Plasma Membrane Of Dendrites Research Articles

MARCKS and PI(4,5)P2 reciprocally regulate actin-based dendritic spine morphology.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P2. We observed that the concentration of free PI(4,5)P2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P2.

5-HT1A and 5-HT2B receptor interaction and co-clustering regulate serotonergic neuron excitability

Many psychiatric diseases have been associated with serotonin (5-HT) neuron dysfunction. The firing of 5-HT neurons is known to be under 5-HT1A receptor-mediated autoinhibition, but functional consequences of coexpressed receptors are unknown. Using co-immunoprecipitation, BRET, confocal, and super-resolution microscopy in hippocampal and 5-HT neurons, we present evidence that 5-HT1A and 5-HT2B receptors can form heterodimers and co-cluster at the plasma membrane of dendrites. Selective agonist stimulation of coexpressed 5-HT1A and 5-HT2B receptors prevents 5-HT1A receptor internalization and increases 5-HT2B receptor membrane clustering. Current clamp recordings of 5-HT neurons revealed that 5-HT1A receptor stimulation of acute slices from mice lacking 5-HT2B receptors in 5-HT neurons increased their firing activity trough Ca2+-activated potassium channel inhibition compared to 5-HT neurons from control mice. This work supports the hypothesis that the relative expression of 5-HT1A and 5-HT2B receptors tunes the neuronal excitability of serotonergic neurons through potassium channel regulation.

Gas7 is a novel dendritic spine initiation factor.

Brain stores new information by modifying connections between neurons. When new information is learnt, a group of neurons gets activated and they are connected to each other via synapses. Dendritic spines are protrusions along neuronal dendrites where excitatory synapses are located. Dendritic spines are the first structures to protrude out from the dendrite to reach out to other neurons and establish a new connection. Thus, it is expected that neuronal activity enhances spine initiation. However, the molecular mechanisms linking neuronal activity to spine initiation are poorly known. Membrane binding BAR domain proteins are involved in spine initiation, but it is not known whether neuronal activity affects BAR domain proteins. Here, we used bicuculline treatment to activate excitatory neurons in organotypic hippocampal slices. With this experimental setup we identified F-BAR domain containing Growth Arrest-Specific Protein (Gas7) as a novel spine initiation factor responding to neuron activity. Upon bicuculline addition, Gas7 clustered to create spine initiation hotspots, thus increasing the probability to form new spines in activated neurons. Gas7 clustering and localization was dependent on PI3-kinase activity and intact F-BAR domain. Gas7 overexpression enhanced N-WASP localization to clusters as well as it increased the clustering of actin. Arp2/3 complex was required for normal Gas7-induced actin clustering. Gas7 overexpression increased and knockdown decreased spine density in hippocampal pyramidal neurons. Taken together, we suggest that Gas7 creates platforms under the dendritic plasma membrane which facilitate spine initiation. These platforms grow upon neuronal activation, increasing the probability of making new spines and new connections between active neurons. As such, we identified a novel molecular mechanism to link neuronal activity to the formation of new connections between neurons.Significance StatementWhen we learn something new, neurons change their connections to other neurons. We know that active neurons connect to each other, but we have poor understanding on the cellular or molecular mechanisms which link neuronal activity to the formation of new connections. Here we show how neuronal activation can facilitate formation of new connections to wire the firing neurons together.

Palmitoylation controls the stability of 190 kDa ankyrin-G in dendritic spines and is regulated by ZDHHC8 and lithium.

AnkG, encoded by the ANK3 gene, is a multifunctional scaffold protein with complex isoform expression: the 480 and 270 kDa isoforms have roles at the axon initial segment and node of Ranvier, whereas the 190 kDa isoform (AnkG-190) has an emerging role in the dendritic shaft and spine heads. All isoforms of AnkG undergo palmitoylation, a post-translational modification regulating protein attachment to lipid membranes. However, palmitoylation of AnkG-190 has not been investigated in dendritic spines. The ANK3 gene and altered expression of AnkG proteins are associated with a variety of neuropsychiatric and neurodevelopmental disorders including bipolar disorder and are implicated in the lithium response, a commonly used mood stabilizer for bipolar disorder patients, although the precise mechanisms involved are unknown. Here, we showed that Cys70 palmitoylation stabilizes the localization of AnkG-190 in spine heads and at dendritic plasma membrane nanodomains. Mutation of Cys70 impairs AnkG-190 function in dendritic spines and alters PSD-95 scaffolding. Interestingly, we find that lithium reduces AnkG-190 palmitoylation thereby increasing its mobility in dendritic spines. Finally, we demonstrate that the palmitoyl acyl transferase ZDHHC8, but not ZDHHC5, increases AnkG-190 stability in spine heads and is inhibited by lithium. Together, our data reveal that palmitoylation is critical for AnkG-190 localization and function and a potential ZDHHC8/AnkG-190 mechanism linking AnkG-190 mobility to the neuronal effects of lithium.

Domain and cell type-specific immunolocalisation of voltage-gated potassium channels in the mouse striatum

Diverse classes of voltage-gated potassium channels (Kv) are integral to the variety of electrical activity patterns that distinguish different classes of neurons in the brain. A feature of their heterogenous expression patterns is the highly precise manner in which specific cell types target their location within functionally specialised sub-cellular domains. Although Kv expression profiles in cortical brain regions are widely reported, their immunolocalisation in sub-cortical areas such as the striatum, and in associated diseases such as Parkinson’s disease (PD), remain less well described. Therefore, the broad aims of this study were to provide a high resolution immunolocalisation analysis of various Kv subtypes within the mouse striatum and assess their potential plasticity in a model of PD. Immunohistochemistry and confocal microscopy revealed that immunoreactivity for Kv1.1, 1.2 and 1.4 overlapped to varying degrees with excitatory and inhibitory axonal marker proteins suggesting these Kv subtypes are targeted to axons innervating striatal medium spiny neurons (MSNs). Immunoreactivity for Kv1.3 strongly overlapped with signal for mitochondrial marker proteins in MSN somata and dendrites. Kv1.5 immunoreactivity was expressed in parvalbumin-immunopositive neurons whereas Kv1.6 was located in cells immunopositive for microglia. Signal for Kv2.1 was concentrated on the somatic and proximal dendritic plasma membrane of MSNs, whilst immunoreactivity for Kv4.2 was targeted to their distal dendritic regions. Finally, striatal Kv2.1 expression, at both the mRNA and protein levels, was decreased in alpha-synuclein overexpressing mice, yet increased in alpha-synuclein knockout mice, compared to wild-type counterparts. The data indicate a variety of Kv expression patterns that are distinctive to the striatum and susceptible to pathology that mirrors PD. Furthermore, these findings advance our understanding of the molecular diversity of various striatal cell types, and potentially have implications for the homeostatic changes of MSN excitability during associated medical conditions such as PD.

Activity-dependent PI4P synthesis by PI4KIIIα regulates long-term synaptic potentiation

Phosphatidylinositol 4-phosphate (PI4P) is a low abundant phospholipid with important roles in lipid transport and membrane trafficking. However, little is known of its metabolism and function in neurons. Here, we investigate its subcellular distribution and functional roles in dendrites of rodent hippocampal neurons during resting state and long-term synaptic potentiation (LTP). We show that neural activity causes dynamic reversible changes in PI4P metabolism in dendrites. Upon LTP induction, PI4KIIIα, a type III phosphatidylinositol 4-kinase, localizes to the dendritic plasma membrane (PM) in a calcium-dependent manner and causes substantial increase in the levels of PI4P. Acute inhibition of PI4KIIIα activity abolishes trafficking of the AMPA-type glutamate receptor to the PM during LTP induction, and silencing of PI4KIIIα expression in the hippocampal CA1 region causes severe impairment of LTP and long-term memory. Collectively, our results identify an essential role for PI4KIIIα-dependent PI4P synthesis in synaptic plasticity of central nervous system neurons.

Endoplasmic reticulum proteins Meigo and Gp93 govern dendrite targeting by regulating Toll-6 localization

Neuronal target recognition is performed by numerous cell-surface transmembrane proteins. Correct folding of these proteins occurs in the endoplasmic reticulum (ER) lumen of the neuronal cells before being transported to the plasma membrane of axons or dendrites. Disturbance in this protein folding process in the ER leads to dysfunction of neuronal cell surface molecules, resulting in abnormal neuronal targeting. In this study, we report that the ER-resident protein Meigo in Drosophila, governs the dendrite targeting of olfactory projection neurons (PNs) along the mediolateral axis of the antennal lobe by regulating Toll-6 localization. Loss of Meigo causes Toll-6 mislocalization in the PNs and mediolateral dendrite targeting defects, which are suppressed by Toll-6 overexpression. Furthermore, we found that the ER-chaperone protein, Gp93, also regulates the mediolateral targeting of PN dendrites by localization of the Toll-6 protein. Gp93 overexpression in the PN homozygous for the meigo mutation, partially rescued the dendrite targeting defect, while meigo knockdown decreased Gp93 expression levels in cultured cells. These results indicate that the ER-proteins Meigo and Gp93 regulate dendrite targeting by attenuating the amount and localization of cell surface receptors, including Toll-6, implying the unexpected but active involvement of ER proteins in neural wiring.

Acute Delta 9-tetrahydrocannabinol administration differentially alters the hippocampal opioid system in adult female and male rats.

Our prior studies demonstrated that the rat hippocampal opioid system can undergo sex-specific adaptations to external stimuli that can influence opioid-associated learning processes. This opioid system extensively overlaps with the cannabinoid system. Moreover, acute administration of Δ9 Tetrahydrocannabinoid (THC), the primary psychoactive constituent of cannabis, can alter cognitive behaviors that involve the hippocampus. Here, we use light and electron microscopic immunocytochemical methods to examine the effects of acute THC (5mg/kg, i.p., 1h) on mossy fiber Leu-Enkephalin (LEnk) levels and the distribution and phosphorylation levels of delta and mu opioid receptors (DORs and MORs, respectively) in CA3 pyramidal cells and parvalbumin dentate hilar interneurons of adult female and male Sprague-Dawley rats. In females with elevated estrogen states (proestrus/estrus stage), acute THC altered the opioid system so that it resembled that seen in vehicle-injected females with low estrogen states (diestrus) and males: (1) mossy fiber LEnk levels in CA2/3a decreased; (2) phosphorylated-DOR levels in CA2/3a pyramidal cells increased; and (3) phosphorylated-MOR levels increased in most CA3b laminae. In males, acute THC resulted in the internalization of MORs in parvalbumin-containing interneuron dendrites which would decrease disinhibition of granule cells. In both sexes, acute THC redistributed DORs to the near plasma membrane of CA3 pyramidal cell dendrites, however, the dendritic region varied with sex. Additionally, acute THC also resulted in a sex-specific redistribution of DORs within CA3 pyramidal cell dendrites which could differentially promote synaptic plasticity and/or opioid-associated learning processes in both females and males.

Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein-membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

SummaryNeurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

Oxycodone injections not paired with conditioned place preference have little effect on the hippocampal opioid system in female and male rats.

Oxycodone (Oxy) conditioned place preference (CPP) in Sprague Dawley rats results in sex-specific alterations in hippocampal opioid circuits in a manner that facilitates opioid-associative learning processes, particularly in females. Here, we examined if Oxy (3mg/kg, I.P.) or saline (Sal) injections not paired with behavioral testing similarly affect the hippocampal opioid system. Sal-injected females compared to Sal-injected males had: (1) higher densities of cytoplasmic delta opioid receptors (DOR) in GABAergic hilar dendrites suggesting higher baseline reserve DOR pools and (2) elevated phosphorylated DOR levels, but lower phosphorylated mu opioid receptor (MOR) levels in CA3a suggesting that the baseline pools of activated opioid receptors vary in females and males. In contrast to CPP studies, Oxy-injections in the absence of behavioral tests resulted in few changes in the hippocampal opioid system in either females or males. Specifically, Oxy-injected males compared to Sal-injected males had fewer DORs near the plasma membrane of CA3 pyramidal cell dendrites and in CA3 dendritic spines contacted by mossy fibers, and lower pMOR levels in CA3a. Oxy-injected females compared to Sal-injected females had higher total DORs in GABAergic dendrites and lower total MORs in parvalbumin-containing dendrites. Thus, unlike Oxy CPP, Oxy-injections redistributed opioid receptors in hippocampal neurons in a manner that would either decrease (males) or not alter (females) excitability and plasticity processes. These results indicate that the majority of changes within hippocampal opioid circuits that would promote opioid-associative learning processes in both females and males do not occur with Oxy administration alone, and instead must be paired with CPP.

Palmitoylated Proteins in Dendritic Spine Remodeling.

Activity-responsive changes in the actin cytoskeleton are required for the biogenesis, motility, and remodeling of dendritic spines. These changes are governed by proteins that regulate the polymerization, depolymerization, bundling, and branching of actin filaments. Thus, processes that have been extensively characterized in the context of non-neuronal cell shape change and migration are also critical for learning and memory. In this review article, we highlight actin regulatory proteins that associate, at least transiently, with the dendritic plasma membrane. All of these proteins have been shown, either in directed studies or in high—throughput screens, to undergo palmitoylation, a potentially reversible, and stimulus-dependent cysteine modification. Palmitoylation increases the affinity of peripheral proteins for the membrane bilayer and contributes to their subcellular localization and recruitment to cholesterol-rich membrane microdomains.

AMPA receptors in the synapse turnover by monomer diffusion

The number and subunit compositions of AMPA receptors (AMPARs), hetero- or homotetramers composed of four subunits GluA1–4, in the synapse is carefully tuned to sustain basic synaptic activity. This enables stimulation-induced synaptic plasticity, which is central to learning and memory. The AMPAR tetramers have been widely believed to be stable from their formation in the endoplasmic reticulum until their proteolytic decomposition. However, by observing GluA1 and GluA2 at the level of single molecules, we find that the homo- and heterotetramers are metastable, instantaneously falling apart into monomers, dimers, or trimers (in 100 and 200 ms, respectively), which readily form tetramers again. In the dendritic plasma membrane, GluA1 and GluA2 monomers and dimers are far more mobile than tetramers and enter and exit from the synaptic regions. We conclude that AMPAR turnover by lateral diffusion, essential for sustaining synaptic function, is largely done by monomers of AMPAR subunits, rather than preformed tetramers.

Plasma Membrane Affiliated AMPA GluA1 in Estrogen Receptor β-containing Paraventricular Hypothalamic Neurons Increases Following Hypertension in a Mouse Model of Post-menopause

Sex and ovarian function contribute to hypertension susceptibility, however, the mechanisms are not well understood. Prior studies show that estrogens and neurogenic factors, including hypothalamic glutamatergic NMDA receptor plasticity, play significant roles in rodent hypertension. Here, we investigated the role of sex and ovarian failure on AMPA receptor plasticity in estrogen-sensitive paraventricular nucleus (PVN) neurons in naïve and angiotensin II (AngII) infused male and female mice and female mice at early and late stages of accelerated ovarian failure (AOF). High-resolution electron microscopy was used to assess the subcellular distribution of AMPA GluA1 in age-matched male and female estrogen receptor beta (ERβ) enhanced green fluorescent protein (EGFP) reporter mice as well as female ERβ-EGFP mice treated with 4-vinylcyclohexene diepoxide. In the absence of AngII, female mice at a late stage of AOF displayed higher levels of GluA1 on the plasma membrane, indicative of functional protein, in ERβ-expressing PVN dendrites when compared to male, naïve female and early stage AOF mice. Following slow-pressor AngII infusion, males, as well as early and late stage AOF females had elevated blood pressure. Significantly, only late stage-AOF female mice infused with AngII had an increase in GluA1 near the plasma membrane in dendrites of ERβ-expressing PVN neurons. In contrast, prior studies reported that plasmalemmal NMDA GluN1 increased in ERβ-expressing PVN dendrites in males and early, but not late stage AOF females. Together, these findings reveal that early and late stage AOF female mice display unique molecular signatures of long-lasting synaptic strength prior to, and following hypertension.

Group I metabotropic glutamate receptors in the primate motor thalamus: subsynaptic association with cortical and sub-cortical glutamatergic afferents.

Preclinical evidence indicates that mGluR5 is a potential therapeutic target for Parkinson's disease and L-DOPA-induced dyskinesia. However, the mechanisms through which these therapeutic benefits are mediated remain poorly understood. Although the regulatory role of mGluR5 on glutamatergic transmission has been examined in various basal ganglia nuclei, very little is known about the localization and function of mGluR5 in the ventral motor and intralaminar thalamic nuclei, the main targets of basal ganglia output in mammals. Thus, we used immuno-electron microscopy to map the cellular and subcellular localization of group I mGluRs (mGluR1a and mGluR5) in the ventral motor and caudal intralaminar thalamic nuclei in rhesus monkeys. Furthermore, using double immuno-electron microscopy, we examined the subsynaptic localization of mGluR5 in relation to cortical and sub-cortical glutamatergic afferents. Four major conclusions can be drawn from these data. First, mGluR1a and mGluR5 are expressed postsynaptically on the plasma membrane of dendrites of projection neurons and GABAergic interneurons in the basal ganglia- and cerebellar-receiving regions of the ventral motor thalamus and in CM. Second, the plasma membrane-bound mGluR5 immunoreactivity is preferentially expressed perisynaptically at the edges of cortical and sub-cortical glutamatergic afferents. Third, the mGluR5 immunoreactivity is more strongly expressed in the lateral than the medial tiers of CM, suggesting a preferential association with thalamocortical over thalamostriatal neurons in the primate CM. Overall, mGluR5 is located to subserve powerful modulatory role of cortical and subcortical glutamatergic transmission in the primate ventral motor thalamus and CM.

Ultrastructural localization of DREADDs in monkeys.

Designer receptors exclusively activated by designer drugs (DREADDs) are extensively used to modulate neuronal activity in rodents, but their use in primates remains limited. An essential need that remains is the demonstration that DREADDs are efficiently expressed on the plasma membrane of primate neurons. To address this issue, electron microscopy immunogold was used to determine the subcellular localization of the AAV vector-induced DREADDs hM4Di and hM3Dq fused to different tags in various brain areas of rhesus monkeys and mice. When hM4Di was fused to mCherry, the immunogold labelling was mostly confined to the intracellular space, and poorly expressed at the plasma membrane in monkey dendrites. In contrast, the hM4Di-mCherry labelling was mostly localized to the dendritic plasma membrane in mouse neurons, suggesting species differences in the plasma membrane expression of these exogenous proteins. The lack of hM4Di plasma membrane expression may limit the functional effects of systemic administration of DREADD-actuators in monkey neurons. Removing the mCherry and fusing of hM4Di with the haemagglutinin (HA) tag resulted in strong neuronal plasma membrane immunogold labelling in both monkeys and mice neurons. Finally, hM3Dq-mCherry was expressed mostly at the plasma membrane in monkey neurons, indicating that the fusion of mCherry with hM3Dq does not hamper membrane incorporation of this specific DREADD. Our results suggest that the pattern of ultrastructural expression of DREADDs in monkey neurons depends on the DREADD/tag combination. Therefore, a preliminary characterization of plasma membrane expression of specific DREADD/tag combinations is recommended when using chemogenetic approaches in primates.

Distribution of densin in neurons.

Densin is a scaffold protein known to associate with key elements of neuronal signaling. The present study examines the distribution of densin at the ultrastructural level in order to reveal potential sites that can support specific interactions of densin. Immunogold electron microscopy on hippocampal cultures shows intense labeling for densin at postsynaptic densities (PSDs), but also some labeling at extrasynaptic plasma membranes of soma and dendrites and endoplasmic reticulum. At the PSD, the median distance of label from the postsynaptic membrane was ~27 nm, with the majority of label (90%) confined within 40 nm from the postsynaptic membrane, indicating predominant localization of densin at the PSD core. Depolarization (90 mM K+ for 2 min) promoted a slight shift of densin label within the PSD complex resulting in 77% of label remaining within 40 nm from the postsynaptic membrane. Densin molecules firmly embedded within the PSD may target a minor pool of CaMKII to substrates at the PSD core.

L-Type Voltage-Gated Ca2+ Channels Regulate Synaptic Activity-Triggered Recycling Endosome Fusion in Neuronal Dendrites

The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs) with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca2+ from distinct sources. NMDA receptor Ca2+ initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca2+ channels (L-VGCCs) regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion.

Extracellular Remodeling by Lysosomes: An Inside-Out Mechanism of Spine Plasticity

In this issue of Neuron, Padamsey etal. (2017) demonstrate that neuronal activity triggers lysosomal fusion with the plasma membrane in dendrites. Quite unexpectedly, the lysosomes release signaling molecules that induce extracellular matrix remodeling to support long-lasting dendritic spine plasticity.

Identification of PSD-95 Depalmitoylating Enzymes.

Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. Protein palmitoylation, the most common lipid modification, dynamically regulates neuronal protein localization and function. Its unique reversibility is conferred by DHHC-type palmitoyl acyl transferases (palmitoylating enzymes) and still controversial palmitoyl-protein thioesterases (depalmitoylating enzymes). Here, we identified the membrane-anchored serine hydrolases, ABHD17A, 17B, and 17C, as the physiological PSD-95 depalmitoylating enzymes that regulate PSD-95 palmitoylation cycles in neurons. This study describes the first direct evidence for the neuronal depalmitoylating enzyme and provides a new aspect of the dynamic regulatory mechanisms of synaptic development and synaptic plasticity. In addition, our established APEGS assay, which provides unbiased and quantitative information about the palmitoylation state and dynamics, revealed the distinct regulatory mechanisms for synaptic palmitoylation.