RB1 was altered in patients with neurodegenerative disease.

ARB1 mutation rates by DNA sequence data in blood samples from PD (n = 189), AD (n = 33), HD (n = 69), and ALS (n = 75) patients from the Guangzhou KingMed Diagnostics Group Co. B Detailed information of the six mutation sites in neurodegenerative patients. C The detailed frequencies of the six mutation sites in neurodegenerative patients, normal Eastern Asian population, and frequency in the ALAF project from NCBI. PD Parkinson’s disease, AD Alzheimer’s disease, HD Huntington’s disease, ALS amyotrophic lateral sclerosis; -: no data. (Fisher’s exact test; ***P < 0.001).

Rb1 deficiency impairs swimming behavior in juveniles and cognitive ability in adults.

A Tracking from single zebrafish larvae in siblings and zrb1-KO mutants at 5 dpf for 15 min. A′ The statistical plot of free-swimming distance and free-swimming speed in siblings and zrb1-KO mutants at 5 dpf for 15 min (t-test; mean ± SEM; ****P < 0.0001; n = 12). B The assessment of food stimulus (right arm, EC: enriched chamber) on learning and memory performances of wt and zrb1-KO^+/− heterozygotes adults in the T-maze test. B′. The statistical plot of left and right arm residence time in wt and zrb1-KO^+/− adult heterozygotes (t-test; mean ± SEM; **P < 0.01; ns not significant; n = 10).

Rb1 deficiency induced increased neuronal apoptosis of the hindbrain.

A Apoptotic vesicles in the hindbrain in siblings and zrb1-KO mutants at 3 dpf. The white arrows indicate the location of apoptotic vesicles. The image at the bottom left is a magnification of 400 for the red broken line area. A′ The statistical plot of apoptotic vesicles in the hindbrain of siblings and zrb1-KO mutants (t-test; mean ± SEM; ****P < 0.0001; n = 8). B Stained by NR to visualize microglia in the cerebellum and myelencephalon of siblings and zrb1-KO mutants at 5 dpf. The white dotted line outlines the cerebellum and myelencephalon. B′ The statistical plot of NR⁺ cells in the cerebellum and myelencephalon in siblings and zrb1-KO mutants (t-test; mean ± SEM; ****P < 0.0001; n = 10). C Co-staining of AO signals (green) and Tg(nbt:dsRed) of siblings and zrb1-KO mutants at 3 dpf. The white dotted line outlines the cerebellum and myelencephalon. The white arrows indicate the apoptotic cells. C′ The statistical plot of the number of AO⁺/NBT-dsRed⁺ cells in the cerebellum and myelencephalon of siblings and zrb1-KO mutants (t-test; mean ± SEM; ****P < 0.0001; n = 10). D Co-staining with AO (green) and Tg(nbt:dsRed) in 3 dpf sibling embryos and zrb1-KO mutants after injecting with control and zrb1 mRNA. D′ Quantification of AO⁺/NBT-dsRed⁺ cells of the cerebellum and myelencephalon in all groups of (D) (one-way ANOVA; mean ± SEM; **P < 0.01; ****P < 0.0001; n = 10).

Rb1 regulates the apoptosis of post-mitotic neurons.

A An UMAP plot re-clustered NSPCs and post mitotic cells into 22 clusters, which were further categorized into 6 subgroups (NSPCs, forebrain neurons, midbrain neurons, cerebellum neurons, myelencephalon neurons, and others) based on their respective locations and differentiation characteristics. NSPCs: 1-precursor, 4-retina neuroblasts (r-neuroblasts), 8-radial glia, 9-progenitors, and 15-retina-photoreceptor precursor cells (r-pho-pre); forebrain (F) neurons: 12-ventral forebrain gabaergic (v-f-gaba), 16-pallium glutamatergic (pallium-glu), 17-hypothalamus (hyp), and 20-dorsal habenula (d-ha); midbrain (M) neurons: 2-midbrain gabaergic (m-gaba), 3-midbrain/thalamus (m-th), and 6 midbrain optic tectum (m-optic tectum); cerebellum (C) neurons: 5-granule and 18-Purkinje; myelencephalon (Mye) neurons: 7-mid-hind boundary-gabaergic (mhb-gaba) and 10-hindbrain/cranial nerves; others: 0-neurons, 11-retinal ganglion cells (rgc), 13-vagal, 14-ganglion, 19-retina-Muller glia (r-m-glia) and 21-cornea. The top 20 functionally enriched KEGG pathways were found in the analysis of DEGs in the myelencephalon (B) and cerebellum (C). The red arrows indicate apoptosis pathways. Dorsal views of AO staining after wild-type microinjection of huc:cas9-T2A-mCherry, U6:gRNA(rb1) (D) plasmid and nestin:cas9-T2A-mCherry, U6:gRNA(rb1) (E) plasmid. The white dotted line outlines the cerebellum and myelencephalon. The white arrows indicate the apoptotic cells. D′, E′ The statistical analysis of AO⁺ cells in the cerebellum and myelencephalon between the control group and microinjection group of (D, E) (t-test; mean ± SEM; ****P < 0.0001; ns, not significant; n ≥ 10).

Rb1 regulates post-mitotic neuron apoptosis through the Kmt5b-bcl2a/caspase pathway.

AO staining in the brain at 3 dpf after overexpression of bcl2a in the nervous system (A) or treated Z-VAD-FMK (B) of siblings and zrb1-KO mutants. The white dotted line outlines the cerebellum and myelencephalon. A′, B′ Quantification of AO⁺ cells in all groups of (A, B) (one-way ANOVA; mean ± SEM; ****P < 0.0001; n = 10). C Dot plot showing the expression levels of kmt5b in six brain regions. The gray level represents the average expression; the dot size represents the percentage of cells expressing the marker genes. D Co-immunoprecipitation (Co-IP) analyses on the interaction between zRb1 and Kmt5b. The GPF–Kmt5b and mCherry-zRb1 plasmids were injected into wild-type zebrafish. Immunoprecipitation was performed with an antibody against GPF (upper panel) and confirmed with reciprocal immunoprecipitation experiments with antibodies against mCherry (lower panel). IgG represents a control antibody used for IPs. Input lanes contain lysate equal to one fifth of the amount used for the pull-down assays. IP indicates the antibody used for immunoprecipitation. TUNEL staining in the brain at 3 dpf after injecting huc:kmt5b-egfp plasmid (E) and kmt5b MO (F) in siblings and zrb1-KO mutants. The white dotted line outlines the cerebellum and myelencephalon. Quantification of TUNEL⁺ cells of the hindbrain after injecting huc:kmt5b-egfp plasmid (E′) and kmt5b MO (F′) in siblings and zrb1-KO mutants (one-way ANOVA; mean ± SEM; *P < 0.05; **P < 0.01; ****P < 0.0001; n ≥ 10).

R621S and L819V mutations of RB1 may play a role in neuronal apoptosis.

A AO staining in the brain at 3 dpf after injecting with control, hRB1 mRNA, hRB1^R621S mRNA, and hRB1^L819V mRNA in siblings and zrb1-KO mutants. The white dotted line outlines the cerebellum and myelencephalon. A′ Quantification of AO⁺ cells in all groups of (A) (one-way ANOVA; mean ± SEM; ****P < 0.0001; ns, not significant; n ≥ 10). B Co-immunoprecipitation (Co-IP) analyses on the interaction between hRB1/ hRB1^R621/hRB1^L819V and Kmt5b. 293T cells were transfected with GPF–Kmt5b and mCherry-hRB1, mCherry-hRB1^R621 or mCherry hRB1^L819. Immunoprecipitation was performed with an antibody against GPF (upper panel) and confirmed with reciprocal immunoprecipitation experiments with antibodies against mCherry (lower panel). IgG represents a control antibody used for IPs. Input lanes contain lysate equal to one fifth of the amount used for the pull-down assays. IP indicates the antibody used for immunoprecipitation.