Impairment of the neurotrophic signaling hub B-Raf contributes to motoneuron degeneration in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motoneuron disease caused by deletions of the Survival of Motoneuron 1 gene (SMN1) and low SMN protein levels. SMN restoration is the concept behind a number of recently approved drugs which result in impressive yet limited effects. Since SMN has already been enhanced in treated patients, complementary SMN-independent approaches are needed. Previously, a number of altered signaling pathways which regulate motoneuron degeneration have been identified as candidate targets. However, signaling pathways form networks, and their connectivity is still unknown in SMA. Here, we used presymptomatic SMA mice to elucidate the network of altered signaling in SMA. The SMA network is structured in two clusters with AKT and 14-3-3 ζ/δ in their centers. Both clusters are connected by B-Raf as a major signaling hub. The direct interaction of B-Raf with 14-3-3 ζ/δ is important for an efficient neurotrophic activation of the MEK/ERK pathway and crucial for motoneuron survival. Further analyses in SMA mice revealed that both proteins were down-regulated in motoneurons and the spinal cord with B-Raf being reduced at presymptomatic stages. Primary fibroblasts and iPSC-derived motoneurons from SMA patients both showed the same pattern of down-regulation. This mechanism is conserved across species since a Caenorhabditis elegans SMA model showed less expression of the B-Raf homolog lin-45. Accordingly, motoneuron survival was rescued by a cell autonomous lin-45 expression in a C. elegans SMA model resulting in improved motor functions. This rescue was effective even after the onset of motoneuron degeneration and mediated by the MEK/ERK pathway.

Homeoprotein ENGRAILED-1 promotes motoneuron survival and motor functions

ABSTRACTMotoneuron degeneration leads to skeletal muscle denervation and impaired motor functions, yet the signals involved remain poorly understood. We find that extracellular ENGRAILED-1, a homeoprotein expressed in spinal cord V1 interneurons that synapse on α-motoneurons, has non-cell autonomous activity. Mice heterozygote for Engrailed-1 develop muscle weakness, abnormal spinal reflex and partial neuromuscular junction denervation. A single intrathecal injection of ENGRAILED-1 restores innervation, limb strength, extensor reflex and prevents lumbar α-motoneuron death for several months. The autophagy gene p62, which was found to network with Engrailed-1 and amyotrophic lateral sclerosis genes, is misregulated in Engrailed-1 heterozygote mice α-motoneurons and is rescued following ENGRAILED-1 injection. These results identify ENGRAILED-1 as an α-motoneuron trophic factor with long-lasting protective activity.

Improved motor unit number estimate when motor unit alternation is addressed

Motor unit number estimation (MUNE) is important for determining motoneuron survival with age or in conditions such as amyotrophic lateral sclerosis or spinal cord injury. The original incremental method and approaches that were introduced to minimize alternation (e.g., multiple-point stimulation) are most commonly used, but one must accept the limitation that alternation of motor units may still inflate the estimate. Alternation occurs because axon thresholds are probabilistic and overlap for different axons; therefore, different combination of motor units may respond at a given stimulus intensity. Our aims were to quantify motor unit alternation systematically in the thenar muscles of 35 healthy adults by digital subtraction of EMG and force, and to compare MUNE with and without alternation. Alternation was prevalent, with one to nine occurrences in the first 7 to 11 steps in EMG in 34 of 35 muscles. It occurred in the first 3 steps in EMG in 49% of muscles. This alternation resulted in fewer units than steps in EMG (3 to 10 units at step 7 to 11). Accounting for alternation using digital subtraction reduced MUNE by up to 50%, day-to-day, and between-participant variability in MUNE. These results highlight the need to quantify alternation to improve the reliability and precision of motor unit number estimates, which will allow for detection of smaller changes in motoneuron survival with age, various health conditions, and/or due to an intervention. NEW & NOTEWORTHY Motor unit alternation was quantified systematically for the first time, addressing a major limitation of motor unit number estimates. Accounting for alternation decreased motor unit number estimates, and improved the reliability and precision of the motor unit number estimate, which will allow smaller, clinically relevant changes in motoneuron survival to be detected.