C9ORF72 Deficiency Results in Neurodegeneration in the Zebrafish Retina.

Hexanucleotide repeat expansions within the gene C9ORF72 are the most common cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This disease-causing expansion leads to a reduction in C9ORF72 expression levels in patients, suggesting loss of C9ORF72 function could contribute to disease. To further understand the consequences of C9ORF72 deficiency in vivo, we generated a c9orf72 mutant zebrafish line. Analysis of the adult female spinal cords revealed no appreciable neurodegenerative pathology such as loss of motor neurons or increased levels of neuroinflammation. However, detailed examination of adult female c9orf72-/- retinas showed prominent neurodegenerative features, including a decrease in retinal thickness, gliosis, and an overall reduction in neurons of all subtypes. Analysis of rod and cone cells within the photoreceptor layer showed a disturbance in their outer segment structure and rhodopsin mislocalization from rod outer segments to their cell bodies and synaptic terminals. Thus, C9ORF72 may play a previously unappreciated role in retinal homeostasis and suggests C9ORF72 deficiency can induce tissue specific neuronal loss.

Regenerative neurogenesis: the integration of developmental, physiological and immune signals.

In fishes and salamanders, but not mammals, neural stem cells switch back to neurogenesis after injury. The signalling environment of neural stem cells is strongly altered by the presence of damaged cells and an influx of immune, as well as other, cells. Here, we summarise our recently expanded knowledge of developmental, physiological and immune signals that act on neural stem cells in the zebrafish central nervous system to directly, or indirectly, influence their neurogenic state. These signals act on several intracellular pathways, which leads to changes in chromatin accessibility and gene expression, ultimately resulting in regenerative neurogenesis. Translational approaches in non-regenerating mammals indicate that central nervous system stem cells can be reprogrammed for neurogenesis. Understanding signalling mechanisms in naturally regenerating species show the path to experimentally promoting neurogenesis in mammals.

CRISPR gRNA phenotypic screening in zebrafish reveals pro-regenerative genes in spinal cord injury.

Zebrafish exhibit robust regeneration following spinal cord injury, promoted by macrophages that control post-injury inflammation. However, the mechanistic basis of how macrophages regulate regeneration is poorly understood. To address this gap in understanding, we conducted a rapid in vivo phenotypic screen for macrophage-related genes that promote regeneration after spinal injury. We used acute injection of synthetic RNA Oligo CRISPR guide RNAs (sCrRNAs) that were pre-screened for high activity in vivo. Pre-screening of over 350 sCrRNAs allowed us to rapidly identify highly active sCrRNAs (up to half, abbreviated as haCRs) and to effectively target 30 potentially macrophage-related genes. Disruption of 10 of these genes impaired axonal regeneration following spinal cord injury. We selected 5 genes for further analysis and generated stable mutants using haCRs. Four of these mutants (tgfb1a, tgfb3, tnfa, sparc) retained the acute haCR phenotype, validating the approach. Mechanistically, tgfb1a haCR-injected and stable mutant zebrafish fail to resolve post-injury inflammation, indicated by prolonged presence of neutrophils and increased levels of il1b expression. Inhibition of Il-1ß rescues the impaired axon regeneration in the tgfb1a mutant. Hence, our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, but can be applied to any biological function of interest.

Truly-Biocompatible Gold Catalysis Enables Vivo-Orthogonal Intra-CNS Release of Anxiolytics.

Being recognized as the best-tolerated of all metals, the catalytic potential of gold (Au) has thus far been hindered by the ubiquitous presence of thiols in organisms. Herein we report the development of a truly-catalytic Au-polymer composite by assembling ultrasmall Au-nanoparticles at the protein-repelling outer layer of a co-polymer scaffold via electrostatic loading. Illustrating the in vivo-compatibility of the novel catalysts, we show their capacity to uncage the anxiolytic agent fluoxetine at the central nervous system (CNS) of developing zebrafish, influencing their swim pattern. This bioorthogonal strategy has enabled -for the first time- modification of cognitive activity by releasing a neuroactive agent directly in the brain of an animal.

Regeneration of Dopaminergic Neurons in Adult Zebrafish Depends on Immune System Activation and Differs for Distinct Populations.

Adult zebrafish, in contrast to mammals, regenerate neurons in their brain, but the extent and variability of this capacity is unclear. Here we ask whether the loss of various dopaminergic neuron populations is sufficient to trigger their functional regeneration. Both sexes of zebrafish were analyzed. Genetic lineage tracing shows that specific diencephalic ependymo-radial glial (ERG) progenitor cells give rise to new dopaminergic [tyrosine hydroxylase-positive (TH+)] neurons. Ablation elicits an immune response, increased proliferation of ERG progenitor cells, and increased addition of new TH+ neurons in populations that constitutively add new neurons (e.g., diencephalic population 5/6). Inhibiting the immune response attenuates neurogenesis to control levels. Boosting the immune response enhances ERG proliferation, but not addition of TH+ neurons. In contrast, in populations in which constitutive neurogenesis is undetectable (e.g., the posterior tuberculum and locus ceruleus), cell replacement and tissue integration are incomplete and transient. This is associated with a loss of spinal TH+ axons, as well as permanent deficits in shoaling and reproductive behavior. Hence, dopaminergic neuron populations in the adult zebrafish brain show vast differences in regenerative capacity that correlate with constitutive addition of neurons and depend on immune system activation.SIGNIFICANCE STATEMENT Despite the fact that zebrafish show a high propensity to regenerate neurons in the brain, this study reveals that not all types of dopaminergic neurons are functionally regenerated after specific ablation. Hence, in the same adult vertebrate brain, mechanisms of successful and incomplete regeneration can be studied. We identify progenitor cells for dopaminergic neurons and show that activating the immune system promotes the proliferation of these cells. However, in some areas of the brain this only leads to insufficient replacement of functionally important dopaminergic neurons that later disappear. Understanding the mechanisms of regeneration in zebrafish may inform interventions targeting the regeneration of functionally important neurons, such as dopaminergic neurons, from endogenous progenitor cells in nonregenerating mammals.

Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy.

Degeneration and loss of lower motor neurons is the major pathological hallmark of spinal muscular atrophy (SMA), resulting from low levels of ubiquitously-expressed survival motor neuron (SMN) protein. One remarkable, yet unresolved, feature of SMA is that not all motor neurons are equally affected, with some populations displaying a robust resistance to the disease. Here, we demonstrate that selective vulnerability of distinct motor neuron pools arises from fundamental modifications to their basal molecular profiles. Comparative gene expression profiling of motor neurons innervating the extensor digitorum longus (disease-resistant), gastrocnemius (intermediate vulnerability), and tibialis anterior (vulnerable) muscles in mice revealed that disease susceptibility correlates strongly with a modified bioenergetic profile. Targeting of identified bioenergetic pathways by enhancing mitochondrial biogenesis rescued motor axon defects in SMA zebrafish. Moreover, targeting of a single bioenergetic protein, phosphoglycerate kinase 1 (Pgk1), was found to modulate motor neuron vulnerability in vivo. Knockdown of pgk1 alone was sufficient to partially mimic the SMA phenotype in wild-type zebrafish. Conversely, Pgk1 overexpression, or treatment with terazosin (an FDA-approved small molecule that binds and activates Pgk1), rescued motor axon phenotypes in SMA zebrafish. We conclude that global bioenergetics pathways can be therapeutically manipulated to ameliorate SMA motor neuron phenotypes in vivo.

Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish.

In adult zebrafish, relatively quiescent progenitor cells show lesion-induced generation of motor neurons. Developmental motor neuron generation from the spinal motor neuron progenitor domain (pMN) sharply declines at 48 hours post-fertilisation (hpf). After that, mostly oligodendrocytes are generated from the same domain. We demonstrate here that within 48 h of a spinal lesion or specific genetic ablation of motor neurons at 72 hpf, the pMN domain reverts to motor neuron generation at the expense of oligodendrogenesis. By contrast, generation of dorsal Pax2-positive interneurons was not altered. Larval motor neuron regeneration can be boosted by dopaminergic drugs, similar to adult regeneration. We use larval lesions to show that pharmacological suppression of the cellular response of the innate immune system inhibits motor neuron regeneration. Hence, we have established a rapid larval regeneration paradigm. Either mechanical lesions or motor neuron ablation is sufficient to reveal a high degree of developmental flexibility of pMN progenitor cells. In addition, we show an important influence of the immune system on motor neuron regeneration from these progenitor cells.

Reduce, reuse, recycle - Developmental signals in spinal cord regeneration.

Anamniotes, fishes and amphibians, have the capacity to regenerate spinal cord tissue after injury, generating new neurons that mature and integrate into the spinal circuitry. Elucidating the molecular signals that promote this regeneration is a fundamental question in regeneration research. Model systems, such as salamanders and larval and adult zebrafish are used to analyse successful regeneration. This shows that many developmental signals, such as Notch, Hedgehog (Hh), Bone Morphogenetic Protein (BMP), Wnt, Fibroblast Growth Factor (FGF), Retinoic Acid (RA) and neurotransmitters are redeployed during regeneration and activate resident spinal progenitor cells. Here we compare the roles of these signals in spinal cord development and regeneration of the much larger and fully patterned adult spinal cord. Understanding how developmental signalling systems are reactivated in successfully regenerating species may ultimately lead to ways to reactivate similar systems in mammalian progenitor cells, which do not show neurogenesis after spinal injury.

Neural development and regeneration: it's all in your spinal cord.

The spinal cord constitutes an excellent model system for studying development and regeneration of a functional nervous system, from specification of its precursors to circuit formation. The latest advances in the field of spinal cord development and its regeneration following damage were discussed at a recent EMBO workshop 'Spinal cord development and regeneration' in Sitges, Spain (October, 2014), highlighting the use of direct visualization of cellular processes, genome-wide molecular techniques and the development of methods for directed stem cell differentiation and regeneration.

Chondrolectin affects cell survival and neuronal outgrowth in in vitro and in vivo models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is characterized by the selective loss of spinal motor neurons owing to reduced levels of survival motor neuron (Smn) protein. In addition to its well-established role in assembling constituents of the spliceosome, diverse cellular functions have been proposed for Smn, but the reason why low levels of this widely expressed protein result in selective motor neuron pathology is still debated. In longitudinal studies of exon-level changes in SMA mouse model tissues, designed to determine the contribution of splicing dysfunction to the disease, we have previously shown that a generalized defect in splicing is unlikely to play a causative role in SMA. Nevertheless, we identified a small subset of genes that were alternatively spliced in the spinal cord compared with control mice before symptom onset, indicating a possible mechanistic role in disease. Here, we have performed functional studies of one of these genes, chondrolectin (Chodl), known to be highly expressed in motor neurons and important for correct motor axon outgrowth in zebrafish. Using in vitro and in vivo models of SMA, we demonstrate altered expression of Chodl in SMA mouse spinal motor neurons, show that Chodl has distinct effects on cell survival and neurite outgrowth and that increasing the expression of chodl can rescue motor neuron outgrowth defects in Smn-depleted zebrafish. Our findings thus link the dysregulation of Chodl to the pathophysiology of motor neuron degeneration in SMA.

Notch signaling controls generation of motor neurons in the lesioned spinal cord of adult zebrafish.

In mammals, increased Notch signaling is held partly responsible for a lack of neurogenesis after a spinal injury. However, this is difficult to test in an essentially nonregenerating system. We show that in adult zebrafish, which exhibit lesion-induced neurogenesis, e.g., of motor neurons, the Notch pathway is also reactivated. Although apparently compatible with neuronal regeneration in zebrafish, forced activity of the pathway significantly decreased progenitor proliferation and motor neuron generation. Conversely, pharmacological inhibition of the pathway increased proliferation and motor neuron numbers. This demonstrates that Notch is a negative signal for regenerative neurogenesis, and, importantly, that spinal motor neuron regeneration can be augmented in an adult vertebrate by inhibiting Notch signaling.

Chondrolectin mediates growth cone interactions of motor axons with an intermediate target.

The C-type lectin chondrolectin (chodl) represents one of the major gene products dysregulated in spinal muscular atrophy models in mice. However, to date, no function has been determined for the gene. We have identified chodl and other novel genes potentially involved in motor axon differentiation, by expression profiling of transgenically labeled motor neurons in embryonic zebrafish. To enrich the profile for genes involved in differentiation of peripheral motor axons, we inhibited the function of LIM-HDs (LIM homeodomain factors) by overexpression of a dominant-negative cofactor, thereby rendering labeled axons unable to grow out of the spinal cord. Importantly, labeled cells still exhibited axon growth and most cells retained markers of motor neuron identity. Functional tests of chodl, by overexpression and knockdown, confirm crucial functions of this gene for motor axon growth in vivo. Indeed, knockdown of chodl induces arrest or stalling of motor axon growth at the horizontal myoseptum, an intermediate target and navigational choice point, and reduced muscle innervation at later developmental stages. This phenotype is rescued by chodl overexpression, suggesting that correct expression levels of chodl are important for interactions of growth cones of motor axons with the horizontal myoseptum. Combined, these results identify upstream regulators and downstream functions of chodl during motor axon growth.

Rapid Testing of Gene Function in Axonal Regeneration After Spinal Cord Injury Using Larval Zebrafish.

Larval zebrafish show axonal regrowth over a complex spinal injury site and recovery of function within days after injury. Here we describe a simple protocol to disrupt gene function in this model using acute injections of highly active synthetic gRNAs to rapidly detect loss-of-function phenotypes without the need for breeding.

Drug screening in zebrafish larvae reveals inflammation-related modulators of secondary damage after spinal cord injury in mice.

Prolonged inflammation after spinal cord injury is detrimental to recovery. To find pharmacological modulators of the inflammation response, we designed a rapid drug screening paradigm in larval zebrafish followed by testing of hit compounds in a mouse spinal cord injury model. Methods: We used reduced il-1ß linked green fluorescent protein (GFP) reporter gene expression as a read-out for reduced inflammation in a screen of 1081 compounds in larval zebrafish. Hit drugs were tested in a moderate contusion model in mice for cytokine regulation, and improved tissue preservation and locomotor recovery. Results: Three compounds robustly reduced il-1ß expression in zebrafish. Cimetidine, an over-the-counter H2 receptor antagonist, also reduced the number of pro-inflammatory neutrophils and rescued recovery after injury in a zebrafish mutant with prolonged inflammation. Cimetidine action on il-1ß expression levels was abolished by somatic mutation of H2 receptor hrh2b, suggesting specific action. In mice, systemic treatment with Cimetidine led to significantly improved recovery of locomotor behavior as compared to controls, accompanied by decreased neuronal tissue loss and a shift towards a pro-regenerative profile of cytokine gene expression. Conclusion: Our screen revealed H2 receptor signaling as a promising target for future therapeutic interventions in spinal cord injury. This work highlights the usefulness of the zebrafish model for rapid screening of drug libraries to identify therapeutics to treat mammalian spinal cord injury.

SSDP cofactors regulate neural patterning and differentiation of specific axonal projections.

The developmental activity of LIM homeodomain transcription factors (LIM-HDs) is critically controlled by LIM domain-interacting cofactors of LIM-HDs (CLIM, also known as NLI or LDB). CLIM cofactors associate with single-stranded DNA binding proteins (SSDPs, also known as SSBPs) thereby recruiting SSDP1 and/or SSDP2 to LIM-HD/CLIM complexes. Although evidence has been presented that SSDPs are important for the activity of specific LIM-HD/CLIM complexes, the developmental roles of SSDPs are unclear. We show that SSDP1a and SSDP1b mRNAs are widely expressed early during zebrafish development with conspicuous expression of SSDP1b in sensory trigeminal and Rohon-Beard neurons. SSDP1 and CLIM immunoreactivity co-localize in these neuronal cell types and in other structures. Over-expression of the N-terminal portion of SSDP1 (N-SSDP1), which contains the CLIM-interaction domain, increases endogenous CLIM protein levels in vivo and impairs the formation of eyes and midbrain-hindbrain boundary. In addition, manipulation of SSDP1 via N-SSDP1 over-expression or SSDP1b knock down impairs trigeminal and Rohon-Beard sensory axon growth. We show that N-SSDP1 is able to partially rescue the inhibition of axon growth induced by a dominant-negative form of CLIM (DN-CLIM). These results reveal specific functions of SSDP in neural patterning and sensory axon growth, in part due to the stabilization of LIM-HD/CLIM complexes.

Claudin k is specifically expressed in cells that form myelin during development of the nervous system and regeneration of the optic nerve in adult zebrafish.

The zebrafish has become an important model organism to study myelination during development and after a lesion of the adult central nervous system (CNS). Here, we identify Claudin k as a myelin-associated protein in zebrafish and determine its localization during development and adult optic nerve regeneration. We find Claudin k in subcellular compartments consistent with location in autotypic tight junctions of oligodendrocytes and myelinating Schwann cells. Expression starts in the hindbrain at 2 days (mRNA) and 3 days (protein) postfertilization and is maintained in adults. A newly generated claudin k:green fluorescent protein (GFP) reporter line allowed us to characterize oligodendrocytes in the adult retina that express Claudin k and olig2, but not P0 and uniquely only form loose wraps of membrane around axons. After a crush of the adult optic nerve, Claudin k protein levels were first reduced and then recovered within 4 weeks postlesion, concomitant with optic nerve myelin de- and regeneration. During optic nerve regeneration, oligodendrocytes, many of which were newly generated, repopulated the lesion site and exhibited increasing morphological complexity over time. Thus, Claudin k is a novel myelin-associated protein expressed by oligodendrocytes and Schwann cells from early stages of wrapping and myelin formation in zebrafish development and adult regeneration, suggesting important functions of the gene for myelin formation and maintenance. Our Claudin k antibodies and claudin k:GFP reporter line represent excellent ways to visualize oligodendrocyte and Schwann cell differentiation in vivo.

An exception to the rule? Regeneration of the injured spinal cord in the spiny mouse.

The capacity for long-distance axon regeneration and functional recovery after spinal cord injury in the adult has long been thought to be a unique feature of certain non-mammalian vertebrates. However, in this issue of Developmental Cell, Nogueira-Rodrigues et al. report an astonishingly high regenerative ability in the spiny mouse.

Truly-Biocompatible Gold Catalysis Enables Vivo-Orthogonal Intra-CNS Release of Anxiolytics.

Being recognized as the best-tolerated of all metals, the catalytic potential of gold (Au) has thus far been hindered by the ubiquitous presence of thiols in organisms. Herein we report the development of a truly-catalytic Au-polymer composite by assembling ultrasmall Au-nanoparticles at the protein-repelling outer layer of a co-polymer scaffold via electrostatic loading. Illustrating the in vivo-compatibility of the novel catalysts, we show their capacity to uncage the anxiolytic agent fluoxetine at the central nervous system (CNS) of developing zebrafish, influencing their swim pattern. This bioorthogonal strategy has enabled -for the first time- modification of cognitive activity by releasing a neuroactive agent directly in the brain of an animal.

Analysis of the astray/robo2 zebrafish mutant reveals that degenerating tracts do not provide strong guidance cues for regenerating optic axons.

During formation of the optic projection in astray/robo2 mutant zebrafish, optic axons exhibit rostrocaudal pathfinding errors, ectopic midline crossing and increased terminal arbor size. Here we show that these errors persist into adulthood, even when robo2 function is conditionally reduced only during initial formation of the optic projection. Adult errors include massive ectopic optic tracts in the telencephalon. During optic nerve regeneration in astray/robo2 animals, these tracts are not repopulated and ectopic midline crossing is reduced compared with unlesioned mutants. This is despite a comparable macrophage/microglial response and upregulation of contactin1a in oligodendrocytes of entopic and ectopic tracts. However, other errors, such as expanded termination areas and ectopic growth into the tectum, were frequently recommitted by regenerating optic axons. Retinal ganglion cells with regenerating axons reexpress robo2 and expression of slit ligands is maintained in some areas of the adult optic pathway. However, slit expression is reduced rostral and caudal to the chiasm, compared with development and ubiquitous overexpression of Slit2 did not elicit major pathfinding phenotypes. This shows that (1) there is not an efficient correction mechanism for large-scale pathfinding errors of optic axons during development; (2) degenerating tracts do not provide a strong guidance cue for regenerating optic axons in the adult CNS, unlike the PNS; and (3) robo2 is less important for pathfinding of optic axons during regeneration than during development.

Automated in vivo drug screen in zebrafish identifies synapse-stabilising drugs with relevance to spinal muscular atrophy.

Synapses are particularly vulnerable in many neurodegenerative diseases and often the first to degenerate, for example in the motor neuron disease spinal muscular atrophy (SMA). Compounds that can counteract synaptic destabilisation are rare. Here, we describe an automated screening paradigm in zebrafish for small-molecule compounds that stabilize the neuromuscular synapse in vivo. We make use of a mutant for the axonal C-type lectin chondrolectin (chodl), one of the main genes dysregulated in SMA. In chodl-/- mutants, neuromuscular synapses that are formed at the first synaptic site by growing axons are not fully mature, causing axons to stall, thereby impeding further axon growth beyond that synaptic site. This makes axon length a convenient read-out for synapse stability. We screened 982 small-molecule compounds in chodl chodl-/- mutants and found four that strongly rescued motor axon length. Aberrant presynaptic neuromuscular synapse morphology was also corrected. The most-effective compound, the adenosine uptake inhibitor drug dipyridamole, also rescued axon growth defects in the UBA1-dependent zebrafish model of SMA. Hence, we describe an automated screening pipeline that can detect compounds with relevance to SMA. This versatile platform can be used for drug and genetic screens, with wider relevance to synapse formation and stabilisation.