Adaptor Protein APPL1 Couples Synaptic NMDA Receptor with Neuronal Prosurvival Phosphatidylinositol 3-Kinase/Akt Pathway

Abstract

It is well known that NMDA receptors (NMDARs) can both induce neurotoxicity and promote neuronal survival under different circumstances. Recent studies show that such paradoxical responses are related to the receptor location: the former to the extrasynaptic and the latter to the synaptic. The phosphoinositide 3-kinase (PI3K)/Akt kinase cascade is a key pathway responsible for the synaptic NMDARdependent neuroprotection. However, it is still unknown how synaptic NMDARs are coupled with the PI3K/Akt pathway. Here, we explored the role of an adaptor protein-adaptor protein containing pH domain, PTB domain, and leucine zipper motif (APPL1)-in this signal coupling using rat cortical neurons. We found that APPL1 existed in postsynaptic densities and associated with the NMDAR complex through binding to PSD95 at its C-terminal PDZ-binding motif. NMDARs, APPL1, and the PI3K/Akt cascade formed a complex in rat cortical neurons. Synaptic NMDAR activity increased the association of this complex, induced activation of the PI3K/Akt pathway, and consequently protected neurons against starvation-induced apoptosis. Perturbing APPL1 interaction with PSD95 by a peptide comprising the APPL1 C-terminal PDZ-binding motif dissociated the PI3K/Akt pathway from NMDARs. Either the peptide or lentiviral knockdown of APPL1 blocked synaptic NMDAR-dependent recruitment and activation of PI3K/Akt pathway, and consequently blocked synaptic NMDAR-dependent neuroprotection. These results suggest that APPL1 contributes to connecting synaptic NMDARs with the intracellular PI3K/Akt cascade and the downstream prosurvival signaling pathway in rat cortical neurons.

Huntington’s disease: Neural dysfunction linked to inositol polyphosphate multikinase

“…The BDNF pathway is altered in HD because of decreased transcription and release of BDNF at the corticostriatal synapses (4,38) as well as impaired TrkB receptor signaling (39). Similarly, synaptic (but not extrasynaptic) NMDARs enhance Akt phosphorylation and neuroprotection (40).…”

Serine 707 of APPL1 is Critical for the Synaptic NMDA Receptor-Mediated Akt Phosphorylation Signaling Pathway

“…In a previous study, we also reported a key role of APPL1 in the CNS [11]. We found that APPL1 is partially co-localized with PSD95 and N-methyl-D-aspartate receptors (NMDARs) in cultured neurons, and couples synaptic NMDARs with the PI3K/Akt pathway through its direct interaction with PSD95.…”

“…Cyan fluorescent protein (CFP)-tagged APPL1 (CFP-APPL1), C-terminal 4-amino-acid-truncated APPL1 (CFP-APPL1 D4 ), and mCherry-tagged PSD95 (mCherry-PSD95) have been described in a previous study [11]. CFP-or green fluorescent protein (GFP)-tagged APPL1 with serine 707 replaced by alanine (CFP-APPL1 S707A and GFP-APPL1 S707A ), mCherry-tagged PDZ1-or both PDZ1-and 2-deleted PSD95 (mCherry-PSD95 DPDZ1 or mCherry-PSD95 DPDZ1/2 ) were constructed by conventional molecular cloning.…”

“…The primary neuron culture method was as described previously [11][12][13][14]. Briefly, hippocampal and cortical tissues were removed from rat pups on embryonic day 17 under a dissecting microscope in ice-cold Hanks' balanced salt solution (Gibco, Grand Island, NY).…”

“…The protocol for co-IP was as previously described [11,15]. The cortex was dissociated from adult C57BL/6 mice, homogenized quickly on ice, and lysed in buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mmol/L PMSF, and 1 lg/mL aprotinin.…”