No 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...

The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the γ-aminobutyric acid–synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH2-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1–23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH2-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters.

The efficacy and success of many cellular processes is dependent on a tight orchestration of proteins trafficking to and from their site(s) of action in a time-controlled fashion. Recently, a dynamic cycle of palmitoylation/de-palmitoylation has been shown to regulate shuttling of several proteins, including the small GTPases H-Ras and N-Ras, and the GABA-synthesizing enzyme GAD65, between the Golgi compartment and either the plasma membrane or synaptic vesicle membranes. These proteins are peripheral membrane proteins that in the depalmitoylated state cycle rapidly on and off the cytosolic face of ER/Golgi membranes. Palmitoylation of one or more cysteines, by a Golgi localized palmitoyl transferase (PAT) results in trapping in Golgi membranes, and sorting to a vesicular pathway in route to the plasma membrane or synaptic vesicles. A depalmitoylation step by an acyl protein thioesterase (APT) releases the protein from membranes in the periphery of the cell resulting in retrograde trafficking back to Golgi membranes by a non-vesicular pathway. The proteins can then enter a new cycle of palmitoylation and depalmitoylation. This inter-compartmental trafficking is orders of magnitude faster than vesicular trafficking. Recent advances in identifying a large family of PATs, their protein substrates, and single PAT mutants with severe phenotypes, reveal their critical importance in development, synaptic transmission, and regulation of signaling cascades. The emerging knowledge of enzymes involved in adding and removing palmitate is that they provide an intricate regulatory network involved in timing of protein function and transport that responds to intracellular and extracellular signals.

Accumulation of the GABA inhibitory neurotransmitter for rapid delivery into synapses can be accomplished by GAD65 dependent and independent membrane-targeting of GAD67.