Evaluation of intra-cortical administration of AAV expressing an shRNA to PTEN alone and in combination with salmon fibrin as a therapy for spinal cord injury

Program#/Poster#: 270.14/FF6
Location: Hall F-J Neuroscience 2012, SfN’s 42nd annual meeting, is scheduled for Oct. 13 -17 in New Orleans at the Ernest N. Morial Convention Center.
Presentation time: Sunday, Oct 14, 2012, 2:00 PM – 3:00 PM
Authors: *G. LEWANDOWSKI1, A. F. CHAN1, L. A. FLANAGAN-MONUKI2,3, O. STEWARD1;
1Reeve-Irvine Res. Ctr., 2Neurol., 3Sue and Bill Gross Stem Cell Res. Ctr., Univ. California, Irvine, Irvine, CA
Abstract: Recent studies have shown that conditional genetic deletion of the tumor suppressor gene PTEN in cortical motoneurons enables them to regenerate their axons after spinal cord injury (Liu et al., 2010, Nat. Neurosci. 13, 1075). Here, our goal is to develop an intervention that would allow PTEN to be targeted at the time of, or after a spinal cord injury. Towards this end we developed, in conjunction with the University of Pennsylvania Vector Core, an adeno-associated virus (AAV) vector that expresses an shRNA specific for PTEN (AAV2/9-shPTEN). In the first experiment, adult Sprague Dawley rats received injections of either AAV2/9-shPTEN or AAV2/9-shLuc into 5 sites of the sensorimotor cortex (109 genome copies/site). At 4 weeks post-injection the rats were subjected to either 45 min. of activity in an enriched environment or kept quite in their home cage. All rats were then humanely killed by cardiac perfusion. The brains were immunostained for PTEN and phosphorylated ribosomal protein S6 (pS6). The sphere of PTEN suppression in AAV2/9-shPTEN injected brains was ~2 mm. In individual AAV2/9-shPTEN transduced neurons there was almost 100% inhibition of PTEN expression. Neurons with silenced PTEN showed increased immunostaining for pS6, indicating persistent activation of mTOR-dependent protein synthesis. There were no changes in immunostaining for PTEN or pS6 in AAV2/9-shLuc injected brains. Additionally, in ongoing analysis we are assessing the neural response and synaptic changes in neurons with suppressed PTEN by evaluating Arc, PSD95, neurofilament and GAP43 proteins by immunohistochemistry. These data will provide information regarding any abnormal neural responses in PTEN-silenced neurons as well as changes in the cortical dendritic field corresponding to the area of PTEN suppression.
In a second experiment, rats were trained in a skilled forepaw reaching task (Montoya Staircase) and then AAV2/9-shPTEN or AAV2/9-shLuc was administered as above. One week post-administration, the rats received a C6 dorsal hemisection spinal cord injury. Because it has recently been shown that salmon fibrin treatment promotes functional recovery (Sharp et al., 2012, Exp. Neurol. 235, 345) half of the rats in each group received salmon fibrin in the lesion site, at the time of injury. Forepaw motor function is being measured using the Montoya staircase task for 10 weeks post-injury. Axon regeneration will then be evaluated using anterograde tracer, BDA. From These data we will determine whether suppression of PTEN in the cortex of adult rats alone, or in combination with salmon fibrin, promotes axon regeneration and improved motor function after spinal cord injury.

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