Neuronal functions of the ubiquitin-proteasome pathway

The morphological basis for signalling in the nervous system is formed by an extensive network of cellular processes required for interconnecting nerve cells. We study basic mechanisms underlying the development of this network in rat pheochromocytoma (PC12) cells and sympathetic neurons. Both cell types are widely used for the investigation of two fundamental aspects of neuronal differentiation: neurotransmission and neurite outgrowth. The development of a neuronal phenotype is mediated by growth factors through a variety of effects on protein and mRNA expression. In order to identify proteins involved in neuronal signalling and process formation, gene expression profiles of PC12 cells differentiated by nerve growth factor (NGF) were obtained. Serial analysis of gene expression (SAGE) and differential display PCR (DD-PCR) suggested that the regulation of the ubiquitin/proteasome pathway may play an important role in the NGF response. Until now, little is known about the biological significance of this pathway in neuronal transmission, survival, differentiation or regeneration. We have identified two ubiquitin-conjugating enzymes (E2-14kD, UbcE2A) and several proteasomal subunits which are regulated by NGF on the mRNA level. Although some proteasomal subunit mRNAs are increased, the overall activity of the proteasome is reduced by growth factors and complete pharmacological inhibition of the proteasome is accompanied by neuronal differentiation of PC12 cells. One goal of this project is to extend these findings to the nervous system. We will analyze the expression and regulation of E2-14kD, UbcE2A and of selected proteasomal subunits in cultured neurons and during brain development. Overexpression or blockade of the endogenous protein by antisense or dominant negative strategies will be used to examine the functional significance of the selected candidate genes in vitro. The modification of genes involved in ubiquitination and proteolytic cleavage of specific proteins could promote or inhibit differentiation into the neuronal phenotype and provide new insight into the molecular basis of neurite formation and axonal regeneration. This may lead to new therapeutic targets for the treatment of traumatic or degenerative diseases in the nervous system.

Pheochromocytoma (PC12) cells represent a suitable model for the investigation of the molecular mechanisms underlying neurite outgrowth and neuronal differentiation. The mRNA profile of PC12 cells treated with different growth factors was investigated by differential display-PCR (DD-PCR) und serial analysis of gene expression (SAGE). We identified a number of genes regulating various aspects of neuronal survival and axonal growth. Some of them encode proteins involved in ubiquitin-proteasome mediated proteolysis. Ubiquitin represents a 76 residue peptide that is attached as a marker to normal and abnormal (e.g., mutated) proteins for degradation in the proteasome, a large protein complex required for energy-dependent cleavage of peptides that need to be inactivated. The ubiquitin-conjugating enzyme, HR6 (previously named E2-14kD), and the catalytic proteasomal subunit ß5 were found to be up-regulated by NGF treatment of PC12 cells. The aim of the project was to verify differential expression in this neuronal cell line using alternative techniques and to elucidate possible functional aspects of this regulation. The expression pattern of HR6 was investigated in the nervous system and in various other organs using immunofluorescence techniques. Moreover, the regulation of HR6 by neurotrophic factors was carefully analyzed. The requirement of HR6 expression for neurite outgrowth was confirmed in vitro applying antisense and siRNA techniques. All catalytic subunits of the proteasome (the molecular scissors`) were investigated with regard to their regulation and functional relevance in PC12 cells and primary neurons using pharmacological inhibitors. It became clear that the proteasome is absolutely required for axonal initiation, elongation and maintenance. Taken together, this project significantly contributes to the deeper understanding of the functional significance of ubiquitination and proteasomal protein degradation in neurons. This may lead to the development of novel therapeutic strategies targeting degenerative and traumatic diseases of the nervous system.