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"The ROCK-LIMK-cofilin pathway regulates the structural plasticity of rod photoreceptor synaptic terminals"

by
Weiwei Wang
Pharmacology and Physiology Program
M.A. 2005, Xiamen University, China
B.S. 2002, Xiamen University, China


Thesis Advisor: Ellen Townes-Anderson, Ph.D.
Professor
Department of Pharmacology, Physiology and Neuroscience

Monday, August 24, 2015
2:00 P.M., MSB Room H609


Abstract

In response to injury, rod photoreceptors display structural synaptic plasticity, including axon retraction and neuritic sprouting. Previous studies demonstrated that the mechanism of retraction involves activation of RhoA and its downstream effector Rho kinase (ROCK). Here, we pursued further downstream events, the role of the ROCK-LIMK-cofilin pathway (Part-I) and the contribution of actin filament turnover (Part-II), in rod synaptic terminal plasticity. A better understanding of the basic mechanisms involved in the photoreceptor injury response may help in the development of therapeutic strategies for retinal injury.

Part-I. Confocal microscopy demonstrated the presence of phosphorylated-LIMK (p-LIMK), the active form of LIMK, in axon terminals and varicosity-bearing processes of isolated salamander rod cells. Inhibition of LIMK significantly decreased retraction. Furthermore, inhibition of ROCK and p21-activated kinase (Pak), the upstream regulators of LIMK, also reduced retraction; together they had an additive effect. Process growth (sprouting) in 3 day-old cultures was also reduced by inhibition of LIMK or Pak, especially at the basal (axon-bearing) region of the rod cells. Combining Ca2+ channel and LIMK inhibition had no additional effect on retraction, but did further inhibit sprouting. In porcine retinal explants, which are similar to human retina, incubated for 24 hours after detachment, LIMK inhibition reduced rod axon retraction and improved retinal morphology. Thus, our results demonstrate that both retraction and growth require LIMK activity. The relationship between LIMK and Ca2+ is likely to be complex. Nonetheless, LIMK inhibition may have therapeutic potential for reducing pathological rod synaptic plasticity after retinal injury.

Part-II. Cofilin, which is regulated by LIMK and promotes actin filament turnover through severing of the filaments, was examined in detached porcine retina by western blot and confocal microscopy. P-cofilin, present in the synaptic terminal of rod photoreceptors, increased over 24 hours whereas total cofilin remained relatively stable. Turnover of the actin cytoskeleton was assessed with a barbed end assay and phalloidin staining in isolated salamander rod cells. The barbed end assay revealed that severing of actin filaments was highest about 1 hr after retinal detachment and decreased over the next 3 hrs. Quantification of phalloidin florescence demonstrated a 30% decrease of actin filaments in the axonal region of rod cells in culture. The increase of p-cofilin and barbed end labeling and the decrease of actin filaments in the axonal region, were all reduced by LIMK inhibition. Cytochalasin D (CytoD), which prevents turnover of actin filaments, significantly reduced axon retraction by rod photoreceptor as well by more than 60%.

To summarize, our data show that the ROCK-LIMK-cofilin pathway is involved in both axon retraction and neuritic sprouting of rod photoreceptors after retinal detachment and works in part by up-regulating actin depolymerization. Inhibition of this pathway can prevent axon retraction and stabilize synaptic structure presumably by stabilizing the actin filament cytoskeleton. Treatment to reduce activity in this pathway may help preserve vision after retinal injury.


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