Abstract
Purpose:
Rho GTPases function as intracellular switches integrating signals from the extracellular environment and play critical roles in morphological neuroplasticity. We have shown that blocking RhoA or Rho kinase (ROCK) activity can prevent rod photoreceptor axon terminal retraction after retinal detachment, indicating treatments that manipulate RhoA signaling may promote synaptic stability (Fontainhas and Townes-Anderson, IOVS, 2011). In the present work, we focus on the time course of changes in the activities of the Rho GTPases after detachment.
Methods:
Neural retinas were detached from porcine eyecups, cultured in serum-free medium for up to 24 hrs, and then frozen and lysed for activity assays and western blot analysis. RhoA activation was determined with a Rhotekin binding assay, while Rac1 and Cdc42 activations were determined with a p21 activated kinase 1 (PAK) binding assay. Determinations were repeated in more than one animal.
Results:
Activation of Rho GTPases was compared to control, attached retina from the same eye. RhoA was activated quickly, within 1 min after detachment, rose several fold by 2 hrs, in some cases, and remained activated after 24 hours in vitro albeit at a lower level. Both Rac1 and Cdc42 activities were transiently decreased 1 min after detachment; then Rac1 activity gradually recovered to a level higher than that of attached retina, while Cdc42 activity was up-regulated by 2 hrs and then decreased to a level lower than control. Phosphorylated cofilin, regulated by LIM kinase (LIMK), a down stream effector of both RhoA and Rac, was steadily and significantly increased over 24 hrs. Other protein levels also changed: 1) phosphorylated CREB increased immediately but decreased to normal levels 2 hrs after detachment; whereas 2) the level of PKCα was down-regulated; and 3) GFAP was up-regulated at 24 hrs. These changes at 24 hrs could be prevented by the ROCK inhibitor Y27632.
Conclusions:
Rac1, Cdc42 and the downstream LIMK activities are all changed 24 hrs after detachment, a time co-incident with active rod cell axon retraction. Thus, in addition to RhoA, other elements of the Rho pathways appear to play a role in the plasticity of the photoreceptor synapse. Understanding the time course of Rho GTPase activities will help in devising effective therapies to preserve the morphology of the outer plexiform layer and normal protein expression after retinal detachment.
Keywords: 697 retinal detachment •
714 signal transduction •
648 photoreceptors