Shift from extracellular signal-regulated kinase to AKT/cAMP response element-binding protein pathway increases survival-motor-neuron expression in spinal-muscular-atrophy-like mice and patient cells

Abstract

Spinal muscular atrophy (SMA), a recessive neurodegenerative disease, is characterized by the selective loss of spinal motor neurons. No available therapy exists for SMA, which represents one of the leading genetic causes of death in childhood. SMA is caused by a mutation of the survival-of-motor-neuron 1 (SMN1) gene, leading to a quantitative defect in the survival-motor-neuron (SMN) protein expression. All patients retain one or more copies of the SMN2 gene, which modulates the disease severity by producing a small amount of stable SMN protein. We reported recently that NMDA receptor activation, directly in the spinal cord, significantly enhanced the transcription rate of the SMN2 genes in a mouse model of very severe SMA (referred as type 1) by a mechanism that involved AKT/CREB pathway activation. Here, we provide the first compelling evidence for a competition between the MEK/ERK/Elk-1 and the phosphatidylinositol 3-kinase/AKT/CREB signaling pathways for SMN2 gene regulation in the spinal cord of type 1 SMA-like mice. The inhibition of the MEK/ERK/Elk-1 pathway promotes the AKT/CREB pathway activation, leading to (1) an enhanced SMN expression in the spinal cord of SMA-like mice and in human SMA myotubes and (2) a 2.8-fold lifespan extension in SMA-like mice. Furthermore, we identified a crosstalk between ERK and AKT signaling pathways that involves the calcium-dependent modulation of CaMKII activity. Together, all these data open new perspectives to the therapeutic strategy for SMA patients.

Figure 1.

ERK pathway inhibition by U0126 induces a significant increase in SMN expression in the motor neurons of severe SMA-like mice and in myogenic cells of severe-type SMA patients. A, B, Western blot analysis and quantification of SMN protein expression in the ventral lumbar spinal cord of vehicle- and U0126-treated control mice at 2 and 6 d of age, vehicle-treated SMA-like mice at 2 d of age, and U0126-treated SMA-like mice at 2 and 6 d of age (n = 3). C, D, Western blot analysis and quantification of SMN protein expression in brain, heart, kidneys, lungs, liver, diaphragm, and tibialis of U0126-treated SMA-like mice at 2 d of age (n = 3). E–I, Immunodetection of gems in ChAT-positive motor neurons in the lumbar spinal cord (L1–L5) of vehicle-treated (E) and U0126-treated control (F) mice at 6 d of age, vehicle-treated SMA-like mice at 2 d of age (G), and U0126-treated SMA-like mice at 6 d of age (H). Scale bar, 25 μm. Quantitative analysis of gems per motor neuron (I). J, K, Western blot analysis and quantification of SMN protein expression in vehicle- and U0126-treated human SMA cultured myotubes from two type 2 SMA patients (n = 3 each patient). L, M, Western blot analysis and quantification of SMN protein expression in vehicle- and U0126-treated human SMA cultured myotubes and myoblasts from a type 2 SMA patient (n = 3). N, O, Quantification by real-time RT-PCR of exon 7–exon 8 (E7-E8) segment containing SMN transcripts normalized either by exon 4–exon 5 (E4-E5) segment containing SMN transcripts (N) or by 18S transcripts (O) in the ventral lumbar spinal cord of vehicle- and U0126-treated control and SMA-like mice at 2 d of age (n = 9). Data are displayed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Activated Elk-1 and CREB transcription factors compete for binding the SMN2 promoter in the spinal cord of severe SMA-like mice. A, B, SMN promoter site 1 (A) and site 2 (B) sequences. C–F, Western blot analysis and quantification of Elk-1 protein (C, D) phosphorylation and CREB protein (E, F) phosphorylation in the ventral lumbar spinal cord of vehicle- and U0126-treated control mice at 2 and 6 d of age, vehicle-treated SMA-like mice at 2 d of age, and U0126-treated SMA-like mice at 2 and 6 d of age (n = 3). G–J, ChIP analysis of phospho-Elk-1 (G, H) and phospho-CREB (I, J) in the ventral lumbar spinal cord of vehicle- and U0126-treated control mice at 2 and 6 d of age, vehicle-treated SMA-like mice at 2 d of age, and U0126-treated SMA-like mice at 2 and 6 d of age (n = 9). Quantitative real-time PCR was performed to detect SMN2 promoter site 1 (G, I) and site 2 (H, J). Data are displayed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

ERK pathway inhibition by U0126 alters H3 and H4 acetylation profiles in the spinal cord of severe SMA-like mice. A–D, ChIP analysis of histone H3 (A, B) and H4 (C, D) acetylation in the ventral lumbar spinal cord of vehicle- and U0126-treated control mice at 2 and 6 d of age, vehicle-treated SMA-like mice at 2 d of age, and U0126-treated SMA-like mice at 2 and 6 d of age (n = 9). Quantitative real-time PCR was performed to detect SMN2 promoter site 1 (A, C) and site 2 (B, D). Data are displayed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

ERK pathway inhibition by U0126 leads to CaMKII activation and triggers the AKT/CREB pathway in the spinal cord of SMA-like mice. A–D, Western blot analysis and quantification of AKT protein in the ventral lumbar spinal cord of vehicle- and U0126-treated control mice at 2 and 6 d of age, vehicle-treated SMA-like mice at 2 d of age, and U0126-treated SMA-like mice at 2 and 6 d of age (A, B) (n = 3) and in vehicle- and U0126-treated human SMA cultured myotubes from two type 2 SMA patients (C, D) (n = 3). E, F, Western blot analysis and quantification of CREB protein phosphorylation in the ventral lumbar spinal cord of control mice at 6 d of age and SMA-like mice at 2 d of age, treated by vehicle, ERK inhibitor U0126 with or without CaMKII inhibitor KN-93 (n = 3). G–J, Kinetic analysis by Western blot of CaMKII protein activation (G), AKT protein activation (H), and CREB protein activation (I) after in vivo ERK inhibition by U0126 in spinal cords of control mice at 6 d of age and quantifications (n = 2) (J). K–N, Kinetic analysis by Western blot of CaMKII protein activation (K), AKT protein activation (L), and CREB protein activation (M) after in vivo ERK inhibition by U0126 in spinal cords of SMA-like mice at 2 d of age and quantifications (n = 2) (N). Data are displayed as mean ± SEM. *p < 0.05.

Figure 5.

The alteration of intracellular pathways induced by ERK pathway inhibition is Ca²⁺ influx dependent. A–D, Western blot analysis and quantification of SMN protein expression in control (A, B) and SMA (C, D) spinal cord explants in the presence or absence of U0126 or the calcium chelators EGTA and BAPTA-AM (n = 3). E–H, Western blot analysis and quantification of CaMKII protein phosphorylation in control (E, F) and SMA (G, H) spinal cord explants in the presence or absence of U0126 or either the calcium chelators EGTA and BAPTA-AM (n = 3). I–L, Western blot analysis and quantification of CREB protein phosphorylation in control (I, J) and SMA (K, L) spinal cord explants in the presence or absence of U0126 or either the calcium chelators EGTA and BAPTA-AM (n = 3). M, Time course of the U0126 effect on an Oregon Green 488 BAPTA-1-loaded motor neurons recorded in spinal cord section of a control mouse. Images represent either the basal fluorescence level (left side) or the fluorescence at the peak of the response (right side). Scale bars, 20 μm. Inset shows spikes recorded on the same cell under the cell-attached configuration of the patch clamp. Data are displayed as mean ± SEM. *p < 0.05.

Figure 6.

The crosstalk between ERK and AKT signaling pathways is reciprocal in SMA spinal cord. A, B, Western blot analysis and quantification of ERK protein phosphorylation in control and SMA spinal cord explants in the presence or absence of either NMDA or the CREB inhibitor KG-501 (n = 3). C, D, Western blot analysis and quantification of Elk-1 protein phosphorylation in control and SMA spinal cord explants in the presence or not of NMDA and of the CREB inhibitor KG-501 (n = 3). E, F, Western blot analysis and quantification of AKT protein phosphorylation in control and SMA spinal cord explants in the presence or absence of either NMDA or the CREB inhibitor KG-501 (n = 3). G, H, Western blot analysis and quantification of SMN protein expression in control and SMA spinal cord explants in the presence or absence of NMDA or the CREB inhibitor KG-501 (n = 3). Data are displayed as mean ± SEM. *p < 0.05.

Figure 7.

Inhibition of ERK pathway by U0126 induces a significant motor neuron protection, increases lifespan, and improves weight curve in severe SMA-like mice. A–G, Immunodetection of ChAT-positive motor neurons in the lumbar spinal cord (L1–L5) of vehicle-treated (A) and U0126-treated control (B) mice at 2 d of age, vehicle-treated (C) and U0126-treated control (D) mice at 6 d of age, vehicle-treated (E) and U0126-treated SMA-like mice at 2 d of age (F), and U0126-treated SMA-like mice at 6 d of age (G). Scale bars, 100 μm. H–J, Quantitative analysis of the number (H) and the cell body area (I, J) of motor neurons per ventral horn in the ventral lumbar spinal cord of vehicle- and U0126-treated control mice at 2 and 6 d of age, vehicle-treated SMA-like mice at 2 d of age, and vehicle- and U0126-treated SMA-like mice at 6 d of age (n = 4). K, Lifespan of U0126-treated SMA-like mice (n = 22) compared with vehicle-treated (n = 12) and untreated SMA-like mice (n = 10). L, Weight curve in U0126-treated (n = 22) and vehicle-treated SMA-like mice (n = 12) compared with U0126-treated (n = 15) and vehicle-treated control (n = 15) mice. M, Time spent to stand up after being placed on the side for vehicle-treated (n = 5) and U0126-treated control (n = 5) mice compared with vehicle-treated (n = 5) and U0126-treated SMA-like mice (n = 5). N, O, Total number of crossings (N) and mobility (O) during 5 min in the open-field test for vehicle-treated (n = 5) and U0126-treated control (n = 5) mice compared with vehicle-treated (n = 5) and U0126-treated SMA-like (n = 5) mice. Data are displayed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8.

Inhibition of ERK pathway by AZD6244 (Selumetinib) significantly enhances SMN expression and AKT activation, increases lifespan, and improves weight curve in severe SMA-like mice. A, B, Western blot analysis and quantification of SMN protein expression in the ventral lumbar spinal cord of vehicle- and AZD6244-treated control mice at 2 and 6 d of age, vehicle-treated SMA-like mice at 2 d of age, and AZD6244-treated SMA-like mice at 2 and 6 d of age (n = 3). C, D, Western blot analysis and quantification of SMN protein expression in brain, heart, kidneys, lungs, liver, diaphragm, and tibialis of AZD6244-treated SMA-like mice at 2 d of age (n = 3). E, F, Western blot analysis and quantification of AKT protein phosphorylation in the ventral lumbar spinal cord of vehicle- and AZD6244-treated control mice at 2 and 6 d of age, vehicle-treated SMA-like mice at 2 d of age, and AZD6244-treated SMA-like mice at 2 and 6 d of age (n = 3). G, Lifespan of AZD6244-treated SMA-like mice (n = 11) compared with vehicle-treated (n = 13) and untreated (n = 10) SMA-like mice. H, Weight curve in AZD6244-treated (n = 11) and vehicle-treated SMA-like (n = 13) mice compared with AZD6244-treated (n = 10) and vehicle-treated control (n = 10) mice. Data are displayed as mean ± SEM. *p < 0.05, ***p < 0.001.

Figure 9.

Proposed mechanisms involved in the increase of SMN expression induced by ERK inhibition in the spinal cord of severe SMA-like mice. A, Activation profile of ERK/Elk-1 and AKT/CREB signaling pathways in lumbar spinal cord of vehicle-treated SMA-like mice compared with control. Large characters indicate activated molecules. B, Activation profile of ERK/Elk-1 and AKT/CREB signaling pathways in lumbar spinal cord of U0126-treated SMA-like mice compared with vehicle-treated SMA-like mice. Large characters indicate activated molecules.