Differential effect of Fas activation on spinal muscular atrophy motoneuron death and induction of axonal growth.

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the most common motoneuron diseases affecting adults and infants, respectively. ALS and SMA are both characterized by the selective degeneration of motoneurons. Although different in their genetic etiology, growing evidence indicates that they share molecular and cellular pathogenic signatures that constitute potential common therapeutic targets. We previously described a motoneuron-specific death pathway elicited by the Fas death receptor, whereby vulnerable ALS motoneurons show an exacerbated sensitivity to Fas activation. However, the mechanisms that drive the loss of SMA motoneurons remains poorly understood. Here, we describe an in vitro model of SMA-associated degeneration using primary motoneurons derived from Smn2B/- SMA mice and show that Fas activation selectively triggers death of the proximal motoneurons. Fas-induced death of SMA motoneurons has the molecular signature of the motoneuron-selective Fas death pathway that requires activation of p38 kinase, caspase-8, -9 and -3 as well as upregulation of collapsin response mediator protein 4 (CRMP4). In addition, Rho-associated Kinase (ROCK) is required for Fas recruitment. Remarkably, we found that exogenous activation of Fas also promotes axonal elongation in both wildtype and SMA motoneurons. Axon outgrowth of motoneurons promoted by Fas requires the activity of ERK, ROCK and caspases. This work defines a dual role of Fas signaling in motoneurons that can elicit distinct responses from cell death to axonal growth.

Expression of ALS-linked SOD1 Mutation in Motoneurons or Myotubes Induces Differential Effects on Neuromuscular Function In vitro.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects upper and lower motoneurons. Dismantlement of the neuromuscular junction (NMJ) is an early pathological hallmark of the disease whose cellular origin remains still debated. We developed an in vitro NMJ model to investigate the differential contribution of motoneurons and muscle cells expressing ALS-causing mutation in the superoxide dismutase 1 (SOD1) to neuromuscular dysfunction. The primary co-culture system allows the formation of functional NMJs and fosters the expression of the ALS-sensitive fast fatigable type II-b myosin heavy chain (MHC) isoform. Expression of SOD1G93A in myotubes does not prevent the formation of a functional NMJ but leads to decreased contraction frequency and lowers the slow type I MHC isoform transcript levels. Expression of SOD1G93A in both motoneurons and myotubes or in motoneurons alone however alters the formation of a functional NMJ. Our results strongly suggest that motoneurons are a major factor involved in the process of NMJ dismantlement in an experimental model of ALS.

Identification of DmTTLL5 as a Major Tubulin Glutamylase in the Drosophila Nervous System.

Microtubules (MTs) play crucial roles during neuronal life. They are formed by heterodimers of alpha and beta-tubulins, which are subjected to several post-translational modifications (PTMs). Amongst them, glutamylation consists in the reversible addition of a variable number of glutamate residues to the C-terminal tails of tubulins. Glutamylation is the most abundant MT PTM in the mammalian adult brain, suggesting that it plays an important role in the nervous system (NS). Here, we show that the previously uncharacterized CG31108 gene encodes an alpha-tubulin glutamylase acting in the Drosophila NS. We show that this glutamylase, which we named DmTTLL5, initiates MT glutamylation specifically on alpha-tubulin, which are the only glutamylated tubulin in the Drosophila brain. In DmTTLL5 mutants, MT glutamylation was not detected in the NS, allowing for determining its potential function. DmTTLL5 mutants are viable and we did not find any defect in vesicular axonal transport, synapse morphology and larval locomotion. Moreover, DmTTLL5 mutant flies display normal negative geotaxis behavior and their lifespan is not altered. Thus, our work identifies DmTTLL5 as the major enzyme responsible for initiating neuronal MT glutamylation specifically on alpha-tubulin and we show that the absence of MT glutamylation is not detrimental for Drosophila NS function.

KCC3 loss-of-function contributes to Andermann syndrome by inducing activity-dependent neuromuscular junction defects.

Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na+/K+-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na+/K+-ATPase α1 localization and reduces NMJ denervation in Slc12a6-/- mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.