Defective Acetylcholine Receptor Subunit Switch Precedes Atrophy of Slow-Twitch Skeletal Muscle Fibers Lacking ERK1/2 Kinases in Soleus Muscle

To test the role of extracellular-signal regulated kinases 1 and 2 (ERK1/2) in slow-twitch, type 1 skeletal muscle fibers, we studied the soleus muscle in mice genetically deficient for myofiber ERK1/2. Young adult mutant soleus was drastically wasted, with highly atrophied type 1 fibers, denervation at most synaptic sites, induction of “fetal” acetylcholine receptor gamma subunit (AChRγ), reduction of “adult” AChRε, and impaired mitochondrial biogenesis and function. In weanlings, fiber morphology and mitochondrial markers were mostly normal, yet AChRγ upregulation and AChRε downregulation were observed. Synaptic sites with fetal AChRs in weanling muscle were ~3% in control and ~40% in mutants, with most of the latter on type 1 fibers. These results suggest that: (1) ERK1/2 are critical for slow-twitch fiber growth; (2) a defective γ/ε-AChR subunit switch, preferentially at synapses on slow fibers, precedes wasting of mutant soleus; (3) denervation is likely to drive this wasting, and (4) the neuromuscular synapse is a primary subcellular target for muscle ERK1/2 function in vivo

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

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 Hb9(Cre) as a driver, we selectively raised SMN expression in motor neurons in conditional SMAΔ7 mice. Unlike a previous study that used choline acetyltransferase (ChAT(Cre+) ) as a driver on the same mice, and another report that used Hb9(Cre) 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 ChAT(Cre+) 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 Hb9(Cre+) SMA mice relative to ChAT(Cre+) SMA mice. This suggests that our lack of overall phenotypic improvement is most likely due to an unexpectedly poor recombination efficiency driven by Hb9(Cre) . 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

Emerging roles for MAP kinases in agrin signaling

Information between neurons and the target cells they innervate passes through sites of functional contact called synapses. How synapses form and are altered by sensory or cognitive experience is central to understand nervous system function. Studies of synapse formation and plasticity have concentrated on a few “model” synapses. The vertebrate neuromuscular junction (NMJ), the synapse between a motoneuron in the spinal cord and a skeletal muscle fiber, is one such model synapse. The extracellular matrix proteoglycan agrin plays an essential organizing role at the NMJ. Agrin is also present at some synapses in the brain and in other organs in the periphery, but its function outside the NMJ is unclear. The core signaling pathway for agrin at the NMJ, which is still incompletely defined, includes molecules specifically involved in this cascade and molecules used in other signaling pathways in many cells. Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved components of intracellular signaling modules that control a myriad of cellular processes. This article reviews emerging evidence that suggests that MAPKs are involved in agrin signaling at the NMJ and in the putative functions of agrin in the formation of a subset of synapses in the brain

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I dedicate this body of work to my MomAcknowledgements I am grateful to the members of my dissertation committee, Drs. Wesley J

Schematic representation of the <i>Smn</i> WT allele and the <i>Smn^Res</i> conditional hybrid mutant allele before and after Hb9-Cre recombination.

<p>Blue boxes represent mouse <i>Smn</i> exons. Red boxes represent human <i>SMN2</i> exons. Arrows within boxes display orientation relative to transcription start. Green stars show approximate location of <i>loxP</i> sites (<i>lox71, lox 66</i>) in <i>Smn^Res</i>. Black lines connecting exons show splicing pattern for the predominant transcript encoded by each allele. Protein products are named to the right. <i>SMN67m8h</i> is the transcript encoded by the repaired <i>Smn^Res</i> allele. Not drawn to scale.</p

Hb9-Cre recombination fails to improve lifespan, weight gain and motor behavior in SMAΔ7 mice.

<p><b>A.</b> Kaplan-Meier curves demonstrate no increase in survival, p = 0.6436, log-rank test. Mean life span: Hb9(Cre⁺)SMA: 13.41±3.73 days, n = 37. Hb9(Cre⁻)SMA mice: 14.6±2.64 days; n = 20. Controls: n = 130. <b>B</b>. While controls (n = 107) gained weight as expected, no statistical differences in weight were found during the lifespan of Hb9(Cre⁺)SMA and Hb9(Cre⁻)SMA mice except at P12 (*p = 0.004, F = 9.78, ANOVA). Hb9(Cre⁺)SMA: n = 10–27 per time point. Hb9(Cre⁻)SMA: n = 3–16 per time point. <b>C and </b><b>D</b>. Motor behavior was assessed by the righting reflex (C) and hindlimb (tube) suspension (D) tests. Only the latency to fall was recorded for the tube assays. While controls improved their performance with age, SMA mice motor behavior deteriorated as they approached end stage. For the righting reflex assays: Controls: n = 107 per time point. Hb9(Cre⁺)SMA: n = 7–27 per time point. Hb9(Cre⁻)SMA: n = 4–16 per time point. For the tube test: Controls: n = 148 per time point. Hb9(Cre⁺)SMA: n = 7–28 per time point. Hb9(Cre⁻)SMA: n = 5–17 per time point.</p