The specificity of the Aβo-induced current of PrPC-mGluR5 oocytes was examined. Although mGluR1 expression leads to equally strong Glu-induced current (Figures 3A and 3B), there is no detectable
Aβo-induced current (Figures 3A and 3C). PrPC lacking the Aβo binding domain, PrPΔ23–111 (Chen et al., 2010, Laurén et al., 2009 and Um et al., 2012), fails to support Aβo-induced signaling through mGluR5 (Figure 3C). The anti-PrPC antibody, 6D11, binds to residues 95–105 and prevents Aβo interaction (Chung et al., 2010, Laurén et al., 2009 and Um et al., 2012). Preincubation with 6D11 blocks Aβo responses, but not Glu responses, in PrPC-mGluR5 oocytes (Figures 3B and 3C). The Aβo-induced response has an XAV-939 solubility dmso EC50 of 1 μM monomer equivalent, an estimated 10 nM oligomer concentration (Figure 3D). A characteristic of G protein-mediated responses in Xenopus oocytes is strong desensitization. Maximal Glu stimulation nearly eliminates subsequent responses to Glu for 10–15 min. Consistent with the Aβo-PrPC-mGluR5 responses
sharing this pathway, pretreatment with Glu eliminates the response to subsequent Aβo ( Figure 3E). In addition, pretreatment with cell Vorinostat supplier permeable BAPTA-AM to chelate intracellular calcium abrogated the Aβo-induced signal ( Figure 3E), as for Glu ( Saugstad et al., 1996). Thus, Aβo interaction with a PrPC-mGluR5 complex mobilizes calcium stores. Although mGluR5-mediated signaling to Fyn is as robust with Aβo-PrPC as with Glu, signaling to calcium mobilization is substantially less effective for Aβo-PrPC than with Glu as the mGluR5 ligand, so Aβo does not mimic Glu precisely. We considered whether Aβo regulates neuronal calcium signaling through mGluR5 directly and acutely. Chronic else Aβo-PrPC-Fyn signaling can indirectly alter NMDA receptor (NMDAR) trafficking to modulate NMDA-induced calcium responses (Um et al., 2012). We used a calcium-sensitive fluorescent
dye to assess direct immediate response to Aβo in 21 days in vitro (DIV) cortical cultures. In low-density cultures, there is little direct calcium response to Aβo under the conditions that modulate NMDAR responses (data not shown; Um et al., 2012). With microscopic imaging, Aβo occasionally induces local calcium transients, but there is no generalization and measurement across a microtiter well does not detect a change (not shown). Higher density cultures exhibit spontaneous synchronized calcium increases (Figure S3A) that depend on network connectivity being suppressed by tetrodotoxin (TTX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or 2-amino-5-phosphonopentanoic acid (APV) (Figures S3B and S3C) (Dravid and Murray, 2004). Under these conditions, Aβo induces an increase of intracellular calcium (Figure 4A). Averaging multiple wells smoothes random spontaneous signals (Figure S3A), and Aβo-induced responses are apparent (Figure 4A). This response is oligomer specific; no response is detected with monomeric Aβ (Figure 4C).