Jonathan G. Hanley

Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis.

The dynamic regulation of actin polymerization plays crucial roles in cell morphology and endocytosis. The mechanistic details of these processes and the proteins involved are not fully understood, especially in neurons. PICK1 is a PDZ–BAR-domain protein involved in regulated AMPA receptor (AMPAR) endocytosis in neurons. Here, we demonstrate that PICK1 binds filamentous (F)-actin and the actin-nucleating Arp2/3 complex, and potently inhibits Arp2/3-mediated actin polymerization. RNA interference (RNAi) knockdown of PICK1 in neurons induces a reorganization of the actin cytoskeleton resulting in aberrant cell morphology. Wild-type PICK1 rescues this phenotype, but a mutant PICK1, PICK1W413A, that does not bind or inhibit Arp2/3 has no effect. Furthermore, this mutant also blocks NMDA-induced AMPAR internalization. This study identifies PICK1 as a negative regulator of Arp2/3-mediated actin polymerization that is critical for a specific form of vesicle trafficking, and also for the development of neuronal architecture.

PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking

Regulation of AMPA receptor (AMPAR) trafficking results in changes in receptor number at the postsynaptic membrane, and hence modifications in synaptic strength, which are proposed to underlie learning and memory. NMDA receptor‐mediated postsynaptic Ca2+ influx enhances AMPAR internalisation, but the molecular mechanisms that trigger such trafficking are not well understood. We investigated whether AMPAR‐associated protein–protein interactions known to regulate receptor surface expression may be directly regulated by Ca2+. PICK1 binds the AMPAR GluR2 subunit and is involved in AMPAR internalisation and LTD. We show that PICK1 is a Ca2+‐binding protein, and that PICK1–GluR2 interactions are enhanced by the presence of 15 μM Ca2+. Deletion of an N‐terminal acidic domain in PICK1 reduces its ability to bind Ca2+, and renders the GluR2–PICK1 interaction insensitive to Ca2+. Overexpression of this Ca2+‐insensitive mutant occludes NMDA‐induced AMPAR internalisation in hippocampal neurons. This work reveals a novel postsynaptic Ca2+‐binding protein that provides a direct mechanistic link between NMDAR‐mediated Ca2+ influx and AMPAR endocytosis.

PICK1: A multi-talented modulator of AMPA receptor trafficking

AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor trafficking is a fundamental mechanism for regulating synaptic strength, and hence may underlie cellular processes involved in learning and memory. PICK1 (protein interacting with C-kinase) is a PDZ and BAR domain-containing protein that has recently emerged as a key regulator of AMPA receptor traffic. Via the PDZ domain, PICK1 interacts directly with AMPA receptor subunits and is involved in the regulated removal of AMPA receptors from the synaptic plasma membrane. PICK1 has the ability to functionally interact with a number of cellular processes, including calcium signaling, actin polymerisation and phospholipid membrane architecture. In this review, I summarize recent findings that describe the importance of PICK1 in neurons and its specific molecular characteristics that enable it to regulate AMPA receptor trafficking.

PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity

Activity‐dependent remodelling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the precise molecular mechanisms that regulate this process are unclear. Activators of Arp2/3‐mediated actin polymerisation are required for spine enlargement; however, during long‐term depression (LTD), spines shrink via actin depolymerisation and Arp2/3 inhibitors in this process have not yet been identified. Here, we show that PICK1 regulates spine size in hippocampal neurons via inhibition of the Arp2/3 complex. PICK1 knockdown increases spine size, whereas PICK1 overexpression reduces spine size. NMDA receptor activation results in spine shrinkage, which is blocked by PICK1 knockdown or overexpression of a PICK1 mutant that cannot bind Arp2/3. Furthermore, we show that PICK1–Arp2/3 interactions are required for functional hippocampal LTD. This work demonstrates that PICK1 is a novel regulator of spine dynamics. Via Arp2/3 inhibition, PICK1 has complementary yet distinct roles during LTD to regulate AMPA receptor trafficking and spine size, and therefore functions as a crucial factor in both structural and functional plasticity.

The small GTPase Arf1 modulates Arp2/3-mediated actin polymerization via PICK1 to regulate synaptic plasticity.

Summary Inhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1.

PICK1-mediated Glutamate Receptor Subunit 2 (GluR2) Trafficking Contributes to Cell Death in Oxygen/Glucose-deprived Hippocampal Neurons

Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca2+/Zn2+-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.

Oxygen/glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors.

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+, resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A3 receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca2+ also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A3 receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection.

Molecular mechanisms for regulation of AMPAR trafficking by PICK1

AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor trafficking is a fundamental mechanism for regulating synaptic strength, and hence may underlie cellular processes involved in learning and memory. PICK1 (protein that interacts with protein C-kinase) has recently emerged as a key regulator of AMPAR (AMPA receptor) traffic, and the precise molecular mechanisms of PICK1s action are just beginning to be unravelled. In this review, I summarize recent findings that describe some important molecular characteristics of PICK1 with respect to AMPAR cell biology.

Subunit-specific trafficking mechanisms regulating the synaptic expression of Ca2+-permeable AMPA receptors.

AMPA receptors are the main excitatory neurotransmitter receptor in the brain, and hence regulating the number or properties of synaptic AMPA receptors brings about critical changes in synaptic transmission. Synaptic plasticity is thought to underlie learning and memory, and can be brought about by decreasing or increasing the number of AMPA receptors localised to synaptic sites by precisely regulating AMPA receptor trafficking. AMPA receptors are tetrameric assemblies of subunits GluA1-4, and the vast majority are GluA1/2 and GluA2/3 heteromers. The inclusion of GluA2 subunit is critical because it renders the AMPA receptor channel impermeable to Ca(2+) ions. The vast majority of synaptic AMPA receptors in the brain contain GluA2, but relatively recent discoveries indicate that an increasing number of specific forms of synaptic plasticity involve not only an alteration of the number of synaptic AMPA receptors, but also changes to their GluA2 content. The resulting change in AMPA receptor Ca(2+) permeability clearly has profound consequences for synaptic transmission and intracellular signalling events. The subunit-specific trafficking mechanisms that cause such changes represent an emerging field of research with implications for an increasing number of physiological or pathological situations, and are the topic of this review.

PICK1 Mediates Transient Synaptic Expression of GluA2-Lacking AMPA Receptors during Glycine-Induced AMPA Receptor Trafficking

The number and subunit composition of postsynaptic AMPA receptors (AMPARs) is a key determinant of synaptic transmission. The vast majority of AMPARs contain GluA2 subunit, which renders the channel impermeable to calcium. However, a small proportion are GluA2 lacking and therefore calcium permeable (CP-AMPARs). It has been proposed recently that long-term potentiation (LTP) involves not only an increase in the total number of AMPARs at the synapse but also a transient switch to CP-AMPARs in the first few minutes after LTP induction. The molecular mechanisms that underlie this switch to CP-AMPARs and the subsequent switch back to calcium-impermeable AMPARs are unknown. Here, we show that endogenous GluA1 is rapidly inserted at the synaptic plasma membrane of rat hippocampal neurons immediately after stimulation with elevated glycine, a treatment known to induce LTP. In contrast, GluA2 is restricted from trafficking to the cell surface by a glycine-induced increase in PICK1–GluA2 binding on endosomal compartments. Between 5 and 20 min after stimulus, activation of CP-AMPARs triggers a release of GluA2 from PICK1, allowing GluA2-containing AMPARs to traffic to the synaptic plasma membrane. These results define a PICK1-dependent mechanism that underlies transient alterations in the subunit composition and calcium permeability of synaptic AMPARs that is important during the early phase after stimulation with glycine and therefore is likely to be important during the expression of LTP.