Activity-dependent post-translational regulation of palmitoylating and depalmitoylating enzymes in the hippocampus.

Activity-induced changes in protein palmitoylation can regulate the plasticity of synaptic connections, critically impacting learning and memory. Palmitoylation is a reversible post-translational modification regulated by both palmitoyl-acyl transferases that mediate palmitoylation and palmitoyl thioesterases that depalmitoylate proteins. However, it is not clear how fluctuations in synaptic activity can mediate the dynamic palmitoylation of neuronal proteins. Using primary hippocampal cultures, we demonstrate that synaptic activity does not impact the transcription of palmitoylating and depalmitoylating enzymes, changes in thioesterase activity, or post-translational modification of the depalmitoylating enzymes of the ABHD17 family and APT2 (also known as LYPLA2). In contrast, synaptic activity does mediate post-translational modification of the palmitoylating enzymes ZDHHC2, ZDHHC5 and ZDHHC9 (but not ZDHHC8) to influence protein-protein interactions, enzyme stability and enzyme function. Post-translational modifications of the ZDHHC enzymes were also observed in the hippocampus following fear conditioning. Taken together, our findings demonstrate that signaling events activated by synaptic activity largely impact activity of the ZDHHC family of palmitoyl-acyl transferases with less influence on the activity of palmitoyl thioesterases.

Regulation of hippocampal excitatory synapses by the Zdhhc5 palmitoyl acyltransferase.

Palmitoylation is the most common post-translational lipid modification in the brain; however, the role of palmitoylation and palmitoylating enzymes in the nervous system remains elusive. One of these enzymes, Zdhhc5, has previously been shown to regulate synapse plasticity. Here, we report that Zdhhc5 is also essential for the formation of excitatory, but not inhibitory, synapses both in vitro and in vivo. We demonstrate in vitro that this is dependent on the enzymatic activity of Zdhhc5, its localization at the plasma membrane and its C-terminal domain, which has been shown to be truncated in a patient with schizophrenia. Loss of Zdhhc5 in mice results in a decrease in the density of excitatory hippocampal synapses accompanied by alterations in membrane capacitance and synaptic currents, consistent with an overall decrease in spine number and silent synapses. These findings reveal an important role for Zdhhc5 in the formation and/or maintenance of excitatory synapses.

Activity-dependent regulation of NMDA receptors in substantia nigra dopaminergic neurones.

N-Methyl-d-aspartate receptors (NMDARs) are Ca(2+)-permeable glutamate receptors that play a critical role in synaptic plasticity and promoting cell survival. However, overactive NMDARs can trigger cell death signalling pathways and have been implicated in substantia nigra pars compacta (SNc) pathology in Parkinson's disease. Calcium ion influx through NMDARs recruits Ca(2+)-dependent proteins that can regulate NMDAR activity. The surface density of NMDARs can also be regulated dynamically in response to receptor activity via Ca(2+)-independent mechanisms. We have investigated the activity-dependent regulation of NMDARs in SNc dopaminergic neurones. Repeated whole-cell agonist applications resulted in a decline in the amplitude of NMDAR currents (current run-down) that was use dependent and not readily reversible. Run-down was reduced by increasing intracellular Ca(2+) buffering or by reducing Ca(2+) influx but did not appear to be mediated by the same regulatory proteins that cause Ca(2+)-dependent run-down in hippocampal neurones. The NMDAR current run-down may be mediated in part by a Ca(2+)-independent mechanism, because intracellular dialysis with a dynamin-inhibitory peptide reduced run-down, suggesting a role for clathrin-mediated endocytosis in the regulation of the surface density of receptors. Synaptic NMDARs were also subject to current run-down during repeated low-frequency synaptic stimulation in a Ca(2+)-dependent but dynamin-independent manner. Thus, we report, for the first time, regulation of NMDARs in SNc dopaminergic neurones by changes in intracellular Ca(2+) at both synaptic and extrasynaptic sites and provide evidence for activity-dependent changes in receptor trafficking. These mechanisms may contribute to intracellular Ca(2+) homeostasis in dopaminergic neurones by limiting Ca(2+) influx through the NMDAR.

CellPalmSeq: A curated RNAseq database of palmitoylating and de-palmitoylating enzyme expression in human cell types and laboratory cell lines.

The reversible lipid modification protein S-palmitoylation can dynamically modify the localization, diffusion, function, conformation and physical interactions of substrate proteins. Dysregulated S-palmitoylation is associated with a multitude of human diseases including brain and metabolic disorders, viral infection and cancer. However, the diverse expression patterns of the genes that regulate palmitoylation in the broad range of human cell types are currently unexplored, and their expression in commonly used cell lines that are the workhorse of basic and preclinical research are often overlooked when studying palmitoylation dependent processes. We therefore created CellPalmSeq (https://cellpalmseq.med.ubc.ca), a curated RNAseq database and interactive webtool for visualization of the expression patterns of the genes that regulate palmitoylation across human single cell types, bulk tissue, cancer cell lines and commonly used laboratory non-human cell lines. This resource will allow exploration of these expression patterns, revealing important insights into cellular physiology and disease, and will aid with cell line selection and the interpretation of results when studying important cellular processes that depend on protein S-palmitoylation.

Exploring the expression patterns of palmitoylating and de-palmitoylating enzymes in the mouse brain using the curated RNA-seq database BrainPalmSeq.

Protein S-palmitoylation is a reversible post-translational lipid modification that plays a critical role in neuronal development and plasticity, while dysregulated S-palmitoylation underlies a number of severe neurological disorders. Dynamic S-palmitoylation is regulated by a large family of ZDHHC palmitoylating enzymes, their accessory proteins, and a small number of known de-palmitoylating enzymes. Here, we curated and analyzed expression data for the proteins that regulate S-palmitoylation from publicly available RNAseq datasets, providing a comprehensive overview of their distribution in the mouse nervous system. We developed a web-tool that enables interactive visualization of the expression patterns for these proteins in the nervous system (http://brainpalmseq.med.ubc.ca/), and explored this resource to find region and cell-type specific expression patterns that give insight into the function of palmitoylating and de-palmitoylating enzymes in the brain and neurological disorders. We found coordinated expression of ZDHHC enzymes with their accessory proteins, de-palmitoylating enzymes and other brain-expressed genes that included an enrichment of S-palmitoylation substrates. Finally, we utilized ZDHHC expression patterns to predict and validate palmitoylating enzyme-substrate interactions.

Synaptic activity-dependent changes in the hippocampal palmitoylome.

Dynamic protein S-palmitoylation is critical for neuronal function, development, and synaptic plasticity. Synaptic activity-dependent changes in palmitoylation have been reported for a small number of proteins. Here, we characterized the palmitoylome in the hippocampi of male mice before and after context-dependent fear conditioning. Of the 121 differentially palmitoylated proteins identified, just over half were synaptic proteins, whereas others were associated with metabolic functions, cytoskeletal organization, and signal transduction. The synapse-associated proteins generally exhibited increased palmitoylation after fear conditioning. In contrast, most of the proteins that exhibited decreased palmitoylation were associated with metabolic processes. Similar results were seen in cultured rat hippocampal neurons in response to chemically induced long-term potentiation. Furthermore, we found that the palmitoylation of one of the synaptic proteins, plasticity-related gene-1 (PRG-1), also known as lipid phosphate phosphatase-related protein type 4 (LPPR4), was important for synaptic activity-induced insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynaptic membrane. The findings identify proteins whose dynamic palmitoylation may regulate their role in synaptic plasticity, learning, and memory.

Synapse-to-Nucleus Communication through NFAT Is Mediated by L-type Ca2+ Channel Ca2+ Spike Propagation to the Soma.

Long-term information storage in the brain requires continual modification of the neuronal transcriptome. Synaptic inputs located hundreds of micrometers from the nucleus can regulate gene transcription, requiring high-fidelity, long-range signaling from synapses in dendrites to the nucleus in the cell soma. Here, we describe a synapse-to-nucleus signaling mechanism for the activity-dependent transcription factor NFAT. NMDA receptors activated on distal dendrites were found to initiate L-type Ca2+ channel (LTCC) spikes that quickly propagated the length of the dendrite to the soma. Surprisingly, LTCC propagation did not require voltage-gated Na+ channels or back-propagating action potentials. NFAT nuclear recruitment and transcriptional activation only occurred when LTCC spikes invaded the somatic compartment, and the degree of NFAT activation correlated with the number of somatic LTCC Ca2+ spikes. Together, these data support a model for synapse to nucleus communication where NFAT integrates somatic LTCC Ca2+ spikes to alter transcription during periods of heightened neuronal activity.

Potential for therapeutic targeting of AKAP signaling complexes in nervous system disorders.

A common feature of neurological and neuropsychiatric disorders is a breakdown in the integrity of intracellular signal transduction pathways. Dysregulation of ion channels and receptors in the cell membrane and the enzymatic mediators that link them to intracellular effectors can lead to synaptic dysfunction and neuronal death. However, therapeutic targeting of these ubiquitous signaling elements can lead to off-target side effects due to their widespread expression in multiple systems of the body. A-kinase anchoring proteins (AKAPs) are multivalent scaffolding proteins that compartmentalize a diverse range of receptor and effector proteins to streamline signaling within nanodomain signalosomes. A number of essential neurological processes are known to critically depend on AKAP-directed signaling and an understanding of the role AKAPs play in nervous system disorders has emerged in recent years. Selective targeting of AKAP protein-protein interactions may be a means to uncouple pathologically active signaling pathways in neurological disorders with a greater degree of specificity. In this review we will discuss the role of AKAPs in both regulating normal nervous system function and dysfunction associated with disease, and the potential for therapeutic targeting of AKAP signaling complexes.

STIM1 Ca2+ Sensor Control of L-type Ca2+-Channel-Dependent Dendritic Spine Structural Plasticity and Nuclear Signaling.

Potentiation of synaptic strength relies on postsynaptic Ca2+ signals, modification of dendritic spine structure, and changes in gene expression. One Ca2+ signaling pathway supporting these processes routes through L-type Ca2+ channels (LTCC), whose activity is subject to tuning by multiple mechanisms. Here, we show in hippocampal neurons that LTCC inhibition by the endoplasmic reticulum (ER) Ca2+ sensor, stromal interaction molecule 1 (STIM1), is engaged by the neurotransmitter glutamate, resulting in regulation of spine ER structure and nuclear signaling by the NFATc3 transcription factor. In this mechanism, depolarization by glutamate activates LTCC Ca2+ influx, releases Ca2+ from the ER, and consequently drives STIM1 aggregation and an inhibitory interaction with LTCCs that increases spine ER content but decreases NFATc3 nuclear translocation. These findings of negative feedback control of LTCC signaling by STIM1 reveal interplay between Ca2+ influx and release from stores that controls both postsynaptic structural plasticity and downstream nuclear signaling.