Electrophysiological Biomarkers in Spinal Muscular Atrophy: Preclinical Proof of Concept

Abstract

Objective: Preclinical therapies that restore survival motor neuron (SMN) protein levels can dramatically extend survival in spinal muscular atrophy (SMA) mouse models. Biomarkers are needed to effectively translate these promising therapies to clinical trials. Our objective was to investigate electrophysiological biomarkers of compound muscle action potential (CMAP), motor unit number estimation (MUNE) and electromyography (EMG) using an SMA mouse model.

Methods: Sciatic CMAP, MUNE, and EMG were obtained in SMNΔ7 mice at ages 3-13 days and at 21 days in mice with SMN selectively reduced in motor neurons (ChAT^Cre ). To investigate these measures as biomarkers of treatment response, measurements were obtained in SMNΔ7 mice treated with antisense oligonucleotide (ASO) or gene therapy.

Results: CMAP was significantly reduced in SMNΔ7 mice at days 6-13 (p<0.01), and MUNE was reduced at days 7-13 (p<0.01). Fibrillations were present on EMG in SMNΔ7 mice but not controls (p=0.02). Similar findings were seen at 21 days in ChAT^Cre mice. MUNE in ASO-treated SMNΔ7 mice were similar to controls at day 12 and 30. CMAP reduction persisted in ASO-treated SMNΔ7 mice at day 12 but was corrected at day 30. Similarly, CMAP and MUNE responses were corrected with gene therapy to restore SMN.

Interpretation: These studies confirm features of preserved neuromuscular function in the early postnatal period and subsequent motor unit loss in SMNΔ7 mice. SMN restoring therapies result in preserved MUNE and gradual repair of CMAP responses. This provides preclinical evidence for the utilization of CMAP and MUNE as biomarkers in future SMA clinical trials.

Figure 1

Sciatic CMAP, incremental MUNE, and electromyography. (A) Anesthesia rig with adapted nose cone and electrode placement for sciatic CMAP and MUNE (a, recording electrodes; b, ground electrode; c, stimulating electrodes). (B) Sciatic CMAP response with expected biphasic, initial negative morphology. (C) Ten superimposed incremental MUNE responses with similar biphasic morphology. (D) Fibrillation potentials from an end-stage SMNΔ7 mouse (13 days). CMAP, compound muscle action potential; MUNE, motor unit number estimation; SMN, survival motor neuron.

Figure 2

Transformed longitudinal CMAP (square root) and MUNE (logarithm) measurements in untreated SMNΔ7 and control mice. (A) Sciatic CMAP amplitude shows a divergent drop in SMNΔ7 mice and amplitude is significantly reduced at 6–13 days (P < 0.01). There is partial recovery of CMAP amplitude at days 11–13. (B) Sciatic MUNE is similar in SMNΔ7 mice and controls at early postnatal time points. There is progressive motor unit loss beginning at PND 6 that reaches statistical significance reduced on days 7–13 (P < 0.01). CMAP, compound muscle action potential; MUNE, motor unit number estimation; SMN, survival motor neuron; PND, postnatal day.

Figure 3

Polyneuronal innervation effect on MUNE response. (A) Correlation of the rate of polyneuronal innervation pruning with increasing MUNE responses in control mice. (B) Plot of MUNE ratio (SMNΔ7 vs. control), to correct for polyneuronal innervation, to demonstrate onset and progression of motor unit degeneration in SMNΔ7 mice. CMAP, compound muscle action potential; MUNE, motor unit number estimation; SMN, survival motor neuron.

Figure 4

CMAP and MUNE at 12 and 30 days following SMN restoration with ASO. (A) At 12 days, CMAP amplitudes are significantly reduced in untreated and treated SMNΔ7 mice compared with controls. There is no significant difference between treated and untreated SMNΔ7 mice. (B) At 12 days, MUNE is significantly higher in treated SMNΔ7 mice compared with untreated SMNΔ7 mice. There is no significant difference between treated SMNΔ7 mice and controls. *P < 0.01. (C) At 30 days, CMAP amplitudes are similar between treated SMNΔ7 mice and controls (P = 0.4822). (D) At 30 days, MUNE is slightly reduced in treated SMNΔ7 mice, but this does not reach statistical significance (P = 0.0733). CMAP, compound muscle action potential; MUNE, motor unit number estimation; SMN, survival motor neuron; ASO, antisense oligonucleotide; SMNΔ7, untreated SMNΔ7; Tr-SMNΔ7, antisense oligonucleotide treated SMNΔ7; Tr-Ctr, antisense oligonucleotide treated controls; Untr-Ctr, untreated controls.