Overexpression of LPP enhances degeneration in rdgA: To test the ability of LPP to dephosphorylate PA generated during PI(4,5)P2 hydrolysis, the rdgA mutant was used. In rdgA3, a hypomorphic allele, photoreceptors look ultrastructurally normal on eclosion and degenerate with time when grown in laboratory incubator conditions. Transgenic flies were generated and whether overexpression of one of the Drosophila LPPs, CG11426 (encoding an LPP - closely linked to laza), affected the phenotype of rdgA, was tested. When CG11426 was overexpressed in rdgA3 using Rh1-GAL4, there was obvious degeneration of the rhabdomeres in newly eclosed flies. Over a period of 72 hr post-eclosion, rdgA3+ LPP photoreceptors degenerated more rapidly than rdgA3 alone. By contrast, when CG11426 was overexpressed in otherwise wild-type photoreceptors, there was no detectable effect on photoreceptor ultrastructure. Similar results were obtained with three other Drosophila LPPs, wun, wun2, and laza. These results provide strong genetic evidence that LPP can functionally antagonise the DGK activity encoded by rdgA (Garcia-Murillas, 2006).
The enhancement of rdgA3 by LPPs suggests that these enzymes can dephosphorylate PA in vivo. This was tested by measuring retinal PA levels using liquid chromatography followed by mass spectrometry, comparing rdgA3 with rdgA3 + CG11426. It was found that levels of PA in rdgA3 retinae were ~60% of wild-type. Importantly, this reduction was dramatically enhanced in rdgA3+ LPP: these retinae had ~20% of total wild-type PA levels. These results demonstrate that PA levels in rdgA3 are reduced and that when overexpressed, LPP enhances this reduction in vivo (Garcia-Murillas, 2006).
Retinal degeneration in rdgA3 can be completely blocked by norpAP24, a strong hypomorph in PLCβ essential for phototransduction, suggesting that the enhancement of rdgA by LPP is most likely mediated by PA derived from PI(4,5)P2. To confirm this idea, CG11426 was overexpressed in norpAP24, rdgA3. This analysis revealed normal rhabdomeres in norpAP24, rdgA3 + LPP and shows that the enhancement of rdgA3 by LPP requires ongoing light-induced PI(4,5)P2 hydrolysis. These results demonstrate that LPP can antagonize DGK function, most likely by dephosphorylating PA generated by PI(4,5)P2 hydrolysis (Garcia-Murillas, 2006).
Changes in retinal PA in rdgA are associated with depletion of PI: While analysis of laza genetic interaction with rdgA demonstrate the concerted action of rdgA and laza in regulating phosphatidic acid levels during phototransduction, they raise the question of how PA levels are linked to abnormal Trp channel activity seen in rdgA. One possibility is that PA directly regulates Trp channels, although it has previously been reported that supplementation of PA during whole-cell recording failed to suppress constitutive Trp activity in rdgA. To test the possibility that there might be changes in other phospholipid classes that could account for the rdgA phenotype, a lipidomic analysis was performed studying three other classes of phospholipids, including PI, phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Since the reduction in absolute levels of phospholipids presumably partly reflects the ongoing degeneration process and associated loss of membranes, the levels of each phospholipid was expressed as a fraction of the level of PC + PE that are major building blocks of membranes. This analysis revealed a significant reduction in the levels of PI in rdgA3 that was further enhanced on overexpression of LPP. These findings demonstrate that the reductions in PA levels during phototransduction are associated with a reduction in at least one other phospholipid class, namely PI, and raise the possibility that reduction in photoreceptor PI levels might contribute to the rdgA phenotype (Garcia-Murillas, 2006).
Mutants in cds enhance degeneration of rdgA3: To test the role of PI depletion in the rdgA phenotype, mutants in CDP-DAG synthase (cds1), the enzyme that converts PA to CDP-DAG, were used. cds1 has been shown to affect the rate of PI(4,5)P2 resynthesis during the light response (Hardie, 2001). In addition, it has been found that cds1 mutants show reductions in the abundance of the two molecular species that constitute the major fraction of PI in wild-type photoreceptors. rdgA3; cds1 double mutants were generated and the rate of degeneration was compared to rdgA3 and cds1. It was found that cds1 enhances the rate of degeneration in rdgA3. To understand the biochemical basis of this degeneration, phospholipid levels were analyzed comparing rdgA3 retinae with those from rdgA3; cds1. This study revealed that although PA and DAG levels were not different in the two genotypes, there were significant changes in the levels of PI and PIP, suggesting that reduced levels of PI and PIP may contribute to the degeneration phenotype of rdgA3 (Garcia-Murillas, 2006).
PA regulates levels of PI synthase transcripts: An immediate metabolic fate of PA is conversion to PI by the sequential activity of CDP-DAG synthase and PI synthase. Thus, it is likely that the reduction of PI levels in rdgA3 and rdgA3 + LPP are partly explained by the reduced levels of PA available as substrate for CDP-DAG synthase. However, recently it has been shown that in yeast PA can transcriptionally regulate the levels of PI synthase (Loewen, 2004). To test whether this was also the case in Drosophila photoreceptors, RT-PCR was used to compare levels of PI synthase (CG9245- CDP-diacylglycerol-inositol 3-phosphatidyltransferase) transcript in wild-type retinae with those from rdgA3 and rdgA3 + LPP. This analysis revealed that CG9245 transcript levels in rdgA3 were reduced and were virtually obliterated in RNA from rdgA3 + LPP retinae. Thus, the levels of PI synthase transcript are directly correlated with the levels of retinal PA (Garcia-Murillas, 2006).
laza modulates the degeneration of rdgB: Since biochemical analysis strongly indicated that PA levels generated during phototransduction are linked to PI resynthesis, it was of interest to ask whether this might also impact on the PI(4,5)P2 resynthesis. To test this in vivo, the rdgB mutant, defective in the Drosophila homolog of PI transfer protein, was used. Loss-of-function mutants in rdgB show (1) light-dependent retinal degeneration and (2) a reduced rate of PI(4,5)P2 resynthesis during the light response. LPP was overexpressed in rdgB mutants and rdgBKS222 was compared with rdgBKS222 + LPP. This analysis showed that overexpression of LPP enhances degeneration in rdgBKS222. Conversely, loss-of-function mutants in laza were able to slow the rate of degeneration of rdgBKS222 (Garcia-Murillas, 2006).
sktl enhances degeneration in rdgA3: To test the role of reduced PI(4,5)P2 synthesis consequent to the reduced PA and PI levels in rdgA, the effect was analyzed of reduced type 1 PIP kinase activity on the rdgA phenotype. For this mutants were used in sktl that encode one of the two type I PIP kinases in Drosophila. Since null mutants in sktl are cell-lethal in photoreceptors, a heteroallelic combination of sktlΔ20 (a null allele) and sktlΔ1-1 (a hypomorphic insertion in the upstream region of the sktl gene) were used. sktlΔ20/sktlΔ1-1 itself does not show any degeneration on the timescale of the experiments described. rdgA3; sktlΔ20/sktlΔ1-1 photoreceptors were generated and the rate of degeneration was compared to that of the rdgA3 single mutant. This analysis revealed that sktlΔ20/sktlΔ1-1 enhances the rate of degeneration in rdgA3 (Garcia-Murillas, 2006).
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