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S. M. Hauck, P. Ekstrom, F. Paquet-Durand, P. Ahuja-Jensen, S. Suppmann, I. Kameshita, T. van Veen, M. Ueffing; Increased Phosphorylation of Phosducin in Degenerating rd1 Retina Is Linked to Constitutively Active CaMKII in Rod Outer Segments. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3750.
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© ARVO (1962-2015); The Authors (2016-present)
The rd1 mouse is an RP model that carries a mutation in a rod photoreceptor specific phosphodiesterase gene, leading to rapid degeneration of these cells. Elucidation of the molecular differences between rd1 and healthy retina is crucial for explaining this degeneration and could assist in suggesting therapies.
We used high-resolution proteomics to compare the proteomes of the rd1 mouse retina and its congenic, wild-type, counterpart at postnatal day 11 (PN11), when photoreceptor death is profound. Proteins were resolved by 2-dimensional gel electrophoresis. Significantly differentially expressed proteins were excised from gels and identified by mass spectrometry (MALDI-TOF). Results were confirmed by immunostaining on retinal sections. An in vitro assay for calcium-calmodulin activated protein kinase II (CaMKII) was established to screen for rod outer segment specific proteins targeted by active CaMKII.
Retinal morphology was unaltered in rd1 retina at PN11 but photoreceptor apoptosis is abundant. Stringent comparative proteomics analyses led to the identification of five candidate proteins differentially expressed in the rd1 model. The difference in one such protein, phosducin, related to an altered modification pattern in the rd1 retina rather than to changed expression levels. Additional experiments showed phosducin in healthy retinae to be highly phosphorylated in the dark but not in the light-adapted phase. In contrast, rd1 phosducin was highly phosphorylated irrespective of light status, indicating a dysfunctional rd1 light/dark response. The increased rd1 phosducin phosphorylation coincided with increased activation of CaMKII, known to utilize phosducin as a substrate. In vitro CaMKII activity led to phosphorylation of several proteins in photoreceptor outer segment preparations (ROS). By mass spectrometry, we could confirm phosducin as a target for active CaMKII in ROS.
Given the increased rod calcium levels present in the rd1 mutation, calcium-evoked over-activation of CaMKII may be an early and long sought for step in events leading to photoreceptor degeneration in the rd1 mouse. Identification of additional targets of active CaMKII in ROS in rd1 genotype may resolve the pathway to degeneration in this model.
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