The Daam2-VHL-Nedd4 axis governs developmental and regenerative oligodendrocyte differentiation.

Dysregulation of the ubiquitin-proteasomal system (UPS) enables pathogenic accumulation of disease-driving proteins in neurons across a host of neurological disorders. However, whether and how the UPS contributes to oligodendrocyte dysfunction and repair after white matter injury (WMI) remains undefined. Here we show that the E3 ligase VHL interacts with Daam2 and their mutual antagonism regulates oligodendrocyte differentiation during development. Using proteomic analysis of the Daam2-VHL complex coupled with conditional genetic knockout mouse models, we further discovered that the E3 ubiquitin ligase Nedd4 is required for developmental myelination through stabilization of VHL via K63-linked ubiquitination. Furthermore, studies in mouse demyelination models and white matter lesions from patients with multiple sclerosis corroborate the function of this pathway during remyelination after WMI. Overall, these studies provide evidence that a signaling axis involving key UPS components contributes to oligodendrocyte development and repair and reveal a new role for Nedd4 in glial biology.

Daam2 Regulates Myelin Structure and the Oligodendrocyte Actin Cytoskeleton through Rac1 and Gelsolin.

Myelin is essential to neuronal health and CNS function, and oligodendrocytes (OLs) undergo a complex process of cytoskeletal remodeling to form compact myelin sheaths. We previously discovered that a formin protein, Dishevelled associated activator of morphogenesis 2 (Daam2), suppresses OL differentiation through Wnt signaling; however, its role in cytoskeletal control remains unknown. To investigate this, we used OL-specific Daam2 conditional knockout (Daam2 cKO) mice of either sex and found myelin decompaction during an active period of myelination in postnatal development and motor coordination deficits in adulthood. Using primary OL cultures, we found Daam2-depleted OLs showed morphologic dysregulation during differentiation, suggesting that Daam2 regulates the OL cytoskeleton. In vivo screening identified the actin regulators Rac1 and Gelsolin as possible effectors in Daam2-deficient OL cytoskeletal regulation. Using gain-of-function and loss-of-function (LOF) experiments in primary OLs, we found that Rac1 and Gelsolin operate downstream of Daam2 in OL differentiation, with Gelsolin and Daam2 promoting and inhibiting membrane spreading during late differentiation, respectively. In vivo experiments using Daam2 cKO mice revealed increased protein levels of Gelsolin in the developing white matter with no change in RNA levels, suggesting that Daam2 acts in a posttranslational manner to suppress Gelsolin levels. In vitro biochemical studies show Daam2 induces Gelsolin ubiquitination and degradation in OLs. Together, our studies show Daam2 is essential for formation of functional myelin through modulation of Gelsolin levels to regulate the OL cytoskeleton. These findings further demonstrate the critical role of cytoskeletal dynamics in myelination and reveal novel avenues for treatment of a variety of white matter diseases.SIGNIFICANCE STATEMENT Proper myelin formation is essential to CNS function, and oligodendrocytes (OLs) require extensive changes in the actin cytoskeleton to form myelin sheaths. Here, we show that the formin protein Dishevelled associated activator of morphogenesis 2 (Daam2) is necessary for myelin compaction during development and motor learning in adulthood. Further, we demonstrate that Daam2 regulates OL differentiation and morphology through actin regulators Rac1 and Gelsolin. Lastly, we find that Daam2 may control myelin compaction by modulating the ubiquitination and degradation of Gelsolin through recruitment of the E3 ubiquitin ligase Nedd4. These findings reveal novel pathways for regulating myelin structure and function during white matter development.

Daam2 couples translocation and clustering of Wnt receptor signalosomes through Rac1.

Wnt signaling plays a critical role in development across species and is dysregulated in a host of human diseases. A key step in signal transduction is the formation of Wnt receptor signalosomes, during which a large number of components translocate to the membrane, cluster together and amplify downstream signaling. However, the molecular processes that coordinate these events remain poorly defined. Here, we show that Daam2 regulates canonical Wnt signaling via the PIP2-PIP5K axis through its association with Rac1. Clustering of Daam2-mediated Wnt receptor complexes requires both Rac1 and PIP5K, and PIP5K promotes membrane localization of these complexes in a Rac1-dependent manner. Importantly, the localization of Daam2 complexes and Daam2-mediated canonical Wnt signaling is dependent upon actin polymerization. These studies - in chick spinal cord and human and monkey cell lines - highlight novel roles for Rac1 and the actin cytoskeleton in the regulation of canonical Wnt signaling and define Daam2 as a key scaffolding hub that coordinates membrane translocation and signalosome clustering.

Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function.

Astrocytes display extraordinary morphological complexity that is essential to support brain circuit development and function. Formin proteins are key regulators of the cytoskeleton; however, their role in astrocyte morphogenesis across diverse brain regions and neural circuits is unknown. Here, we show that loss of the formin protein Daam2 in astrocytes increases morphological complexity in the cortex and olfactory bulb, but elicits opposing effects on astrocytic calcium dynamics. These differential physiological effects result in increased excitatory synaptic activity in the cortex and increased inhibitory synaptic activity in the olfactory bulb, leading to altered olfactory behaviors. Proteomic profiling and immunoprecipitation experiments identify Slc4a4 as a binding partner of Daam2 in the cortex, and combined deletion of Daam2 and Slc4a4 restores the morphological alterations seen in Daam2 mutants. Our results reveal new mechanisms regulating astrocyte morphology and show that congruent changes in astrocyte morphology can differentially influence circuit function.

Development of myelinating glia: An overview.

Myelin is essential to nervous system function, playing roles in saltatory conduction and trophic support. Oligodendrocytes (OLs) and Schwann cells (SCs) form myelin in the central and peripheral nervous systems respectively and follow different developmental paths. OLs are neural stem-cell derived and follow an intrinsic developmental program resulting in a largely irreversible differentiation state. During embryonic development, OL precursor cells (OPCs) are produced in distinct waves originating from different locations in the central nervous system, with a subset developing into myelinating OLs. OPCs remain evenly distributed throughout life, providing a population of responsive, multifunctional cells with the capacity to remyelinate after injury. SCs derive from the neural crest, are highly dependent on extrinsic signals, and have plastic differentiation states. SC precursors (SCPs) are produced in early embryonic nerve structures and differentiate into multipotent immature SCs (iSCs), which initiate radial sorting and differentiate into myelinating and non-myelinating SCs. Differentiated SCs retain the capacity to radically change phenotypes in response to external signals, including becoming repair SCs, which drive peripheral regeneration. While several transcription factors and myelin components are common between OLs and SCs, their differentiation mechanisms are highly distinct, owing to their unique lineages and their respective environments. In addition, both OLs and SCs respond to neuronal activity and regulate nervous system output in reciprocal manners, possibly through different pathways. Here, we outline their basic developmental programs, mechanisms regulating their differentiation, and recent advances in the field.

A Combined Conduit-Bioactive Hydrogel Approach for Regeneration of Transected Sciatic Nerves.

Transected peripheral nerve injury (PNI) affects the quality of life of patients, which leads to socioeconomic burden. Despite the existence of autografts and commercially available nerve guidance conduits (NGCs), the complexity of peripheral nerve regeneration requires further research in bioengineered NGCs to improve surgical outcomes. In this work, we introduce multidomain peptide (MDP) hydrogels, as intraluminal fillers, into electrospun poly(ε-caprolactone) (PCL) conduits to bridge 10 mm rat sciatic nerve defects. The efficacy of treatment groups was evaluated by electromyography and gait analysis to determine their electrical and motor recovery. We then studied the samples' histomorphometry with immunofluorescence staining and automatic axon counting/measurement software. Comparison with negative control group shows that PCL conduits filled with an anionic MDP may improve functional recovery 16 weeks postoperation, displaying higher amplitude of compound muscle action potential, greater gastrocnemius muscle weight retention, and earlier occurrence of flexion contracture. In contrast, PCL conduits filled with a cationic MDP showed the least degree of myelination and poor functional recovery. This phenomenon may be attributed to MDPs' difference in degradation time. Electrospun PCL conduits filled with an anionic MDP may become an attractive tissue engineering strategy for treating transected PNI when supplemented with other bioactive modifications.

Self-assembling multidomain peptide hydrogels accelerate peripheral nerve regeneration after crush injury.

Multidomain peptide (MDP) hydrogels are a class of self-assembling materials that have been shown to elicit beneficial responses for soft tissue regeneration. However, their capacity to promote nervous system regeneration remains unknown. The peripheral nervous system (PNS) substantially recovers after injury, partly due to the abundance of extracellular matrix (ECM) components in its basal lamina. However, severe peripheral nerve injuries that significantly damage the ECM continue to be a major clinical challenge as they occur at a high rate and can be extremely detrimental to patients' quality of life. In this study, a panel of eight MDPs were designed to contain various motifs mimicking extracellular matrix components and growth factors and successfully self-assembled into injectable, nanofibrous hydrogels. Using an in vitro screening system, various lysine based MDPs were found to enhance neurite outgrowth. To test their capacity to promote nerve regeneration in vivo, rat sciatic nerve crush injury was performed with MDP hydrogels injected directly into the injury sites. MDP hydrogels were found to enhance macrophage recruitment to the injury site and degrade efficiently over time. Rats that were injected with the MDP hydrogel K2 and laminin motif-containing MDPs K2-IIKDI and K2-IKVAV were found to have significantly accelerated functional recovery and remyelination compared to those injected with HBSS or other MDPs. These results demonstrate that MDPs enhance neurite outgrowth and promote a multicellular pro-regenerative response in peripheral nerve injury. This study provides important insights into the potential of MDPs as biomaterials for nerve regeneration and other clinical applications.

Modeling IKZF1 lesions in B-ALL reveals distinct chemosensitivity patterns and potential therapeutic vulnerabilities.

IKAROS family zinc finger 1 (IKZF1) alterations represent a diverse group of genetic lesions that are associated with an increased risk of relapse in B-cell acute lymphoblastic leukemia. Due to the heterogeneity of concomitant lesions, it remains unclear how IKZF1 abnormalities directly affect cell function and therapy resistance, and whether their consideration as a prognostic indicator is valuable in improving outcome. CRISPR/Cas9 strategies were used to engineer multiple panels of isogeneic lymphoid leukemia cell lines with a spectrum of IKZF1 lesions to measure changes in chemosensitivity, gene expression, cell cycle, and in vivo engraftment that can be linked to loss of IKAROS protein. IKZF1 knockout and heterozygous null cells displayed relative resistance to a number of common therapies for B-cell acute lymphoblastic leukemia, including dexamethasone, asparaginase, and daunorubicin. Transcription profiling revealed a stem/myeloid cell-like phenotype and JAK/STAT upregulation after IKAROS loss. A CRISPR homology-directed repair strategy was also used to knock-in the dominant-negative IK6 isoform into the endogenous locus, and a similar drug resistance profile, with the exception of retained dexamethasone sensitivity, was observed. Interestingly, IKZF1 knockout and IK6 knock-in cells both have significantly increased sensitivity to cytarabine, likely owing to marked downregulation of SAMHD1 after IKZF1 knockout. Both types of IKZF1 lesions decreased the survival time of xenograft mice, with higher numbers of circulating blasts and increased organ infiltration. Given these findings, exact specification of IKZF1 status in patients may be a beneficial addition to risk stratification and could inform therapy.