Fig. 7

Hyperhomocysteinemic zebrafish develops brain damage associated with reduced autophagy, constitutive ER stress and ubiquitinated protein load. A Immunoblot of zebrafish embryos injected with Cntl MO or CBS MO at 24 hpf. confirminging downregulation of CBS. β-actin was used as a loading control. B Concentration of Hcy (mM) at 24 hpf in Cntl MO and CBS MO zebrafish embryos as measured by HPLC. CBS morphants showing HHcy. C Representative confocal images (10X) showing brain defects in a brain specific Kaede (green) expressing transgenic fish Tg(HuC:Kaede) embryos injected with Control MO or CBS MO. Arrows showing neuronal damage in CBS morphants. Scale bar 200 µm. D Bar diagram represents percentage of embryos with brain defect in the CBS MO compared to Cntl MO. More than 200 embryos were counted phenotypically. E Representative images (10X) of TUNEL staining in the brain of Cntl MO and CBS MO embryos. Left panel showing fluorescent images only and right panel showing bright field and fluorescent image overlap. Arrowheads indicate excess number of apoptotic cells in the head region of CBS morphants. Scale bar 25 µm. The area highlighted by the dotted lines indicating the brain region of the embryos. Representative western blots showing markers for intrinsic suppression of autophagy (F) and increase in ER stress markers (G) as well as polyubiquitinated protein load (H) in CBS morphants compared to controls. β-actin was used as a loading control. Densitometric values in the blots represent the ratio of respective protein signal to β-actin signal normalized to control. I Representative confocal images showing rescue of apoptotic cell death in the brain region of CBS morphant upon autophagic activation by overexpressing Beclin1. Left panel showing fluorescent images only and right panel showing bright field and fluorescent image overlap. Scale bar 20 µm. Arrowheads indicate attenuation of excess apoptosis in Beclin1 mRNA and CBS MO co-injected embryos as compared to only CBS MO injected ones. The area highlighted by the dotted lines indicating the brain region of the embryos. J Schematic showing pathways of Hcy induced neurotoxicity (1). Rescue of neurotoxicity either by autophagic activation upstream (2) or by blocking ER stress downstream (3). Data are shown as mean ± SEM with n ≥ 3. **P < 0.01. ****P < 0.0001