Recombinant prion protein induces rapid polarization and development of synapses in embryonic rat hippocampal neurons in vitro

Abstract

While a b-sheet-rich form of the prion protein (PrP Sc ) causes neurodegeneration, the biological activity of its precursor, the cellular prion protein (PrP C ), has been elusive. We have studied the effect of purified recombinant prion protein (recPrP) on rat fetal hippocampal neurons in culture. Overnight exposure to Syrian hamster or mouse recPrP, folded into an a-helical-rich conformation similar to that of PrP C , resulted in a 1.9-fold increase in neurons with a differentiated axon, a 13.5-fold increase in neurons with differentiated dendrites, a fivefold increase in axon length, and the formation of extensive neuronal circuitry. Formation of synaptic-like contacts was increased by a factor of 4.6 after exposure to recPrP for 7 days. Neither the N-terminal nor C-terminal domains of recPrP nor the PrP paralogue doppel (Dpl) enhanced the polarization of neurons. Inhibitors of protein kinase C (PKC) and of Src kinases, including p59Fyn, blocked the effect of recPrP on axon elongation, while inhibitors of phosphatidylinositol 3-kinase showed a partial inhibition, suggesting that signaling cascades involving these kinases are candidates for transduction of recPrP-mediated signals. The results predict that full-length PrP C functions as a growth factor involved in development of neuronal polarity. Keywords: axon development, cell polarity, function of prion protein, neuritogenesis, neuronal differentiation. Conversion of PrP C into an alternatively folded isoform, PrP Sc , is a fundamental event in transmissible spongiform encephalopathies or prion diseases (Prusiner 1998). PrP C is a ubiquitous cell-surface membrane protein, which is anchored to the membrane by a C-terminal glycosyl phosphatidyl inositol moiety, but can also acquire a transmembrane topology or be secreted (Hegde et al. 1998;Ermonval et al. 2003; for review). The chromosomal PrP gene, Prnp, is a member of the Prn gene family that also includes the gene Prnd encoding Dpl. Dpl shares 25% sequence similarity with PrP but lacks the N-terminally located octameric repeats and hydrophobic regions present in PrP (Moore et al. 1999). In contrast to PrP, Dpl has a very low expression in the adult brain, is expressed primarily in testis, and less so in other peripheral tissues (Li et al. 2000). Address correspondence and reprint requests to Steinunn Baekkeskov, Diabetes Center, University of California San Francisco, 513 Parnassus Ave., HSW-1090, San Francisco, CA 94143-0534, USA. E-mail: s_baekkeskov@biochem.ucsf.edu Abbreviations used: CD, circular dichroism; DMSO, dimethylsulfoxide; Dpl, doppel; ERK, extracellular signal-regulated kinase; MAP2, microtubule-associated protein 2; MoDpl(27-155), full-length recombinant mouse doppel protein; MoPrP(23-230), full-length recombinant mouse prion protein; PBS, phosphate-buffered saline; PI3-kinase, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PrP C , cellular prion protein; PrP Sc , scrapie form of the prion protein; recPrP, recombinant prion protein; SDS-PAGE, sodium d...

Prion protein (PrP c ) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis

“…Our observations about neural development are further supported by recent studies of neuronal maturation, which demonstrated that folded recombinant PrP c added to cultured rat hippocampal neurons induces neuronal differentiation and process outgrowth (5). Studies such as these indicate that in vitro neural cell preparations are effective systems with which to study the role of PrP c in discrete stages of neuronal development and to determine what mechanisms might compensate for the lack of PrP c in precursors and their neuronal progeny.…”

“…Despite these putative roles for PrP c , mice that are null for PrP c exhibit no consistent, overt phenotype other than resistance to infection with prions, the infectious agent in the transmissible spongiform encephalopathies (4). Recent work has shown that PrP c induces polarization in synapse development as well as in neuritogenesis in embryonic hippocampal neuron cultures (5,6). PrP c is also up-regulated after focal cerebral ischemia (7), and PrP c overexpression reduces the extent of neuronal loss after ischemic insult, suggesting that PrP c might confer a neuroprotective effect in certain contexts (8).…”

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

“…And more relevantly, if increased PrP c expression in neuronal cultures potentiates neuronal differentiation, polarization and synapse formation, does it increase cell proliferation in the subventricular zone? (Kanaani et al, 2005). Answers to these questions have not been given, but we can hypothesize that PrP c acts as an "inducer" of the intrinsic proliferative properties of these cells in "neuronal niches", probably by forming part of or acting on a "neurotrophic" complex, and that PrP c expression inhibits cell proliferation in other neural cell types that do not show this "neurotrophic" complex.…”

“…Intrinsic Prnp expression is developmentally regulated (Miele et al, 2003) and takes place postnatally in neural tissue (Steele et al, 2006), especially during the fate restriction of multipotential cells towards the neural lineage (Mouillet-Richard et al, 1999), stages characterized by neuronal polarization and synapse formation. Conversely, extracellular PrP c may act via specific receptors in neighboring neurons to promote synapse formation and neuronal maturation (Graner et al, 2000;Kanaani et al, 2005).…”

Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes

“…PrP Sc but not PrP C accumulation stimulates the Notch and Hes pathways, resulting in the loss of dendritic processes (34,35). PrP C seems to function in signal transduction and neurite outgrowth in conjunction with the neural cell adhesion molecule, N-CAM (36,37). Moreover, PrP C -deficient mice exhibit defects in spatial memory tasks (38).…”