Non-inhibitory effects of neuroserpin on neuronal development

Abstract

Neuroserpin is a serine protease inhibitor (serpin) that is expressed in the developing and adult nervous systems. Neuroserpin knockout mice show increased phobic responses to novel situations. However, relatively few details of neuroserpin function at the cellular and molecular levels are known. While neuroserpin inhibits the protease tissue plasminogen activator (tPA) in vitro, it is not clear whether the main physiological role of neuroserpin is to regulate tPA activity levels. The overall goal of this thesis was to investigate the function of neuroserpin at the cellular level using two different model systems and to determine whether neuroserpin’s inhibitory activity was required for its cellular effects. Experiments with PC12 cells, which are commonly used as a neuronal model, revealed that neuroserpin regulated cell-cell adhesion and neurite outgrowth through a mechanism that was not dependent on its inhibitory activity. In cultures of dissociated embryonic hippocampal neurons, neuroserpin was found to be located in both the axons and dendrites, including the growth cones and tips of these processes, and co-localised with synapsin I-positive presynaptic puncta. Detailed investigations of the effects of exogenous neuroserpin on both short-term and long-term neuronal cultures were carried out using an automated image analysis methodology developed as part of this thesis. These experiments showed that neuroserpin regulated the development of axons, dendrites and synapses in these neurons. Neuroserpin treatment resulted in a reduction in the size of lamellipodia-like areas in the axon growth cones, developing dendrites and cell body. Neuroserpin also accelerated axon formation and had complex effects on dendrite branching, promoting branching at early stages but inhibiting branching at later stages. In long-term neurons, neuroserpin treatment increased the size of both presynaptic and postsynaptic puncta without affecting their density. Similar effects were found to occur with non-inhibitory forms of neuroserpin. Additional investigations suggested that some, but not all, of these effects could be modulated by an antagonist to the LDL-related protein 1 (LRP1) receptor, which is known to bind to neuroserpin. Together, these results highlight a novel cellular function of neuroserpin as an extrinsic factor that exhibits neurorotrophic-like effects independently of its inhibitory activity.