Psychiatric drugs bind to classical targets within early exocytotic pathways: therapeutic effects

Abstract

The classical targets for antipsychotic and antidepressant drugs are G protein-coupled receptors and neurotransmitter transporters, respectively. Full therapeutic actions of these drugs require several weeks. We show how therapeutic effects may eventually accrue after existing therapeutic ligands bind to these classical targets, not on the plasma membrane but rather within endoplasmic reticulum (ER) and cis-Golgi. Consequences of such binding may include pharmacological chaperoning: the nascent drug targets are stabilized against degradation and can therefore exit the ER more readily. Another effect may be matchmaking: heterodimers and homodimers of the target form and can more readily exit the ER. Summarizing recent data for nicotinic receptors, we explain how such effects could lead to reduced ER stress and to a decreased unfolded protein response, including changes in gene activation and protein synthesis. In effects not directly related to cellular stress, escorting would allow increased ER exit and trafficking of known associated proteins, as well as other proteins such as growth factors and their receptors, producing both cell-autonomous and non-cell-autonomous effects. Axonal transport of relevant proteins may underlie the several weeks required for full therapy. In contrast, the antidepressant effects of ketamine and other N-methyl-D-aspartate receptor ligands, which occur within <2 hours, could arise from dendritically localized intracellular binding, followed by chaperoning, matchmaking, escorting, and reduced ER stress. Thus, the effects of intracellular binding extend beyond proteostasis of the targets themselves and involve pathways distinct from ion channel and G protein activation. We propose experimental tests and note pathophysiological correlates.

Figure 1.

Insights from intracellular nicotine actions on the α4β2 nicotinic acetylcholine receptors (nAChR). (A) Chaperoning, matchmaking, reduction of endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) (5,16,88). Nicotine enters the neuron, permeates into the ER, and serves as a chaperone that favors assembly and stabilization of α4β2 nAChRs (shown in the insert at bottom). This decreases interactions with immunoglobulin binding protein (BiP), modulating protein kinase R-like ER-localized eukaryotic initiation factor 2α kinase (PERK)-activating transcription factor 4 (ATF4) and inositol-requiring enzyme 1 (IRE1)-X-box binding protein 1 (XBP1) (also shown in the insert at bottom). The insert at top shows that during ER stress, activating transcription factor 6 (ATF6) leaves the ER and enters the Golgi, where a fragment is cleaved; this then translocates to the nucleus and becomes a transcription factor. The UPR influences gene activation, via transcription factor binding to at least three unfolded protein response elements (UPRE). Experiments in our lab have not yet explored the IRE1 branch of the UPR, and it is shown as a dashed line. The M3-M4 loop of some nAChR subunits (purple) mediates ER retention and export via interactions with vesicle coat protein I and II complex proteins (COPI and COPII) (Figure S1 in Supplement 1). Ribosomes bound to the ER membrane are shown as gray dots. (B) An escort mechanism. The prototoxin lynx is synthesized, then transported to the ER lumen, guided by the usual signal sequence. Lynx resembles nAChR toxins from snake venom and is thought to bind like these toxins, at the interface between nAChR subunits (6,93). Unlike the snake venom toxins, lynx has a Glycophosphatidylinositol (GPI) anchor in the membrane. Lynx can therefore guide nAChRs toward cholesterol-rich regions of intracellular membranes (cholesterol molecules are shown in the membranes that anchor lynx). Nicotine binds at the same interface. One postulated consequence of nicotine binding would be displacement of lynx, abducting nAChRs from cholesterol-rich regions. This mechanism has not been tested. CSF, cerebrospinal fluid; p-eIF2α, phosphorylated eukaryotic initiation factor 2α.

Figure 2.

(A, B) Intracellular pharmacological chaperoning and matchmaking of G protein-coupled receptors (GPCRs) and downstream effects. (A) The conventional outside-in assumptions that GPCR antagonists manipulate second messengers and kinase cascades, resulting in gene activation. The diagram includes receptor trafficking in the late exocytotic/endocytotic pathway and signaling by p-arrestin. The diagram omits known facts about homodimerization and heterodimerization of GPCRs. (B) The inside-out view. Intracellular pharmacological chaperoning and matchmaking of GPCR and downstream effects, including reduction of endoplasmic reticulum (ER) stress and suppression of unfolded protein response. See also Figure 1 and Figure SI in Supplement 1. ATF4, activating transcription factor 4; ATF6, activating transcription factor 6; BiP, immunoglobulin binding protein; CSF, cerebrospinal fluid; IRE1, inositol-requiring enzyme 1; p-eIF2α, phosphorylated eukaryotic initiation factor 2α; PERK, protein kinase R-like endoplasmic reticulum-localized eukaryotic initiation factor 2α kinase; UPRE, unfolded protein response elements; XBP1, X-box binding protein 1.

Figure 3.

Diagrams of a dendrite, showing possible explanations for the 1- to 2-hour delay before antidepressant actions of ketamine and related N-methyl-D-aspartate (NMDA) receptor blockers. Ketamine exerts at least some of these antidepressant effects via increases in brain-derived neurotrophic factor (BDNF) secretion (76,77,94,95), and we therefore diagram mechanisms leading to BDNF secretion. Brain-derived neurotrophic factor secretion presumably requires depolarization, which may explain the requirement for 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptor activation (82). Note that dendritic endoplasmic reticulum (ER) and Golgi are thought to be simpler than the somatically located organelles shown in previous figures. The selective NMDA receptor 2B antagonist Ro 25-6981 had similar effects to ketamine (77). The transcription of BDNF is not required (as shown by insensitivity to actinomycin D), but its synthesis is required (as shown by sensitivity to anisomycin). In hippocampus, ketamine and NMDA, in the absence of neuronal activity, led to dephosphorylation of eukaryotic elongation factor (eEF2) (also called calcium/calmodulin-dependent eukaryotic elongation factor 2 kinase), but only ketamine produced this effect in cortex. Rottlerin and NH125, which inhibit several kinases including eEF2 kinase, also had BDNF-dependent antidepressant activity. (A) An outside-in mechanism, downstream from NMDA receptor block. Ketamine binds within the NMDA receptor pore but does not enter the neuron. The data have generally been interpreted in light of the knowledge that ketamine blocks spontaneous miniature excitatory postsynaptic currents through NMDA receptors (76). It is implied that decreased Ca²⁺ influx through NMDA receptors begins the transduction pathway leading to effects on presynaptic or postsynaptic efficiency. (B) Possible inside-out mechanisms, resulting from intracellular binding to nascent NMDA receptors. Existing data show that ligands can act as pharmacological chaperones for glutamate receptors within the ER (96), analogous to experiments in which nicotine acts as a pharmacological chaperone for nicotinic acetylcholine receptors. Two possible sequelae could lead to increased BDNF secretion. First, enhanced ER exit would decrease ER stress, for instance by decreasing phosphorylated protein kinase R-like ER-localized eukaryotic initiation factor 2α kinase (pPERK). This would decrease phosphorylated eukaryotic initiation factor 2α (p-eIF2α), increasing synthesis of ER proteins, including BDNF, thus producing the observed BDNF increase. Activation of NMDA receptors increases ER stress markers such as eIF2α phosphorylation and CCAAT/enhancer-binding protein homologous protein (CHOP) (97). However, it is not known whether blockade of NMDA receptors by ketamine has any effect on eIF2α phosphorylation in the absence of ER stress. This would be a key test of the chaperoning-ER stress hypothesis. Blockade by MK-801 did decrease caspase-12 activation, even in the absence of ER stress; however, caspase-12 activation may occur in a pathway distinct from ER stress (98). Second, an escort effect of intracellular ketamine-NMDA receptor binding arises from the fact that both NMDA receptors and BDNF are trafficked via a nonstandard ER and Golgi vesicle pathway involving synapse-associated protein 97 (SAP97) and calcium/calmodulin-dependent serine protein kinase (CASK) (99,100). Additional knowledge about the synapse-associated protein 97-calcium/calmodulin-dependent serine protein kinase trafficking pathway is crucial for evaluating the escort hypothesis. Chaperoning and/or matchmaking would occur in the ER. Escorting would occur when both the NMDA receptor and BDNF bind to Sec24 or at a later step. BiP, immunoglobulin binding protein; COPI and COPII, vesicle coat protein I and II complex; CSF, cerebrospinal fluid; IRE1, inositol-requiring enzyme 1; mRNA, messenger RNA; p-eEF2, phosphorylated eukaryotic elongation factor.