Background Congenital Mirror Movement Syndrome (CMMS) involves involuntary movements on one side of the body while voluntary movements are performed on the other side. They disrupt left-right coordination and can be caused by a pathogenic variant in the DCC gene. CMMS have been extensively studied in the upper limb, but its impact on lower limb during gait in humans is unclear.Objectives Determine the impact of CMMS on locomotor patterns in individuals with a DCC pathogenic variant.Methods The gait pattern of seven individuals with a DCC pathogenic variant and ten healthy controls was assessed using 3D motion capture (kinematics), electromyographic recordings (EMG) of the soleus (SOL) and tibialis anterior (TA) muscles and foot pressure distribution during comfortable walking, fast walking, and running.Results In comparison to controls, the DCC group exhibited prolonged soleus (SOL) EMG activity and overlapping left-right SOL activation at a comfortable walking speed. No change was noted in TA. Both groups demonstrated increased pressure at the toe during the push-off phase; however, individuals with the most severe CMMS also displayed an early stance-phase pressure peak, reflecting contralateral push-off. These features diminished at higher walking speeds. Kinematic analysis revealed delayed knee flexion and prolonged double-limb support in the DCC group. Group differences were more pronounced when comparing control participants to DCC individuals with observable CMMS (Wood and Tauber scale).Conclusions DCC variants impair left-right coordination, particularly affecting distal extensor muscle timing during gait. These disruptions are most evident during slow walking and correlate with mirror movement severity.

Specific and biologically informed treatments for medulloblastoma, especially for the highly lethal TP53-mutant SHH subgroup, remain elusive, where radiotherapy is the primary treatment modality. Leveraging genome-wide CRISPR-Cas9 dropout screening in combination with lethal doses of radiotherapy, we identify loss of p53 as the main driver of radiation resistance in SHH medulloblastoma. A negative-selection CRISPR-Cas9 screen across multiple models of Trp53-deficient SHH medulloblastoma reveals a strong synthetic lethal interaction between components of the non-homologous end-joining pathway and radiation, particularly DNA-dependent protein kinase (DNA-PK) and its binding partners. Both genetic and pharmacological perturbation of DNA-PK enhance radiosensitivity in TP53-deficient SHH medulloblastoma, leading to cell death. In vivo treatment of both somatic and germline TP53-mutant SHH medulloblastoma models with peposertib, a small-molecule inhibitor of DNA-PK, significantly improves survival when combined with radiotherapy, strongly supporting further clinical investigation.

Sonic hedgehog (Shh) is an axon guidance molecule that can act as either a chemorepellent or a chemoattractant, depending on the neuron type and their developmental stage. In the developing spinal cord, Shh initially attracts commissural axons to the floor plate and later repels them after they cross the midline. In the developing visual system, Shh repels ipsilateral retinal ganglion cell (iRGC) axons at the optic chiasm. Although Shh requires the endocytic adaptor Numb for attraction of spinal commissural axons, the molecular mechanisms underlying Shh dual function in attraction and repulsion are still unclear. In this study, we show that Numb is essential for two Shh-mediated repulsion processes: iRGC axon repulsion at the optic chiasm and antero-posterior commissural axon repulsion in the spinal cord. Therefore, Numb is required for Shh-mediated attraction and repulsion. These results position Numb as a central player in the non-canonical Shh signaling pathway mediating axon repulsion.

During cerebellar development, granule cell precursors (GCPs) undergo a series of tightly regulated events, including proliferation, migration, and differentiation. Arhgef7, a guanine nucleotide exchange factor for Rac1 and Cdc42, plays a crucial role in these processes. This study investigates the role of Arhgef7 in cerebellar development using conditional knock-out (cKO) mice. We demonstrate that Arhgef7 is expressed in GCPs. Loss of Arhgef7 in GCPs results in severe cerebellar hypoplasia and foliation defects, particularly affecting lobules VI/VII. Arhgef7 cKO mice exhibit reduced postnatal GCP proliferation, disorganized cell layers, delayed differentiation, and impaired tangential and radial migration of GCPs. Our findings highlight the essential role of Arhgef7 in regulating multiple aspects of GCP development, thereby ensuring proper cerebellar morphogenesis.

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. (c) 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle that serves as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identify Numb as a ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss impedes Sonic hedgehog (Shh)-induced Ptch1 exit from the cilium, resulting in reduced Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into cell fates reliant on high Hh activity. Genetic ablation of Numb in the developing cerebellum impairs the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a regulator of ciliary Ptch1 levels during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in cell signaling.

The axon guidance cue netrin-1 signals through its receptor DCC (deleted in colorectal cancer) to attract commissural axons to the midline. Variants in DCC are frequently associated with congenital mirror movements (CMMs). A CMM-associated variant in the cytoplasmic tail of DCC is located in a conserved motif predicted to bind to a regulator of actin dynamics called the WAVE (Wiskott-Aldrich syndrome protein-family verprolin homologous protein) regulatory complex (WRC). Here, we explored how this variant affects DCC function and may contribute to CMM. We found that a conserved WRC-interacting receptor sequence (WIRS) motif in the cytoplasmic tail of DCC mediated the interaction between DCC and the WRC. This interaction was required for netrin-1-mediated axon guidance in cultured rodent commissural neurons. Furthermore, the WIRS motif of Fra, the Drosophila DCC ortholog, was required for attractive signaling in vivo at the Drosophila midline. The CMM-associated R1343H variant of DCC, which altered the WIRS motif, prevented the DCC-WRC interaction and impaired axon guidance in cultured commissural neurons and in Drosophila. The findings reveal the WRC as a pivotal component of netrin-1-DCC signaling and uncover a molecular mechanism explaining how a human genetic variant in the cytoplasmic tail of DCC may lead to CMM.

During development, Shh attracts axons of spinal cord commissural neurons to the floor plate. Shh-mediated attraction of commissural axons requires the receptor Boc. How Boc regulates cytoskeletal changes in growth cones in response to Shh is not fully understood. To identify effectors of Boc in Shh-mediated axon guidance, we used BioID to screen for proteins in proximity to Boc. Top hits included members of the WAVE regulatory complex (WRC), which acts downstream of Rac1 to promote actin cytoskeleton assembly. Therefore, we hypothesized that the WRC is important for Shh-mediated growth cone turning. Using biochemical and cellular assays, we found that Boc directly interacts with the WRC and that this interaction can occur in live cells. Moreover, we found that knockdown of Nckap1 and Cyfip1/2, two subunits of the WRC, in commissural neurons, impairs axon attraction toward a Shh gradient. Our results demonstrate that the WRC is for Shh-mediated axon attraction.

Neuronal wiring in the developing nervous system relies on axon guidance, a process which enables proper axon pathfinding to target cells. Disruption of axon guidance results in a wide spectrum of defects including congenital mirror movements (CMM) and corpus callosum agenesis (CCA). Axon guidance signaling pathways, such as Netrin-1/DCC, act through guanine nucleotide exchange factors (GEFs) to activate Rho GTPases, which regulate axon outgrowth and pathfinding. MCF2 is an X-linked gene that encodes a GEF which activates the GTPases Rac1 and Cdc42. We identify a recurrent hemizygous MCF2 variant [NM_001171876.2: c.31C > T p.(R11W)] in two unrelated cases displaying axon guidance and corticospinal defects: an adult male with CMM and an unrelated male fetus with CCA and abnormal corticospinal tract decussation on autopsy. Through biochemical and cellular studies, we demonstrate that MCF2 interacts physically with DCC and is able to induce DCC subcellular relocalization. The MCF2 p.(R11W) variant disrupts the interaction with DCC, is less efficient at relocalizing DCC, and has decreased GEF activity. Together, our results link an MCF2 variant to axon guidance defects in humans. Furthermore, our data suggests that this MCF2 variant may impair axon guidance by impacting DCC signaling, a key regulator of commissural and corticospinal axon guidance.

During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its receptor Boc and activates downstream effectors such as Smoothened (Smo) and Src family kinases (SFKs). SFK activation requires Smo activity and is also required for Shh-mediated axon guidance. Here we report that β-arrestin1 and β-arrestin2 (β-arrestins) serve as scaffolding proteins that link Smo and SFKs in Shh-mediated axon guidance. We found that β-arrestins are expressed in rat commissural neurons. We also found that Smo, β-arrestins, and SFKs form a tripartite complex, with the complex formation dependent on β-arrestins. β-arrestin knockdown blocked the Shh-mediated increase in Src phosphorylation, demonstrating that β-arrestins are required to activate Src kinase downstream of Shh. β-arrestin knockdown also led to the loss of Shh-mediated attraction of rat commissural axons in axon turning assays. Expression of two different dominant-negative β-arrestins, β-arrestin1 V53D which blocks the internalization of Smo and β-arrestin1 P91G-P121E which blocks its interaction with SFKs, also led to the loss of Shh-mediated attraction of commissural axons. In vivo, the expression of these dominant-negative β-arrestins caused defects in commissural axon guidance in the spinal cord of chick embryos of mixed sexes. Thus we show that β-arrestins are essential scaffolding proteins that connect Smo to SFKs and are required for Shh-mediated axon guidance.

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.

SUMMARYThe axon guidance cue, Netrin-1, signals through its receptor DCC to attract commissural axons to the midline. Pathogenic variants in DCC frequently lead to congenital mirror movements (CMM), but how these variants impact DCC function is largely unknown. Screening ofDCCin individuals with CMM recently revealed a novel variant located in a conserved motif in the cytoplasmic tail of DCC that is predicted to bind to a central actin nucleation promoting factor, the WAVE regulatory complex (WRC). Here, we use biochemical and axon guidance assays to show that this CMM-associated DCC variant is pathogenic by disrupting the interaction between DCC and the WRC. This DCC-WRC interaction is evolutionarily conserved and is required for Netrin-1 mediated commissural axon outgrowth and guidance. Together, we identify the WRC as a pivotal component of Netrin-1/DCC signaling and further provide a molecular mechanism explaining how genetic variants in DCC may lead to CMM.

SUMMARYThe transduction of Hedgehog (Hh) signaling relies on the primary cilium, a cell surface organelle serving as a signaling hub for the cell. Using proximity labeling and quantitative proteomics, we identified Numb as a new ciliary protein that positively regulates Hh signaling. Numb localizes to the ciliary pocket and acts as an endocytic adaptor to incorporate Ptch1 into clathrin-coated vesicles, thereby promoting Ptch1 exit from the cilium, a key step in Hh signaling activation. Numb loss hampers Sonic Hedgehog (Shh)-induced Ptch1 departure from the cilium, resulting in reduced activation of Hh signaling. Numb loss in spinal neural progenitors reduces Shh-induced differentiation into Nkx2.2-positive progenitors, a process reliant on high Hh signaling activity. Genetic ablation of Numb in the developing cerebellum impaired the proliferation of granule cell precursors, a Hh-dependent process, resulting in reduced cerebellar size. This study highlights Numb as a critical regulator of Ptch1 levels in the cilium during Hh signal activation and demonstrates the key role of ciliary pocket-mediated endocytosis in modulating the transduction of cell signaling.