The microtubule regulator ringer functions downstream from the RNA repair/splicing pathway to promote axon regeneration.

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro- and antiregeneration factors are being identified. We previously reported that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Here, through transcriptome profiling, we show that the tubulin polymerization-promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca-deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell-autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule-associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, ringer lies downstream from and is negatively regulated by the microtubule-associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, our findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration.

Bi-allelic Loss-of-Function Variants in NUP188 Cause a Recognizable Syndrome Characterized by Neurologic, Ocular, and Cardiac Abnormalities.

Nucleoporins (NUPs) are an essential component of the nuclear-pore complex, which regulates nucleocytoplasmic transport of macromolecules. Pathogenic variants in NUP genes have been linked to several inherited human diseases, including a number with progressive neurological degeneration. We present six affected individuals with bi-allelic truncating variants in NUP188 and strikingly similar phenotypes and clinical courses, representing a recognizable genetic syndrome; the individuals are from four unrelated families. Key clinical features include congenital cataracts, hypotonia, prenatal-onset ventriculomegaly, white-matter abnormalities, hypoplastic corpus callosum, congenital heart defects, and central hypoventilation. Characteristic dysmorphic features include small palpebral fissures, a wide nasal bridge and nose, micrognathia, and digital anomalies. All affected individuals died as a result of respiratory failure, and five of them died within the first year of life. Nuclear import of proteins was decreased in affected individuals' fibroblasts, supporting a possible disease mechanism. CRISPR-mediated knockout of NUP188 in Drosophila revealed motor deficits and seizure susceptibility, partially recapitulating the neurological phenotype seen in affected individuals. Removal of NUP188 also resulted in aberrant dendrite tiling, suggesting a potential role of NUP188 in dendritic development. Two of the NUP188 pathogenic variants are enriched in the Ashkenazi Jewish population in gnomAD, a finding we confirmed with a separate targeted population screen of an international sampling of 3,225 healthy Ashkenazi Jewish individuals. Taken together, our results implicate bi-allelic loss-of-function NUP188 variants in a recessive syndrome characterized by a distinct neurologic, ophthalmologic, and facial phenotype.

Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam.

Both cell-intrinsic and extrinsic pathways govern axon regeneration, but only a limited number of factors have been identified and it is not clear to what extent axon regeneration is evolutionarily conserved. Whether dendrites also regenerate is unknown. Here we report that, like the axons of mammalian sensory neurons, the axons of certain Drosophila dendritic arborization (da) neurons are capable of substantial regeneration in the periphery but not in the CNS, and activating the Akt pathway enhances axon regeneration in the CNS. Moreover, those da neurons capable of axon regeneration also display dendrite regeneration, which is cell type-specific, developmentally regulated, and associated with microtubule polarity reversal. Dendrite regeneration is restrained via inhibition of the Akt pathway in da neurons by the epithelial cell-derived microRNA bantam but is facilitated by cell-autonomous activation of the Akt pathway. Our study begins to reveal mechanisms for dendrite regeneration, which depends on both extrinsic and intrinsic factors, including the PTEN-Akt pathway that is also important for axon regeneration. We thus established an important new model system--the fly da neuron regeneration model that resembles the mammalian injury model--with which to study and gain novel insights into the regeneration machinery.

A naturally monomeric infrared fluorescent protein for protein labeling in vivo.

Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.

All hands on deck: Adipocytes lept-in to drive nerve regeneration.

The glial metabolic state instructs nerve regeneration. In this issue of Cell Metabolism, Sundaram, Schütza, Schröter, et al. demonstrate that nerve injury induces adipocytes-glia signaling via leptin, thereby modulating glial metabolism and driving nerve regeneration.

Mechanosensitive Ion Channels, Axonal Growth, and Regeneration.

Cells sense and respond to mechanical stimuli by converting those stimuli into biological signals, a process known as mechanotransduction. Mechanotransduction is essential in diverse cellular functions, including tissue development, touch sensitivity, pain, and neuronal pathfinding. In the search for key players of mechanotransduction, several families of ion channels were identified as being mechanosensitive and were demonstrated to be activated directly by mechanical forces in both the membrane bilayer and the cytoskeleton. More recently, Piezo ion channels were discovered as a bona fide mechanosensitive ion channel, and its characterization led to a cascade of research that revealed the diverse functions of Piezo proteins and, in particular, their involvement in neuronal repair.

Drosophila Laser Axotomy Injury Model to Investigate RNA Repair and Splicing in Axon Regeneration.

The limited axon regeneration capacity of mature neurons often leads to insufficient functional recovery after damage to the central nervous system (CNS). To promote CNS nerve repair, there is an urgent need to understand the regeneration machinery in order to develop effective clinical therapies. To this aim, we developed a Drosophila sensory neuron injury model and the accompanying behavioral assay to examine axon regeneration competence and functional recovery after injury in the peripheral and central nervous systems. Specifically, we used a two-photon laser to induce axotomy and performed live imaging to assess axon regeneration, combined with the analysis of the thermonociceptive behavior as a readout of functional recovery. Using this model, we found that the RNA 3'-terminal phosphate cyclase (Rtca), which acts as a regulator for RNA repair and splicing, responds to injury-induced cellular stress and impedes axon regeneration after axon breakage. Here we describe how we utilize our Drosophila model to assess the role of Rtca during neuroregeneration.

Glia instruct axon regeneration via a ternary modulation of neuronal calcium channels in Drosophila.

A neuron's regenerative capacity is governed by its intrinsic and extrinsic environment. Both peripheral and central neurons exhibit cell-type-dependent axon regeneration, but the underlying mechanism is unclear. Glia provide a milieu essential for regeneration. However, the routes of glia-neuron signaling remain underexplored. Here, we show that regeneration specificity is determined by the axotomy-induced Ca2+ transients only in the fly regenerative neurons, which is mediated by L-type calcium channels, constituting the core intrinsic machinery. Peripheral glia regulate axon regeneration via a three-layered and balanced modulation. Glia-derived tumor necrosis factor acts through its neuronal receptor to maintain calcium channel expression after injury. Glia sustain calcium channel opening by enhancing membrane hyperpolarization via the inwardly-rectifying potassium channel (Irk1). Glia also release adenosine which signals through neuronal adenosine receptor (AdoR) to activate HCN channels (Ih) and dampen Ca2+ transients. Together, we identify a multifaceted glia-neuron coupling which can be hijacked to promote neural repair.

A designer peptide against the EAG2-Kvß2 potassium channel targets the interaction of cancer cells and neurons to treat glioblastoma.

Glioblastoma (GBM) is an incurable brain cancer that lacks effective therapies. Here we show that EAG2 and Kvß2, which are predominantly expressed by GBM cells at the tumor-brain interface, physically interact to form a potassium channel complex due to a GBM-enriched Kvß2 isoform. In GBM cells, EAG2 localizes at neuron-contacting regions in a Kvß2-dependent manner. Genetic knockdown of the EAG2-Kvß2 complex decreases calcium transients of GBM cells, suppresses tumor growth and invasion and extends the survival of tumor-bearing mice. We engineered a designer peptide to disrupt EAG2-Kvß2 interaction, thereby mitigating tumor growth in patient-derived xenograft and syngeneic mouse models across GBM subtypes without overt toxicity. Neurons upregulate chemoresistant genes in GBM cells in an EAG2-Kvß2-dependent manner. The designer peptide targets neuron-associated GBM cells and possesses robust efficacy in treating temozolomide-resistant GBM. Our findings may lead to the next-generation therapeutic agent to benefit patients with GBM.

mll ortholog containing functional domains of human MLL is expressed throughout the zebrafish lifespan and in haematopoietic tissues.

Infant leukaemia is an embryonal disease in which the underlying MLL translocations initiate in utero. Zebrafish offer unique potential to understand how MLL impacts haematopoiesis from the earliest embryonic timepoints and how translocations cause leukaemia as an embryonal process. In this study, a zebrafish mll cDNA syntenic to human MLL spanning the 5' to 3' UTRs, was cloned from embryos, and mll expression was characterized over the zebrafish lifespan. The protein encoded by the 35-exon ORF exhibited 46·4% overall identity to human MLL and 68-100% conservation in functional domains (AT-hooks, SNL, CXXC, PHD, bromodomain, FYRN, taspase1 sites, FYRC, SET). Maternally supplied transcripts were detected at 0-2 hpf. Strong ubiquitous early zygotic expression progressed to a cephalo-caudal gradient during later embryogenesis. mll was expressed in the intermediate cell mass (ICM) where primitive erythrocytes are produced and in the kidney where definitive haematopoiesis occurs in adults. mll exhibits high cross species conservation, is developmentally regulated in haematopoietic and other tissues and is expressed from the earliest embryonic timepoints throughout the zebrafish lifespan. Haematopoietic tissue expression validates using zebrafish for MLL haematopoiesis and leukaemia models.

Signalling and crosstalk of Rho GTPases in mediating axon guidance.

Axon extension during development of the nervous system is guided by many factors, but the signalling mechanisms responsible for triggering this extension remain mostly unknown. Here we have examined the role of Rho family small guanosine triphosphatases (GTPases) in mediating axon guidance by diffusible factors. Expression of either dominant-negative or constitutively active Cdc42 in cultured Xenopus laevis spinal neurons, at a concentration that does not substantially affect filopodial formation and neurite extension, abolishes the chemoattractive growth cone turning induced by a gradient of brain-derived neurotrophic factor that can activate Cdc42 and Rac in cultured neurons. Chemorepulsion induced by a gradient of lysophosphatidic acid is also abolished by the expression of dominant-negative RhoA. We also show that an asymmetry in Rho kinase or filopodial initiation across the growth cone is sufficient to trigger the turning response and that there is a crosstalk between the Cdc42 and RhoA pathways through their converging actions on the myosin activity essential for growth cone chemorepulsion.

The chromatin remodeler ISWI acts during Drosophila development to regulate adult sleep.

Sleep disruptions are among the most commonly reported symptoms across neurodevelopmental disorders (NDDs), but mechanisms linking brain development to normal sleep are largely unknown. From a Drosophila screen of human NDD-associated risk genes, we identified the chromatin remodeler Imitation SWItch/SNF (ISWI) to be required for adult fly sleep. Loss of ISWI also results in disrupted circadian rhythms, memory, and social behavior, but ISWI acts in different cells and during distinct developmental times to affect each of these adult behaviors. Specifically, ISWI expression in type I neuroblasts is required for both adult sleep and formation of a learning-associated brain region. Expression in flies of the human ISWI homologs SMARCA1 and SMARCA5 differentially rescues adult phenotypes, while de novo SMARCA5 patient variants fail to rescue sleep. We propose that sleep deficits are a primary phenotype of early developmental origin in NDDs and point toward chromatin remodeling machinery as critical for sleep circuit formation.

The Atr-Chek1 pathway inhibits axon regeneration in response to Piezo-dependent mechanosensation.

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.

Pathogenic variants in SMARCA5, a chromatin remodeler, cause a range of syndromic neurodevelopmental features.

Intellectual disability encompasses a wide spectrum of neurodevelopmental disorders, with many linked genetic loci. However, the underlying molecular mechanism for more than 50% of the patients remains elusive. We describe pathogenic variants in SMARCA5, encoding the ATPase motor of the ISWI chromatin remodeler, as a cause of a previously unidentified neurodevelopmental disorder, identifying 12 individuals with de novo or dominantly segregating rare heterozygous variants. Accompanying phenotypes include mild developmental delay, frequent postnatal short stature and microcephaly, and recurrent dysmorphic features. Loss of function of the SMARCA5 Drosophila ortholog Iswi led to smaller body size, reduced sensory dendrite complexity, and tiling defects in larvae. In adult flies, Iswi neural knockdown caused decreased brain size, aberrant mushroom body morphology, and abnormal locomotor function. Iswi loss of function was rescued by wild-type but not mutant SMARCA5. Our results demonstrate that SMARCA5 pathogenic variants cause a neurodevelopmental syndrome with mild facial dysmorphia.

Optical control of ERK and AKT signaling promotes axon regeneration and functional recovery of PNS and CNS in Drosophila.

Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple signaling circuits. Channelrhodopsin-based optogenetics shows the feasibility of stimulating neural repair but does not pin down specific signaling cascades. Here, we utilized optogenetic systems, optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to neuroregeneration in live Drosophila larvae. We showed that optoRaf or optoAKT activation not only enhanced axon regeneration in both regeneration-competent and -incompetent sensory neurons in the peripheral nervous system but also allowed temporal tuning and proper guidance of axon regrowth. Furthermore, optoRaf and optoAKT differ in their signaling kinetics during regeneration, showing a gated versus graded response, respectively. Importantly in the central nervous system, their activation promotes axon regrowth and functional recovery of the thermonociceptive behavior. We conclude that non-neuronal optogenetics targets damaged neurons and signaling subcircuits, providing a novel strategy in the intervention of neural damage with improved precision.

Most cells have a built-in regeneration signaling program that allows them to divide and repair. But, in the cells of the central nervous system, which are called neurons, this program is ineffective. This is why accidents and illnesses affecting the brain and spinal cord can cause permanent damage. Reactivating regeneration in neurons could help them repair, but it is not easy. Certain small molecules can switch repair signaling programs back on. Unfortunately, these molecules diffuse easily through tissues, spreading around the body and making it hard to target individual damaged cells. This both hampers research into neuronal repair and makes treatments directed at healing damage to the nervous system more likely to have side-effects. It is unclear whether reactivating regeneration signaling in individual neurons is possible. One way to address this question is to use optogenetics. This technique uses genetic engineering to fuse proteins that are light-sensitive to proteins responsible for relaying signals in the cell. When specific wavelengths of light hit the light-sensitive proteins, the fused signaling proteins switch on, leading to the activation of any proteins they control, for example, those involved in regeneration. Wang et al. used optogenetic tools to determine if light can help repair neurons in fruit fly larvae. First, a strong laser light was used to damage an individual neuron in a fruit fly larva that had been genetically modified so that blue light would activate the regeneration program in its neurons. Then, Wang et al. illuminated the cell with dim blue light, switching on the regeneration program. Not only did this allow the neuron to repair itself, it also allowed the light to guide its regeneration. By focusing the blue light on the damaged end of the neuron, it was possible to guide the direction of the cell's growth as it regenerated. Regeneration programs in flies and mammals involve similar signaling proteins, but blue light does not penetrate well into mammalian tissues. This means that further research into LEDs that can be implanted may be necessary before neuronal repair experiments can be performed in mammals. In any case, the ability to focus treatment on individual neurons paves the way for future work into the regeneration of the nervous system, and the combination of light and genetics could reveal more about how repair signals work.

Glial Metabolic Rewiring Promotes Axon Regeneration and Functional Recovery in the Central Nervous System.

Axons in the mature central nervous system (CNS) fail to regenerate after axotomy, partly due to the inhibitory environment constituted by reactive glial cells producing astrocytic scars, chondroitin sulfate proteoglycans, and myelin debris. We investigated this inhibitory milieu, showing that it is reversible and depends on glial metabolic status. We show that glia can be reprogrammed to promote morphological and functional regeneration after CNS injury in Drosophila via increased glycolysis. This enhancement is mediated by the glia derived metabolites: L-lactate and L-2-hydroxyglutarate (L-2HG). Genetically/pharmacologically increasing or reducing their bioactivity promoted or impeded CNS axon regeneration. L-lactate and L-2HG from glia acted on neuronal metabotropic GABAB receptors to boost cAMP signaling. Local application of L-lactate to injured spinal cord promoted corticospinal tract axon regeneration, leading to behavioral recovery in adult mice. Our findings revealed a metabolic switch to circumvent the inhibition of glia while amplifying their beneficial effects for treating CNS injuries.

Chloride intracellular channel 1 cooperates with potassium channel EAG2 to promote medulloblastoma growth.

Ion channels represent a large class of drug targets, but their role in brain cancer is underexplored. Here, we identify that chloride intracellular channel 1 (CLIC1) is overexpressed in human central nervous system malignancies, including medulloblastoma, a common pediatric brain cancer. While global knockout does not overtly affect mouse development, genetic deletion of CLIC1 suppresses medulloblastoma growth in xenograft and genetically engineered mouse models. Mechanistically, CLIC1 enriches to the plasma membrane during mitosis and cooperates with potassium channel EAG2 at lipid rafts to regulate cell volume homeostasis. CLIC1 deficiency is associated with elevation of cell/nuclear volume ratio, uncoupling between RNA biosynthesis and cell size increase, and activation of the p38 MAPK pathway that suppresses proliferation. Concurrent knockdown of CLIC1/EAG2 and their evolutionarily conserved channels synergistically suppressed the growth of human medulloblastoma cells and Drosophila melanogaster brain tumors, respectively. These findings establish CLIC1 as a molecular dependency in rapidly dividing medulloblastoma cells, provide insights into the mechanism by which CLIC1 regulates tumorigenesis, and reveal that targeting CLIC1 and its functionally cooperative potassium channel is a disease-intervention strategy.

The Mechanosensitive Ion Channel Piezo Inhibits Axon Regeneration.

Neurons exhibit a limited ability of repair. Given that mechanical forces affect neuronal outgrowth, it is important to investigate whether mechanosensitive ion channels may regulate axon regeneration. Here, we show that DmPiezo, a Ca2+-permeable non-selective cation channel, functions as an intrinsic inhibitor for axon regeneration in Drosophila. DmPiezo activation during axon regeneration induces local Ca2+ transients at the growth cone, leading to activation of nitric oxide synthase and the downstream cGMP kinase Foraging or PKG to restrict axon regrowth. Loss of DmPiezo enhances axon regeneration of sensory neurons in the peripheral and CNS. Conditional knockout of its mammalian homolog Piezo1 in vivo accelerates regeneration, while its pharmacological activation in vitro modestly reduces regeneration, suggesting the role of Piezo in inhibiting regeneration may be evolutionarily conserved. These findings provide a precedent for the involvement of mechanosensitive channels in axon regeneration and add a potential target for modulating nervous system repair.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron - dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

Targeted Delivery and Tolerability of MRI-Guided CED Infusion into the Cerebellum of Nonhuman Primates.

This study explored the feasibility of intraparenchymal delivery (gadoteridol and/or Serotype 5 Adeno-Associated Viral Vector-enhanced Green Fluorescent Protein [AAV5-eGFP]) into the cerebellum of nonhuman primates using real-time magnetic resonance imaging-guided convection enhanced delivery (MRI-CED) technology. All animals tolerated the neurosurgical procedure without any clinical sequela. Gene expression was detected within the cerebellar parenchyma at the site of infusion and resulted in transduction of neuronal cell bodies and fibers. Histopathology indicated localized damage along the stem of the cannula tract. These findings demonstrate the potential of real-time MRI-CED to deliver therapeutics into the cerebellum, which has extensive reciprocal connections and may be used as a target for the treatment of neurological disorders.