Aberrant neuregulin 1 signaling in amyotrophic lateral sclerosis

Abstract

Neuregulin 1 (NRG1) is a neuron-derived trophic molecule that supports axoglial and neuromuscular development through several alternatively spliced isoforms; its possible role in the pathogenesis and progression of amyotrophic lateral sclerosis (ALS) is not known. We analyzed the relationship of NRG1 isoform expression to glial cell activation and motor neuron loss in spinal cords of ALS patients and during disease progression in the superoxide dismutase 1 (SOD1) ALS mouse model. Microgliosis, astrocytosis, and motor neuron loss were observed in the ventral horns in ALS patients and were increased in SOD1 mice along with disease progression. Type III (membrane-bound) NRG1 expression was reduced in parallel with motor neuron loss, but Type I (secreted) NRG1 expression was increased and was associated with glial activation. Increased NRG1 receptor activation was observed on activated microglia in both ALS patients and in SOD1 mice. This activation was observed at the time of disease onset and before upregulation of NRG1 gene expression in the mice. The downregulation of membrane-bound Type III NRG1 forms may reflect motor neuron loss, but increased signaling by secreted-type NRG1 isoforms could contribute to disease pathogenesis through glial cell activation. NRG1 might, therefore, represent a novel therapeutic target against disease progression in ALS.

Figure 1

Spinal cords of amyotrophic lateral sclerosis (ALS) patients and superoxide dismutase 1 (SOD1) mice. Spinal cords stained with Luxol fast blue-periodic acid Schiff demonstrate myelin loss (top and second rows, pink areas) within the anterior and lateral corticospinal tracts (LCSTs) in ALS, but not in the mouse dorsal corticospinal tract (DCST). Cresyl violet stains show motor neuron loss (large cells) in the ventral horn in both ALS and SOD1 mice along with increased cellularity compared to control (third row). Scale bars: top row, 500 μm for human and 200 μm for mouse; second and third rows, 50 μm for human and mouse.

Figure 2

Activated microglia accumulate in the ventral horn in amyotrophic lateral sclerosis (ALS) and in superoxide dismutase 1 (SOD1) mice. Human (CD68-positive: green, top row) and mouse (CD11b-positive: green, bottom row) microglia are increased in the ventral horns of an ALS patient and an end-stage SOD1 mouse compared to a control patient and a non-transgenic (non-Tg) control mouse. Reactive astrocytes (glial fibrillary acidic protein [GFAP]-positive, red, top and bottom rows) are increased in the ventral horn of the SOD1 mouse (bottom row), but not markedly in the ALS case (top row). Scale bars: 20 μm.

Figure 3

Motor neuron loss and glial changes are associated with changes in neuregulin1 (NRG) isoform expression in superoxide dismutase 1 (SOD1) mice. (A, B) There were significant reductions in motor neurons in SOD1 mice (A) and increases in activated microglia and reactive astrocytes (B) in the ventral horns of SOD1 mice compared to their non-transgenic (non-Tg) littermates (n = 4-8 mice/group/time point; high-power field (HPF) = 0.28 mm² for A; HPF = 0.14 mm^{2 for} B; *p < 0.05, **p < 0.01, ***p < 0.001 for SOD1 mice vs. non -Tg mice at the same time point for A and B). (C) There was an increased in type I NRG1 mRNA at end stage and a corresponding decrease in type III NRG1 mRNA in SOD1 mice vs. age- and gender-matched non Tg littermates (n = 4/group). (D) The same samples showed little change in brain-derived neurotrophic factor (BDNF) and glial cell-line derived neurotrophic factor (GDNF) mRNA (n = fold change from 4 SOD1 mice vs. 4 non-Tg mice at each time point. *p < 0.05, **p < 0.01 for the fold changes at day 56, day 90 or day >112<126 vs. day 35 for C and D.

Figure 4

Characterization of neuregulin1 (NRG1) isoform-specific antibodies. (A) Schematic of type I and type III NRG1 domain structure shows that both types of NRG1 are expressed as transmembrane precursor proteins that can be recognized by a pan-NRG1 antibody directed against the cytoplasmic tail (SC384). Following cleavage, “released” forms of NRG1(including the type I form) contain 2 extracellular domains: an immunoglobulin-like (IG) domain that functions as a heparin-binding domain (HBD) and an EGF-like domain (EGF) that is sufficient to bind and activate the erbB receptors. The type III NRG1 “membrane-associated form” precursor can also be cleaved, however, the hydrophobic cysteine-rich domain (CRD) remains tethered in the membrane. (B) We generated a highly specific anti-CRD domain NRG1 antibody that recognizes the intracellular portion of type III NRG1. The antibody is specific on Western blots for a 42-kDa protein in Chinese hamster ovary cells transfected with human type III NRG1, but not for type I NRG1 or enhanced green fluorescent protein (EGFP). Western blots of human and mouse spinal cords show that the CRD NRG1 antibody also recognizes the type III NRG1 of approximately 50 kDa. The difference in size could be due to glycosylation or alternative splicing. (C) Immunostaining of the same transfected Chinese hamster ovary cells with either human type III NRG1 or human type I NRG show that the anti-CRD NRG1 antibody specifically recognizes only type III NRG1.

Figure 5

Reduced Type III neuregulin1 (NRG1) protein expression correlates with motor neuron loss in the ventral horn in amyotrophic lateral sclerosis (ALS) and in superoxide dismutase 1 (SOD1) mice. (A, B) A pan-NRG1 antibody stains motor neurons and additional small cells in the ventral horn of both ALS and SOD1 mice (A), whereas type III NRG1 protein expression is only expressed in motor neurons (B). Scale bars: top rows, 50 μm; bottom rows, 10 μm. (C) Reduction in type III NRG1 mRNA expression in SOD1 mice (from Fig. 3C) correlated significantly with motor neuron number (n= 4 SOD1 mice / time point; data taken from Fig. 3A; n = fold change of type III NRG1 from 4 SOD1 mice vs. 4 non-transgenic (non-Tg) mice at each time point. #: p < 0.05 for the fold changes at day >112<126 vs. day 35). (n = 4-8 SOD1 mice / time point; *: p < 0.05, **: p < 0.01, for motor neuron number at day 90 or day >112<126 vs. day 35). (D) Type III (CRD) NRG1 protein is reduced in spinal cords from end-stage SOD1 mice vs. littermate non-Tg mice by Western blotting.

Figure 6

ErbB2 receptor expression and activation (p-erbB2) is increased in the ventral horn of end stage superoxide dismutase 1 (SOD1) mice. (A) Neuregulin1 (NRG1) receptors (erbB2) and erbB2 receptor activation (p-erbB2) are both increased in the lumbar ventral spinal cord of end-stage SOD1 mice compared to non-transgenic (non-Tg) littermates. Scale bar: 50 μm. (B) Western blots show the specificity of anti-erbB2 and anti-p-erbB2 antibodies as a series of bands at 185 kDa in spinal cords from non-Tg mice.

Figure 7

Neuregulin1 (NRG1) receptor activation occurs primarily on microglia and is present from clinical disease onset in superoxide dismutase 1 (SOD1) mice. (A) Confocal images show colocalization of activated p-erbB2 (green, first and second rows) mostly with microglia (CD11b-positive, red, first row), but less so on astrocytes (glial fibrillary acidic protein [GFAP]-positive, red, second row). Scale bar: 10 μm. (B) Quantitation of p-erbB2 positive microglia and astrocytes shows that while most microglia show receptor activation, most astrocytes do not (n = 4 mice/group/time point; results are expressed as percentage of total microglia or astrocytes). (C) NRG1 receptor activation is seen in microglia at disease onset and is increased as the disease progresses in SOD1 mice. (n = 4-8 mice/group/time point; *p < 0.05, **p < 0.01, ***p < 0.001, for SOD1 mice vs. non-transgenic (non-Tg) mice at the same time point for B and C).

Figure 8

Neuregulin1 (NRG1) receptor activation is present on activated microglia in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, but not controls. NRG1 receptor activation (p-erbB2-positive, green) colocalizes with activated microglia (CD68-positive, red) in the ventral horn of an ALS patient. Quantitation of the percent overlapping signal revealed 25.00% ± 6.25% overlap vs. 2.67 ± 1.15% for controls. The blue channel shows nuclear staining with 4’, 6-diamidino-2-phenylindole (DAPI) to demonstrate presence of cells. This was similar in the no-primary control shown in the lower panel. Similar results were seen in 3 ALS and 3 control patients (p < 0.01). Scale bars: 10 μm.