Defining the Genetic Landscape of Congenital Mirror Movements in 80 Affected Individuals.

BACKGROUND: Congenital mirror movements (CMM) is a rare neurodevelopmental disorder characterized by involuntary movements from one side of the body that mirror voluntary movements on the opposite side. To date, five genes have been associated with CMM, namely DCC, RAD51, NTN1, ARHGEF7, and DNAL4. OBJECTIVE: The aim of this study is to characterize the genetic landscape of CMM in a large group of 80 affected individuals. METHODS: We screened 80 individuals with CMM from 43 families for pathogenic variants in CMM genes. In large CMM families, we tested for presence of pathogenic variants in multiple affected and unaffected individuals. In addition, we evaluated the impact of three missense DCC variants on binding between DCC and Netrin-1 in vitro. RESULTS: Causal pathogenic/likely pathogenic variants were found in 35% of probands overall, and 70% with familial CMM. The most common causal gene was DCC, responsible for 28% of CMM probands and 80% of solved cases. RAD51, NTN1, and ARHGEF7 were rare causes of CMM, responsible for 2% each. Penetrance of CMM in DCC pathogenic variant carriers was 68% and higher in males than females (74% vs. 54%). The three tested missense variants (p.Ile164Thr; p.Asn176Ser; and p.Arg1343His) bind Netrin-1 similarly to wild type DCC. CONCLUSIONS: A genetic etiology can be identified in one third of CMM individuals, with DCC being the most common gene involved. Two thirds of CMM individuals were unsolved, highlighting that CMM is genetically heterogeneous and other CMM genes are yet to be discovered. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Genetics of mirror movements identifies a multifunctional complex required for Netrin-1 guidance and lateralization of motor control.

Mirror movements (MM) disorder is characterized by involuntary movements on one side of the body that mirror intentional movements on the opposite side. We performed genetic characterization of a family with autosomal dominant MM and identified ARHGEF7, a RhoGEF, as a candidate MM gene. We found that Arhgef7 and its partner Git1 bind directly to Dcc. Dcc is the receptor for Netrin-1, an axon guidance cue that attracts commissural axons to the midline, promoting the midline crossing of axon tracts. We show that Arhgef7 and Git1 are required for Netrin-1-mediated axon guidance and act as a multifunctional effector complex. Arhgef7/Git1 activates Rac1 and Cdc42 and inhibits Arf1 downstream of Netrin-1. Furthermore, Arhgef7/Git1, via Arf1, mediates the Netrin-1-induced increase in cell surface Dcc. Mice heterozygous for Arhgef7 have defects in commissural axon trajectories and increased symmetrical paw placements during skilled walking, a MM-like phenotype. Thus, we have delineated how ARHGEF7 mutation causes MM.

Heterozygous Dcc Mutant Mice Have a Subtle Locomotor Phenotype.

Axon guidance receptors such as deleted in colorectal cancer (DCC) contribute to the normal formation of neural circuits, and their mutations can be associated with neural defects. In humans, heterozygous mutations in DCC have been linked to congenital mirror movements, which are involuntary movements on one side of the body that mirror voluntary movements of the opposite side. In mice, obvious hopping phenotypes have been reported for bi-allelic Dcc mutations, while heterozygous mutants have not been closely examined. We hypothesized that a detailed characterization of Dcc heterozygous mice may reveal impaired corticospinal and spinal functions. Anterograde tracing of the Dcc+/- motor cortex revealed a normally projecting corticospinal tract, intracortical microstimulation (ICMS) evoked normal contralateral motor responses, and behavioral tests showed normal skilled forelimb coordination. Gait analyses also showed a normal locomotor pattern and rhythm in adult Dcc+/- mice during treadmill locomotion, except for a decreased occurrence of out-of-phase walk and an increased duty cycle of the stance phase at slow walking speed. Neonatal isolated Dcc+/- spinal cords had normal left-right and flexor-extensor coupling, along with normal locomotor pattern and rhythm, except for an increase in the flexor-related motoneuronal output. Although Dcc+/- mice do not exhibit any obvious bilateral impairments like those in humans, they exhibit subtle motor deficits during neonatal and adult locomotion.

Sonic hedgehog accelerates DNA replication to cause replication stress promoting cancer initiation in medulloblastoma.

The mechanisms generating cancer-initiating mutations are not well understood. Sonic hedgehog (SHH) pathway activation is frequent in medulloblastoma (MB), with PTCH1 mutations being a common initiating event. Here we investigated the role of the developmental mitogen SHH in initiating carcinogenesis in the cells of origin: granule cell progenitors (GCPs). We delineate a molecular mechanism for tumor initiation in MB. Exposure of GCPs to Shh causes a distinct form of DNA replication stress, increasing both origin firing and fork velocity. Shh promotes DNA helicase loading and activation, with increased Cdc7-dependent origin firing. The S-phase duration is reduced and hyper-recombination occurs, causing copy number neutral loss of heterozygosity-a frequent event at the PTCH1/ptch1 locus. Moreover, Cdc7 inhibition to attenuate origin firing reduces recombination and preneoplastic tumor formation in mice. Therefore, tissue-specific replication stress induced by Shh promotes loss of heterozygosity, which in tumor-prone Ptch1+/- GCPs results in loss of this tumor suppressor-an early cancer-initiating event.

The Ciliary Protein Arl13b Functions Outside of the Primary Cilium in Shh-Mediated Axon Guidance.

The small GTPase Arl13b is enriched in primary cilia and regulates Sonic hedgehog (Shh) signaling. During neural development, Shh controls patterning and proliferation through a canonical, transcription-dependent pathway that requires the primary cilium. Additionally, Shh controls axon guidance through a non-canonical, transcription-independent pathway whose connection to the primary cilium is unknown. Here we show that inactivation of Arl13b results in defective commissural axon guidance in vivo. In vitro, we demonstrate that Arl13b functions autonomously in neurons for their Shh-dependent guidance response. We detect Arl13b protein in axons and growth cones, far from its well-established ciliary enrichment. To test whether Arl13b plays a non-ciliary function, we used an engineered, cilia-localization-deficient Arl13b variant and found that it was sufficient to mediate Shh axon guidance in vitro and in vivo. Together, these results indicate that, in addition to its ciliary role in canonical Shh signaling, Arl13b plays a cilia-independent role in Shh-mediated axon guidance.

Recent advances in SHH medulloblastoma progression: tumor suppressor mechanisms and the tumor microenvironment.

Medulloblastoma, the most common of the malignant pediatric brain tumors, is a group of four molecularly and clinically distinct cancers with different cells of origin. One of these medulloblastoma groups displays activation of Sonic hedgehog (SHH) signaling and originates from granule cell precursors of the developing cerebellum. Ongoing basic and clinical research efforts are tailored to discover targeted and safer therapies, which rely on the identification of the basic mechanisms regulating tumor initiation, progression, and metastasis. In SHH medulloblastoma, the mechanisms regulating neural progenitor transformation and progression to advanced tumors have been studied in some detail. The present review discusses recent advances on medulloblastoma progression derived from studies using mouse models of SHH medulloblastoma. We focus on mechanisms that regulate progression from precancerous lesions to medulloblastoma, describing novel roles played by tumor suppressor mechanisms and the tumor microenvironment.

De Novo Pathogenic Variants in N-cadherin Cause a Syndromic Neurodevelopmental Disorder with Corpus Collosum, Axon, Cardiac, Ocular, and Genital Defects.

Cadherins constitute a family of transmembrane proteins that mediate calcium-dependent cell-cell adhesion. The extracellular domain of cadherins consists of extracellular cadherin (EC) domains, separated by calcium binding sites. The EC interacts with other cadherin molecules in cis and in trans to mechanically hold apposing cell surfaces together. CDH2 encodes N-cadherin, whose essential roles in neural development include neuronal migration and axon pathfinding. However, CDH2 has not yet been linked to a Mendelian neurodevelopmental disorder. Here, we report de novo heterozygous pathogenic variants (seven missense, two frameshift) in CDH2 in nine individuals with a syndromic neurodevelopmental disorder characterized by global developmental delay and/or intellectual disability, variable axon pathfinding defects (corpus callosum agenesis or hypoplasia, mirror movements, Duane anomaly), and ocular, cardiac, and genital anomalies. All seven missense variants (c.1057G>A [p.Asp353Asn]; c.1789G>A [p.Asp597Asn]; c.1789G>T [p.Asp597Tyr]; c.1802A>C [p.Asn601Thr]; c.1839C>G [p.Cys613Trp]; c.1880A>G [p.Asp627Gly]; c.2027A>G [p.Tyr676Cys]) result in substitution of highly conserved residues, and six of seven cluster within EC domains 4 and 5. Four of the substitutions affect the calcium-binding site in the EC4-EC5 interdomain. We show that cells expressing these variants in the EC4-EC5 domains have a defect in cell-cell adhesion; this defect includes impaired binding in trans with N-cadherin-WT expressed on apposing cells. The two frameshift variants (c.2563_2564delCT [p.Leu855Valfs∗4]; c.2564_2567dupTGTT [p.Leu856Phefs∗5]) are predicted to lead to a truncated cytoplasmic domain. Our study demonstrates that de novo heterozygous variants in CDH2 impair the adhesive activity of N-cadherin, resulting in a multisystemic developmental disorder, that could be named ACOG syndrome (agenesis of corpus callosum, axon pathfinding, cardiac, ocular, and genital defects).

The Shh receptor Boc is important for myelin formation and repair.

Myelination leads to the formation of myelin sheaths surrounding neuronal axons and is crucial for function, plasticity and repair of the central nervous system (CNS). It relies on the interaction of the axons and the oligodendrocytes: the glial cells producing CNS myelin. Here, we have investigated the role of a crucial component of the Sonic hedgehog (Shh) signalling pathway, the co-receptor Boc, in developmental and repairing myelination. During development, Boc mutant mice display a transient decrease in oligodendroglial cell density together with delayed myelination. Despite recovery of oligodendroglial cells at later stages, adult mutants still exhibit a lower production of myelin basic protein correlated with a significant decrease in the calibre of callosal axons and a reduced amount of the neurofilament NF-M. During myelin repair, the altered OPC differentiation observed in the mutant is reminiscent of the phenotype observed after blockade of Shh signalling. In addition, Boc mutant microglia/macrophages unexpectedly exhibit the apparent inability to transition from a highly to a faintly ramified morphology in vivo Altogether, these results identify Boc as an important component of myelin formation and repair.

Boc Acts via Numb as a Shh-Dependent Endocytic Platform for Ptch1 Internalization and Shh-Mediated Axon Guidance.

During development, Shh attracts commissural axons toward the floor plate through a non-canonical, transcription-independent signaling pathway that requires the receptor Boc. Here, we find that Shh induces Boc internalization into early endosomes and that endocytosis is required for Shh-mediated growth-cone turning. Numb, an endocytic adaptor, binds to Boc and is required for Boc internalization, Shh-mediated growth-cone turning in vitro, and commissural axon guidance in vivo. Similar to Boc, Ptch1 is also internalized by Shh in a Numb-dependent manner; however, the binding of Shh to Ptch1 alone is not sufficient to induce Ptch1 internalization nor growth-cone turning. Therefore, the binding of Shh to Boc is required for Ptch1 internalization and growth-cone turning. Our data support a model where Boc endocytosis via Numb is required for Ptch1 internalization and Shh signaling in axon guidance. Thus, Boc acts as a Shh-dependent endocytic platform gating Ptch1 internalization and Shh signaling.

Long-Range Guidance of Spinal Commissural Axons by Netrin1 and Sonic Hedgehog from Midline Floor Plate Cells.

An important model for axon pathfinding is provided by guidance of embryonic commissural axons from dorsal spinal cord to ventral midline floor plate (FP). FP cells produce a chemoattractive activity, comprised largely of netrin1 (FP-netrin1) and Sonic hedgehog (Shh), that can attract the axons at a distance in vitro. netrin1 is also produced by ventricular zone (VZ) progenitors along the axons' route (VZ-netrin1). Recent studies using region-specific netrin1 deletion suggested that FP-netrin1 is dispensable and VZ-netrin1 sufficient for netrin guidance activity in vivo. We show that removing FP-netrin1 actually causes guidance defects in spinal cord consistent with long-range action (i.e., over hundreds of micrometers), and double mutant analysis supports that FP-netrin1 and Shh collaborate to attract at long range. We further provide evidence that netrin1 may guide via chemotaxis or haptotaxis. These results support the model that netrin1 signals at both short and long range to guide commissural axons in spinal cord.

Overexpression of Desmoglein 2 in a Mouse Model of Gorlin Syndrome Enhances Spontaneous Basal Cell Carcinoma Formation through STAT3-Mediated Gli1 Expression.

Activation of the hedgehog pathway is causative of virtually all sporadic and Gorlin syndrome-related basal cell carcinomas (BCCs), with loss of function of Ptc1 being the most common genomic lesion. Sporadic BCCs also overexpress Dsg2, a desmosomal cadherin normally found in the basal layer. Using a mouse model of Gorlin syndrome (Ptc1+/lacZ mice), we found that overexpressing Dsg2 in the basal layer (K14-Dsg2/Ptc1+/lacZ mice) or the superficial epidermis (Inv-Dsg2/Ptc1+/lacZ mice) resulted in increased spontaneous BCC formation at 3 and 6 months, respectively. The tumors did not show loss of heterozygosity of Ptc1, despite high levels of Gli1 and phosphorylated Stat3. A panel of sporadic human BCCs showed increased staining of both Dsg2 and phosphorylated Stat3 in all nine samples. Overexpression of Dsg2 in ASZ001 cells, a Ptc1-/- BCC cell line, induced Stat3 phosphorylation and further increased Gli1 levels, in both an autocrine and paracrine manner. Three different Stat3 inhibitors reduced viability and Gli1 expression in ASZ001 cells but not in HaCaT cells. Conversely, stimulation of Stat3 in ASZ001 cells with IL-6 increased Gli1 expression. Our results indicate that Dsg2 enhances canonical hedgehog signaling downstream of Ptc1 to promote BCC development through the activation of phosphorylated Stat3 and regulation of Gli1 expression.

Polarized Dock Activity Drives Shh-Mediated Axon Guidance.

In the developing spinal cord, Sonic hedgehog (Shh) attracts commissural axons toward the floorplate. How Shh regulates the cytoskeletal remodeling that underlies growth cone turning is unknown. We found that Shh-mediated growth cone turning requires the activity of Docks, which are unconventional GEFs. Knockdown of Dock3 and 4, or their binding partner ELMO1 and 2, abolished commissural axon attraction by Shh in vitro. Dock3/4 and ELMO1/2 were also required for correct commissural axon guidance in vivo. Polarized Dock activity was sufficient to induce axon turning, indicating that Docks are instructive for axon guidance. Mechanistically, we show that Dock and ELMO interact with Boc, the Shh receptor, and that this interaction is reduced upon Shh stimulation. Furthermore, Shh stimulation translocates ELMO to the growth cone periphery and activates Rac1. This identifies Dock/ELMO as an effector complex of non-canonical Shh signaling and demonstrates the instructive role of GEFs in axon guidance.

Axon Guidance: Gained in Translation.

In this issue of Neuron, Cagnetta et al. (2018) describe a novel method to identify, in an unbiased manner, newly synthesized axonal proteins in response to axon guidance cues. They find that axons stimulated by different guidance cues (Netrin-1, BDNF, and Sema3A) show distinct and common signatures.

MIO-TCD: A new benchmark dataset for vehicle classification and localization.

The ability to train on a large dataset of labeled samples is critical to the success of deep learning in many domains. In this paper, we focus on motor vehicle classification and localization from a single video frame and introduce the "MIOvision Traffic Camera Dataset" (MIO-TCD) in this context. MIO-TCD is the largest dataset for motorized traffic analysis to date. It includes 11 traffic object classes such as cars, trucks, buses, motorcycles, bicycles, pedestrians. It contains 786,702 annotated images acquired at different times of the day and different periods of the year by hundreds of traffic surveillance cameras deployed across Canada and the United States. The dataset consists of two parts: a "localization dataset", containing 137,743 full video frames with bounding boxes around traffic objects, and a "classification dataset", containing 648,959 crops of traffic objects from the 11 classes. We also report results from the 2017 CVPR MIO-TCD Challenge, that leveraged this dataset, and compare them with results for state-of-the-art deep learning architectures. These results demonstrate the viability of deep learning methods for vehicle localization and classification from a single video frame in real-life traffic scenarios. The topperforming methods achieve both accuracy and Kappa score above 96% on the classification dataset and mean-average precision of 77% on the localization dataset. We also identify scenarios in which state-of-the-art methods still fail and we suggest avenues to address these challenges. Both the dataset and detailed results are publicly available on-line [1].

Sonic Hedgehog Is a Remotely Produced Cue that Controls Axon Guidance Trans-axonally at a Midline Choice Point.

At the optic chiasm choice point, ipsilateral retinal ganglion cells (RGCs) are repelled away from the midline by guidance cues, including Ephrin-B2 and Sonic Hedgehog (Shh). Although guidance cues are normally produced by cells residing at the choice point, the mRNA for Shh is not found at the optic chiasm. Here we show that Shh protein is instead produced by contralateral RGCs at the retina, transported anterogradely along the axon, and accumulates at the optic chiasm to repel ipsilateral RGCs. In vitro, contralateral RGC axons, which secrete Shh, repel ipsilateral RGCs in a Boc- and Smo-dependent manner. Finally, knockdown of Shh in the contralateral retina causes a decrease in the proportion of ipsilateral RGCs in a non-cell-autonomous manner. These findings reveal a role for axon-axon interactions in ipsilateral RGC guidance, and they establish that remotely produced cues can act at axon guidance midline choice points.

Loss of Dcc in the spinal cord is sufficient to cause a deficit in lateralized motor control and the switch to a hopping gait.

BACKGROUND: Humans with heterozygous mutations in the axon guidance receptor DCC display congenital mirror movements (MMs), which are involuntary movements of body parts, such as fingers, on one side of the body that mirror voluntary movement of the opposite side. In mice, the homozygous Dcckanga mutant allele causes synchronous MM-like hindlimb movements during locomotion, resulting in hopping. In both human and mice, the neuroanatomical defect responsible for the deficit in lateralized motor control remains to be elucidated. RESULTS: Using the HoxB8-Cre line to specifically remove Dcc from the spinal cord, we found misrouting of commissural axons during their migration toward the floor plate, resulting in fewer axons crossing the midline. These mice also have a hopping gait, indicating that spinal cord guidance defects alone are sufficient to cause hopping. CONCLUSIONS: Dcc plays a role in the development of local spinal networks to ensure proper lateralization of motor control during locomotion. Local spinal cord defects following loss of Dcc cause a hopping gait in mice and may contribute to MM in humans. Developmental Dynamics 247:620-629, 2018. © 2017 Wiley Periodicals, Inc.

Linking Hedgehog, Translation, and mTORC1 in Medulloblastoma.

In this issue of Developmental Cell, Wu et al. (2017) report that mTORC1-dependent mRNA translation is required for Hedgehog signaling. They show that this process is essential for proliferation of cerebellum granule neuron precursor cells in response to Hedgehog signaling and for the formation of medulloblastoma, a cerebellum brain tumor.