An over-expression system for characterizing Ppt1 function in Drosophila

Abstract

Background: The infantile onset form of Neuronal Ceroid Lipofuscinoses (INCL) is the earliest and most severe form of NCL, with neurological symptoms that reflect massive neurodegeneration in the CNS and retina. INCL is due to recessively inherited mutations at the CLN1 locus. This locus encodes the evolutionarily conserved enzyme palmitoyl-protein thioesterase 1 (PPT1), indicating an essential role for protein palmitoylation in normal neuronal function.

Results: To begin to elucidate the specific role that Ppt1 plays in neuronal cells, we have developed a Ppt1 over-expression system in Drosophila. We report that over-expression of DmPpt1 in the developing Drosophila visual system leads to the loss of cells through apoptotic cell death. This DmPpt1 over-expression phenotype is suppressed by DmPpt1 genomic deficiencies. Moreover, over-expression of DmPpt1S123A, which bears a catalytic site serine 123 to alanine mutation, does not lead to the severe eye phenotype observed with over-expression of wild-type DmPpt1. Thus, cell loss in DmPpt1 flies is directly related to the dosage of wildtype DmPpt1.

Conclusions: Although INCL is due to the loss of PPT1; increased levels of DmPpt1 also lead to neurodegeneration possibly via a detrimental effect on some aspect of PPT1's normal function. This suggests that the precise levels of PPT1 activity are important for neuronal cell survival. The Drosophila DmPpt1 over-expression system provides a resource for genetic experiments that aim to identify the processes by which PPT1 regulates the palmitoylation-state of its essential protein substrates.

Figure 1

Analysis of DmPpt1 over-expression phenotypes. A-D, Scanning Electron Micrographs showing the external surface of control and DmPpt1 over-expression eyes (200×). E-H, Semi-thin sections through fixed retinal tissue showing the internal structure of control and DmPpt1 over-expression retinas. Scale bar = 2 μm. A and E, GMR:Gal4/CyO. B and F, UAS:DmPpt1^8.1/CyO. C and G, GMR:Gal4/UAS:DmPpt1^8.1. D and H, GMR:Gal4/UAS:DmPpt1^2.1. I, A light microscope image of a GMR:Gal4/UAS:DmPpt1^8.1 eye showing the black ommatidia present in the fully differentiated eye. J. Quantification of the loss of rhabdomeres in GMR:Gal4/UAS:DmPpt1^8.1 eyes in relation to the GMR:Gal4/CyO and UAS:DmPpt1^8.1/CyO controls. *p < .00001 using Ttest.

Figure 2

DmPpt1 over-expressing eyes undergo apoptotic cell death. A-C, Transmission electron micrographs. A, UAS:DmPpt1^8.1/CyO control showing normal ommatidial structure. Scale bar = 2 μm. B, An image of GMR:Gal4/UAS:DmPpt1^8.1 over-expression ommatidium showing individual degenerating photoreceptors (labeled with an asterisk). Scale bar = 1 μm. C, An image of GMR:Gal4/UAS:DmPpt1^2.1over-expression ommatidium showing all photoreceptor undergoing cell death with no rhabdomeres present. Scale bar = 1 μm. D, Scanning electron micrograph (200×) of a GMR:Gal4, UAS:DmPpt1^8.1/+; UAS:DmPpt1^2.1/+ eye. E, Scanning electron micrograph (200×) of a GMR:Gal4, UAS:DmPpt1^8.1/pGMR:p35 ; UAS:DmPpt1^2.1/+ eye.

Figure 3

Deficiency chromosome suppression of the DmPpt1 over-expression phenotype. A-C, Scanning electron micrographs (200×). A, +/+; GMR:Gal4, UAS:DmPpt1^8.1/+; UAS:DmPpt1^2.1/+ control. B, Df(1) RA2/+; GMR:Gal4, UAS:DmPpt1^8.1/+; UAS:DmPpt1^2.1/+. C, Df(1) KA12/+; GMR:Gal4, UAS:DmPpt1^8.1/+; UAS:DmPpt1^2.1/+.

Figure 4

Analysis of DmPpt1S123A catalytic mutant over-expression. A-B, Scanning electron micrographs (200×). C-D, Light microscope images of semi-thin retinal sections. Scale bar= 2 μm. A and C, GMR:Gal4/UAS:DmPpt1S123A¹. B and D, GMR:Gal4/UAS:DmPpt1S123A⁴. E. Quantification of the number of rhabdomeres per ommatidia in DmPpt1S123A over-expression eyes compared to DmPpt1^8.1 over-expression eyes. *p < .00001 by Ttest.