The Rac-GAP alpha2-Chimaerin Signals via CRMP2 and Stathmins in the Development of the Ocular Motor System.

A precise sequence of axon guidance events is required for the development of the ocular motor system. Three cranial nerves grow toward, and connect with, six extraocular muscles in a stereotyped pattern, to control eye movements. The signaling protein alpha2-chimaerin (α2-CHN) plays a pivotal role in the formation of the ocular motor system; mutations in CHN1, encoding α2-CHN, cause the human eye movement disorder Duane Retraction Syndrome (DRS). Our research has demonstrated that the manipulation of α2-chn signaling in the zebrafish embryo leads to ocular motor axon wiring defects, although the signaling cascades regulated by α2-chn remain poorly understood. Here, we demonstrate that several cytoskeletal regulatory proteins-collapsin response mediator protein 2 (CRMP2; encoded by the gene dpysl2), stathmin1, and stathmin 2-bind to α2-CHN. dpysl2, stathmin1, and especially stathmin2 are expressed by ocular motor neurons. We find that the manipulation of dpysl2 and of stathmins in zebrafish larvae leads to defects in both the axon wiring of the ocular motor system and the optokinetic reflex, impairing horizontal eye movements. Knockdowns of these molecules in zebrafish larvae of either sex caused axon guidance phenotypes that included defasciculation and ectopic branching; in some cases, these phenotypes were reminiscent of DRS. chn1 knock-down phenotypes were rescued by the overexpression of CRMP2 and STMN1, suggesting that these proteins act in the same signaling pathway. These findings suggest that CRMP2 and stathmins signal downstream of α2-CHN to orchestrate ocular motor axon guidance and to control eye movements.SIGNIFICANCE STATEMENT The precise control of eye movements is crucial for the life of vertebrate animals, including humans. In humans, this control depends on the arrangement of nerve wiring of the ocular motor system, composed of three nerves and six muscles, a system that is conserved across vertebrate phyla. Mutations in the protein alpha2-chimaerin have previously been shown to cause eye movement disorders (squint) and axon wiring defects in humans. Our recent work has unraveled how alpha2-chimaerin coordinates axon guidance of the ocular motor system in animal models. In this article, we demonstrate key roles for the proteins CRMP2 and stathmin 1/2 in the signaling pathway orchestrated by alpha2-chimaerin, potentially giving insight into the etiology of eye movement disorders in humans.

Upregulation of miR-205 induces CHN1 expression, which is associated with the aggressive behaviour of cervical cancer cells and correlated with lymph node metastasis.

BACKGROUND: Cervical cancer is the leading cause of cancer-related death in women worldwide. However, the mechanisms mediating the development and progression of cervical cancer are unclear. In this study, we aimed to elucidate the roles of microRNAs and a1-chimaerin (CHN1) protein in cervical cancer progression. METHODS: The expression of miR-205 and CHN1 protein was investigated by in situ hybridisation and immunohistochemistry. We predicted the target genes of miR-205 using software prediction and dual luciferase assays. The expression of mRNAs and proteins was tested by qRT-PCR and western blotting respectively. The ability of cell growth, migration and invasion was evaluated by CCK-8 and transwell. Cell apoptosis was analysed by flow cytometry analysis. RESULTS: We found that miR-205 and CHN1 were highly expressed in human cervical cancer tissue compared with paired normal cervical tissues. The CHN1 gene was shown to be targeted by miR-205 in HeLa cells. Interestingly, transfection with miR-205 mimic upregulated CHN1 mRNA and protein, while miR-205 inhibitor downregulated CHN1 in high-risk and human papilloma virus (HPV)-negative human cervical cancer cells in vitro,. These data suggested that miR-205 positively regulated the expression of CHN1. Furthermore, the miR-205 mimic promoted cell growth, apoptosis, migration, and invasion in high-risk and HPV-negative cervical cancer cells, while the miR-205 inhibitor blocked these biological processes. Knockdown of CHN1 obviously reduced the aggressive cellular behaviours induced by upregulation of miR-205, suggesting that miR-205 positively regulated CHN1 to mediate these cell behaviours during the development of cervical cancer. Furthermore, CHN1 was correlated with lymph node metastasis in clinical specimens. CONCLUSIONS: Our findings showed that miR-205 positively regulated CHN1 to mediate cell growth, apoptosis, migration, and invasion during cervical cancer development, particularly for high-risk HPV-type cervical cancer. These findings suggested that dysregulation of miR-205 and subsequent abnormalities in CHN1 expression promoted the oncogenic potential of human cervical cancer.

Spinal RacGAP α-Chimaerin Is Required to Establish the Midline Barrier for Proper Corticospinal Axon Guidance.

In the developing CNS, the midline barrier, which comprises guidance molecule-expressing midline glial somata and processes, plays a pivotal role in midline axon guidance. Accumulating evidence has revealed the molecular mechanisms by which the midline barrier ensures proper midline guidance for axons. In contrast, the mechanisms for establishing the midline barrier remain obscure. Here, we report that Rac-specific GTPase-activating protein (RacGAP) α-chimaerin is required for both axonal repulsion at and establishment of the midline barrier in the spinal cord. We generated cortex-specific and spinal-cord-specific α-chimaerin gene (Chn1) knock-out mice (Cx-Chn1KO and Sp-Chn1KO mice, respectively) and found that both showed aberrant corticospinal tract (CST) axon midline crossing in the spinal cord. Strikingly, Sp-Chn1KO mice had breaks (holes) in the ephrinB3(+) spinal midline barrier and EphA4(+) CST axons aberrantly crossed the midline through these holes. During normal embryonic development, EphA4(+) spinal cells are located in juxta-midline areas but are excluded from the midline. In contrast, in Chn1KO embryos, several EphA4(+) cells were aberrantly relocated into the midline and the midline barrier was broken around these cells. Similarly, the spinal cord midline of Epha4KO mice was invaded by juxta-midline EphA4 cells (i.e., Epha4 promoter-active cells) during the embryonic stage and holes were formed in the midline barrier. Juxta-midline EphA4 cells in the spinal cord expressed α-chimaerin. We propose that spinal α-chimaerin aids in establishing an intact spinal midline barrier by mediating juxta-midline EphA4(+) cell repulsion, thus preventing these cells from breaking into the ephrinB3(+) midline barrier.SIGNIFICANCE STATEMENT The midline barrier plays a critical role in midline axon guidance, which is fundamental to the formation of neural circuits that are responsible for proper left-right coordination of the body. Studies have revealed some of the mechanisms underlying how the midline barrier navigates axons. In contrast, the establishment of the midline barrier during embryonic development remains unclear. In this study, we determined that α-chimaerin is required for the formation of an intact midline barrier. Spinal-cord-specific α-chimaerin knock-out mice had spinal midline barriers with numerous breaks (holes), through which corticospinal axons aberrantly crossed the midline. We propose that α-chimaerin protects the midline barrier by mediating cell-repulsive signaling in juxta-midline cells, which prevents these cells from invading the midline.

The Rac-GAP alpha2-chimaerin regulates hippocampal dendrite and spine morphogenesis.

Dendritic spines are fine neuronal processes where spatially restricted input can induce activity-dependent changes in one spine, while leaving neighboring spines unmodified. Morphological spine plasticity is critical for synaptic transmission and is thought to underlie processes like learning and memory. Significantly, defects in dendritic spine stability and morphology are common pathogenic features found in several neurodevelopmental and neuropsychiatric disorders. The remodeling of spines relies on proteins that modulate the underlying cytoskeleton, which is primarily composed of filamentous (F)-actin. The Rho-GTPase Rac1 is a major regulator of F-actin and is essential for the development and plasticity of dendrites and spines. However, the key molecules and mechanisms that regulate Rac1-dependent pathways at spines and synapses are not well understood. We have identified the Rac1-GTPase activating protein, α2-chimaerin, as a critical negative regulator of Rac1 in hippocampal neurons. The loss of α2-chimaerin significantly increases the levels of active Rac1 and induces the formation of aberrant polymorphic dendritic spines. Further, disruption of α2-chimaerin signaling simplifies dendritic arbor complexity and increases the presence of dendritic spines that appear poly-innervated. Our data suggests that α2-chimaerin serves as a "brake" to constrain Rac1-dependent signaling to ensure that the mature morphology of spines is maintained in response to network activity.

α2-chimaerin is required for Eph receptor-class-specific spinal motor axon guidance and coordinate activation of antagonistic muscles.

Axonal guidance involves extrinsic molecular cues that bind growth cone receptors and signal to the cytoskeleton through divergent pathways. Some signaling intermediates are deployed downstream of molecularly distinct axon guidance receptor families, but the scope of this overlap is unclear, as is the impact of embryonic axon guidance fidelity on adult nervous system function. Here, we demonstrate that the Rho-GTPase-activating protein α2-chimaerin is specifically required for EphA and not EphB receptor signaling in mouse and chick spinal motor axons. Reflecting this specificity, the loss of α2-chimaerin function disrupts the limb trajectory of extensor-muscle-innervating motor axons the guidance of which depends on EphA signaling. These embryonic defects affect coordinated contraction of antagonistic flexor-extensor muscles in the adult, indicating that accurate embryonic motor axon guidance is critical for optimal neuromuscular function. Together, our observations provide the first functional evidence of an Eph receptor-class-specific intracellular signaling protein that is required for appropriate neuromuscular connectivity.

Developmental RacGAP α2-Chimaerin Signaling Is a Determinant of the Morphological Features of Dendritic Spines in Adulthood.

Morphological characteristics of dendritic spines form the basis of cognitive ability. However, molecular mechanisms involved in fine-tuning of spine morphology during development are not fully understood. Moreover, it is unclear whether, and to what extent, these developmental mechanisms determine the normal adult spine morphological features. Here, we provide evidence that α2-isoform of Rac-specific GTPase-activating protein α-chimaerin (α2-chimaerin) is involved in spine morphological refinement during late postnatal period, and furthermore show that this developmental α2-chimaerin function affects adult spine morphologies. We used a series of mice with global and conditional knock-out of α-chimaerin isoforms (α1-chimaerin and α2-chimaerin). α2-Chimaerin disruption, but not α1-chimaerin disruption, in the mouse results in an increased size (and density) of spines in the hippocampus. In contrast, overexpression of α2-chimaerin in developing hippocampal neurons induces a decrease of spine size. Disruption of α2-chimaerin suppressed EphA-mediated spine morphogenesis in cultured developing hippocampal neurons. α2-Chimaerin disruption that begins during the juvenile stage results in an increased size of spines in the hippocampus. Meanwhile, spine morphologies are unaltered when α2-chimaerin is deleted only in adulthood. Consistent with these spine morphological results, disruption of α2-chimaerin beginning in the juvenile stage led to an increase in contextual fear learning in adulthood; whereas contextual learning was recently shown to be unaffected when α2-chimaerin was deleted only in adulthood. Together, these results suggest that α2-chimaerin signaling in developmental stages contributes to determination of the morphological features of adult spines and establishment of normal cognitive ability. SIGNIFICANCE STATEMENT: Recent studies of neurodevelopmental disorders in humans and their animal models have led to an attractive hypothesis that spine morphogenesis during development forms the basis of adult cognition. In particular, the roles of Rac and its regulators, such as Rac-specific GTPase-activating proteins (RacGAPs) and Rac guanine nucleotide exchange factors, are a topic of focus in spine morphogenesis and cognitive ability. Using a series of mice with global and conditional knock-out (KO) of RacGAP α-chimaerin isoforms (α1-chimaerin and α2-chimaerin), we provide compelling evidence demonstrating that α2-chimaerin is involved in spine morphological refinement during late postnatal development and that this developmental α2-chimaerin function affects adult spine morphologies. Furthermore, our results clearly showed that α2-chimaerin signaling during late postnatal development contributes to normal cognitive ability in adult mice.

Spinal glutamatergic neurons defined by EphA4 signaling are essential components of normal locomotor circuits.

EphA4 signaling is essential for the spatiotemporal organization of neuronal circuit formation. In mice, deletion of this signaling pathway causes aberrant midline crossing of axons from both brain and spinal neurons and the complete knock-outs (KOs) exhibit a pronounced change in motor behavior, where alternating gaits are replaced by a rabbit-like hopping gait. The neuronal mechanism that is responsible for the gait switch in these KO mice is not known. Here, using intersectional genetics, we demonstrate that a spinal cord-specific deletion of EphA4 signaling is sufficient to generate the overground hopping gait. In contrast, selective deletion of EphA4 signaling in forebrain neurons, including the corticospinal tract neurons, did not result in a change in locomotor pattern. The gait switch was attributed to the loss of EphA4 signaling in excitatory Vglut2+ neurons, which is accompanied by an increased midline crossing of Vglut2+ neurons in the ventral spinal cord. Our findings functionally define spinal EphA4 signaling in excitatory Vglut2+ neurons as required for proper organization of the spinal locomotor circuitry, and place these cells as essential components of the mammalian locomotor network.

Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin.

Eye movements depend on correct patterns of connectivity between cranial motor axons and the extraocular muscles. Despite the clinical importance of the ocular motor system, little is known of the molecular mechanisms underlying its development. We have recently shown that mutations in the Chimaerin-1 gene encoding the signaling protein α2-chimaerin (α2-chn) perturb axon guidance in the ocular motor system and lead to the human eye movement disorder, Duane retraction syndrome (DRS). The axon guidance cues that lie upstream of α2-chn are unknown; here we identify candidates to be the Semaphorins (Sema) 3A and 3C, acting via the PlexinA receptors. Sema3A/C are expressed in and around the developing extraocular muscles and cause growth cone collapse of oculomotor neurons in vitro. Furthermore, RNAi knockdown of α2-chn or PlexinAs in oculomotor neurons abrogates Sema3A/C-dependent growth cone collapse. In vivo knockdown of endogenous PlexinAs or α2-chn function results in stereotypical oculomotor axon guidance defects, which are reminiscent of DRS, whereas expression of α2-chn gain-of-function constructs can rescue PlexinA loss of function. These data suggest that α2-chn mediates Sema3-PlexinA repellent signaling. We further show that α2-chn is required for oculomotor neurons to respond to CXCL12 and hepatocyte growth factor (HGF), which are growth promoting and chemoattractant during oculomotor axon guidance. α2-chn is therefore a potential integrator of different types of guidance information to orchestrate ocular motor pathfinding. DRS phenotypes can result from incorrect regulation of this signaling pathway.

α2-Chimaerin regulates a key axon guidance transition during development of the oculomotor projection.

The ocular motor system consists of three nerves which innervate six muscles to control eye movements. In humans, defective development of this system leads to eye movement disorders, such as Duane Retraction Syndrome, which can result from mutations in the α2-chimaerin signaling molecule. We have used the zebrafish to model the role of α2-chimaerin during development of the ocular motor system. We first mapped ocular motor spatiotemporal development, which occurs between 24 and 72 h postfertilization (hpf), with the oculomotor nerve following an invariant sequence of growth and branching to its muscle targets. We identified 52 hpf as a key axon guidance "transition," when oculomotor axons reach the orbit and select their muscle targets. Live imaging and quantitation showed that, at 52 hpf, axons undergo a switch in behavior, with striking changes in the dynamics of filopodia. We tested the role of α2-chimaerin in this guidance process and found that axons expressing gain-of-function α2-chimaerin isoforms failed to undergo the 52 hpf transition in filopodial dynamics, leading to axon stalling. α2-chimaerin loss of function led to ecotopic and misguided branching and hypoplasia of oculomotor axons; embryos had defective eye movements as measured by the optokinetic reflex. Manipulation of chimaerin signaling in oculomotor neurons in vitro led to changes in microtubule stability. These findings demonstrate that a correct level of α2-chimaerin signaling is required for key oculomotor axon guidance decisions, and provide a zebrafish model for Duane Retraction Syndrome.

Special clinical features with a novel mutation site of CHN1 gene in a Chinese family with Duane retraction syndrome.

PURPOSE: This study is to describe the special clinical and genotypic features of a Chinese family with variant types of Duane retraction syndrome and to present our experience on managing these cases. METHODS: Four individuals from one family were reviewed by ophthalmologic examinations, in which two affected and two unaffected individuals were revealed. MRI scans were performed on the two patients. Relevant gene mutations were screened by the next-generation sequencing technology and confirmed by Sanger sequencing technology. RESULTS: The six-year-old proband presented with special clinical features of severe horizontal gaze dysfunction, exotropia and mild scoliosis. His mother showed significantly limited binocular abductions, with retraction of eyeballs in adduction. From MRI scans, abducens nerves were not observed in both patients and the oculomotor nerve was slightly thin in the proband. The proband and his mother shared the same CHN1 gene mutation site (c. 62A>G; p.Y21C). Strabismus surgery was performed on the proband to correct the primary gaze exotropia.(NM_001822: exon3 or NM_001025201: exon4: c. 62A>G; p.Y21C). CONCLUSIONS: A novel CHN1 gene mutation was revealed from a Chinese family with Duane retraction syndrome. Remarkably, the proband and his mother presented different clinical features of ocular motility disorder. Strabismus correction surgery and amblyopia training helped to improve the appearance and visual function of the proband.

There's more than one way to skin a chimaerin.

In two manuscripts published in Neuron (Beg et al. and Wegmeyer et al.) and one published in Cell (Iwasato et al.), investigators have found that a particular GAP, alpha-chimaerin, is required in vivo for ephrinB3/EphA4-dependent motor circuit formation.

EphA4-dependent axon guidance is mediated by the RacGAP alpha2-chimaerin.

Neuronal network formation in the developing nervous system is dependent on the accurate navigation of nerve cell axons and dendrites, which is controlled by attractive and repulsive guidance cues. Ephrins and their cognate Eph receptors mediate many repulsive axonal guidance decisions by intercellular interactions resulting in growth cone collapse and axon retraction of the Eph-presenting neuron. We show that the Rac-specific GTPase-activating protein alpha2-chimaerin binds activated EphA4 and mediates EphA4-triggered axonal growth cone collapse. alpha-Chimaerin mutant mice display a phenotype similar to that of EphA4 mutant mice, including aberrant midline axon guidance and defective spinal cord central pattern generator activity. Our results reveal an alpha-chimaerin-dependent signaling pathway downstream of EphA4, which is essential for axon guidance decisions and neuronal circuit formation in vivo.

alpha2-Chimaerin is an essential EphA4 effector in the assembly of neuronal locomotor circuits.

The assembly of neuronal networks during development requires tightly controlled cell-cell interactions. Multiple cell surface receptors that control axon guidance and synapse maturation have been identified. However, the signaling mechanisms downstream of these receptors have remained unclear. Receptor signals might be transmitted through dedicated signaling lines defined by specific effector proteins. Alternatively, a single cell surface receptor might couple to multiple effectors with overlapping functions. We identified the neuronal RacGAP alpha2-chimaerin as an effector for the receptor tyrosine kinase EphA4. alpha2-Chimaerin interacts with activated EphA4 and is required for ephrin-induced growth cone collapse in cortical neurons. alpha2-Chimaerin mutant mice exhibit a rabbit-like hopping gait with synchronous hindlimb movements that phenocopies mice lacking EphA4 kinase activity. Anatomical and functional analyses of corticospinal and spinal interneuron projections reveal that loss of alpha2-chimaerin results in impairment of EphA4 signaling in vivo. These findings identify alpha2-chimaerin as an indispensable effector for EphA4 in cortical and spinal motor circuits.

Genome-wide association study of diabetic retinopathy in a Taiwanese population.

PURPOSE: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a complex multifactorial pathogenesis. The aim of this study was to identify the susceptibility genes that increase the risk of DR in type 2 diabetes (T2D) and to further elucidate the underlying mechanism of DR pathogenesis. DESIGN: A case-control study. PARTICIPANTS: We included 749 unrelated individuals with T2D (174 with DR and 575 without DR) and 100 nondiabetic controls. METHODS: We conducted a genome-wide association study using Illumina HumanHap550-Duo BeadChips. MAIN OUTCOME MEASURES: Compared with the genotypic distribution of single nucleotide polymorphisms (SNPs) between subjects with DR and without DR. RESULTS: Using statistical models, we selected a total of 12 SNPs with P-values <1 × 10(-6) that were associated with DR. After controlling for diabetes duration and hemoglobin A(1C), 9 of the 12 SNPs located on 5 chromosomal regions were found to be associated with DR. Five loci not previously associated with DR susceptibility were identified in and around the following genes: MYSM1 (Myb-like, SWIRM, and MPN domains 1) located on chromosome 1p (odds ratio [OR], 1.50; 95% confidence interval [CI], 1.03-2.20); PLXDC2 (plexin domain-containing 2) located on the chromosome 10p (OR, 1.67; 95% CI, 1.06-2.65); ARHGAP22 (Rho GTPase-activating protein 22) located on chromosome 10q (OR, 1.65; 95% CI, 1.05-2.60); and HS6ST3 (heparan sulfate 6-O-sulfotransferase 3) located on chromosome 13q (OR, 2.33; 95% CI, 1.13-4.77). The SNPs rs13163610 and rs17376456 located in the unknown gene on chromosome 5q were also associated with DR (OR, 3.63; 95% CI, 1.38-9.58). CONCLUSIONS: We identified a genetic association for susceptibility to DR in 5 novel chromosomal regions and PLXDC2 and ARHGAP22, the latter 2 of which are genes implicated in endothelial cell angiogenesis and increased capillary permeability. These findings suggest unsuspected pathways in the pathogenesis of DR.

CHN1 and duane retraction syndrome: Expanding the phenotype to cranial nerves development disease.

Duane retraction syndrome is a congenital eye movement disorder characterized by a failure of abducens nerve to develop normally, resulting in restriction or absence of abduction, adduction, or both, and narrowing of the palpebral fissure and retraction of the globe on attempted adduction. There is a genetic heterogeneity in Duane retraction syndrome (DURS). DURS maps to chromosome 8q13 in some patients, and pathogenic variants in CHN1 and MAFB genes are known to lead to DURS. We report here a child and his father with Duane retraction syndrome, associated to swallowing difficulties and unilateral trapeze aplasia. A whole exome sequencing revealed a heterozygous missense variant in CHN1 gene. This gene encodes GTPase-activating protein and is involved in the assembly of neuronal locomotor circuits. A patient with a 8q deletion has previously been described with a Duane retraction syndrome associated to trapeze aplasia. We provide an additional description to support the role in cranial nerves development of the CHN1 gene.

Bioinformatics-based analysis of the association between the A1-chimaerin (CHN1) gene and gastric cancer.

Gastric cancer (GC) is one of the most common causes of cancer-related deaths worldwide and the identification of additional therapeutic targets and biomarkers has become vital. The A1-chimaerin (CHN1) gene encodes a ras-related protein that can be activated or inactivated by binding to GTP or GDP. The present study aimed to assess the expression of CHN1 in GC tissue and cells, to explore its relationship with GC progression, and to discover the potential mechanisms underlying these associations. The ONCOMINE database and The Cancer Genome Atlas (TCGA) were used to determine the transcriptional levels of CHN1 in GC. Western blot and immunohistochemistry were used for detecting protein expression. Correlations between CHN1 levels and the clinical outcomes of GC patients were examined using Kaplan-Meier and Cox regression analyses. Moreover, the CIBERSORT algorithm was used to estimate immune cell infiltration. In GC patients, CHN1 transcription and CHN1 protein expression were upregulated, and a high expression of CHN1 was remarkably linked to poor survival in GC patients. CHN1 expression was associated with immune infiltrates and this gene showed potential involvement in multiple cancer-related pathways. Furthermore, the expression of CHN1 was correlated with the immunotherapeutic response. Finally, our results indicated that the pro-carcinogenic role of CHN1 may involve DNA methylation. To our knowledge, this is the first report characterizing CHN1 expression in GC. Our results show that high CHN1 levels could be used as a clinical biomarker for poor prognosis and that CHN1 inhibitors may have potential as anti-cancer drugs.

Cortical control of adaptive locomotion in wild-type mice and mutant mice lacking the ephrin-Eph effector protein alpha2-chimaerin.

In voluntary control, supraspinal motor systems select the appropriate response and plan movement mechanics to match task constraints. Spinal circuits translate supraspinal drive into action. We studied the interplay between motor cortex (M1) and spinal circuits during voluntary movements in wild-type (WT) mice and mice lacking the α2-chimaerin gene (Chn1(-/-)), necessary for ephrinB3-EphA4 signaling. Chn1(-/-) mice have aberrant bilateral corticospinal systems, aberrant bilateral-projecting spinal interneurons, and disordered voluntary control because they express a hopping gait, which may be akin to mirror movements. We addressed three issues. First, we determined the role of the corticospinal system in adaptive control. We trained mice to step over obstacles during treadmill locomotion. We compared performance before and after bilateral M1 ablation. WT mice adaptively modified their trajectory to step over obstacles, and M1 ablation increased substantially the incidence of errant steps over the obstacle. Chn1(-/-) mice randomly stepped or hopped during unobstructed locomotion but hopped over the obstacle. Bilateral M1 ablation eliminated this obstacle-dependent hop selection and increased forelimb obstacle contact errors. Second, we characterized the laterality of corticospinal action in Chn1(-/-) mice using pseudorabies virus retrograde transneuronal transport and intracortical microstimulation. We showed bilateral connections between M1 and forelimb muscles in Chn1(-/-) and unilateral connections in WT mice. Third, in Chn1(-/-) mice, we studied adaptive responses before and after unilateral M1 ablation. We identified a more important role for contralateral than ipsilateral M1 in hopping over the obstacle. Our findings suggest an important role for M1 in the mouse in moment-to-moment adaptive control, and further, using Chn1(-/-) mice, a role in mediating task-dependent selection of mirror-like hopping movements over the obstacle. Our findings also stress the importance of subcortical control during adaptive locomotion because key features of the trajectory remained largely intact after M1 ablation.

Analysis of the CHN1 gene in patients with various types of congenital ocular motility disorders.

BACKGROUND: Mutations in the gene CHN1 have been described in autosomal dominant Duane's retraction syndrome (DRS) and mutations have been shown to interfere with normal innervation of target eye muscles by oculomotor axons in chick embryos. We screened for CHN1 mutations in patients with various congenital ocular motility disorders. METHODS: Altogether, 29 patients with different congenital ocular motility disorders and a positive family history of congenital ocular motility disturbances or strabismus or bilateral affection or accompanying congenital disorders were enrolled in this study. DNA samples of patients suffering from DRS (n = 5), Brown syndrome (n = 13), other congenital motility disorders of the oblique eye muscles (n = 6), double elevator palsy (n = 4), and vertical retraction syndrome (n = 1) were investigated by direct sequencing of all coding exons of CHN1. RESULTS: In the families of our index patients with DRS, other family members displayed DRS, see-saw nystagmus, infantile esotropia, microtropia, or Brown syndrome, respectively. In the families of our patients with cases of Brown syndrome, bilateral abduction deficiency, infantile esotropia, and unspecified strabismus occurred. The patients with congenital disorders of the oblique muscles and with congenital elevation deficiencies other than Brown syndrome had relatives with ptosis, infantile esotropia, DRS, congenital abduction deficiency, and unspecified forms of strabismus. Thus a considerable intrafamilial overlap between different types of congenital forms of motility disorders and strabismus does exist. No mutations were detected in the CHN1 gene in our patients. In addition to known polymorphisms, we identified four novel heterozygous single-nucleotide substitutions, one in the 5'UTR, two in intronic regions, and one in the coding region leading to a synonymous amino acid substitution. CONCLUSIONS: We found no evidence for a causative involvement of CHN1 mutations in congenital ocular motor anomalies different from autosomal dominant Duane's retraction syndrome and provide further evidence for genetic heterogeneity in familial forms of DRS.

Identification of a novel CHN1 p.(Phe213Val) variant in a large Han Chinese family with congenital Duane retraction syndrome.

Duane retraction syndrome (DRS) is a neuromuscular dysfunction of the eyes. Although many causative genes of DRS have been identified in Europe and the United States, few reports have been published in regard to Chinese DRS. The aim of the present study was to explore the genetic defect of DRS in a Chinese family. Exome sequencing was used to identify the disease-causing gene for the two affected family members. Ophthalmic and physical examinations, as well as genetic screenings for variants in chimerin 1 (CHN1), were performed for all family members. Functional analyses of a CHN1 variant in 293T cells included a Rac-GTP activation assay, α2-chimaerin translocation assay, and co-immunoprecipitation assay. Genetic analysis revealed a NM_001822.7: c.637T > G variant in the CHN1 gene, which resulted in the substitution of a highly conserved C1 domain with valine at codon 213 (NP_001813.1: p.(Phe213Val)) (ClinVar Accession Number: SCV001335305). In-silico analysis revealed that the p.(Phe213Val) substitution affected the protein stability and connections among the amino acids of CHN1 in terms of its tertiary protein structure. Functional studies indicated that the p.(Phe213Val) substitution reduced Rac-GTP activity and enhanced membrane translocation in response to phorbol-myristoyl acetate (PMA). Together with previous studies, our present findings demonstrate that CHN1 may be an important causative gene for different ethnicities with DRS.