Mechanisms and functions of protein S-acylation.

Over the past two decades, protein S-acylation (often referred to as S-palmitoylation) has emerged as an important regulator of vital signalling pathways. S-Acylation is a reversible post-translational modification that involves the attachment of a fatty acid to a protein. Maintenance of the equilibrium between protein S-acylation and deacylation has demonstrated profound effects on various cellular processes, including innate immunity, inflammation, glucose metabolism and fat metabolism, as well as on brain and heart function. This Review provides an overview of current understanding of S-acylation and deacylation enzymes, their spatiotemporal regulation by sophisticated multilayered mechanisms, and their influence on protein function, cellular processes and physiological pathways. Furthermore, we examine how disruptions in protein S-acylation are associated with a broad spectrum of diseases from cancer to autoinflammatory disorders and neurological conditions.

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.

Regulation of dendrite morphology and excitatory synapse formation by zDHHC15.

Protein palmitoylation is the most common post-translational lipid modification in the brain and is mediated by a family of 24 zDHHC enzymes. There has been growing interest in zDHHCs due to mounting evidence that these enzymes play key roles in the development and function of neuronal connections, and the fact that a number of zDHHCs have been associated with neurodevelopmental and neurodegenerative diseases. Loss-of-function variants in several zDHHCs, including zDHHC15, have been identified in patients with intellectual disabilities; however, the function of zDHHC15 in the brain has not been well studied. Here, we demonstrate that knocking down zDHHC15 in primary rat hippocampal cultures reduces dendritic outgrowth and arborization, as well as spine maturation. Moreover, knockdown of zDHHC15 reduces palmitoylation of PSD-95 and its trafficking into dendrites, resulting in an overall decrease in the density of excitatory synapses being formed onto mutant cells.

Consumption of palatable food primes food approach behavior by rapidly increasing synaptic density in the VTA.

In an environment with easy access to highly palatable and energy-dense food, food-related cues drive food-seeking regardless of satiety, an effect that can lead to obesity. The ventral tegmental area (VTA) and its mesolimbic projections are critical structures involved in the learning of environmental cues used to predict motivationally relevant outcomes. Priming effects of food-related advertising and consumption of palatable food can drive food intake. However, the mechanism by which this effect occurs, and whether these priming effects last days after consumption, is unknown. Here, we demonstrate that short-term consumption of palatable food can prime future food approach behaviors and food intake. This effect is mediated by the strengthening of excitatory synaptic transmission onto dopamine neurons that is initially offset by a transient increase in endocannabinoid tone, but lasts days after an initial 24-h exposure to sweetened high-fat food (SHF). This enhanced synaptic strength is mediated by a long-lasting increase in excitatory synaptic density onto VTA dopamine neurons. Administration of insulin into the VTA, which suppresses excitatory synaptic transmission onto dopamine neurons, can abolish food approach behaviors and food intake observed days after 24-h access to SHF. These results suggest that even a short-term exposure to palatable foods can drive future feeding behavior by "rewiring" mesolimbic dopamine neurons.

Cognitive flexibility and long-term depression (LTD) are impaired following ß-catenin stabilization in vivo.

The cadherin/ß-catenin adhesion complex is a key mediator of the bidirectional changes in synapse strength which are believed to underlie complex learning and memory. In the present study, we demonstrate that stabilization of ß-catenin in the hippocampus of adult mice results in significant impairments in cognitive flexibility and spatial reversal learning, including impaired extinction during the reversal phase of the Morris water maze and deficits in a delayed nonmatch to place T-maze task. In accordance with these deficits, ß-catenin stabilization was found to abolish long-term depression by stabilizing cadherin at the synaptic membrane and impairing AMPA receptor endocytosis, while leaving basal synaptic transmission and long-term potentiation unaffected. These results demonstrate that the ß-catenin/cadherin adhesion complex plays an important role in learning and memory and that aberrant increases in synaptic adhesion can have deleterious effects on cognitive function.

Use-dependent activation of neuronal Kv1.2 channel complexes.

In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as "prepulse potentiation," in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons.

Activity-dependent secretion of progranulin from synapses.

The secreted growth factor progranulin (PGRN) has been shown to be important for regulating neuronal survival and outgrowth, as well as synapse formation and function. Mutations in the PGRN gene that result in PGRN haploinsufficiency have been identified as a major cause of frontotemporal dementia (FTD). Here we demonstrate that PGRN is colocalized with dense-core vesicle markers and is co-transported with brain-derived neurotrophic factor (BDNF) within axons and dendrites of cultured hippocampal neurons in both anterograde and retrograde directions. We also show that PGRN is secreted in an activity-dependent manner from synaptic and extrasynaptic sites, and that the temporal profiles of secretion are distinct in axons and dendrites. Neuronal activity is also shown to increase the recruitment of PGRN to synapses and to enhance the density of PGRN clusters along axons. Finally, treatment of neurons with recombinant PGRN is shown to increase synapse density, while decreasing the size of the presynaptic compartment and specifically the number of synaptic vesicles per synapse. Together, this indicates that activity-dependent secretion of PGRN can regulate synapse number and structure.

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.

ß-Pix modulates actin-mediated recruitment of synaptic vesicles to synapses.

Presynaptic compartments are formed through the recruitment of preassembled clusters of proteins to points of cell-cell contact, however, the molecular mechanism(s) underlying this process remains unclear. We demonstrate that clusters of polymerized actin can recruit and maintain synaptic vesicles to discrete sites along the axon, and that cadherin/ß-catenin/scribble/ß-pix complexes play an important role in this event. Previous work has demonstrated that ß-catenin and scribble are important for the clustering of vesicles at synapses. We demonstrate that ß-pix, a Rac/Cdc42 guanine nucleotide exchange factor (GEF), forms a complex with cadherin, ß-catenin, and scribble at synapses and enhances localized actin polymerization in rat hippocampal neurons. In cells expressing ß-pix siRNA or dominant-negative ß-pix that lacks its GEF activity, actin polymerization at synapses is dramatically reduced, and synaptic vesicle localization is disrupted. This ß-pix phenotype can be rescued by cortactin overexpression, suggesting that ß-pix-mediated actin polymerization at synapses regulates vesicle localization.

Progranulin deficiency decreases gross neural connectivity but enhances transmission at individual synapses.

Frontotemporal dementia (FTD) has been linked to mutations in the progranulin gene (GRN) that lead to progranulin (PGRN) haploinsufficiency. Thus far, our understanding of the effects of PGRN depletion in the brain has been derived from investigation of gross pathology, and more detailed analyses of cellular function have been lacking. We report that knocking down PGRN levels in rat primary hippocampal cultures reduces neural connectivity by decreasing neuronal arborization and length as well as synapse density. Despite this, the number of synaptic vesicles per synapse and the frequency of mEPSCs are increased in PGRN knockdown cells, suggesting an increase in the probability of release at remaining synapses. Interestingly, we demonstrate that the number of vesicles per synapse is also increased in postmortem brain sections from FTD patients with PGRN haploinsufficiency, relative to controls. Our observations show that PGRN knockdown severely alters neuronal connectivity in vitro and that the synaptic vesicle phenotype observed in culture is consistent with that observed in the hippocampus of FTD patients.

N-cadherin and neuroligins cooperate to regulate synapse formation in hippocampal cultures.

Cadherins and neuroligins (NLs) represent two families of cell adhesion proteins that are essential for the establishment of synaptic connections in vitro; however, it remains unclear whether these proteins act in concert to regulate synapse density. Using a combination of overexpression and knockdown analyses in primary hippocampal neurons, we demonstrate that NL1 and N-cadherin promote the formation of glutamatergic synapses through a common functional pathway. Analysis of the spatial relationship between N-cadherin and NL1 indicates that in 14-day in vitro cultures, almost half of glutamatergic synapses are associated with both proteins, whereas only a subset of these synapses are associated with N-cadherin or NL1 alone. This suggests that NL1 and N-cadherin are spatially distributed in a manner that enables cooperation at synapses. In young cultures, N-cadherin clustering and its association with synaptic markers precede the clustering of NL1. Overexpression of N-cadherin at this time point enhances NL1 clustering and increases synapse density. Although N-cadherin is not sufficient to enhance NL1 clustering and synapse density in more mature cultures, knockdown of N-cadherin at later time points significantly attenuates the density of NL1 clusters and synapses. N-cadherin overexpression can partially rescue synapse loss in NL1 knockdown cells, possibly due to the ability of N-cadherin to recruit NL2 to glutamatergic synapses in these cells. We demonstrate that cadherins and NLs can act in concert to regulate synapse formation.

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.

Distinct but overlapping roles of LRRTM1 and LRRTM2 in developing and mature hippocampal circuits.

LRRTMs are postsynaptic cell adhesion proteins that have region-restricted expression in the brain. To determine their role in the molecular organization of synapses in vivo, we studied synapse development and plasticity in hippocampal neuronal circuits in mice lacking both Lrrtm1 and Lrrtm2. We found that LRRTM1 and LRRTM2 regulate the density and morphological integrity of excitatory synapses on CA1 pyramidal neurons in the developing brain but are not essential for these roles in the mature circuit. Further, they are required for long-term-potentiation in the CA3-CA1 pathway and the dentate gyrus, and for enduring fear memory in both the developing and mature brain. Our data show that LRRTM1 and LRRTM2 regulate synapse development and function in a cell-type and developmental-stage-specific manner, and thereby contribute to the fine-tuning of hippocampal circuit connectivity and plasticity.

BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin-beta-catenin interactions.

Neurons of the vertebrate central nervous system have the capacity to modify synapse number, morphology, and efficacy in response to activity. Some of these functions can be attributed to activity-induced synthesis and secretion of the neurotrophin brain-derived neurotrophic factor (BDNF); however, the molecular mechanisms by which BDNF mediates these events are still not well understood. Using time-lapse confocal analysis, we show that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles "splitting" away from synaptic sites. We demonstrate that BDNF's ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin-beta-catenin adhesion complexes that occurs after tyrosine phosphorylation of beta-catenin. Artificially maintaining cadherin-beta-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility, as well as the longer-term BDNF-mediated increase in synapse number. Together, this data demonstrates that the disruption of cadherin-beta-catenin complexes is an important molecular event through which BDNF increases synapse density in cultured hippocampal neurons.

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction.

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

Cadherins: actin with the cytoskeleton to form synapses.

Classic cadherins are calcium-dependent homophilic cell adhesion molecules that are enriched at synapses and thought to function in target recognition and adhesion at synaptic junctions. This brief review highlights evidence that cadherins and their associated catenins play a role in directing the development of pre- and postsynaptic specializations. In particular, the question of whether cadherin regulation of the actin cytoskeleton at discrete contact sites translates into the assembly of synaptic compartments will be explored.

Astrocytes drive cortical vasodilatory signaling by activating endothelial NMDA receptors.

Astrocytes express neurotransmitter receptors that serve as sensors of synaptic activity and initiate signals leading to activity-dependent local vasodilation and increases in blood flow. We previously showed that arteriolar vasodilation produced by activation of cortical astrocytes is dependent on endothelial nitric oxide synthase (eNOS) and endogenous agonists of N-methyl-D-aspartate (NMDA) receptors. Here, we tested the hypothesis that these effects are mediated by NMDA receptors expressed by brain endothelial cells. Primary endothelial cultures expressed NMDA receptor subunits and produced nitric oxide in response to co-agonists, glutamate and D-serine. In cerebral cortex in situ, immunoelectron microscopy revealed that endothelial cells express the GluN1 NMDA receptor subunit at basolateral membrane surfaces in an orientation suitable for receiving intercellular messengers from brain cells. In cortical slices, activation of astrocytes by two-photon flash photolysis of a caged Ca2+ compound or application of a metabotropic glutamate receptor agonist caused endothelial NO generation and local vasodilation. These effects were mitigated by NMDA receptor antagonists and conditional gene silencing of endothelial GluN1, indicating at least partial dependence on endothelial NMDA receptors. Our observations identify a novel astrocyte-endothelial vasodilatory signaling axis that could contribute to endothelium-dependent vasodilation in brain functional hyperemia.

The X-Linked Intellectual Disability Gene Zdhhc9 Is Essential for Dendrite Outgrowth and Inhibitory Synapse Formation.

Palmitoylation is a reversible post-translational lipid modification that facilitates vesicular transport and subcellular localization of modified proteins. This process is catalyzed by ZDHHC enzymes that are implicated in several neurological and neurodevelopmental disorders. Loss-of-function mutations in ZDHHC9 have been identified in patients with X-linked intellectual disability (XLID) and associated with increased epilepsy risk. Loss of Zdhhc9 function in hippocampal cultures leads to shorter dendritic arbors and fewer inhibitory synapses, altering the ratio of excitatory-to-inhibitory inputs formed onto Zdhhc9-deficient cells. While Zdhhc9 promotes dendrite outgrowth through the palmitoylation of the GTPase Ras, it promotes inhibitory synapse formation through the palmitoylation of another GTPase, TC10. Zdhhc9 knockout mice exhibit seizure-like activity together with increased frequency and amplitude of both spontaneous and miniature excitatory and inhibitory postsynaptic currents. These findings present a plausible mechanism for how the loss of ZDHHC9 function may contribute to XLID and epilepsy.