The C57BL/6J Mouse Strain Background Modifies the Effect of a Mutation in Bcl2l2

Bcl2l2 encodes BCL-W, an antiapoptotic member of the BCL-2 family of proteins. Intercross of Bcl2l2 +/− mice on a mixed C57BL/6J, 129S5 background produces Bcl2l2 −/− animals with the expected frequency. In contrast, intercross of Bcl2l2 +/− mice on a congenic C57BL/6J background produces relatively few live-born Bcl2l2 −/− animals. Genetic modifiers alter the effect of a mutation. C57BL/6J mice (Mus musculus) have a mutant allele of nicotinamide nucleotide transhydrogenase (Nnt) that can act as a modifier. Loss of NNT decreases the concentration of reduced nicotinamide adenine dinucleotide phosphate within the mitochondrial matrix. Nicotinamide adenine dinucleotide phosphate is a cofactor for glutathione reductase, which regenerates reduced glutathione, an important antioxidant. Thus, loss of NNT activity is associated with increased mitochondrial oxidative damage and cellular stress. To determine whether loss of Bcl2l2 −/− mice on the C57BL/6J background was mediated by the Nnt mutation, we outcrossed Bcl2l2 congenic C57BL/6J (Nnt −/−) mice with the closely related C57BL/6JEiJ (Nnt +/+) strain to produce Bcl2l2 +/− ; Nnt +/+ and Bcl2l2 +/− ; Nnt −/− animals. Intercross of Bcl2l2 +/− ; Nnt +/+ mice produced Bcl2l2 −/− with the expected frequency, whereas intercross of Bcl2l2 +/− ; Nnt −/− animals did not. This finding indicates the C57BL/6J strain background, and possibly the Nnt mutation, modifies the Bcl2l2 mutant phenotype. This and previous reports highlight the importance of knowing the genetic composition of mouse strains used in research studies as well as the accurate reporting of mouse strains in the scientific literature

Prolyl isomerase Pin1 regulates axon guidance by stabilizing CRMP2A selectively in distal axons

SummaryAxon guidance relies on precise translation of extracellular signal gradients into local changes in cytoskeletal dynamics, but the molecular mechanisms regulating dose-dependent responses of growth cones are still poorly understood. Here, we show that during embryonic development in growing axons, a low level of Semaphorin3A stimulation is buffered by the prolyl isomerase Pin1. We demonstrate that Pin1 stabilizes CDK5-phosphorylated CRMP2A, the major isoform of CRMP2 in distal axons. Consequently, Pin1 knockdown or knockout reduces CRMP2A levels specifically in distal axons and inhibits axon growth, which can be fully rescued by Pin1 or CRMP2A expression. Moreover, Pin1 knockdown or knockout increases sensitivity to Sema3A-induced growth cone collapse in vitro and in vivo, leading to developmental abnormalities in axon guidance. These results identify an important isoform-specific function and regulation of CRMP2A in controlling axon growth and uncover Pin1-catalyzed prolyl isomerization as a regulatory mechanism in axon guidance

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