Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Apoptotic pathways involved in TSEs

NCAM interaction with PrP enhances NCAM-mediated neurite outgrowth

Redistribution of NCAM to lipid rafts in response to NCAM homophilic binding is required for NCAM-mediated neurite outgrowth (Niethammer et al., 2002; Leshchyns'ka et al., 2003). In this paradigm substrate-coated or soluble NCAM interacts with and signals through NCAM at the neuronal cell surface. Since the binding of PrP-Fc to NCAM also redistributes NCAM lipid rafts, we investigated whether PrP-Fc promotes NCAM-mediated neurite outgrowth. PrP-Fc was thus applied to cultured hippocampal neurons and neurite length was measured after 24 hours. PrP-Fc increased neurite lengths when compared to the control group as previously observed (Chen et al., 2003), suggesting that PrP-Fc induced redistribution of NCAM to lipid rafts promotes neurite outgrowth. Alternatively, clustering of GPI-anchored raft associated proteins may also activate intracellular signalling cascades leading to enhanced neurite outgrowth (Doherty et al., 1993).

To exclude the possibility that PrP-Fc acts via clustering of PrP at the neuronal cell surface and not via NCAM, we incubated neurons with different concentrations of polyclonal antibodies against PrPC thereby clustering PrP at the cell surface. Unexpectedly, we found that antibodies against PrPC inhibited neurite outgrowth, indicating that clustering of PrP is not sufficient to induce neurite outgrowth. Furthermore, it suggested that PrP antibodies inhibit cis-interactions between PrP and a binding partner at the cell surface that was required for neurite outgrowth. To directly assess the role of NCAM in PrP-Fc induced neurite outgrowth, we treated NCAM-/- neurons with PrP-Fc and found that, in contrast to NCAM+/+ neurons, NCAM-/- neurons did not respond to PrP-Fc by increased neurite outgrowth, confirming that NCAM is a major receptor for PrP in PrP-Fc induced neurite outgrowth.

To analyse the role of PrP-to-NCAM trans-interaction in NCAM-mediated neurite outgrowth, we estimated neurite outgrowth in response to PrP-Fc in PrP-/- neurons, thereby abolishing cis-interactions between NCAM and PrP. PrP-Fc enhanced neurite lengths, indicating that trans-interactions between NCAM and PrP are involved in promoting neurite outgrowth.

To analyse the role of cis-interactions between NCAM and PrP in NCAM-mediated neurite outgrowth, we compared neurite outgrowth in response to NCAM activation in PrP-/- neurons transfected with GFP alone (control) or GFP together with PrP. Transfected PrP was delivered to the cell surface and partially co-localised with NCAM. In GFP transfected neurons treatment with NCAM-Fc enhanced neurite outgrowth when compared to untreated GFP transfected cells, indicating a response to NCAM.

However, PrP transfected neurons treated with NCAM-Fc produced even longer neurites neurons transfected only with GFP. This increase in the NCAM-Fc elicited response thus evolves from an NCAM-to-PrP cis-interaction. Furthermore, application of PrP antibodies completely abolished the NCAM-Fc induced response in wild type neurons, probably by interfering with cis-interactions between NCAM and PrP at the neuronal cell surface and fyn activation.

The combined observations indicate that NCAM is a major neuronal receptor for PrP presented in a trans-fashion. We also conclude that cis-interactions between NCAM and PrP at the neuronal cell surface enhance NCAM-induced neurite outgrowth when NCAM is presented to neurons in a trans-fashion.

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