AAV-Mediated Overexpression of Neuroserpin in the Hippocampus Decreases PSD-95 Expression but Does Not Affect Hippocampal-Dependent Learning and Memory

Vicky W K Tsang,Nigel P Birch,Deborah Young,Matthew J During

doi:10.1371/journal.pone.0091050

Abstract

Neuroserpin is a serine protease inhibitor, or serpin, that is expressed in the nervous system and inhibits the protease tissue plasminogen activator (tPA). Neuroserpin has been suggested to play a role in learning and memory but direct evidence for such a role is lacking. Here we have used an adeno-associated virus (AAV) vector expression system to investigate the effect of neuroserpin on hippocampal-dependent learning and memory in the young adult rat. A FLAG-tagged neuroserpin construct was initially characterized by in vitro transcription/translation and transfection into HEK293 cells and shown to interact with tPA and be targeted to the secretory pathway. Targeted injection of a chimeric AAV1/2 vector expressing FLAG-neuroserpin resulted in localized overexpression in the dorsal hippocampus. Neuroserpin overexpression led to the appearance of an unstable neuroserpin:tPA complex in zymographic assays consistent with interaction with endogenous tPA in vivo. Rats overexpressing neuroserpin also showed a significant decrease in the levels of postsynaptic density protein 95, a major postsynaptic scaffolding protein. Three weeks after injection, a range of behavioural tests was performed to measure spatial and associative learning and memory, as well as innate and acquired fear. These tests provided no evidence of a role for neuroserpin in hippocampal-dependent learning and memory. In summary this study does not support a role for neuroserpin in hippocampal-dependent learning and memory in young adult rats but does suggest an involvement of neuroserpin in hippocampal synaptic plasticity.

Highlights

Neuroserpin was discovered as an axonally-secreted protein in chicken dorsal root ganglion cells and subsequently shown to be a member of the serpin superfamily of serine protease inhibitors [1]
Neuroserpin expression is increased in the developing visual cortex during the critical period of experiencedependent refinement of neuronal synaptic connectivity and decreased following monocular deprivation [12]. tissue plasminogen activator (tPA) activity is increased under the same conditions [13] raising the possibility that the balance between neuroserpin and tPA levels plays a role in the remodelling of these synaptic connections
The coding sequence was subcloned into an associated virus (AAV) expression plasmid under control of a chicken-beta actin/CMV hybrid (CBA) promoter and containing a woodchuck post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation sequence flanked by AAV2 inverted terminal repeats to generate pAAVCBA-FLAG-NS-WPRE-bGHpA

Summary

Introduction

Neuroserpin was discovered as an axonally-secreted protein in chicken dorsal root ganglion cells and subsequently shown to be a member of the serpin superfamily of serine protease inhibitors [1]. Neuroserpin transcripts and protein are largely restricted to the nervous system, and expressed in neurons both during development and in the adult [1,2,3]. The expression of neuroserpin in these regions which are known to display a high degree of plasticity has led to speculation that neuroserpin may play a role in the modulation of synaptic efficacy or structural changes associated with learning and memory [3]. Such changes could be through inhibition of its target protease tissue plasminogen activator (tPA) [2,4]. Neuroserpin expression is increased in the developing visual cortex during the critical period of experiencedependent refinement of neuronal synaptic connectivity and decreased following monocular deprivation [12]. tPA activity is increased under the same conditions [13] raising the possibility that the balance between neuroserpin and tPA levels plays a role in the remodelling of these synaptic connections

Methods

Results

Conclusion