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 phosphorylation by CK2α negatively regulates Wnt activity during white matter development and injury.

Wnt signaling plays an essential role in developmental and regenerative myelination in the central nervous system. The Wnt signaling pathway is composed of multiple regulatory layers; thus, how these processes are coordinated to orchestrate oligodendrocyte (OL) development remains unclear. Here, we show CK2α, a Wnt/ß-catenin signaling Ser/Thr kinase, phosphorylates Daam2, inhibiting its function and Wnt activity during OL development. Intriguingly, we found Daam2 phosphorylation differentially impacts distinct stages of OL development, accelerating early differentiation followed by decelerating maturation and myelination. Application toward white matter injury revealed CK2α-mediated Daam2 phosphorylation plays a protective role for developmental and behavioral recovery after neonatal hypoxia, while promoting myelin repair following adult demyelination. Together, our findings identify a unique regulatory node in the Wnt pathway that regulates OL development via protein phosphorylation-induced signaling complex instability and highlights a new biological mechanism for myelin restoration.

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.

Loss of Oxidation Resistance 1, OXR1, Is Associated with an Autosomal-Recessive Neurological Disease with Cerebellar Atrophy and Lysosomal Dysfunction.

We report an early-onset autosomal-recessive neurological disease with cerebellar atrophy and lysosomal dysfunction. We identified bi-allelic loss-of-function (LoF) variants in Oxidative Resistance 1 (OXR1) in five individuals from three families; these individuals presented with a history of severe global developmental delay, current intellectual disability, language delay, cerebellar atrophy, and seizures. While OXR1 is known to play a role in oxidative stress resistance, its molecular functions are not well established. OXR1 contains three conserved domains: LysM, GRAM, and TLDc. The gene encodes at least six transcripts, including some that only consist of the C-terminal TLDc domain. We utilized Drosophila to assess the phenotypes associated with loss of mustard (mtd), the fly homolog of OXR1. Strong LoF mutants exhibit late pupal lethality or pupal eclosion defects. Interestingly, although mtd encodes 26 transcripts, severe LoF and null mutations can be rescued by a single short human OXR1 cDNA that only contains the TLDc domain. Similar rescue is observed with the TLDc domain of NCOA7, another human homolog of mtd. Loss of mtd in neurons leads to massive cell loss, early death, and an accumulation of aberrant lysosomal structures, similar to what we observe in fibroblasts of affected individuals. Our data indicate that mtd and OXR1 are required for proper lysosomal function; this is consistent with observations that NCOA7 is required for lysosomal acidification.

IRF2BPL Is Associated with Neurological Phenotypes.

Interferon regulatory factor 2 binding protein-like (IRF2BPL) encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals who carry damaging heterozygous variants in IRF2BPL and are affected with neurological symptoms. Five individuals who carry IRF2BPL nonsense variants resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The IRF2BPL bioinformatics signature based on population genomics is consistent with a gene that is intolerant to variation. We show that the fruit-fly IRF2BPL ortholog, called pits (protein interacting with Ttk69 and Sin3A), is broadly detected, including in the nervous system. Complete loss of pits is lethal early in development, whereas partial knockdown with RNA interference in neurons leads to neurodegeneration, revealing a requirement for this gene in proper neuronal function and maintenance. The identified IRF2BPL nonsense variants behave as severe loss-of-function alleles in this model organism, and ectopic expression of the missense variants leads to a range of phenotypes. Taken together, our results show that IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

Novel role of dynamin-related-protein 1 in dynamics of ER-lipid droplets in adipose tissue.

Dynamin-Related-Protein 1 (DRP1) critically regulates mitochondrial and peroxisomal fission in multicellular organisms. However, the impact of DRP1 on other organelles, especially its direct influence on ER functions remains largely unclear. Here, we report that DRP1 translocates to endoplasmic reticulum (ER) in response to ß-adrenergic stimulation. To further investigate the function of DRP1 on ER-lipid droplet (LD) dynamics and the metabolic subsequences, we generated an adipose tissue-specific DRP1 knockout model (Adipo-Drp1flx/flx ). We found that the LDs in adipose tissues of Adipo-Drp1flx/flx mice exhibited more unilocular morphology with larger sizes, and formed less multilocular structures upon cold exposure. Mechanistically, we discovered that abnormal LD morphology occurs because newly generated micro-LDs fail to dissociate from the ER due to DRP1 ablation. Conversely, the ER retention of LDs can be rescued by the overexpressed DRP1 in the adipocytes. The alteration of LD dynamics, combined with abnormal mitochondrial and autophagy functions in adipose tissue, ultimately lead to abnormalities in lipid metabolism in Adipo-Drp1flx/flx mice.

Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially.

Dominant mutations in CACNA1A, encoding the α-1A subunit of the neuronal P/Q type voltage-dependent Ca2+ channel, can cause diverse neurological phenotypes. Rare cases of markedly severe early onset developmental delay and congenital ataxia can be due to de novo CACNA1A missense alleles, with variants affecting the S4 transmembrane segments of the channel, some of which are reported to be loss-of-function. Exome sequencing in five individuals with severe early onset ataxia identified one novel variant (p.R1673P), in a girl with global developmental delay and progressive cerebellar atrophy, and a recurrent, de novo p.R1664Q variant, in four individuals with global developmental delay, hypotonia, and ophthalmologic abnormalities. Given the severity of these phenotypes we explored their functional impact in Drosophila. We previously generated null and partial loss-of-function alleles of cac, the homolog of CACNA1A in Drosophila. Here, we created transgenic wild type and mutant genomic rescue constructs with the two noted conserved point mutations. The p.R1673P mutant failed to rescue cac lethality, displayed a gain-of-function phenotype in electroretinograms (ERG) recorded from mutant clones, and evolved a neurodegenerative phenotype in aging flies, based on ERGs and transmission electron microscopy. In contrast, the p.R1664Q variant exhibited loss of function and failed to develop a neurodegenerative phenotype. Hence, the novel R1673P allele produces neurodegenerative phenotypes in flies and human, likely due to a toxic gain of function.

Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster.

One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminate in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events are unclear. Here, we show that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. We additionally use CRISPR/Cas9 technology to N-terminally tag Rab8, which is then used to address endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. We also demonstrate that Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome protein Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, whereas GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through interactions with the exocyst tethering complex.

A rapid, membrane-dependent pathway directs furrow formation through RalA in the early Drosophila embryo.

Plasma membrane furrow formation is crucial in cell division and cytokinesis. Furrow formation in early syncytial Drosophila embryos is exceptionally rapid, with furrows forming in as little as 3.75 min. Here, we use 4D imaging to identify furrow formation, stabilization, and regression periods, and identify a rapid, membrane-dependent pathway that is essential for plasma membrane furrow formation in vivo. Myosin II function is thought to provide the ingression force for cytokinetic furrows, but the role of membrane trafficking pathways in guiding furrow formation is less clear. We demonstrate that a membrane trafficking pathway centered on Ras-like protein A (RalA) is required for fast furrow ingression in the early fly embryo. RalA function is absolutely required for furrow formation and initiation. In the absence of RalA and furrow function, chromosomal segregation is aberrant and polyploid nuclei are observed. RalA localizes to syncytial furrows, and mediates the movement of exocytic vesicles to the plasma membrane. Sec5, which is an exocyst complex subunit and localizes to ingressing furrows in wild-type embryos, becomes punctate and loses its cortical association in the absence of RalA function. Rab8 also fails to traffic to the plasma membrane and accumulates aberrantly in the cytoplasm in RalA disrupted embryos. RalA localization precedes F-actin recruitment to the furrow tip, suggesting that membrane trafficking might function upstream of cytoskeletal remodeling. These studies identify a pathway, which stretches from Rab8 to RalA and the exocyst complex, that mediates rapid furrow formation in early Drosophila embryos.

Cdk8/CDK19 promotes mitochondrial fission through Drp1 phosphorylation and can phenotypically suppress pink1 deficiency in Drosophila.

Cdk8 in Drosophila is the orthologue of vertebrate CDK8 and CDK19. These proteins have been shown to modulate transcriptional control by RNA polymerase II. We found that neuronal loss of Cdk8 severely reduces fly lifespan and causes bang sensitivity. Remarkably, these defects can be rescued by expression of human CDK19, found in the cytoplasm of neurons, suggesting a non-nuclear function of CDK19/Cdk8. Here we show that Cdk8 plays a critical role in the cytoplasm, with its loss causing elongated mitochondria in both muscles and neurons. We find that endogenous GFP-tagged Cdk8 can be found in both the cytoplasm and nucleus. We show that Cdk8 promotes the phosphorylation of Drp1 at S616, a protein required for mitochondrial fission. Interestingly, Pink1, a mitochondrial kinase implicated in Parkinson's disease, also phosphorylates Drp1 at the same residue. Indeed, overexpression of Cdk8 significantly suppresses the phenotypes observed in flies with low levels of Pink1, including elevated levels of ROS, mitochondrial dysmorphology, and behavioral defects. In summary, we propose that Pink1 and Cdk8 perform similar functions to promote Drp1-mediated fission.

CK2α-dependent regulation of Wnt activity governs white matter development and repair.

Wnt signaling plays an essential role in developmental and regenerative myelination in the CNS. The Wnt signaling pathway is comprised of multiple regulatory layers; thus, how these processes are coordinated to orchestrate oligodendrocyte development remains unclear. Here we show CK2α, a Wnt/ß-catenin signaling Ser/Thr kinase, phosphorylates Daam2, inhibiting its function and Wnt-activity during oligodendrocyte development. Intriguingly, we found Daam2 phosphorylation differentially impacts distinct stages of oligodendrocyte development, accelerating early differentiation followed by decelerating maturation and myelination. Application towards white matter injury revealed CK2α-mediated Daam2 phosphorylation plays a protective role for developmental and behavioral recovery after neonatal hypoxia, while promoting myelin repair following adult demyelination. Together, our findings identify a novel regulatory node in the Wnt pathway that regulates oligodendrocyte development via protein phosphorylation-induced signaling complex instability and highlights a new biological mechanism for myelin restoration. Significance: Wnt signaling plays a vital role in OL development and has been implicated as an adverse event for myelin repair after white matter injury. Emerging studies have shed light on multi-modal roles of Wnt effectors in the OL lineage, but the underlying molecular mechanisms and modifiable targets in OL remyelination remain unclear. Using genetic mouse development and injury model systems, we delineate a novel stage-specific function of Daam2 in Wnt signaling and OL development via a S704/T7-5 phosphorylation mechanism, and determine a new role of the kinase CK2α in contributing to OL development. In-depth understanding of CK2α-Daam2 pathway regulation will allow us to precisely modulate its activity in conjunction with Wnt signaling and harness its biology for white matter pathology.

Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14).

Infantile neuroaxonal dystrophy (INAD) is caused by recessive variants in PLA2G6 and is a lethal pediatric neurodegenerative disorder. Loss of the Drosophila homolog of PLA2G6, leads to ceramide accumulation, lysosome expansion, and mitochondrial defects. Here, we report that retromer function, ceramide metabolism, the endolysosomal pathway, and mitochondrial morphology are affected in INAD patient-derived neurons. We show that in INAD mouse models, the same features are affected in Purkinje cells, arguing that the neuropathological mechanisms are evolutionary conserved and that these features can be used as biomarkers. We tested 20 drugs that target these pathways and found that Ambroxol, Desipramine, Azoramide, and Genistein alleviate neurodegenerative phenotypes in INAD flies and INAD patient-derived neural progenitor cells. We also develop an AAV-based gene therapy approach that delays neurodegeneration and prolongs lifespan in an INAD mouse model.

Very-long-chain fatty acids induce glial-derived sphingosine-1-phosphate synthesis, secretion, and neuroinflammation.

VLCFAs (very-long-chain fatty acids) are the most abundant fatty acids in myelin. Hence, during demyelination or aging, glia are exposed to higher levels of VLCFA than normal. We report that glia convert these VLCFA into sphingosine-1-phosphate (S1P) via a glial-specific S1P pathway. Excess S1P causes neuroinflammation, NF-κB activation, and macrophage infiltration into the CNS. Suppressing the function of S1P in fly glia or neurons, or administration of Fingolimod, an S1P receptor antagonist, strongly attenuates the phenotypes caused by excess VLCFAs. In contrast, elevating the VLCFA levels in glia and immune cells exacerbates these phenotypes. Elevated VLCFA and S1P are also toxic in vertebrates based on a mouse model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Indeed, reducing VLCFA with bezafibrate ameliorates the phenotypes. Moreover, simultaneous use of bezafibrate and fingolimod synergizes to improve EAE, suggesting that lowering VLCFA and S1P is a treatment avenue for MS.

A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels.

In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. Here we show that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. We observe a defect in Fe-S cluster biogenesis and increased iron levels in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and we show that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.

Neuronal activity induces glucosylceramide that is secreted via exosomes for lysosomal degradation in glia.

Recessive variants in GBA1 cause Gaucher disease, a prevalent form of lysosome storage disease. GBA1 encodes a lysosomal enzyme that hydrolyzes glucosylceramide (GlcCer) into glucose and ceramide. Its loss causes lysosomal dysfunction and increased levels of GlcCer. We generated a null allele of the Drosophila ortholog Gba1b by inserting the Gal4 using CRISPR-Cas9. Here, we show that Gba1b is expressed in glia but not in neurons. Glial-specific knockdown recapitulates the defects found in Gba1b mutants, and these can be rescued by glial expression of human GBA1. We show that GlcCer is synthesized upon neuronal activity, and it is transported from neurons to glia through exosomes. Furthermore, we found that glial TGF-ß/BMP induces the transfer of GlcCer from neurons to glia and that the White protein, an ABCG transporter, promotes GlcCer trafficking to glial lysosomes for degradation.

Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies.

Large-scale insecticide application is a primary weapon in the control of insect pests in agriculture. However, a growing body of evidence indicates that it is contributing to the global decline in population sizes of many beneficial insect species. Spinosad emerged as an organic alternative to synthetic insecticides and is considered less harmful to beneficial insects, yet its mode of action remains unclear. Using Drosophila, we show that low doses of spinosad antagonize its neuronal target, the nicotinic acetylcholine receptor subunit alpha 6 (nAChRα6), reducing the cholinergic response. We show that the nAChRα6 receptors are transported to lysosomes that become enlarged and increase in number upon low doses of spinosad treatment. Lysosomal dysfunction is associated with mitochondrial stress and elevated levels of reactive oxygen species (ROS) in the central nervous system where nAChRα6 is broadly expressed. ROS disturb lipid storage in metabolic tissues in an nAChRα6-dependent manner. Spinosad toxicity is ameliorated with the antioxidant N-acetylcysteine amide. Chronic exposure of adult virgin females to low doses of spinosad leads to mitochondrial defects, severe neurodegeneration, and blindness. These deleterious effects of low-dose exposures warrant rigorous investigation of its impacts on beneficial insects.

Two neuronal peptides encoded from a single transcript regulate mitochondrial complex III in Drosophila.

Naturally produced peptides (<100 amino acids) are important regulators of physiology, development, and metabolism. Recent studies have predicted that thousands of peptides may be translated from transcripts containing small open-reading frames (smORFs). Here, we describe two peptides in Drosophila encoded by conserved smORFs, Sloth1 and Sloth2. These peptides are translated from the same bicistronic transcript and share sequence similarities, suggesting that they encode paralogs. Yet, Sloth1 and Sloth2 are not functionally redundant, and loss of either peptide causes animal lethality, reduced neuronal function, impaired mitochondrial function, and neurodegeneration. We provide evidence that Sloth1/2 are highly expressed in neurons, imported to mitochondria, and regulate mitochondrial complex III assembly. These results suggest that phenotypic analysis of smORF genes in Drosophila can provide a wealth of information on the biological functions of this poorly characterized class of genes.

An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.

Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.

Loss of IRF2BPL impairs neuronal maintenance through excess Wnt signaling.

De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like (IRF2BPL) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, we examined its function in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits, the Drosophila ortholog, represses Wnt transcription in flies. In contrast, neuronal depletion of Pits leads to increased wingless (wg) levels in the brain and is associated with axonal loss, whereas inhibition of Wg signaling is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects with an associated increase in wnt1 transcription and downstream signaling. WNT1 is also increased in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Last, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt transcription and signaling.