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Feng He, Melina A Agosto, Ralph Nichols, Theodore G Wensel; Rod bipolar cell degeneration in Pik3c3/Vps34 conditional knockout mice. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1750.
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© ARVO (1962-2015); The Authors (2016-present)
The type III phosphoinositide 3-kinase (Pik3c3/Vps34) participates in various cellular functions, including intracellular trafficking and cell survival. Our previous studies showed aggressive rod degeneration in rod-specific Vps34 conditional knockout mice due to impairment of autophagy and endosomal pathways. In this study, we investigated Vps34 function in bipolar cells, which are the downstream neurons of the visual phototransduction pathway, using bipolar-specific Vps34 conditional knockout mice.
Electroporation of plasmid DNA directing expression of DsRed fused to a phosphoinositide binding domain (DsRed-2xHrs) was used to localize PI(3)P in bipolar cells. A mouse line with a conditional functional deletion of Vps34 in bipolar cells was generated by crossing Vps34 floxed mice with a transgenic mouse line that expresses Cre recombinase in bipolar cells using the Purkinje cell protein-2 (PCP2) promoter. Structural changes in the retina were determined by immunofluorecence and electron microscopy.
PI(3P) was localized to discrete puncta of various sizes in bipolar cells. Loss of Vps34 function in bipolar cells caused significant degeneration of the inner retina. The number of rod ON-bipolar cells, determined by immunostaining with PCP2 and PKCa antibodies, was significantly reduced in Vps34 knockout mice at 3 months, while there were no significant changes in cone ON- or OFF-bipolar cells, horizontal cells, or amacrine cells. The thickness of the inner nuclear and inner plexiform layers was remarkably reduced. No significant degeneration was observed in the photoreceptor cell or ganglion cell layers. Transmission electron microscopy showed an accumulation of vesicles in bipolar cell bodies and a lack of invaginating rod bipolar dendrites in synaptic triads of the outer plexiform layer. Autophagy markers LC3 and p62, as well as ubiquitinated proteins, accumulated and co-localized in rod ON-bipolar cells in Vps34 KO mice. LAMP-1 also accumulated, but did not co-localize with the p62/LC3 puncta, indicating abnormal autophagy in bipolar cells in the absence of Vps34.
Vps34 is essential for rod ON bipolar cell survival. The mechanisms of degeneration in the absence of Vps34 may involve disruption of autophagy and/or related pathways.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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