Abstract

Injuries to the spinal cord and brain often lead to irreversible damage. Central Nervous System (CNS) neurons have difficulty regenerating axons, due to both environmental and intrinsic factors. To better understand the intrinsic factors limiting the regenerative ability of CNS neurons, we conducted three high content screens (HCS) in primary neurons. We over-expressed genes in the neurons and then analyzed the changes in neurite growth. Using automated image acquisition and analysis, we tested 20-90 genes per day, with multiple morphological parameters (e.g. neurite number, length, branching, etc.) measured from 100,000 to 300,000 neurons in a single experiment. For the first screen we compared patterns of gene expression of CNS neurons with Peripheral Nervous System (PNS) neurons, which have a greater intrinsic capacity for regeneration. This comparison yielded 1300 candidate genes for functional testing. For the second screen we compared gene expression in mature corticospinal tract neurons to their embryonic counterparts, which also display a high intrinsic capacity for regeneration, and generated a list of about 800 genes. The third screen examined over 600 kinases, phosphatases and their related enzymes and adaptors. We identified a number of genes known to regulate axon growth, including ERK2 , GSK3b, EphA7, FGFR1 , PI3K, PKC and CAM K1a. This validates the effectiveness of our screens. We have also identified a number of novel genes, not previously associated with neurite growth. We used bioinformatics approaches to cluster genes with different aspects of neurite growth, and found that that there is a poor correlation between genes associated with neurite extension and those involved in formation of neurites emerging from the cell body. By testing the genes on permissive substrates such as laminin, and inhibitory substrates such as chondroitin sulfate proteoglycans (CSPG), we identified specific cDNAs that increase growth in primary neuron cultures only on the CSP Gs, and others that decrease growth only on laminin. Our data show that phenotypic screening provides a robust approach for identifying novel genes and signaling pathways that function in axon growth, and will lead to a greater understanding of the mechanisms needed for achieving neuron regeneration after injury in vivo.