An allelic series of spontaneous Rorb mutant mice exhibit a gait phenotype, changes in retina morphology and behavior, and gene expression signatures associated with the unfolded protein response.

The Retinoid-related orphan receptor beta (RORβ) gene encodes a developmental transcription factor and has 2 predominant isoforms created through alternative first exon usage; one specific to the retina and another present more broadly in the central nervous system, particularly regions involved in sensory processing. RORβ belongs to the nuclear receptor family and plays important roles in cell fate specification in the retina and cortical layer formation. In mice, loss of RORβ causes disorganized retina layers, postnatal degeneration, and production of immature cone photoreceptors. Hyperflexion or high-stepping of rear limbs caused by reduced presynaptic inhibition by Rorb-expressing inhibitory interneurons of the spinal cord is evident in RORβ-deficient mice. RORβ variants in patients are associated with susceptibility to various neurodevelopmental conditions, primarily generalized epilepsies, but including intellectual disability, bipolar, and autism spectrum disorders. The mechanisms by which RORβ variants confer susceptibility to these neurodevelopmental disorders are unknown but may involve aberrant neural circuit formation and hyperexcitability during development. Here we report an allelic series in 5 strains of spontaneous Rorb mutant mice with a high-stepping gait phenotype. We show retinal abnormalities in a subset of these mutants and demonstrate significant differences in various behavioral phenotypes related to cognition. Gene expression analyses in all 5 mutants reveal a shared over-representation of the unfolded protein response and pathways related to endoplasmic reticulum stress, suggesting a possible mechanism of susceptibility relevant to patients

A Spontaneous Mutation in Contactin 1 in the Mouse

Mutations in the gene encoding the immunoglobulin-superfamily member cell adhesion molecule contactin1 (CNTN1) cause lethal congenital myopathy in human patients and neurodevelopmental phenotypes in knockout mice. Whether the mutant mice provide an accurate model of the human disease is unclear; resolving this will require additional functional tests of the neuromuscular system and examination of Cntn1 mutations on different genetic backgrounds that may influence the phenotype. Toward these ends, we have analyzed a new, spontaneous mutation in the mouse Cntn1 gene that arose in a BALB/c genetic background. The overt phenotype is very similar to the knockout of Cntn1, with affected animals having reduced body weight, a failure to thrive, locomotor abnormalities, and a lifespan of 2–3 weeks. Mice homozygous for the new allele have CNTN1 protein undetectable by western blotting, suggesting that it is a null or very severe hypomorph. In an analysis of neuromuscular function, neuromuscular junctions had normal morphology, consistent with previous studies in knockout mice, and the muscles were able to generate appropriate force when normalized for their reduced size in late stage animals. Therefore, the Cntn1 mutant mice do not show evidence for a myopathy, but instead the phenotype is likely to be caused by dysfunction in the nervous system. Given the similarity of CNTN1 to other Ig-superfamily proteins such as DSCAMs, we also characterized the expression and localization of Cntn1 in the retinas of mutant mice for developmental defects. Despite widespread expression, no anomalies in retinal anatomy were detected histologically or using a battery of cell-type specific antibodies. We therefore conclude that the phenotype of the Cntn1 mice arises from dysfunction in the brain, spinal cord or peripheral nervous system, and is similar in either a BALB/c or B6;129;Black Swiss background, raising a possible discordance between the mouse and human phenotypes resulting from Cntn1 mutations

DSCAMs: restoring balance to developmental forces.

Many of the models of neurodevelopmental processes such as cell migration, axon outgrowth, and dendrite arborization involve cell adhesion and chemoattraction as critical physical or mechanical aspects of the mechanism. However, the prevention of adhesion or attraction is under-appreciated as a necessary, active process that balances these forces, insuring that the correct cells are present and adhering in the correct place at the correct time. The phenomenon of not adhering is often viewed as the passive alternative to adhesion, and in some cases this may be true. However, it is becoming increasingly clear that active signaling pathways are involved in preventing adhesion. These provide a balancing force during development that prevents overly exuberant adhesion, which would otherwise disrupt normal cellular and tissue morphogenesis. The strength of chemoattractive signals may be similarly modulated. Recent studies, described here, suggest that Down Syndrome Cell Adhesion Molecule (DSCAM), and closely related proteins such as DSCAML1, may play an important developmental role as such balancers in multiple systems

Contact is repulsive, but please note the enclosed .

Previous models of neuronal dendrite arborization suggested that contact-dependent self-avoidance between dendrite branches prevents self-crossings within the arbor. Two papers in Neuron show how integrin-mediated adhesion to the extracellular matrix restricts dendrites to a two-dimensional space to optimize this mechanism (Han et al., 2012; Kim et al., 2012)

Precision mouse models of Yars/dominant intermediate Charcot-Marie-Tooth disease type C and Sptlc1/hereditary sensory and autonomic neuropathy type 1

Animal models of neurodegenerative diseases such as inherited peripheral neuropathies sometimes accurately recreate the pathophysiology of the human disease, and sometimes accurately recreate the genetic perturbations found in patients. Ideally, models achieve both, but this is not always possible; nonetheless, such models are informative. Here we describe two animal models of inherited peripheral neuropathy: mice with a mutation in tyrosyl tRNA-synthetase, YarsE196K, modeling dominant intermediate Charcot-Marie-Tooth disease type C (diCMTC), and mice with a mutation in serine palmitoyltransferase long chain 1, Sptlc1C133W, modeling hereditary sensory and autonomic neuropathy type 1 (HSAN1). YarsE196K mice develop disease-relevant phenotypes including reduced motor performance and reduced nerve conduction velocities by 4 months of age. Peripheral motor axons are reduced in size, but there is no reduction in axon number and plasma neurofilament light chain levels are not increased. Unlike the dominant human mutations, the YarsE196K mice only show these phenotypes as homozygotes, or as compound heterozygotes with a null allele, and no phenotype is observed in E196K or null heterozygotes. The Sptlc1C133W mice carry a knockin allele and show the anticipated increase in 1-deoxysphingolipids in circulation and in a variety of tissues. They also have mild behavioral defects consistent with HSAN1, but do not show neurophysiological defects or axon loss in peripheral nerves or in the epidermis of the hind paw or tail. Thus, despite the biochemical phenotype, the Sptlc1C133W mice do not show a strong neuropathy phenotype. Surprisingly, these mice were lethal as homozygotes, but the heterozygous genotype studied corresponds to the dominant genetics seen in humans. Thus, YarsE196K homozygous mice have a relevant phenotype, but imprecisely reproduce the human genetics, whereas the Sptlc1C133W mice precisely reproduce the human genetics, but do not recreate the disease phenotype. Despite these shortcomings, both models are informative and will be useful for future research

Neuromuscular analysis of <i>Cntn1</i> mutant mice.

<p>A) Neuromuscular junctions in wild type P11 mice have a plaque-like field of postsynaptic acetylcholine receptors (red) that is beginning to become convoluted. This is completely overlapped by the presynaptic motor nerve terminal (green). B) A similar NMJ morphology is seen in <i>Cntn1</i> mutant mice. Histological examination of longitudinal and cross sections of control (C,D), and mutant (E,F) hind limb muscles at P13 did not reveal hallmarks of myopathy. G,H) Transmission electron microscopy was used to evaluate sarcomere anatomy in the tibialis anterior muscle. The structure was not adversely affected by the mutation. I) The absolute maximal contractile force of the extensor digitorum longus muscle was reduced in mutant mice. J) When normalized for muscle weight, contractile force of mutant muscle was not significantly different than control. The scale bar in H represents 14 µm in A, B, 72 µm in C–F, and 3 µm in G, H.</p

Additional <i>in vitro</i> contractile properties of directly stimulated EDL muscles.

<p>*<0.03.</p><p>**<0.0001.</p><p>Muscle twitches recorded in mutant mice are smaller and have a longer time-to-peak tension. Reduced absolute values are due to smaller muscles in mutants because normalized twitch forces are not different from wild-type. Similar outcome is shown for tetanic force in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029538#pone-0029538-g003" target="_blank">Figure 3</a>. MW:BW ratio%, percent muscle to body-weight; Pt(g), absolute twitch force; Pt (N/g), twitch force normalized to muscle weight; TPT, time-to-peak twitch tension; 1/2RT-twitch half-relaxation time; N, Newtons (force); ms, milliseconds.</p

Dystrobrevin and Syntrophin localization in <i>Cntn1</i> mutant muscle.

<p>Cross sections of triceps surae from three mutant and three littermate control mice (ages P14, P15, and P16) were labeled with antibodies recognizing dystrobrevins and syntrophins (green). NMJs were counterstained with α-bungarotoxin (red). Merged images as well as the green channel with NMJs denoted by arrowheads are shown. Immunolabels are noted at left, genotypes are noted at top (control left, mutant right). The scale bar in the lower right is 25 µm.</p

Contactin1 as a candidate gene for this mutation.

<p>A) The genetic interval of Chromosome 15 between <i>D15Mit242</i> and <i>D15Mit192</i> (2.5 Mb) is shown. Genomic features including protein-coding genes are indicated. The image is from the Ensembl genome browser (<a href="http://ensembl.org/index.html" target="_blank">http://ensembl.org/index.html</a>). B) The structure of CNTN1 is schematized; the protein has an N-terminal signal peptide for secretion, 6 immunoglobulin domains (Ig), 4 fibronectin repeats (FN), and a GPI-linked carboxy-terminal modification for attachment to membranes. C) Immunoblot of CNTN1 in spinal cord and brain from two unaffected littermate control mice and two affected mice reveals an absence of CNTN1 protein in the affected mice. β-actin was used as a loading control.</p

Expression and localization of <i>Cntn1</i> in the retina.

<p>A–C <i>In situ</i> hybridization for <i>Cntn1</i> expression in the wild type mouse retina at P5 (A), P10 (B), and P21 (C). A subset of cells in the retinal ganglion cell layer (bottom) and inner nuclear layer are positive for <i>Cntn1</i> expression. Photoreceptors in the outer nuclear layer (top) do not have signals above background. D) Double label <i>in situ</i> hybridization with <i>Cntn1</i> and syntaxin1a at P21 demonstrates that some amacrine cells in the inner nuclear layer are positive for <i>Cntn1</i> expression (arrowheads). Other <i>Cntn1</i>-positive cells are likely to be bipolar cells based on their position (arrows). E) In the retinal ganglion cell layer, a majority of cells expressing <i>Cntn1</i> at P21 also express <i>Thy1</i>, a marker of ganglion cells. F) Immunolabeling of retinas with anti-CNTN1 antibodies at P14 revealed strong labeling of the synaptic plexiform layers, as well as immunoreactivity in the cellular layers, particularly the inner nuclear layer. G) Immunolabeling retinas from <i>Cntn1</i> mutant mice revealed a marked reduction, but not an elimination of signal intensity in images collected with equivalent parameters.</p