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Recent evidence suggests neurogenesis is on-going throughout life but the relevance of these findings for neurodegenerative disorders such as Parkinson’s disease (PD) is poorly understood. Biallelic PINK1 mutations cause early onset, Mendelian inherited PD. We studied the effect of PINK1 deficiency on adult neurogenesis of dopaminergic (DA) neurons in two complementary model systems. Zebrafish are a widely-used model to study neurogenesis in development and through adulthood. Using EdU analyses and lineage-tracing studies, we first demonstrate that a subset of ascending DA neurons and adjacent local-projecting DA neurons are each generated into adulthood in wild type zebrafish at a rate that decreases with age. Pink1-deficiency impedes DA neurogenesis in these populations, most significantly in early adult life. Pink1 already exerts an early effect on Th1+ progenitor cells rather than on differentiated DA neurons only. In addition, we investigate the effect of PINK1 deficiency in a human isogenic organoid model. Global neuronal differentiation in PINK1-deficient organoids and isogenic controls is similar, but PINK1-deficient organoids display impeded DA neurogenesis. The observation of impaired adult dopaminergic neurogenesis in Pink1 deficiency in two complementing model systems may have significant consequences for future therapeutic approaches in human PD patients with biallelic PINK1 mutations.

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafishgch1mutant (gch1–/–), using CRISPR/Cas technology.gch1–/–zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment ofgch1–/–larvae improved survival without ameliorating the motor phenotype. RNAseq ofgch1–/–larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation ingch1–/–. The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENTGenome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.

Bi-allelic mutations in the glucocerebrosidase gene (GBA1) cause Gaucher's disease, the most common human lysosomal storage disease. We previously reported a marked increase in miR-155 transcript levels and early microglial activation in a zebrafish model of Gaucher's disease (gba1-/-). miR-155 is a master regulator of inflammation and has been implicated in a wide range of different neurodegenerative disorders. The observed miR-155 upregulation preceded the subsequent development of widespread pathology with marked neuroinflammation, closely resembling human Gaucher's disease pathology. We now report similar increases of miR-155 expression in mammalian models of GD, confirming that miR-155 upregulation is a shared feature in glucocerebrosidase (GCase) deficiency across different species. Using CRISPR/Cas9 mutagenesis we then generated a miR-155 mutant zebrafish line (miR-155-/-) with completely abolished miR-155 expression. Unexpectedly, loss of miR-155 did not mitigate either the reduced lifespan or the robust inflammatory phenotypes of gba1-/- mutant zebrafish. Our data demonstrate that neither neuroinflammation nor disease progression in GCase deficiency are dependent on miR-155 and suggest that miR-155 inhibition would not be a promising therapeutic target in Gaucher's disease.

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-1β 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., Author summary Nerve connections that are severed in spinal cord injury do not heal, which can lead to permanent paralysis. Lack of repair may in part be due to prolonged inflammation of the injury site. In contrast, zebrafish show excellent repair of nerve connections after spinal injury and this is associated with controlling inflammation. Due to recent advances in genetic technology (CRISPR/Cas9) we can now determine the function of genes that influence regeneration in the living zebrafish in a matter of days. Here we devise a very rapid screening method for the function of inflammation-related genes in zebrafish larvae after spinal cord injury. We find a number of genes that are necessary for repair of nerve connections and control of the inflammation after injury. This provides important leads to improve our understanding of the role of inflammation in spinal cord injury. Moreover, our fast and robust screening method can be adopted by other researchers to screen for gene functions in a whole animal, which was previously not easily possible.

BackgroundThe additive mechanistic effect of genetic risk variants for Parkinson’s disease (PD) is a plausible but largely unproven hypothesis. We investigated the mechanistic interaction between the two lysosomal PD risk genes glucocerebrosidase 1 (GBA1) and sphingomyelinase 1 (SMPD1) in complementing model systems.MethodsUsing CRISPR/Cas gene editing, smpd1 mutant (smpd1-/-) zebrafish were generated and crossed to our previously characterised gba1-/- zebrafish line, generating double knockouts (gba1-/-;smpd1-/-). Spontaneous motor behaviour and survival were assessed in WT, single mutants and double mutants. HPLC-based sphingolipid quantification was combined with RNAseq based pathway analysis, assessment of the mitochondrial respiratory chain and quantification of lipid membrane oxidation for in-depth assessment of cellular health across all four genotypes. We also determined the effect of combined glucocerebrosidase (GCase) and acid sphingomyelinase (ASM) inactivation on autophagy and alpha-synuclein homeostasis in the human neuronal cell line SH-SY5Y.ResultsUnexpectedly, ASM deficiency rescued the marked behavioural phenotype and prolonged survival in gba1-/-;smpd1-/- double-mutant zebrafish compared to gba1-/-. RNAseq-based pathway analysis confirmed a profound rescue of neuronal function and intracellular homeostasis. We identified complete reciprocal rescue of mitochondrial respiratory chain function and abolished lipid membrane oxidation in gba1-/-;smpd1-/- compared to gba1-/- or smpd1-/- as the underlying rescue mechanism. The complementing in vitro experiments demonstrated an unexpected reduction of α-synuclein levels in human cell lines with combined GCase and ASM deficiency.ConclusionsOur study highlights the importance of functional validation for any putative mechanistic interactions between genetic risk factors and their overall effect on disease-relevant mechanisms rather than readily assuming an additive effect.

Acute CRISPR/Cas9 targeting offers the opportunity for scalable phenotypic genetic screening in zebrafish. However, the unpredictable efficiency of CRISPR gRNA (CrRNA) activity is a limiting factor. Here we describe how to resolve this by prescreening CrRNAs for high activity in vivo, using a simple standardised assay based on restriction fragment length polymorphism analysis (RFLP). We targeted 350 genomic sites with synthetic RNA Oligo guide RNAs (sCrRNAs) in zebrafish embryos and found that almost half exhibited > 90% efficiency in our RFLP assay. Having the ability to preselect highly active sCrRNAs (haCRs), we carried out a focussed phenotypic screen of 30 macrophage-related genes in spinal cord regeneration and found 10 genes whose disruption impaired axonal regeneration. Four (tgfb1a, tgfb3, tnfa, sparc) out of 5 stable mutants subsequently analysed retained the acute haCR phenotype, validating the efficiency of this approach. Mechanistically, lack of tgfb1a leads to a prolonged immune response after injury, which inhibits regeneration. Our rapid and scalable screening approach has identified functional regulators of spinal cord regeneration, and can be applied to study any biological function of interest.HIGHLIGHTS- Synthetic CRISPR gRNAs are highly active- in vivo pre-screening allows rapid assessment of CRISPR gRNA activity- Phenotypic CRISPR screen reveals crucial genes for spinal cord regeneration- tgfb1a promotes spinal regeneration by controlling inflammation

While mitochondrial dysfunction is emerging as key in Parkinson's disease (PD), a central question remains whether mitochondria are actual disease drivers and whether boosting mitochondrial biogenesis and function ameliorates pathology. We address these questions using patient-derived induced pluripotent stem cells and Drosophila models of GBA-related PD (GBA-PD), the most common PD genetic risk. Patient neurons display stress responses, mitochondrial demise, and changes in NAD+ metabolism. NAD+ precursors have been proposed to ameliorate age-related metabolic decline and disease. We report that increasing NAD+ via the NAD+ precursor nicotinamide riboside (NR) significantly ameliorates mitochondrial function in patient neurons. Human neurons require nicotinamide phosphoribosyltransferase (NAMPT) to maintain the NAD+ pool and utilize NRK1 to synthesize NAD+ from NAD+ precursors. Remarkably, NR prevents the age-related dopaminergic neuronal loss and motor decline in fly models of GBA-PD. Our findings suggest NR as a viable clinical avenue for neuroprotection in PD and other neurodegenerative diseases.

During ontogeny, neural stem cells in the spinal cord cease production of neurons. Spinal cord injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. It is unclear whether regenerative neurogenesis from spinal progenitor cells simply depends on recapitulation of developmental signals and intracellular signal transduction or is driven by signals and mechanisms that are unique to regeneration. Using single cell RNAseq of progenitor cells and macrophages, as well as cell type-specific manipulations, we provide evidence for a direct signalling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, Tnfa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This demonstrates regeneration-specific signalling mechanisms that provide targets for future interventions in the non-regenerating spinal cord of mammals.

The additive effect of genetic risk variants on overall disease risk is a plausible but frequently unproven hypothesis. To test this hypothesis, we assessed the biological effect of combined glucocerebrosidase (GCase) and acid sphingomyelinase (ASM) deficiency. Variants in both glucocerebrosidase1 (GBA1) and sphingomyelinase (SMPD1) are genetic risk factors for Parkinson’s disease. Unexpectedly, ASM deficiency resulted in normalized behaviour and prolonged survival in gba1−/−;smpd1−/− double-mutant zebrafish compared to gba1−/−. RNAseq-based pathway analysis confirmed a profound rescue of neuronal function and intracellular homeostasis. We identified complete reciprocal rescue of mitochondrial respiratory chain function and abolished lipid membrane oxidation in gba1−/−;smpd1−/− compared to gba1−/− or smpd1−/− as the underlying rescue mechanism. Complementing in vitro experiments demonstrated an unexpected reduction of α-synuclein levels in human cell lines with combined GCase and ASM deficiency. Our study highlights the importance of functional validation for any putative interactions between genetic risk factors and their overall effect on disease-relevant mechanisms rather than readily assuming an additive effect.SummaryThe additive effect of genetic risk variants on disease risk is a popular but typically unproven hypothesis. We investigated this hypothesis mechanistically for Parkinson’s disease risk factors and provide evidence of an unexpected rescue effect on neuronal function and survival.

Moderate or severe traumatic brain injury (TBI) causes widespread neuronal cell death. Microglia, the resident macrophages of the brain, react to injury by migrating to the lesion site, where they phagocytose cellular debris. Microglial phagocytosis can have both beneficial (e.g. debris clearance) and detrimental (e.g. respiratory burst, phagoptosis) consequences. Hence, whether the overall effect of microglial phagocytosis after brain injury in vivo is neuroprotective or neurotoxic is not known. Here, we establish a system with which to carry out dynamic real-time analyses of the mechanisms regulating cell death after brain injury in vivo. We show that mechanical injury to the larval zebrafish brain induces distinct phases of primary and secondary cell death. Excitotoxicity contributes to secondary cell death in zebrafish, reflecting findings from mammals. Microglia arrive at the lesion site within minutes of injury, where they rapidly engulf dead cells. Importantly, the rate of secondary cell death is increased when the rapid removal of cellular debris by microglia is reduced pharmacologically or genetically. In summary, our results provide evidence that microglial debris clearance is neuroprotective after brain injury in vivo.

Central nervous system injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. Activation of the innate immune system promotes regenerative neurogenesis, but it is fundamentally unknown whether this is indirect through the activation of known developmental signaling pathways or whether immune cells directly signal to progenitor cells using mechanisms that are unique to regeneration. Using single-cell RNA-seq of progenitor cells and macrophages, as well as cell-type-specific manipulations, we provide evidence for a direct signaling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, TNFa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This establishes the principle that macrophages directly communicate to spinal progenitor cells via non-developmental signals after injury, providing potential targets for future interventions in the regeneration-deficient spinal cord of mammals.

Spinal cord injury leads to a massive response of innate immune cells (microglia, macrophages, neutrophils) both, in non-regenerating mammals and in successfully regenerating zebrafish, but the role of these immune cells in functional spinal cord regeneration in zebrafish has not been addressed. Here we show that inhibiting inflammation reduces and promoting it accelerates axonal regeneration in larval zebrafish. Mutant analyses show that peripheral macrophages, but not neutrophils or microglia, are necessary and sufficient for full regeneration. Macrophage-lessirf8mutants show prolonged inflammation with elevated levels of Il-1β and Tnf-α. Decreasing Il-1β levels or number of Il-1β+neutrophils rescues functional regeneration inirf8mutants. However, during early regeneration, interference with Il-1β function impairs regeneration inirf8and wildtype animals. Inhibiting Tnf-α does not rescue axonal growth inirf8mutants, but impairs it in wildtype animals, indicating a pro-regenerative role of Tnf-α. Hence, inflammation is tightly and dynamically controlled by macrophages to promote functional spinal cord regeneration in zebrafish.

Astrocytes are key players in the progression of amyotrophic lateral sclerosis (ALS). Previously, gene expression profiling of astrocytes from the pre-symptomatic stage of the SOD1G93A model of ALS has revealed reduced lactate metabolism and altered trophic support. Here, we have performed microarray analysis of symptomatic and late-stage disease astrocytes isolated by laser capture microdissection (LCM) from the lumbar spinal cord of the SOD1G93A mouse to complete the picture of astrocyte behaviour throughout the disease course. Astrocytes at symptomatic and late-stage disease show a distinct up-regulation of transcripts defining a reactive phenotype, such as those involved in the lysosome and phagocytic pathways. Functional analysis of hexosaminidase B enzyme activity in the spinal cord and of astrocyte phagocytic ability has demonstrated a significant increase in lysosomal enzyme activity and phagocytic activity in SOD1G93A vs. littermate controls, validating the findings of the microarray study. In addition to the increased reactivity seen at both stages, astrocytes from late-stage disease showed decreased expression of many transcripts involved in cholesterol homeostasis and decreased cholesterol synthesis has been confirmed in vitro. Staining for the master regulator of cholesterol synthesis, SREBP2, has revealed an increased localisation to the cytoplasm of motor neurons in late-stage SOD1G93A spinal cord, indicating that motor neurons may attempt to synthesise their own cholesterol in response to decreased astrocytic cholesterol provision. Our data reveal that SOD1G93A astrocytes are characterised more by a loss of supportive function than a toxic phenotype during ALS disease progression and future studies should focus upon restorative therapies.

ObjectivesTo establish and characterise glucocerebrosidase 1 (GBA1) mutant zebrafish line.BackgroundHomozygous GBA1 mutations (GBA1−/−) cause Gaucher disease (GD), heterozygote GBA1 mutations (GBA+/−) are the most common risk factor for Parkinson's disease (PD). The aim of this project was to develop a zebrafish model of GBA1 deficiency and study PD-relevant mechanisms.MethodsGenome editing, RT PCR, behavioural analysis, Immunohistochemistry, H/E staining, in situ hybridisation, biochemical assays.ResultsHomozygous mutants (GBA1−/−) developed normally and did not experience dopaminergic (DA) cell loss during development but displayed loss of DA neurons at 12 weeks. Video tracking revealed a large decrease in spontaneous movements in GBA1−/− compared to WT and an intermediate motor phenotype in GBA1+/− in juvenile zebrafish. All homozygous mutants had to be culled at 3 months of age due to the severity of the phenotype. Established GD biomarkers such as hexosaminidase or chitotriosidase activity were markedly abnormal (pConclusionsGBA1−/− zebrafish share key biochemical and morphological features with GBA1 knockout mice or indeed human GD. This new vertebrate model system for GBA1 deficiency will be useful to study the interaction between GBA1 haploinsufficiency, alpha-synuclein and exogenous toxins.

ObjectiveTo determine if glucocerebrosidase 1 deficiency is a susceptibility factor to 1-methyl-4-phenylpyridinium (MPP+) toxicity using a zebrafish model.Background and hypothesisHeterozygous mutations in the glucocerebrosidase gene (GBA1) are a strong genetic risk factor for Parkinson's disease (PD). Genetic risk factors may increase susceptibility to exogenous neurotoxins. We hypothesized GBA1 haploinsufficiency may increase susceptibility to the classical PD neurotoxin MPTP.MethodsA stable gba1+/− zebrafish line was in-crossed to generate embryos of all genotypes. At 2 days post fertilization (dpf), 100 dechorionated embryos (and 100 controls) were separated into 6 well plates, and exposed to 3 mM MPP+. At 3dpf embryos were fixed in 4% paraformaldehyde. Whole mount in-situ hybridisation was performed using a tyrosine hydroxylase (TH) probe. Embryos were genotyped for gba1 by PCR. TH positive neurons were counted in 10 embryos of each genotype under 20x microscopy.ResultsMPP+ embryos exhibited approximately 25% reduction in TH neurons compared to controls with no differences between genotypes.ConclusionMPP+ exposure results in a loss of TH positive neurons. GBA1 deficiency does not potentiate this. We provide “proof of principle” data for the usefulness of PD mutant zebrafish strains to test possible interactions between PD genetic risk factors and relevant toxins.