Motor behavior and motor neuron phenotype induced by tbk1 knockdown in zebrafish larvae.

A Western blot quantification (ratio to β-actin level) of tbk1 expression after antisense morpholino oligonucleotide (AMO)- and hTBK1 mRNA injections. B Traces representative of the touch-evoked escape response (TEER) in 48 hpf zebrafish injected with tbk1 or mismatch AMO and hTBK1 mRNA injections. C Quantitative analysis of the TEER velocity of 48 hpf zebrafish larvae injected with tbk1 or mismatch AMO and rescued with mRNA encoding human TBK1. One-way ANOVA with Tukey’s multiple comparisons test was used. *p < 0.05, **p < 0.01). D In vivo quantification of spinal motor neuron axonal length and number in 48 hpf transgenic Tg(mnx1:gal4/UAS:RFP) embryos injected with tbk1 or mismatch AMO.

Tbk1 expression and survival in zebrafish larvae with CRISPR-induced deletion of the tbk1 gene.

A Description of CRISPR-Cas9 tandem sgRNA targeting exons 1 and 2 or exons 4 and 5 of the tbk1 gene (top) and genotyping of injected embryos with gel electrophoresis of PCR products amplifying the deleted region. B tbk1 protein quantification by mass spectrometry in zebrafish larvae harboring mosaic tbk1 mutations or their noninjected controls (NIs) (unpaired t test, *p < 0.05). C RT‒qPCR measurement of tbk1 expression in zebrafish larvae with mosaic deletions of the tbk1 gene or NI controls between 6 hpf and 8 dpf (unpaired t test, ***p < 0.001). D Survival rate of zebrafish larvae injected with tbk1 or mismatch AMO (ns), CRISPR-Cas9 sgRNA targeting exons 1 and 2 or exons 4 and 5 of the tbk1 gene or NI controls (log-rank (Mantel‒Cox) test, *** p < 0.001).

Motor behavior of tbk1 mutant zebrafish larvae.

A Traces representative of the touch-evoked escape response (TEER) in 48 hpf tbk1 mutant, tbk1 or mismatch AMO or NI zebrafish larvae. B Quantitative analysis of the TEER velocity of 48 hpf tbk1 mutant, mismatch or tbk1 AMO or NI zebrafish larvae (one-way ANOVA with Tukey’s multiple comparisons test). *p < 0.05, **p < 0.01). C Representative traces suggestive of motor behavior and swimming trajectories of 5 dpf zebrafish larvae, recorded with a semiautomated video tracking system (Viewpoint Zebrabox). D Quantitative analysis of free-swimming (top panel) or stimuli-induced motor behavior (bottom) in 5 (left) or 6 dpf (right) tbk1 mutant or NI control zebrafish larvae. (Unpaired t test for comparative analysis. *p < 0.05).

Metabolomic analysis and nicotinamide riboside treatment of tbk1 mutant zebrafish.

A Metabolite set enrichment analysis showing the enrichment ratio and statistical significance of metabolic pathways in 6 dpf tbk1 mutant zebrafish compared to their noninjected controls (over representation analysis using Metaboanalyst 6.0 with significantly affected metabolites, p < 0.01). B Mass spectrometry quantification of NAD + , quinolinic acid and nicotinamide in tbk1 mutant or NI zebrafish (unpaired t test, p = 0.002, p < 0.001 and p < 0.001, respectively). C Schematic representation of the Nicotinamide riboside (NR) and NAD+ salvage pathway affected in tbk1 mutant zebrafish (NRK, nicotinate riboside kinase; NMNAT, nicotinamide mononucleotide adenylyltransferase). D Schematic timeline used for the treatment of tbk1 CRISPR-Cas9-injected or NI zebrafish with 10 µM NR or 0.1% DMSO in zebrafish embryo water. E Quantitative analysis of larval free-swimming motor behavior upon incubation with NR (one-way ANOVA with Tukey’s multiple comparisons test). *p < 0.05, ***p < 0.001). F Survival rate of tbk1 mutant or NI larvae upon incubation with NR.

Proteomic profile and markers of programmed cell death in tbk1 mutant zebrafish.

A Volcano plot showing protein enrichment measured by mass spectrometry in tbk1 mutant zebrafish at 8 dpf compared to their NI controls (black line = cutoff for statistical significance). B Pathway enrichment analysis based on KEGG 2019 for significantly enriched proteins measured in tbk1 mutant zebrafish compared to their NI controls. C Heatmap representation of protein levels (row z scores) for significantly enriched proteins associated with the KEGG term necroptosis (hsa04217) in tbk1 mutant and NI zebrafish larvae. D Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of cells affected by programmed cell death in the larval brain of tbk1 mutant or NI zebrafish at 6 dpf (OT, optic tectum; Hb, hindbrain; dotted line = outline of hindbrain used for quantification). E Quantification of TUNEL-positive cells in the hindbrain of tbk1 mutant or NI zebrafish (unpaired t test, p = 0.0098). F Survival rate of tbk1 mutant or NI larvae upon incubation with the necroptosis inhibitor necrosulfonamide (NSA) at 1 µM or 0.1% DMSO (log-rank (Mantel Cox) test). ***p < 0.001).

Joint proteomic analysis of tbk1 mutants zebrafish and iMNs from ALS patients with TBK1 mutations.

A Fluorescent immunolabeling of the neuronal marker CHAT in differentiated induced pluripotent stem cell-derived motor neurons (iMNs) at day 21 from an ALS patient harboring the loss of function mutation TBK1^T77fs compared to control iMNs. B Proteomic profiles of ALS-TBK1 iMNs compared to control iMNs. The differentially expressed protein list was compared to the list of zebrafish orthologs differentially expressed in tbk1-deficient zebrafish larvae, and enrichment analysis was performed on the common list of 260 terms. C Network plot showing the relationship between enriched pathways from TBK1-mutant ALS models. A close interaction between the necroptosis pathway and several neurodegenerative pathways, including the amyotrophic lateral sclerosis (ALS) KEGG pathway, is observed. D Heatmap representation of protein levels (row z scores) for significantly enriched proteins in both datasets associated with the KEGG term ALS in tbk1 mutant vs NI zebrafish and ALS-TBK1 iMNs vs control iMNs. E Heatmap representation of protein levels (row z scores) for significantly enriched proteins associated with the KEGG term necroptosis in ALS-TBK1 iMNs vs control iMNs. F Western blotting of STAT1 expression in tbk1 mutant and NI zebrafish. G Mass spectrometry quantification of stat1b protein level in tbk1 mutant and NI zebrafish.