Neuromuscular Junction Defects in Mice with Mutation of dynein heavy chain 1

Disruptions in axonal transport have been implicated in a wide range of neurodegenerative diseases. Cramping 1 (Cra1/+) and Legs at odd angles (Loa/+) mice, with hypomorphic mutations in the dynein heavy chain 1 gene, which encodes the ATPase of the retrograde motor protein dynein, were originally reported to exhibit late onset motor neuron disease. Subsequent, conflicting reports suggested that sensory neuron disease without motor neuron loss underlies the phenotypes of Cra1/+ and Loa/+ mice. Here, we present behavioral and anatomical analyses of Cra1/+ mice. We demonstrate that Cra1/+ mice exhibit early onset, stable behavioral deficits, including abnormal hindlimb posturing and decreased grip strength. These deficits do not progress through 24 months of age. No significant loss of primary motor neurons or dorsal root ganglia sensory neurons was observed at ages where the mice exhibited clear symptomatology. Instead, there is a decrease in complexity of neuromuscular junctions. These results indicate that disruption of dynein function in Cra1/+ mice results in abnormal morphology of neuromuscular junctions. The time course of behavioral deficits, as well as the nature of the morphological defects in neuromuscular junctions, suggests that disruption of dynein function in Cra1/+ mice causes a developmental defect in synapse assembly or stabilization

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders

Neuromuscular junctions of <i>Cra1</i>/+ mice are abnormal at symptomatic ages.

<p>A) α-bungarotoxin labeled NMJs (Scale bar  = 20 µm) B) Quantification of α-bungarotoxin labeling demonstrates no differences in the number of NMJs per section (N = 3 animals per genotype). C) Quantification of α-bungarotoxin labeled NMJs demonstrates no difference in total area (as measured by delineating outer edge and measuring area within that boundary, area analyzed is within white trace of 4Ai) of NMJs in <i>Cra1</i>/+ mice (N = 3 animals per genotype). D) NMJ complexity, as measured by the synaptic area of α-bungarotoxin label (4Aii), is significantly decreased in 2 month and 6 month old <i>Cra1</i>/+ mice (*P<0.05; N = 3 mice per genotype).</p

Synaptic and post-synaptic defects at the neuromuscular junctions in <i>Cra1/+</i> mice.

<p>A) α-bungarotoxin and GFP labeled NMJs. (Scale bar  = 20 µm). B) Motor neuron axons reveal a decrease in the proportion of NMJs with complete overlap between pre and post-synaptic zones and a compensatory increase in the proportion of NMJs with partial overlap in <i>Cra1/+</i> mice (*P<0.05). C) Labeling of nuclei by DAPI stain demonstrates a significant increase in central nuclei in <i>Cra1/+</i> mice, a measure of muscle fiber degeneration and regeneration (central nuclei indicated with red arrow; *P<0.05; N = 3 mice per genotype).</p

<i>Cra1</i>/+ mice show no evidence of motor neuron loss at symptomatic ages.

<p>A) Motor neuron labeling with ChAT of +/+ and <i>Cra1</i>/+ mice at 6 months and 24 months of age (Scale bar  = 50 µm). B) Quantification of ChAT-labeled motor neurons reveals no difference in number between +/+ and <i>Cra1</i>/+ mice (N = 3 animals per genotype). C) Toluidine blue staining of the anterior horn of the spinal cord at 6 months and 24 months of age in +/+ and <i>Cra1/+</i> mice (white arrows indicate motor neurons, Scale bar  = 50 µm). D) Quantification of toluidine blue labeled motor neurons reveals no difference in number between +/+ and <i>Cra1</i>/+ mice (N = 3 animals per genotype).</p

<i>Cra1</i>/+ mice show no evidence of proprioceptive sensory neuron loss at symptomatic ages.

<p>A) Proprioceptive sensory neuron labeling and quantification with parvalbumin, (B) ER81, and (C) TrkC shows no difference between of +/+ and <i>Cra1</i>/+ mice at 6 months of age (Scale bar  = 20 µm). D) Parvalbumin labeling of proprioceptive sensory neuron fibers within the spinal cord shows the central projection of these sensory neurons is intact in <i>Cra1</i>/+ mice (Scale bar  = 20 µm; N = 3 animals per genotype).</p

Sarm1 activation produces cADPR to increase intra-axonal Ca++ and promote axon degeneration in PIPN

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a dying-back axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN)

Sarm1 activation produces cADPR to increase intra-axonal Ca++ and promote axon degeneration in PIPN

Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a dying-back axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN)