On Nogo signaling regulation

Abstract

As neuronal development enters its final stages, axon growth becomes restricted. This lack of regenerative capacity is partly due to the non-permissive environment of growth inhibitory proteins. Three such proteins, Nogo, OMgp and MAG bind to the same receptor, the Nogo receptor (NgR) and induce growth cone collapse and axon growth inhibition. Since NgR is GPI-linked to the cell membrane and lacks a cytoplasmic domain, additional transmembrane molecules are needed for intracellular signaling. The lowaffinity NGF receptor p75, Lingo-1 and TROY serve as coreceptors involved in initiating the intracellular signaling cascade. Together, therefore, NgR, p75, Lingo-1, and TROY have important roles controlling regeneration, growth inhibition and neuronal plasticity. We have found that NgR and Lingo-1 are widely expressed in the brain and that these two gene transcripts are efficiently regulated in the CNS by neuronal activity We hypothesize that the NgR/p75/Lingo-1/TROY system is involved in synaptic plasticity and memory functions.

Aims: To examine expression of components of the Nogo receptor complex during development, in the adult and in the aging brain. To analyze the expression of receptor components. To investigate the role of the NgR in mechanisms of learning, memory and hippocampal neurogenesis in transgenic animals which overexpress NgR in forebrain neurons. To test mechanisms underlying regulation of Nogo receptor components and the possible connection between brain function (e.g. memory formation) and Nogo signaling.

Methods: Histological analysis of different animal models with regard to the expression of transcripts encoding Nogo receptor components using in situ hybridization. Quantitative mRNA analysis using RTPCR. Kainic acid administration and intrahippocampal BDNF injections in adult rats. Generation and characterization of novel transgenic mouse lines that overexpress NgR via a tetracycline inducible CarnKII promoter.

Paper I details the expression of NgR mRNA throughout development and in adult mouse and human tissues. In the adult, NgR is expressed in neurons in specific brain region associated with a high degree of plasticity. Paper II investigates how plasticity is regulated in regions with high NgR expression. We challenged rats with kainic acid and found the NgR was rapidly down-regulated, suggesting activity-dependent regulation. This was confirmed by showing that rats transiently down-regulate NgR during a period of establishing a running behavior, suggesting a role for NgR regulation in learning.

Paper III addresses the NgR coreceptor Lingo-1. We found that Lingo-1 mRNA levels are rapidly and strongly increased in hippocampus by treatments thought to increase neuronal activity. We also describe regulation of NgR: Since BDNF upregulation coincides with the downregulation of NgR we tested a possible causal relationship by intracranial injections of BDNF or BSA. NgR was downregulated by both injections, although BDNF caused a bigger effect than BSA in ipsilateral cortex. Lingo-1 was specifically upregulated by BDNF It is hypothesized that activity-driven structural synaptic plasticity is facilitated by appropriate changes of the levels of Nogo receptor components and trophic factors and that the Nogo system is also involved in stabilizing neuronal networks.

Paper IV addresses the role of the NgR complex in a transgenic mouse model (mceph/mceph) characterized by epilepsy and a markedly enlarged brain. Results suggest that NgR down-regulation coincided with an upregulation of growth promoting proteins. We also found that carbamazepine (CBZ), a commonly used antiepileptic drug, counteracted brain overgrowth in mceph/mceph mice and reduced the number and size of neurons. CBZ normalized brain levels of mRNA encoding BDNF and several components of the Nogo signalling system, which were dramatically upregulated in untreated mceph/mceph brains.

Paper V uses in situ hybridization to map Lingo-1 gene activity patterns in a dult human nervous tissues, as well as inthe developing and adult rat CNS. Lingo-1 mRNA expression was observed in most, but not all neurons of the brain, spinal cord and dorsal root ganglia in developing and adult rats as well as in human adult CNS. We found a good correlation between Lingo-1 mRNA expression and NgR mRNA expression.

Paper VI focuses on the Nogo system in the aging rat brain. We examined the levels of mRNA encoding Nogo, OMgp, MAG, as well as the receptor components NgR, Lingo-1 and Troy in cortex and hippocampus. There were no significant changes of receptor components or the ligands OMgp or MAG. However, Nogo mRNA was significantly, albeit modestly, decreased in hippocampal subregions of aged animals. The specific decrease of Nogo mRNA levels in hippocampus during aging may relate to age-dependent decline of brain plasticity.

Paper VII is an attempt to determine the significance of activity-dependent NgR downregulation by generating transgenic mice with strong inducible NgR overexpression. The goal was to overexpress NgR in forebrain neurons and test if this may decrease plastic changes in vivo. A tetracycline inducible system was employed. The hypothesis was that NgR over-expressing mice should have impaired learning abilities due to reduced synaptic plasticity To date, we have generated 3 transgenic mouse lines (CarnKlIpTRE/NgR) with inducible (tet-off) and specific overexpression of NgR in forebrain neurons as analyzed by in situ hybridization and western blot. Levels of Nogo, Lingo-1, Troy, p75 and BDNF mRNA are not altered and the endogenous NgR mRNA levels did not change to compensate for the overexpression. CarnKIIpTRE/NgR have significantly fewer BrdU labeled cells in the dentate gyrus compared to controls. Behavior of NgR overexpressing mice is currently being investigated using open-field, passive avoidance and swim maze tests.

Conclusions: The results contribute to the understanding of the Nogo signaling system in health and disease, focusing on the Nogo receptor components. The dramatic activity-related changes of neuronal levels of NgR and Lingo-1 transcripts suggest fundamental roles in synaptic plasticity. Our transgenic mouse model may become a helpful tool in determining the role of one of the receptor components, NgR, for such plasticity during development and in adulthood. Increased knowledge about components of the Nogo receptor complex may also help the design of more effective means to improve regeneration in CNS.