Portal Semaphorin 3 A in injury and disease : its role at the neuromuscular junction

Amyotrophic Lateral Sclerosis (ALS) is characterised by a progressive degeneration of motor neurons, which is thought to be initiated by presymptomatic pathological changes at the neuromuscular junction (NMJ); the so-called “dying-back” hypothesis. Recent evidence suggests that repulsive guidance molecules, such as Semaphorin 3A (SEMA3A), are upregulated at vulnerable NMJs in the presymptomatic phases of the disease and as such may play a role in the selective degeneration of these synapses. We hypothesized that inhibition of SEMA3A function at the NMJ would enable NMJs to remain innervated for longer, and would improve ALS-related motor function. SEMA3A mediates its repulsive abilities via a receptor complex on the motor neuron membrane; neuropilin-1 (NRP1) being the ligand binding component, and plexinA being the signal transducing component of the receptor complex. We developed soluble versions of the ligand binding component, i.e. NRP1-Fc, which, in vitro, were capable of preventing SEMA3A-mediated growth cone collapse in cultured dorsal root ganglion explants. Since NRP1 is also a receptor for Vascular Endothelial Growth Factor (VEGF), which has been shown to positively modify the ALS phenotype, we also generated NRP1-Fc isoforms to selectively bind either ligand in order to differentiate between the effects of scavenging both or either ligand. In the current study we used an adeno-associated viral (AAV) vector mediated approach to deliver NRP1-Fc, or the mutant isoforms, to the NMJ of ALS mice via intra-muscular injection. We show that AAV6 effectively targets skeletal muscle, and results in sustained and long-term expression of the transgene. ALS mice whereby SEMA3A and VEGF are neutralised, demonstrated an initial attenuation of the transient dip in motor decline usually seen in ALS mice in the presymptomatic stage, presumably due to SEMA3A neutralization, followed by a delay in the onset of the second and gradual decline in motor function which defines the early symptomatic phase of the disease. However, the improvement is not sustained, which we attribute to the neutralization of VEGF, resulting in a rapid decline of motor function in the late stages of the disease consistent with untreated ALS mice. On the other hand, we see a worsening of motor function upon treatment with variants that should only bind to SEMA3A. To explain this paradoxical phenomenon, we speculate that these soluble NRP1Fc isoforms may be acting as “receptor chaperones” by presenting the scavenged ligand (SEMA3A) to the membrane-bound receptor (NRP1), thus potentiating the endogenous SEMA3A-signaling pathway. Our data illustrate the difficulties in translating in vitro effects to an in vivo environment, especially into a system (i.e. the NMJ of ALS mice) where many other factors are playing a role in NMJ stability. We further discuss the potential effects of these other factors, and how we may be (unknowingly) targeting these pathways with the NRP1-Fc receptors, and how this may explain some of the contradictory results we see. Our findings also highlight the fact that in order to obtain a sustained improvement of motor function in ALS, a multifaceted (and perhaps systemic) approach must be taken to target not just one pathway thought to be involved in the destruction of the NMJ, but as many as are currently known in order to amplify the therapeutic effects in treating this disease.