Improvement of Neuromuscular Synaptic Phenotypes without Enhanced Survival and Motor Function in Severe Spinal Muscular Atrophy Mice Selectively Rescued in Motor Neurons

Abstract

In the inherited childhood neuromuscular disease spinal muscular atrophy (SMA), lower motor neuron death and severe muscle weakness result from the reduction of the ubiquitously expressed protein survival of motor neuron (SMN). Although SMA mice recapitulate many features of the human disease, it has remained unclear if their short lifespan and motor weakness are primarily due to cell-autonomous defects in motor neurons. Using Hb9Cre as a driver, we selectively raised SMN expression in motor neurons in conditional SMAΔ7 mice. Unlike a previous study that used choline acetyltransferase (ChATCre+) as a driver on the same mice, and another report that used Hb9Cre as a driver on a different line of conditional SMA mice, we found no improvement in survival, weight, motor behavior and presynaptic neurofilament accumulation. However, like in ChATCre+ mice, we detected rescue of endplate size and mitigation of neuromuscular junction (NMJ) denervation status. The rescue of endplate size occurred in the absence of an increase in myofiber size, suggesting endplate size is determined by the motor neuron in these animals. Real time-PCR showed that the expression of spinal cord SMN transcript was sharply reduced in Hb9Cre+ SMA mice relative to ChATCre+ SMA mice. This suggests that our lack of overall phenotypic improvement is most likely due to an unexpectedly poor recombination efficiency driven by Hb9Cre. Nonetheless, the low levels of SMN were sufficient to rescue two NMJ structural parameters indicating that these motor neuron cell autonomous phenotypes are very sensitive to changes in motoneuronal SMN levels. Our results directly suggest that even those therapeutic interventions with very modest effects in raising SMN in motor neurons may provide mitigation of neuromuscular phenotypes in SMA patients.

Muscle-Derived Extracellular Signal-Regulated Kinases 1 and 2 Are Required for the Maintenance of Adult Myofibers and Their Neuromuscular Junctions

“…Whole-mount staining of sternomastoid (STN) and tibialis anterior (TA) was essentially done as previously described (27). Antisynaptophysin (anti-SYN) (180130; Life Technologies) was used at 1:200, and fluorescein-␣-bungarotoxin (BTX) (F-1176; Life Technologies) was used at 1:1,000.…”

“…Total RNA extraction, reverse transcription, and real-time PCR were done essentially as previously reported (27). Two hundred nanograms of total RNA per sample was used to generate cDNA.…”

“…Dystrophin staining of transverse frozen sections was performed essentially as previously described (27), except that sections were not fixed and individual primary antibodies to myosin heavy chains (MyHCs) were coincubated with the antidystrophin antibody (15277; Abcam). All MyHC antibodies were from the Developmental Studies Hybridoma Bank as follows: type 1, A4.840; type 2A, SC-71; type 2B, 10F5; and type 2X, 6H1.…”

“…Fluorophore-conjugated secondary antibodies that matched the primary isotype (Jackson ImmunoResearch and Sigma) were used at 1:400 and 1:128, respectively. Fiber area analysis was carried out as previously described (27) on overlapping 10ϫ images that covered an entire muscle cross section. Care was taken not to measure the same fibers more than once.…”

Motor Deficit in a Tauopathy Model Is Induced by Disturbances of Axonal Transport Leading to Dying-Back Degeneration and Denervation of Neuromuscular Junctions

“…The innervation status of motor endplates was assessed after labeling with the synaptophysin antibody and BTX by classifying the synaptophysin distribution into three categories: i) perfect superposition of 15 The innervation status of NMJ was also assessed after labeling with NF and BTX by categorizing them as innervated if there was any overlap between the two labels or denervated if NF was completely absent at the synaptic junction. 16 A minimum of 300 junctions was analyzed for each mouse (n Z 3).…”