Studies On Nogo-A Expression In Cultured Hippocampal Neuron

One of the major reasons of the failure of CNS axon regeneration is the presence of many inhibitory molecules, mostly originated from CNS myelin. Nogo protein expressed by CNS myelin is regarded as a major repulsive factor for the failure of CNS axon regeneration. The interest in this protein stems principally from the neurite growth promoting effect of a monoclonal antibody (IN-1) rasied against NI-250 which has been proved later to be a part of Nogo-A. In vivo, this function-blocking antibody has been shown to partially neutralize the growth inhibitory effect of CNS myelin, and induce long-distance fiber regeneration in spinal cord injuries of the adult mammalian CNS with a recovery of specific reflex and locomotor functions. The cloning of nogo gene in 2000 provides a fundamental tool to comprehensively study the structure and function of Nogo protein. Three major transcipts (Nogo-A, -B, -C)originate from the nogo gene by alternative splicing. The three isoforms have a common carboxy-terminal domain of 188 amino acids, and this region is highly homologous to the reticulon protein family. The carboxyl portion of the Nogo protein contains two transmembrane domains, which are seperated by an extracellular 66-amino-acid fragment (Nogo-66). Nogo-66 receptor, NgR, cloned in 2001, is a leucine-rich-repeat glycophosphatidylinositol-anchored membrane protein which mediates Nogo-66 inhibition of axonal outgrowth. Both the long acidic amino-terminal domain and the Nogo-66 fragment have strong neurite growth inhibitory activity suggest that Nogo-A has at least two inhibitory domains.Northern blot, in situ hybridization, Western blot and immunocytochemistry analyses show that in addition to oligodendrocytes, Nogo-A mRNA and Nogo-A protein are also expressed in neurons in developing and adult brain and spinal cord, Nogo-A is also found in peripheral organs such as heart and testis. Nogo-B is widely distributed in CNS, PNS and many peripheral tissues. Nogo-C is expressed in brain, heart, and skeletal muscle. The wide expression of Nogo-A, -B and -C suggests that the Nogo family proteins may have function(s) in addition to neurite growth inhibition.To investigate the potential function of Nogo-A involved in neuronal development and differentiation, this study mainly used hippocampal neuron culture model, both in conventional and low-density condition, and immunohistochernical and Western blot techniques. Nogo-A expression pattern in hippocampal neurons at different stages and its subcellular distribution were explored by using specific antibody raised against Nogo-A. PC12, 3T3 cells were also cultured as controls. Nogo-A was found in hippocampal neurons atdifferent stages and is located in cytoplasm, membrane and neurites. Nogo-A was detected at proximal part of all neurites before axon formation. Then, Nogo-A expression was gradually enriched in the distal segment and growth cone of the putative axon which grew much faster than other neurites. In mature neuronal network, Nogo-A immunoreactive axons showed a string of beads which correspond to the varicosities or growth cones. The results indicate that Nogo-A expressed in neuron may have function(s) in addition to the neurite growth inhibitory activity, and Nogo-A may play a role in the course of neuronal differentiation such as neurite growth and synapse formation.