Gareth M. Thomas

Targeted In Vivo Mutations of the AMPA Receptor Subunit GluR2 and Its Interacting Protein PICK1 Eliminate Cerebellar Long-Term Depression

Cerebellar long-term depression (LTD) is a major form of synaptic plasticity that is thought to be critical for certain types of motor learning. Phosphorylation of the AMPA receptor subunit GluR2 on serine-880 as well as interaction of GluR2 with PICK1 have been suggested to contribute to the endocytic removal of postsynaptic AMPA receptors during LTD. Here, we show that targeted mutation of PICK1, the GluR2 C-terminal PDZ ligand, or the GluR2 PKC phosphorylation site eliminates cerebellar LTD in mice. LTD can be rescued in cerebellar cultures from mice lacking PICK1 by transfection of wild-type PICK1 but not by a PDZ mutant or a BAR domain mutant deficient in lipid binding, indicating the importance of these domains in PICK1 function. These results demonstrate that PICK1-GluR2 PDZ-based interactions and GluR2 phosphorylation are required for LTD expression in the cerebellum.

Dual Palmitoylation of NR2 Subunits Regulates NMDA Receptor Trafficking

Modification of NMDA receptor function and trafficking contributes to the regulation of synaptic transmission and is important for several forms of synaptic plasticity. Here, we report that NMDA receptor subunits NR2A and NR2B have two distinct clusters of palmitoylation sites in their C-terminal region. Palmitoylation within the first cluster on a membrane-proximal region increases tyrosine phosphorylation of tyrosine-based internalization motifs by Src family protein tyrosine kinases, leading to enhanced stable surface expression of NMDA receptors. In addition, palmitoylation of these sites regulates constitutive internalization of the NMDA receptor in developing neurons. In marked contrast, palmitoylation of the second cluster in the middle of C terminus by distinct palmitoyl transferases causes receptors to accumulate in the Golgi apparatus and reduces receptor surface expression. These data suggest that regulated palmitoylation of NR2 subunits differentially modulates receptor trafficking and might be important for NMDA-receptor-dependent synaptic plasticity.

Palmitoylation by DHHC5/8 Targets GRIP1 to Dendritic Endosomes to Regulate AMPA-R Trafficking

Palmitoylation, a key regulatory mechanism controlling protein targeting, is catalyzed by DHHC-family palmitoyl acyltransferases (PATs). Impaired PAT activity is linked to neurodevelopmental and neuropsychiatric disorders, suggesting critical roles for palmitoylation in neuronal function. However, few substrates for specific PATs are known, and functional consequences of palmitoylation events are frequently uncharacterized. Here, we identify the closely related PATs DHHC5 and DHHC8 as specific regulators of the PDZ domain protein GRIP1b. Binding, palmitoylation, and dendritic targeting of GRIP1b require a PDZ ligand unique to DHHC5/8. Palmitoylated GRIP1b is targeted to trafficking endosomes and may link endosomes to kinesin motors. Consistent with this trafficking role, GRIP1bs palmitoylation turnover rate approaches the highest of all reported proteins, and palmitoylation increases GRIP1bs ability to accelerate AMPA-R recycling. To our knowledge, these findings identify the first neuronal DHHC5/8 substrate, define novel mechanisms controlling palmitoylation specificity, and suggest further links between dysregulated palmitoylation and neuropathological conditions.

Rapid and bi‐directional regulation of AMPA receptor phosphorylation and trafficking by JNK

Jun N‐terminal kinases (JNKs) are implicated in various neuropathological conditions. However, physiological roles for JNKs in neurons remain largely unknown, despite the high expression level of JNKs in brain. Here, using bioinformatic and biochemical approaches, we identify the AMPA receptor GluR2L and GluR4 subunits as novel physiological JNK substrates in vitro, in heterologous cells and in neurons. Consistent with this finding, GluR2L and GluR4 associate with specific JNK signaling components in the brain. Moreover, the modulation of the novel JNK sites in GluR2L and GluR4 is dynamic and bi‐directional, such that phosphorylation and de‐phosphorylation are triggered within minutes following decreases and increases in neuronal activity, respectively. Using live‐imaging techniques to address the functional consequence of these activity‐dependent changes we demonstrate that the novel JNK site in GluR2L controls reinsertion of internalized GluR2L back to the cell surface following NMDA treatment, without affecting basal GluR2L trafficking. Taken together, our results demonstrate that JNK directly regulates AMPA‐R trafficking following changes in neuronal activity in a rapid and bi‐directional manner.

Gain-of-function glutamate receptor interacting protein 1 variants alter GluA2 recycling and surface distribution in patients with autism

Glutamate receptor interacting protein 1 (GRIP1) is a neuronal scaffolding protein that interacts directly with the C termini of glutamate receptors 2/3 (GluA2/3) via its PDZ domains 4 to 6 (PDZ4–6). We found an association (P < 0.05) of a SNP within the PDZ4-6 genomic region with autism by genotyping autistic patients (n = 480) and matched controls (n = 480). Parallel sequencing identified five rare missense variants within or near PDZ4–6 only in the autism cohort, resulting in a higher cumulative mutation load (P = 0.032). Two variants correlated with a more severe deficit in reciprocal social interaction in affected sibling pairs from proband families. These variants were associated with altered interactions with GluA2/3 and faster recycling and increased surface distribution of GluA2 in neurons, suggesting gain-of-function because GRIP1/2 deficiency showed opposite phenotypes. Grip1/2 knockout mice exhibited increased sociability and impaired prepulse inhibition. These results support a role for GRIP in social behavior and implicate GRIP1 variants in modulating autistic phenotype.

SynaptomeDB: an ontology-based knowledgebase for synaptic genes

MOTIVATION The synapse is integral to the function of the brain and may be an important source of dysfunction underlying many neuropsychiatric disorders. Consequently, it is an excellent candidate for large-scale genomic and proteomic study. However, while the tools and databases available for the annotation of high-throughput DNA and protein are generally robust, a comprehensive resource dedicated to the integration of information about the synapse is lacking. RESULTS We present an integrated database, called SynaptomeDB, to retrieve and annotate genes comprising the synaptome. These genes encode components of the synapse including neurotransmitters and their receptors, adhesion/cytoskeletal proteins, scaffold proteins, membrane transporters. SynaptomeDB integrates various and complex data sources for synaptic genes and proteins.

GRIP1 and 2 regulate activity-dependent AMPA receptor recycling via exocyst complex interactions.

PSD-95/SAP90/DLG/ZO-1 (PDZ) domain-mediated protein–protein interactions play important roles in regulating AMPA receptor trafficking and neuronal plasticity. GRIP1 and GRIP2 are homologous multi-PDZ domain-containing proteins that bind to the C-termini of AMPA-R GluA2 and GluA3 subunits. Previous attempts to determine the cellular roles of GRIP1 and GRIP2 in neurons have been complicated by nonspecific reagents, and by the embryonic lethality of conventional GRIP1 KO mice. To circumvent these issues we developed a conditional targeted deletion strategy to knock out GRIP1 in postnatal neurons derived from GRIP2 KO mice. Loss of GRIP1 and 2 did not affect normal AMPA-R steady-state trafficking and endocytosis, but strikingly impaired activity-dependent AMPA-R recycling. This previously uncharacterized role for GRIP1 appears to be mediated by novel interactions with the cellular trafficking machinery via the exocyst protein complex. Indeed, disruption of GRIP1-exocyst binding caused a strikingly similar deficit in AMPA-R recycling. Together these findings reveal a previously unidentified role for AMPA-R-GRIP1-exocyst protein complexes in activity-dependent AMPA-R trafficking.

DHHC8-Dependent PICK1 Palmitoylation is Required for Induction of Cerebellar Long-Term Synaptic Depression

The palmitoyl acyltransferase (PAT) DHHC8 is implicated in synaptic regulation but few DHHC8 substrates are known. Here we report that DHHC8 binds and palmitoylates the PDZ domain-containing protein PICK1 at a cysteine residue that is essential for long-term synaptic depression (LTD) in cultured mouse cerebellar Purkinje neurons. Cerebellar LTD is palmitoylation-dependent and induction of LTD requires DHHC8. Furthermore, PICK1 is a critical DHHC8 substrate whose palmitoylation is necessary for LTD. These results identify the first DHHC8 substrate required for a specific form of synaptic plasticity and provide new insights into synaptic roles of palmitoylation.

Palmitoylation controls DLK localization, interactions and activity to ensure effective axonal injury signaling

Significance Dual leucine-zipper kinase (DLK) is essential for responses to nerve injury and subsequent neural regeneration by controlling transfer of signals from damaged distal axons to neuronal cell bodies. However, DLK is predicted to be freely diffusible, raising the question of how it conveys long-distance, directional signals. Here we report that direct modification of DLK with the lipid palmitate, a process called palmitoylation, is critical for retrograde injury signaling. At the molecular level, palmitoylation targets DLK to retrograde trafficking vesicles, assembles DLK-dependent signaling complexes, and is also essential for DLK kinase activity. This “multifunctional” palmitoylation explains how DLK mediates injury responses and may be a previously unappreciated mechanism that ensures the specificity of enzymatic signaling in diverse cell types. Dual leucine-zipper kinase (DLK) is critical for axon-to-soma retrograde signaling following nerve injury. However, it is unknown how DLK, a predicted soluble kinase, conveys long-distance signals and why homologous kinases cannot compensate for loss of DLK. Here, we report that DLK, but not homologous kinases, is palmitoylated at a conserved site adjacent to its kinase domain. Using short-hairpin RNA knockdown/rescue, we find that palmitoylation is critical for DLK-dependent retrograde signaling in sensory axons. This functional importance is because of three novel cellular and molecular roles of palmitoylation, which targets DLK to trafficking vesicles, is required to assemble DLK signaling complexes and, unexpectedly, is essential for DLK’s kinase activity. By simultaneously controlling DLK localization, interactions, and activity, palmitoylation ensures that only vesicle-bound DLK is active in neurons. These findings explain how DLK specifically mediates nerve injury responses and reveal a novel cellular mechanism that ensures the specificity of neuronal kinase signaling.

Identification of ZDHHC14 as a novel human tumour suppressor gene

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase‐dependent apoptotic pathway and heterozygous knockout of ZDHHC14 decreased cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down‐regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.