Here we describe the establishment and application of a microscopy-based approach to identify new proteins involved in neurite initiation and elongation in response to NGF in PC12 cells. The candidate proteins used for the experiments were preselected according to their tissue-specific expression and subcellular localization in Vero cells (Simpson et al., 2000
). On the basis of these criteria we chose from 1057 available proteins 105 that we finally tested in our experiments for a possible involvement in neurite outgrowth. This revealed 21 effector proteins with a potential role as regulators of neurite initiation or elongation. One reason for this high success rate is likely to be the preselection of the candidate proteins based on their expression in nervous tissue and subcellular localization.
We also performed cotransfection experiments, which allowed us to determine the action of new, previously uncharacterized human effector proteins with respect to Gsk3β and Cdc42-Borg2 function. These cotransfection experiments further support our screening data and provide a first glimpse on how these newly identified proteins might interact with established regulators of neurite outgrowth, such as Gsk3β and Cdc42. Further experiments on these effector proteins will be necessary in order to determine if and how they may be part of a network regulating neurite outgrowth. The experimental data presented here should provide an excellent basis for such detailed studies with the aim to obtain a more comprehensive view on the molecular networks underlying neurite outgrowth.
Although it cannot be formally ruled out, several lines of evidence exist to assure that our overexpression approach did not yield unspecific results. First, proteins with an established role in neuronal differentiation and function but not involved in neurite initiation or elongation showed no apparent effect on neurite outgrowth in our experiments. Second, 14 proteins of the 105 tested were identified as localizing to neurite tips, but 11 of them had no effect on neurite number or length when overexpressed. Third, a number of proteins we identified here as affecting neurite outgrowth are homologues or orthologues of well-characterized proteins already implicated in some aspects of neurite outgrowth in the literature. Finally, real-time qRT-PCR showed that the effector proteins identified are indeed expressed in PC12 cells and the relative expression of several of them changed during the course of neurite outgrowth in accordance with our functional data obtained by overexpression. For example, some genes that are down-regulated after NGF stimulation encode proteins whose overexpression blocked neurite outgrowth.
Our choice of time-lapse microscopy to determine when the neurite localized proteins associate with the site of neurite growth is a powerful technique to complement functional screens. Because protein localization is strongly coupled to function, the fact that proteins associating with the site of neurite growth very early could signify a function in neurite initiation or elongation, as opposed to a function for example in synaptic vesicle regulation or synapse formation when one would expect late accumulation in the neurite tip. From the 14 neurite tip proteins discovered, only 3 (Swissprot ID: Q9UFT2, Q9H0V0, and Q9H0W5) had an effect on neurite outgrowth when overexpressed, and all 3 associated with the sites of neurite growth very early (see ). By extending this approach to a larger number of neurite tip–localized proteins it should be possible to generate a temporal map of protein association with neurite tips that could serve as a basis for network construction and more detailed functional studies.
By using high-throughput and high-content microscopy technology in fixed (Liebel et al., 2003
) and living (Neumann et al., 2006
) cells together with fully automated image analysis to quantify neurite outgrowth (Ramm et al., 2003
), it should be possible to scale-up our approach here from low- to high-throughput and might thus become the basis for future large-scale neuronal-based proteomic studies.
It is interesting to note that there appears to be a strong correlation between the subcellular localization of the effector proteins and the phenotype generated. Six of seven proteins that induced an increase in the number of neurites per cell in our experiments localize to the plasma membrane. This finding is consistent with the view that the neurite outgrowth begins when external signals activate specific factors on the plasma membrane, which in turn triggers the formation of a neurite initiation site that should be distinct from the rest of the plasma membrane at the molecular level. Similarly, 9 of 11 proteins identified as blockers of neurite outgrowth localized either to the actin or microtubule cytoskeleton. This is in full agreement with numerous earlier studies establishing a major role of the cytoskeleton in the control of neurite outgrowth (for reviews see Gallo and Letourneau, 2000
; da Silva and Dotti, 2002
). Finally, effector proteins that affected neurite length upon overexpression are all localized to the Golgi or plasma membrane and are involved in membrane traffic regulation. This is consistent with the view that the delivery of new membrane to growing neurites is an important factor for their growth (for reviews see Futerman and Banker, 1996
; Valtorta and Leoni, 1999
Three proteins identified in our screen are of additional interest because they are potentially involved in human neurological disorders. Swissprot IDs: Q96PE5 (Nobile et al., 2002
) and Q9BQJ4 (Christophe-Hobertus et al., 2001
) have a potential role in temporal lobe epilepsy and hereditary X-linked mental retardation, respectively. However, in both cases previous research reported no mutations in these genes in families carrying the disease (Nobile et al., 2002
; Christophe-Hobertus et al., 2004
), and thus both genes are presently considered as “unlikely” to be involved in the respective disorders. However this earlier work did not investigate the 5′-regulatory regions of the respective genes, and therefore mutations occurring in the promoter region of the genes cannot be excluded as the cause of disease. Consistent with our data, it is therefore possible that in fact deregulation of protein expression and not loss of function is what is causing the neurological disorders related to these two genes. Therefore, based on our data here, the involvement of Q96PE5 and Q9BQJ4 in temporal lobe epilepsy and X-linked mental retardation could be possible and should thus be reevaluated with respect to their regulation and expression. The third disease-linked protein identified is SH3P12. It has been shown to interact with Ataxin-7 and huntingtin, both of which are key players in the neurodegenerative diseases Spinocerebellar ataxia and Huntington disease, respectively (Lebre et al., 2001
). Because, overexpression of SH3P12 blocked neurite outgrowth in PC12 cells, nervous system dysfunction may be related to changes in SH3P12 expression.
Extending this approach to a larger number of candidate proteins and complementing it with RNAi experiments and more detailed functional experiments, such as protein-protein interaction studies will ultimately lead a more comprehensive understanding of neurite outgrowth at the molecular level.