A possible role of PrPC in neurite differentiation and growth
Polarization of neurons and compartmentalization into axons and dendrites are fundamental for the function of the nervous system. In this study, we show that incubation of newly plated fetal hippocampal neurons with recPrP rapidly enhances the development of polarized and interconnected neurons. Specifically, overnight incubation of 1-day-old fetal neurons with purified full-length recPrP results in a several-fold increase in neurite differentiation, and a 5-fold increase in axon length. The effect of recPrP is not species-specific as both Syrian hamster and mouse recPrP have a similar effect. The specificity of recPrP is suggested by the lack of effect of purifed mouse doppel protein, NGF, amylin, and fibronectin on neurite outgrowth and/or differentiation in our overnight assay.
While neuronal networks are already visible after overnight culture with recPrP, formation of synaptic-like contacts defined by co-clustering of the presynaptic marker synaptophysin and the postsynaptic marker PSD-95 requires a longer incubation with recPrP. After 7 days in culture with recPrP, synaptic-like contacts are increased 5-fold compared with cultures without recPrP. At this stage in culture, the enhancement of synaptic-like contacts in cultures with recPrP appears to be the result of a combination of an increase in axonal length and branching and a 2–3-fold increase in the number of synaptophysin clusters (results not shown), while length and number of dendrites and number of PSD-95 clusters are similar in cultures with and without recPrP.
In the experimental conditions described here, fetal hippocampal rat neurons, seeded at low density (50 × 104/well) to facilitate accurate analysis of axonal length and synapse formation of individual neurons, are highly viable, and cell death is rare. Moreover, the cultures are highly homogenous for hippocampal neurons. Importantly, the effect of recPrP both on axonal elongation during a 1-day culture and on synaptogenesis during a 7-day culture is substantial (factor of five increase), and easy to observe and quantify. In comparison, previous studies on the effect of MoPrP-Fc on neonatal cerebellar granule and hippocampal neurons did not distinguish axons and dendrites, but reported an increase in total neurite length by less than a factor of two (Chen et al. 2003; Santuccione et al. 2005). An enhanced survival of neonatal mouse neurons in the presence of MoPrP-Fc (Chen et al. 2003) was also reported, an effect that was not observed in our analyses of rat hippocampal neurons, which evidence 96–99% viability both in the presence and absence of recPrP. We have conducted a series of experiments to study the effect of purified SHaPrP(29–231) on embryonic (E18/19) and neonatal (P1) mouse hippocampal neurons in culture. Both embryonic and neonatal mouse cultures were more heterogeneous than rat cultures with a high percentage of glial cells in addition to neuronal cells (supplementary Fig. 2). Embryonic mouse hippocampal neurons required plating at a twofold higher density than rat neurons in order for neurons to survive, a constraint that hampered analyses of individual neurons. Cultures of neonatal mouse hippocampal neurons, in contrast, could be plated at a similar density as rat hippocampal neurons, albeit at lower viability, and were amenable to analyses of individual neurons. In these similar conditions, the effect of SHaPrP(29–231) on cultures of neonatal mouse hippocampal neurons was strikingly different from the effect on embryonic rat neurons (supplementary Fig. 2): recPrP induced the differentiation and growth of multiple neurites with axon-like characteristics (i.e. predominant expression of Tau1 and low expression of MAP2), in a starburst effect, rather than inducing a single axon. These differences in the effect of recPrP on mouse and rat neurons suggest that endogenous cues required for selection of a single axon and the interplay with PrP may vary either between rat and mouse, or between embryonic and neonatal neurons. As reported for mouse cerebellar granule neurons and MoPrP-Fc, incubation with SHaPrP(29–231) significantly enhanced the viability of hippocampal mouse neurons (∼twofold), suggesting that, in conditions where cell viability is compromised, recPrP may indeed rescue neurons from cell death. Thus, cultures of neonatal mouse hippocampal neurons may offer an assay for the effect of PrP on cell survival. However, the assay we present using embryonic rat hippocampal neurons appears far more tractable for studies of neuronal polarization and allows detailed analyses of the effect of recPrP on axonal differentiation and growth and synapse formation.
The nature of the implicit cell-surface receptor mediating the effect of purified recPrP on the development of neuronal polarity in our assay remains to be elucidated. Several cell-surface proteins known to interact with PrPC, including NCAM (Schmitt-Ulms et al. 2001; Santuccione et al. 2005) and the laminin receptor precursor (Rieger et al. 1997; Gauczynski et al. 2001), are implicated in neurite outgrowth. Most importantly, there is evidence to suggest that the interaction of PrP-Fc with NCAM in mouse neurons in culture promotes neurite outgrowth (Santuccione et al. 2005). Whether the association of PrP with the laminin receptor results in a cumulative effect on neurite outgrowth or controls a distinct aspect of neuronal differentiation is currently unknown. A blocking of those candidate receptors for PrP in our assay using antibodies has been hampered by toxicity of such blockade for embryonic rat neurons, independent of the presence of recPrP. The role of those receptors in the effect of recPrP on differentiation of fetal rat neurons remains the subject of further investigations. Another candidate for mediating the effect of recPrP is surface-anchored PrPC itself. Gauczynski et al. (2001) did not observe differences in the binding of human recPrP to the surface of mouse neurons from either wild-type or Prnp0/0 mice, suggesting that membrane-anchored PrPC is not the main receptor for human recPrP. Furthermore, no difference was found in the enhancing effect of exogenous PrP-Fc on total neurite length in cultures of mouse cerebellar granule or hippocampal neurons derived from either wild-type or Prnp0/0 mice (Chen et al. 2003; Santuccione et al. 2005). Our experiment also did not reveal a difference in the response of hippocampal neurons from either wild-type or Prnp0/0 mice to recPrP (results not shown). Taken together, these results suggest that membrane-anchored PrPC is not a receptor for trans interactions with PrP.
Our results with fetal rat neurons confirm previous data suggesting a role of p59Fyn in the effect of PrP-Fc on polarization of neonatal mouse neurons (Chen et al. 2003). Activation of p59Fyn appears to be mediated through association of PrP-Fc with NCAM (Santuccione et al. 2005). However, in contrast to the studies of mouse neurons incubated with PrP-Fc, a role for ERK and/or PKA (Chen et al. 2003) was not detected in our assay using rat hippocampal neurons. The blocking effect of inhibitors to PKC and PI3-kinase in our assay is consistent with their role in polarization of neurons (Shi et al. 2003; Menager et al. 2004), but further studies are needed to assess whether these kinases are directly involved in transduction of recPrP-mediated signals.
PrPC appears to fold into multiple forms with different topology in the endoplasmic reticulum (Hegde et al. 1998). The detection of two transmembrane forms of opposite membrane orientation, a GPI-linked form, a secreted form, as well as differentially glycosylated forms (Ermonval et al. 2003; for review), suggests the possibility of more than one function in vivo, and perhaps interactions with multiple ligands and/or receptors. Furthermore, the ratio of α-helix versus β-sheet in purified recPrP may well be a critical parameter for function. The in vitro assay described here, measuring the effect of recPrP on axonal elongation in 1–2 day-old cultures of primary hippocampal neurons, provides an approach to dissect the functions of different PrPC isoforms/conformers and their interaction with other cellular factors.