor neurons treated with 10 μg/ml control antibody or 5D10 following growth factor removal, MAP2 (green), arrow, punctated structures along axons, scale bar=15 μm. (e) Quantification of axon punctated structures from d, n=10 axons/10 fields/group. (f) ICC images of rat motor neuron axons treated with 10 μg/ml control antibody or 5D10 following growth factor removal, SV2 (green), βIII-tubulin (red), scale bar=10 μm. (g) Quantification of SV2⁺ segmented structures per mm axon from f, n=10 axons/10 fields/group. (h) ICC images of human motor neurons following sodium arsenite, and treated with 10 μg/ml 5D10 or control antibody, NF (green), scale bar=25 μm. (i) Quantification of surviving motor neuron number from h, sodium arsenite minus condition was used as a positive control (100%), n=12 fields/group. (j) Quantification of axon length from h, n=∼24 axons/12 fields/group. (k) ICC images of rat motor neurons in co-cultures with purified astrocytes from WT or SOD1^G93A mice, and treated with 10 μg/ml 5D10 or control antibody. NF (red), GFAP (green), arrows denote axon punctated structures, scale bar=25 μm. (l) Quantification of NF levels from k by MSD analysis, n=2 samples/group. (m) Quantification of axon (longer than 250 μm) punctated structures from k, n=12 axons/12 fields/group. Data in b, c, e, g, i, j, l, and m were shown as mean±S.E.M. P-values in e were determined by two-tailed unpaired t-test, and in b, c, g, i, j, l and m by one-way analysis of variance (ANOVA followed by Bonferroni's test)

Figure 3

Anti-DR6 antibody promotes human motor neuron survival through inhibition of caspase 3 and activation of Akt phosphorylation. (a) Western blot analysis of cleaved casp3 and phosphorylated Akt in human motor neuron cultured in the presence or absence of growth factor and treated with 5D10 or control antibody (duplicate culture samples were analyzed). β-actin and total Akt were used as internal controls. Quantification of cleaved casp3 (b) and P-Akt (c) levels from a by densitometry. (d) Quantification of the number of surviving MAP2⁺ motor neurons purified from WT or Tnfrsf21^−/− mice following growth factor removal, n=15 fields/group. (e) Quantification of casp3⁺ motor neurons from d, n=15 fields/group. (f) Western blot analysis of cleaved casp3 and P-Akt in human motor neurons cultured in the presence or absence of growth factor and treated with or without casp3 inhibitor (duplicate culture samples were analyzed). β-actin was used as an internal control. Quantification of cleaved casp3 (g) and P-Akt (h) levels from f by densitometry. Data in b, c, d, e, g, and h were shown as mean±S.E.M. P-values were determined by two-tailed unpaired t test

Figure 4

Blocking DR6 improves tissue integrity in SOD1^G93A mice. (a) IHC images of gastrocnemius muscle NMJs in 5D10 or control antibody treated SOD1^G93A mice (age 60 days), SV2 and NF (green), BuTx (red), scale bar=25 μm. (b) Quantification of NMJs from a, n=∼300 NMJs/three animals/group. (c) IHC images of GFAP staining (green) in lumbar spinal cord of WT mice and 5D10 or control antibody treated SOD1^G93A mice (age 60 days), scale bar=95 μm. (d) Quantification of GFAP staining from c, by mean fluorescence intensity (MFI) measurements. GFAP levels in control=100%, n=18 fields/3 animals/group. (e) IHC images of Nissl-stained lumbar spinal cord motor neurons in 5D10 or control antibody treated SOD1^G93A mice (age 100 days), scale bar=100 μm. (f) Quantification of motor neuron number from e, ventral horn motor neurons (>20 μm) were counted, n=9–18 sections/3–6 animals/group. (g) IHC images of cleaved casp3 staining (red) in lumbar spinal cord motor neurons of 5D10 or control antibody treated SOD1^G93A mice (age 100 days), arrows denote casp3⁺ cells, DAPI (blue), scale bar=50 μm, inset scale bar=50 μm. (h) Quantification of casp3⁺ cells in ventral horn region from g, n=9 sections/3 animals/group. Data in b, d, f and h were shown as mean±S.E.M. P-values in b, d and h were determined by two-tailed unpaired t-test, and in f were determined by one-way ANOVA followed by Bonferroni's test

Figure 5

5D10 treatment deceases serum pNfH levels and improves hindlimb grip strength in SOD1^G93A mice. (a) Quantification of serum pNfH levels in WT or SOD1^G93A mice by MSD analysis, n=3–11 animals/group. (b) Quantification of serum pNfH levels of 5D10 or control antibody treated SOD1^G93A mice (age 100 days) by MSD analysis, n=8–9 animals/group. (c) Correlation analysis of serum pNfH levels and gastrocnemius muscle NMJs. (d) Hindlimb grip strength analysis (in grams) of 5D10 or control antibody treated SOD1^G93A mice (age 100 days), n=8–9 animals/group. Data in a, b and d were shown as mean±S.E.M. P-values in a, b and d were determined by two-tailed unpaired t test, and in c was determined by Pearson correlation analysis