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- Materials and methods
- Supporting Information
Prion protein (PrPC) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrPC interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrPC co-opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross-talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrPC-mediated axonogenesis in peripheral neurons in response to STI1 and laminin-γ1 chain-derived peptide (Ln-γ1). STI1 and Ln-γ1 promoted robust axonogenesis in wild-type neurons, whereas no effect was observed in neurons from PrPC-null mice. PrPC binding to Ln-γ1 or STI1 led to an increase in intracellular Ca2+ levels via distinct mechanisms: STI1 promoted extracellular Ca2+ influx, and Ln-γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln-γ1, but depends on, C-type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrPC-mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrPC. These results suggest a role for PrPC as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system.
Prion protein (PrPC) is a ubiquitous cell surface protein anchored through a glycosylphosphatidylinositol moiety that is associated with transmissible spongiform encephalopathies (TSEs). The conversion of PrPC to the infectious isoform PrPSc in TSEs, results in neuronal loss and progressive neurodegeneration (Moore et al. 2009; Hajj et al. 2012). Depletion of PrPC in mice abrogates prion infection and, although no gross phenotype was detected in these animals under normal conditions [reviewed by (Weissmann and Flechsig 2003)], increased neuronal sensitivity to brain injuries was observed [for review, see (Linden et al. 2008)]. Notably, studies have demonstrated that PrPC ablation in neurons, but not in Schwann cells, promoted an adult-onset chronic demyelinating polyneuropathy that affected sensory and motor pathways of the peripheral nervous system (PNS) (Bremer et al. 2010). Furthermore, severe neurodegeneration occurred when PrPC deletion mutants were expressed in the absence of the wild-type protein (Shmerling et al. 1998; Li et al. 2007).
Several physiological functions of PrPC have been described during the past decade, but the role of PrPC loss-of-function in neurodegeneration remains unknown (Caughey and Baron 2006; Aguzzi et al. 2008). PrPC accumulates in focal adhesions and regulates focal adhesion formation and filopodia extension (Schrock et al. 2009). Furthermore, PrPC depletion in zebrafish results in impaired cell adhesion, which leads to gastrulation arrest (Malaga-Trillo et al. 2009). Many roles for PrPC have been characterized in the mammalian nervous system, several of which are mediated via interactions with cell surface molecules (Linden et al. 2008; Martins et al. 2010; Biasini et al. 2012). PrPC interacts directly with neural cell adhesion molecule (NCAM) (Schmitt-Ulms et al. 2001), leading to stabilization of NCAM in lipid rafts and activation of p59fyn to induce NCAM-dependent neuritogenesis (Santuccione et al. 2005). Components of the extracellular matrix (ECM) and their cell surface receptors have been demonstrated to interact with PrPC, showing that it may regulate ECM organization and function (Linden et al. 2008). Notably, the ECM is a well-known microenvironment with important roles in cell survival, migration, and differentiation (Venstrom and Reichardt 1993). The 37-kDa/67-kDa laminin receptor precursor (37LRP/67LR), which is involved in dynamic cellular processes (e.g., increased filopodia, directional motility, modulation of gene expression, and facilitation of Ln-integrins interaction) (Nelson et al. 2008), is one of the ECM receptors known to interact with PrPC (Rieger et al. 1997; Gauczynski et al. 2001). The interaction between 37LRP/67LR and PrPC could contribute to cell-to-cell communication essential for cell survival in a physiological context (Gauczynski et al. 2001). PrPC also participates in axonogenesis by modulating αvβ3 and β1 integrin activity through its interactions with vitronectin and fibronectin, respectively (Hajj et al. 2007; Loubet et al. 2012). In contrast, PrPC binds directly to contactin-associated protein (Caspr), an inhibitor of neuritogenesis, preventing its shedding by reelin and potentiating its inhibitory effect during neurite outgrowth (Devanathan et al. 2010).
PrPC directly interacts with the laminin γ1 chain (Ln-γ1) (between amino acids 1575-1584 of Ln and 173-182 of PrPC), inducing neuritogenesis in hippocampal neurons and PC12 cells (Graner et al. 2000a,b; Coitinho et al. 2006; Beraldo et al. 2011). The signal transduction pathway involved in Ln-γ1/PrPC-dependent effects requires the activation of group I metabotropic glutamate receptors (mGluR1/5), which leads to phospholipase C (PLC) activation and an increase in intracellular calcium levels (Beraldo et al. 2011). PrPC also interacts with stress inducible protein 1 (STI1) (Zanata et al. 2002), a co-chaperone that associates with Hsp70 and Hsp90 to facilitate protein folding and maturation (Nicolet and Craig 1989). STI1 is secreted into the extracellular milieu by astrocytes (Lima et al. 2007), and its interaction with PrPC (at amino acids 113-128) recruits α7 nicotinic acetylcholine receptors (α7nAChR), which are responsible for transducing trophic signals in hippocampal neurons (Beraldo et al. 2010). Taken together, the existence of a large number of PrPC-interacting molecules suggests that PrPC is a key member of a multiprotein complex in the plasma membrane that organizes signaling modules (Linden et al. 2008; Martins et al. 2010) and may fine tune neurite outgrowth.
Although a key role for PrPC interaction with Ln or STI1 has been demonstrated, the ability of PrPC to form the multi-protein complexes with more than one ligand was never addressed. In this study, we used simultaneously two PrPC ligands to observe how different combinations affect cellular signaling and axonogenesis in dorsal root ganglia (DRG) neurons.
- Top of page
- Materials and methods
- Supporting Information
The current study tests the hypothesis that multiple signaling activators of PrPC cooperate to trigger physiological responses by this GPI-anchored protein. Herein, we demonstrate that Ln-γ1 and STI1 induce axonogenesis in a PrPC-dependent manner in DRG neurons, similar to previous observations in hippocampal neurons (Graner et al. 2000a; Lopes et al. 2005; Beraldo et al. 2010). Moreover, there was a synergistic effect on axonogenesis when Ln-γ1 and STI1 were combined in concentrations that were unable to promote effects when either molecule was used alone. The interactions between Ln-γ1 and/or STI1 with PrPC trigger Erk1/2 pathways and induce rapid intracellular calcium mobilization by distinct mechanisms. STI1 induces Ca2+ influx through the plasma membrane, whereas the Ln-γ1 peptide increases intracellular Ca2+ by activating InsP3-R and mobilizing Ca2+ from intracellular stores (ER). Similar to hippocampal neurons (Beraldo et al. 2011), in DRG neurons, the signaling mediated by PrPC-Ln-γ1 peptide depends on group I mGluRs. On the other hand, differently from hippocampal neurons (Beraldo et al. 2010), signaling triggered by PrPC-STI1 in DRG is independent of α7nAChR. Remarkably, when combined, STI1 and Ln-γ1 peptide induced PrPC-dependent axonogenesis without participation of mGluRs and α7nAChR.
Several reports have shown that Ln-γ1 has important neurotrophic properties and plays a significant role in development and disease (Chen and Strickland 2003; Liebkind et al. 2003; Wiksten et al. 2003, 2007; Chen et al. 2009), and that the terminal KDI tripeptide is responsible for the biological functions of the Ln-γ1 peptide (Liesi et al. 2001; Wiksten et al. 2004a,b; Vaananen et al. 2006). Recently, we demonstrated that PrPC interacts directly with group I metabotropic glutamate receptors (mGluRs) in these neurons to transduce Ln-γ1 signals, which in turn activates PLC and produces InsP3, promoting intracellular calcium mobilization from the ER (Beraldo et al. 2011). Our present data demonstrated that similar underlying mechanisms are present in peripheral neurons. As demonstrated herein and by other groups (Alvarez et al. 2000; Hofmann et al. 2001; Carlton and Hargett 2007), DRG neurons express mGluRs, which participate in several events, such as mechanoception and nociception (Crawford et al. 2000; Zhou et al. 2001; Neugebauer and Carlton 2002; Li and Pan 2010). These results suggest a widespread and conserved mechanism for the transduction pathway involved in PrPC-Ln-γ1 signaling in different cell types.
As the initial characterization of STI1 as a PrPC ligand (Martins et al. 1997; Zanata et al. 2002), several reports have provided support for a role of STI1 as a soluble neurotrophic factor (Chiarini et al. 2002; Lopes et al. 2005; Lima et al. 2007; Caetano et al. 2008; Arantes et al. 2009; Hajj et al. 2009; Santos et al. 2011). STI1 is secreted by many cell types (Eustace and Jay 2004; Erlich et al. 2007; Lima et al. 2007; Wang et al. 2010; Walsh et al. 2011), which allows it to interact with PrPC at the cell surface (Zanata et al. 2002; Lopes et al. 2005; Caetano et al. 2008). The signaling pathways required for STI1 to transduce neuritogenic signals were also described in hippocampal neurons and involve the activation of PI3K-Akt-mTOR and ERK1/2 and require calcium influx through α7nAChR (Lopes et al. 2005; Beraldo et al. 2010; Roffe et al. 2010). Similar to results from CNS neurons (Beraldo et al. 2010), the present data show that in DRG neurons, PrPC/STI-induced axonogenesis depends on ERK1/2 activation and calcium influx. On the other hand, the PrPC/STI1 mechanism in DRG neurons also differs in some ways from that in CNS neurons, including the involvement of PLC and PKC pathways and the independence of α7nAChR. Importantly, PLC and PKC pathways participate in the inhibition of the proliferation of astrocytes by PrPC-STI1 (Arantes et al. 2009). These data suggest that STI1-PrPC engagement could activate calcium channels modulated by PLC via DAG production. One class of calcium channels that satisfies these criteria is the family of TRPC (Tu et al. 2009; Albert 2011). The TRPC channels are present in peripheral nervous tissue and have a variety of gating mechanisms, which can be activated by ligand binding, voltage gating, and changes in temperature (Patapoutian 2005; Alessandri-Haber et al. 2009; Ciobanu et al. 2009). These channels have also been implicated in diverse pathological states, including neurodegenerative disorders and cancer (Yamamoto et al. 2007; Nilius and Owsianik 2011; Santoni and Farfariello 2011). Our work presents evidence for the involvement of TRPC channels in STI1-mediated responses. However, more experiments will be necessary to characterize the connection between PrPC-STI1 signaling axis with the activation of this large family of channels.
It is also remarkable that Ca+2 plays a key role in PrPC-Ln-γ1 and PrPC-STI1 signaling, emerging as one of the main signals responsible for mediating PrPC function (Sorgato and Bertoli 2009). The first evidence for a link between prion protein and calcium dynamics came from experiments showing a reduction in bradykinin-induced intracellular calcium mobilization in neuronal cell lines infected with prions (Kristensson et al. 1993; Wong et al. 1996). Thereafter, it was shown that PrPC-null mice present altered intracellular calcium homeostasis in cerebellar granule neurons (Herms et al. 2000) and in hippocampal CA1 neurons (Fuhrmann et al. 2006). Moreover, the hyperexcitability attributed to PrPC-null mice (Collinge et al. 1994; Colling et al. 1996; Walz et al. 1999; Mallucci et al. 2002) may be because of the absence of PrPC inhibitory modulation of the ionotropic glutamate receptor (NMDAR) currents, which could lead to higher excitability (Khosravani et al. 2008). Heterologous expression of PrPC in CHO cells also alters intracellular calcium fluctuation in different cell compartments following the activation of purinergic receptors (Brini et al. 2005).
Our previous study mapped different STI1 and Ln-γ1 binding domains on the PrPC molecule. Although STI1 binds to amino acids 113–128 on PrPC (Zanata et al. 2002), Ln-γ1 interacts within the region of amino acids 173–182 of PrPC (Coitinho et al. 2006). In accord to that, present data shown that both molecules can bind to the same PrPC molecule and cooperate to achieve optimal effects on cellular signaling. In addition, the experiments conducted herein showed that combined treatment with Ln and STI1 causes a robust and synergistic effect on axonogenesis, with growth after only 2 h. Strikingly, even though the response to Ln alone is not dependent on PrPC, the combinatory stimuli with STI1 and Ln is dependent on PrPC. These data suggest at least two possibilities: (i) suboptimal activity levels from integrins triggered by Ln could have synergistic effects with PrPC-STI1 pathways; (ii) at suboptimal concentration Ln could binds preferentially to PrPC through its γ1 chain cooperating with STI1 to stimulate axonogenesis. The mechanisms associated with this phenomenon deserve further exploration.
There are numerous examples of synergism occurring between molecules involved in neurotrophic activities in the literature (Krieglstein 2004; Sharma 2007). Neurotrophic factors from the neurotrophin family (e.g., NGF), glial cell-line derived neurotrophic factor family ligands (e.g., GDNF), and neuropoietic cytokines (e.g., ciliary neurotrophic factor, CNTF) are known to have synergistic effects on neuronal differentiation, survival, synaptic function, and axonal outgrowth/regeneration (Chao 2003; Sharma 2007). Furthermore, there are reports showing cooperation between Ln and NGF in increasing Ln/integrin-induced neurite outgrowth in sensory neurons (Tucker et al. 2005) and PC12 cells (Achyuta et al. 2009).
In the current study, STI1 and Ln-γ1 acted synergistically to transduce neurotrophic signals through PrPC. Remarkably, Ca2+ signaling as well as axonogenesis mediated by PrPC binding to Ln-γ1, and STI1 in DRG are independent of mGluRs. Remarkably, our data point that TRPC channels participate in this event.
The results of the current study indicate that complexes formed between PrPC and its ligands vary depending on the cell type and ligand involved. These findings provide support for the idea that PrPC organizes signaling platforms in plasma membrane microdomains (Linden et al. 2008) and also strengthens the scaffold concept, which suggests that PrPC function could be allosterically modulated by distinct binding molecules leading to different signals and multiple biological responses (Linden et al. 2012). According to this concept, the individual or combinatory binding of Ln-γ1 and STI1 could result in different conformational rearrangements of both PrPC and its ligands leading to the recruitment of diverse transmembrane proteins that participate in a number of downstream signaling pathways. Furthermore, these data provide new cellular substrates to study the molecular basis of peripheral nerve development and to understand the process associated with axonal regeneration.