603 - Deficiency of kdm1a induces locomotor abnormalities and learning and memory deficits in zebrafish larvae

Publication Number: 4591.603

Li Zou, Jiangsu Provincial Women and Children's Health Care Hospital, Nanjing, Jiangsu, China (People's Republic)

Background: Kdm1a is the first discovered histone lysine-specific demethylase, which is required for neurogenesis, and has a role in neuron progenitor cell proliferation and terminal differentiation. Mutations in kdm1a have been detected in neurodevelopmental disorders. However, the effect of kdm1a on neurobehaviors and the underlying mechanisms remain largely unknown.

Objective: Explore the effects and mechanisms of kdm1a on the movement and learning and memory of zebrafish Larvae

Design/Methods: CRISPR/Cas9 was applied to generate a kdm1a deficiency in zebrafish. Further, a series of morphological measurements, open field test, y-maze test and other behavioral tests were performed to systematically characterize the behavioral changes in kdm1a KO zebrafish larvae. In this study, two Transgenic zebrafish models (HuC:egfp and hb9:egfp) and multiple molecular biology analyses were used to evaluate the neurodevelopment in kdm1a deficiency mutant zebrafish larvae.

Results: We found that kdm1a knockout zebrafish exhibited developmental toxicity and abnormal neurobehaviors, including locomotor abnormalities, learning and memory deficits. The HE and Nissl staining of zebrafish larval brain tissue revealed that the density of brain cells and Nissl bodies decreased in kdm1a-/- zebrafish. Kdm1a deficiency also suppressed central nervous system (CNS) neurogenesis in Tg (HuC:egfp) zebrafish, and reduced motor neuron axon length in Tg (hb9:egfp) zebrafish. Moreover, immunofluorescent staining indicated significantly lower levels of NeuN and MAP2 (neuron markers) in kdm1a-/- zebrafish. The genes involved in early neurogenesis (nrd, ngn1 and elavl3) and neural maturation (tuba1, gfap, gap43, and syn2a) were downregulated in kdm1a-/- zebrafish. In addition, expression of genes related to autophagy and apoptosis increased significantly in kdm1a knockout zebrafish.

Conclusion(s): These results indicated that kdm1a deficiency induced locomotor abnormalities and learning and memory deficits in zebrafish larvae accompanied by activation of autophagy and apoptosis. This findings indicate the key role of kdm1a in neurodevelopment, providing novel insights into the mechanisms underlying the neurodevelopmental disorders.

Developmental toxicity in kdm1a deficiency zebrafish larvae
(A) Hatching rate. (B) Survival rate. (C) Malformation rate at 96 hpf. (D) Heart rate. (E) Typically morphological alterations. PE: pericardial edema, CB: curved body, YSE: yolk sac edema, LP: low pigment. Data were presented as mean ± SD of at least three independent experiments. ***P < 0.001, compared to wt group.

Neurobehavioral alteration in kdm1a deficiency zebrafish larvae
(A) The records of motion trail, (B) total distance, (C) time in periphery in the open field test at 120 hpf (15 min). (D) The records of motion trail, (E) relative distance and (F) time in mirror area in the mirror attack test at 120 hpf (15 min). (G) The records of motion trail, (H) distance and (I) time in new arm in Y-maze test at 8 dpf (5 min). Data were presented as mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001, compared to wt group.

Kdm1a deficiency inhibited neurodevelopment in zebrafish larvae
(A) HE and (B) Nissl staining of zebrafish brain in zebrafish larvae. Scale bar: 100 μm (left) and 50 μm (right). (C) Representative green fluorescence of CNS neurogenesis in whole zebrafish (left) and corresponding cerebral regions (right: magnification of white box). Scale bars: 100 μm. (D) Representative green fluorescence of motor neuron in whole zebrafish (left) and corresponding spinal regions (right: magnification of white box). Scale bars: 100 μm. (E) Representative immune-stained images of MAP2 and NeuN in the brain. Scale bar: 200 μm. (F) Relative mRNA level of early neurogenesis and neural maturation related genes. Data were presented as mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001, compared to wt group.