Simple Method for the Preparation of Postsynaptic Density Fraction from Mouse Brain.

Postsynaptic density (PSD) is a morphologically and functionally specialized postsynaptic membrane structure of excitatory synapses. It contains hundreds of proteins such as neurotransmitter receptors, adhesion molecules, cytoskeletal proteins, and signaling enzymes. The study of the molecular architecture of the PSD is one of the most intriguing issues in neuroscience research. The isolation of the PSD from the brain of an animal is necessary for subsequent biochemical and morphological analyses. Many laboratories have developed methods to isolate PSD from the animal brain. In this chapter, we present a simple method to isolate PSD from the mouse brain using sucrose density gradient-based purification of synaptosomes followed by detergent extraction.

A concerted neuron-astrocyte program declines in ageing and schizophrenia.

Human brains vary across people and over time; such variation is not yet understood in cellular terms. Here we describe a relationship between people's cortical neurons and cortical astrocytes. We used single-nucleus RNA sequencing to analyse the prefrontal cortex of 191 human donors aged 22-97 years, including healthy individuals and people with schizophrenia. Latent-factor analysis of these data revealed that, in people whose cortical neurons more strongly expressed genes encoding synaptic components, cortical astrocytes more strongly expressed distinct genes with synaptic functions and genes for synthesizing cholesterol, an astrocyte-supplied component of synaptic membranes. We call this relationship the synaptic neuron and astrocyte program (SNAP). In schizophrenia and ageing-two conditions that involve declines in cognitive flexibility and plasticity1,2-cells divested from SNAP: astrocytes, glutamatergic (excitatory) neurons and GABAergic (inhibitory) neurons all showed reduced SNAP expression to corresponding degrees. The distinct astrocytic and neuronal components of SNAP both involved genes in which genetic risk factors for schizophrenia were strongly concentrated. SNAP, which varies quantitatively even among healthy people of similar age, may underlie many aspects of normal human interindividual differences and may be an important point of convergence for multiple kinds of pathophysiology.

FAM81A is a postsynaptic protein that regulates the condensation of postsynaptic proteins via liquid-liquid phase separation.

Proteome analyses of the postsynaptic density (PSD), a proteinaceous specialization beneath the postsynaptic membrane of excitatory synapses, have identified several thousands of proteins. While proteins with predictable functions have been well studied, functionally uncharacterized proteins are mostly overlooked. In this study, we conducted a comprehensive meta-analysis of 35 PSD proteome datasets, encompassing a total of 5,869 proteins. Employing a ranking methodology, we identified 97 proteins that remain inadequately characterized. From this selection, we focused our detailed analysis on the highest-ranked protein, FAM81A. FAM81A interacts with PSD proteins, including PSD-95, SynGAP, and NMDA receptors, and promotes liquid-liquid phase separation of those proteins in cultured cells or in vitro. Down-regulation of FAM81A in cultured neurons causes a decrease in the size of PSD-95 puncta and the frequency of neuronal firing. Our findings suggest that FAM81A plays a crucial role in facilitating the interaction and assembly of proteins within the PSD, and its presence is important for maintaining normal synaptic function. Additionally, our methodology underscores the necessity for further characterization of numerous synaptic proteins that still lack comprehensive understanding.

Phospholipase Modulation of Synaptic Membrane Landscape: Driving Force Behind Memory Formation?

The synapse is the communication unit of the brain, linking billions of neurons through trillions of synaptic connections. The lipid landscape of the synaptic membrane underpins neurotransmitter release through the exocytic fusion of neurotransmitter-containing vesicles, endocytic recycling of these synaptic vesicles, and the postsynaptic response following binding of the neurotransmitter to specialized receptors. How the connected brain can learn and acquire memories through synaptic plasticity is unresolved. Phospholipases, and especially the phospholipase A1 isoform DDHD2, have recently been shown to play a critical role in memory acquisition through the generation of saturated free fatty acids such as myristic and palmitic acids. This emerging synaptic plasticity pathway suggests that phospholipases cannot only respond to synaptic activity by altering the phospholipid landscape but also contribute to the establishment of long-term memories in our brain.

cis-B7:CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity.

B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on T cells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ T cells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ T cell survival, migration, and cytokine production. In mouse tumor models, loss of T cell-intrinsic cis-B7:CD28 interactions decreased intratumoral T cells and accelerated tumor growth. Thus, B7 ligands on CD8+ T cells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.

Brief synaptic inhibition persistently interrupts firing of fast-spiking interneurons.

Neurons perform input-output operations that integrate synaptic inputs with intrinsic electrical properties; these operations are generally constrained by the brevity of synaptic events. Here, we report that sustained firing of CA1 hippocampal fast-spiking parvalbumin-expressing interneurons (PV-INs) can be persistently interrupted for several hundred milliseconds following brief GABAAR-mediated inhibition in vitro and in vivo. A single presynaptic neuron could interrupt PV-IN firing, occasionally with a single action potential (AP), and reliably with AP bursts. Experiments and computational modeling reveal that the persistent interruption of firing maintains neurons in a depolarized, quiescent state through a cell-autonomous mechanism. Interrupted PV-INs are strikingly responsive to Schaffer collateral inputs. The persistent interruption of firing provides a disinhibitory circuit mechanism favoring spike generation in CA1 pyramidal cells. Overall, our results demonstrate that neuronal silencing can far outlast brief synaptic inhibition owing to the well-tuned interplay between neurotransmitter release and postsynaptic membrane dynamics, a phenomenon impacting microcircuit function.

Turnover of synaptic adhesion molecules.

Molecular interactions between pre- and postsynaptic membranes play critical roles during the development, function and maintenance of synapses. Synaptic interactions are mediated by cell surface receptors that may be held in place by trans-synaptic adhesion or intracellular binding to membrane-associated scaffolding and signaling complexes. Despite their role in stabilizing synaptic contacts, synaptic adhesion molecules undergo turnover and degradation during all stages of a neuron's life. Here we review current knowledge about membrane trafficking mechanisms that regulate turnover of synaptic adhesion molecules and the functional significance of turnover for synapse development and function. Based on recent proteomics, genetics and imaging studies, synaptic adhesion molecules exhibit remarkably high turnover rates compared to other synaptic proteins. Degradation occurs predominantly via endolysosomal mechanisms, with little evidence for roles of proteasomal or autophagic degradation. Basal turnover occurs both during synaptic development and maintenance. Neuronal activity typically stabilizes synaptic adhesion molecules while downregulating neurotransmitter receptors based on turnover. In conclusion, constitutive turnover of synaptic adhesion molecules is not a necessarily destabilizing factor, but a basis for the dynamic regulation of trans-synaptic interactions during synapse formation and maintenance.

Isolation of Neuronal Synaptic Membranes by Sucrose Gradient Centrifugation.

Sucrose gradient centrifugation is a very useful technique for isolating specific membrane types based on their size and density. This is especially useful for detecting fatty acids and lipid molecules that are targeted to specialized membranes. Without fractionation, these types of molecules could be below the levels of detection after being diluted out by the more abundant lipid molecules with a more ubiquitous distribution throughout the various cell membranes. Isolation of specific membrane types where these lipids are concentrated allows for their detection and analysis. We describe herein our synaptic membrane isolation protocol that produces excellent yield and clear resolution of five major membrane fractions from a starting neural tissue homogenate: P1 (nuclear), P2 (cytoskeletal), P3 (neurosynaptosomal), PSD (post-synaptic densities), and SV (synaptic vesicle).

The Role of Membrane Affinity and Binding Modes in Alpha-Synuclein Regulation of Vesicle Release and Trafficking.

Alpha-synuclein is a presynaptic protein linked to Parkinson's disease with a poorly characterized physiological role in regulating the synaptic vesicle cycle. Using RBL-2H3 cells as a model system, we earlier reported that wild-type alpha-synuclein can act as both an inhibitor and a potentiator of stimulated exocytosis in a concentration-dependent manner. The inhibitory function is constitutive and depends on membrane binding by the helix-2 region of the lipid-binding domain, while potentiation becomes apparent only at high concentrations. Using structural and functional characterization of conformationally selective mutants via a combination of spectroscopic and cellular assays, we show here that binding affinity for isolated vesicles similar in size to synaptic vesicles is a primary determinant of alpha-synuclein-mediated potentiation of vesicle release. Inhibition of release is sensitive to changes in the region linking the helix-1 and helix-2 regions of the N-terminal lipid-binding domain and may require some degree of coupling between these regions. Potentiation of release likely occurs as a result of alpha-synuclein interactions with undocked vesicles isolated away from the active zone in internal pools. Consistent with this, we observe that alpha-synuclein can disperse vesicles from in vitro clusters organized by condensates of the presynaptic protein synapsin-1.

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain.

Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.

Microtransplantation of Synaptic Membranes to Reactivate Human Synaptic Receptors for Functional Studies.

Excitatory and inhibitory ionotropic receptors are the major gates of ion fluxes that determine the activity of synapses during physiological neuronal communication. Therefore, alterations in their abundance, function, and relationships with other synaptic elements have been observed as a major correlate of alterations in brain function and cognitive impairment in neurodegenerative diseases and mental disorders. Understanding how the function of excitatory and inhibitory synaptic receptors is altered by disease is of critical importance for the development of effective therapies. To gain disease-relevant information, it is important to record the electrical activity of neurotransmitter receptors that remain functional in the diseased human brain. So far this is the closest approach to assess pathological alterations in receptors' function. In this work, a methodology is presented to perform microtransplantation of synaptic membranes, which consists of reactivating synaptic membranes from snap frozen human brain tissue containing human receptors, by its injection and posterior fusion into the membrane of Xenopus laevis oocytes. The protocol also provides the methodological strategy to obtain consistent and reliable responses of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and γ-aminobutyric acid (GABA) receptors, as well as novel detailed methods that are used for normalization and rigorous data analysis.

Local accumulation times in a diffusion-trapping model of receptor dynamics at proximal axodendritic synapses.

The lateral diffusion and trapping of neurotransmitter receptors within the postsynaptic membrane of a neuron play a key role in determining synaptic strength and plasticity. Trapping is mediated by the reversible binding of receptors to scaffolding proteins (slots) within a synapse. In this paper we introduce a method for analyzing the transient dynamics of proximal axodendritic synapses in a diffusion-trapping model of receptor trafficking. Given a population of spatially distributed synapses, each of which has a fixed number of slots, we calculate the rate of relaxation to the steady-state distribution of bound slots (synaptic weights) in terms of a set of local accumulation times. Assuming that the rates of exocytosis and endocytosis are sufficiently slow, we show that the steady-state synaptic weights are independent of each other (purely local). On the other hand, the local accumulation time of a given synapse depends on the number of slots and the spatial location of all the synapses, indicating a form of transient heterosynaptic plasticity. This suggests that local accumulation time measurements could provide useful information regarding the distribution of synaptic weights within a dendrite.

Association of a newly identified lncRNA LNC_000280 with the formation of acetylcholine receptor clusters in vitro.

Peripheral nerve injury (PNI) can disintegrate acetylcholine receptor (AChR) clusters in the postsynaptic membrane. In our previous research, lncRNAs that were differentially expressed in the whole transcriptome sequencing of denervated muscle atrophy after PNI were screened. By utilizing Gene Ontology (GO) analysis and protein-protein interaction (PPI) networks, a novel lncRNA LNC_000280 was predicted to be associated with neuromuscular junction (NMJ). The myotubes were used to assess the connection between LNC_000280 and AChR cluster formation in vitro by overexpression and knockdown of LNC_000280 in the C2C12 cell line. Our findings demonstrated that the overexpression of LNC_000280 repressed the gene expression and protein level of AChR subunits in myotubes and further reduced the area of AChR aggregates on the cell membrane. In contrast, the knockdown of LNC_000280 brought about opposite results. In addition, the transcriptional level of Sorbs2 changed inversely with the quantity change of LNC_000280. In conclusion, LNC_000280 may associate with the formation of AChR clusters.

Genetic overlap between Alzheimer's disease and blood lipid levels.

Late-onset Alzheimer's disease (AD) has a significant genetic component, but the molecular mechanisms through which genetic risk factors contribute to AD pathogenesis are unclear. We screened for genetic sharing between AD and the blood levels of 615 metabolites to elucidate how the polygenic architecture of AD affects metabolomic profiles. We retrieved summary statistics from genome-wide association studies of AD and the metabolite blood levels and assessed for shared genetic etiology, using a polygenic risk score-based approach. For the blood levels of 31 metabolites, all of which were lipids, we identified and replicated genetic sharing with AD. We also found a positive genetic concordance - implying that genetic risk factors for AD are associated with higher blood levels - for 16 of the 31 replicated metabolites. In the brain, lipids and their intermediate metabolites have essential structural and functional roles, such as forming and dynamically regulating synaptic membranes. Our results imply that genetic risk factors for AD affect lipid levels, which may be leveraged to develop novel treatment strategies for AD.

Effects of Zinc, Mercury, or Lead on [3H]MK-801 and [3H]Fluorowillardiine Binding to Rat Synaptic Membranes.

Glutamate (Glu) is considered the most important excitatory amino acid neurotransmitter in the mammalian Central Nervous System. Zinc (Zn) is co-released with Glu during synaptic transmission and interacts with Glutamate receptors and transporters. We performed binding experiments using [3H]MK-801 (NMDA), and [3H]Fluorowillardine (AMPA) as ligands to study Zn-Glutamate interactions in rat cortical synaptic membranes. We also examined the effects of mercury and lead on NMDA or AMPA receptors. Zinc at 1 nM, significantly potentiates [3H]MK-801 binding. Lead inhibits [3H]MK-801 binding at micromolar concentrations. At millimolar concentrations, Hg also has a significant inhibitory effect. These effects are not reversed by Zn (1 nM). Zinc displaces the [3H]FW binding curve to the right. Lead (nM) and Hg (µM) inhibit [3H]FW binding. At certain concentrations, Zn reverses the effects of these metals on [3H]FW binding. These specific interactions serve to clarify the role of Zn, Hg, and Pb in physiological and pathological conditions.

Postsynaptic cleft density changes with combined exercise protocols in an experimental model of muscular hypertrophy.

The vertical ladder-based protocols contribute to the NMJ junction's adaptations, and when combined with and without load, can be potentiated. The present study aimed to investigate postsynaptic regions of the biceps brachii muscle in adult male Wistar rats submitted to different vertical ladder-based protocols (Sedentary - S; Climbing - C; Climbing with Load - LC and Combined Climbing - CC). The protocols (C, LC, CC) were performed in 24 sessions, 3 x/week, for 8 weeks. The myofibrillar ATPase analysis showed an increase in cross-sectional area (CSA) of the muscle fibers Type I in all trained Groups; Type II in C and LC and reduction in CC; Type IIx higher in all trained Groups. In the postsynaptic cleft, the stained area presents smaller in Groups C, LC, and CC; the total area showed smaller than LC and higher in C and CC. The stained and total perimeter, and dispersion showed a reduction in C, LC, and CC, higher maximum diameter in Groups C and CC, and decreased in LC. Regarding the postsynaptic cleft distribution, the stained area presented a decrease in all trained Groups. The integrated density presented higher principally in CC. The NMJ count showed an increase in all trained Groups. We concluded that the vertical ladder-based protocols combined contributed to the postsynaptic region adaptations.

The Golgi-Associated PDZ Domain Protein Gopc/PIST Is Required for Synaptic Targeting of mGluR5.

In neuronal cells, many membrane receptors interact via their intracellular, C-terminal tails with PSD-95/discs large/ZO-1 (PDZ) domain proteins. Some PDZ proteins act as scaffold proteins. In addition, there are a few PDZ proteins such as Gopc which bind to receptors during intracellular transport. Gopc is localized at the trans-Golgi network (TGN) and binds to a variety of receptors, many of which are eventually targeted to postsynaptic sites. We have analyzed the role of Gopc by knockdown in primary cultured neurons and by generating a conditional Gopc knockout (KO) mouse line. In neurons, targeting of neuroligin 1 (Nlgn1) and metabotropic glutamate receptor 5 (mGlu5) to the plasma membrane was impaired upon depletion of Gopc, whereas NMDA receptors were not affected. In the hippocampus and cortex of Gopc KO animals, expression levels of Gopc-associated receptors were not altered, while their subcellular localization was disturbed. The targeting of mGlu5 to the postsynaptic density was reduced, coinciding with alterations in mGluR-dependent synaptic plasticity and deficiencies in a contextual fear conditioning paradigm. Our data imply Gopc in the correct subcellular sorting of its associated mGlu5 receptor in vivo.

AMPA receptors in the synapse: Very little space and even less time.

Glutamate is by far the most abundant neurotransmitter used by excitatory synapses in the vertebrate central nervous system. Once released into the synaptic cleft, it depolarises the postsynaptic membrane and activates downstream signalling pathways resulting in the propagation of the excitatory signal. Initial depolarisation is primarily mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. These ion channels are the first ones to be activated by released glutamate and their kinetics, dynamics and abundance on the postsynaptic membrane defines the strength of the postsynaptic response. This review focuses on native AMPA receptors and synaptic environment they inhabit and considers structural and functional properties of the receptors obtained in heterologous systems in the light of spatial and temporal constraints of the synapse. This article is part of the special Issue on 'Glutamate Receptors - AMPA receptors'.

ß3-adrenoceptor activation exhibits a dual effect on behaviors and glutamate receptor function in the prefrontal cortex.

ß-adrenoceptor (ß-AR), especially the ß1- and ß2-AR subtypes, is known to participate in stress-related behavioral changes. Recently, SR58611A, a brain-penetrant ß3-AR agonist, exhibits anxiolytic- and antidepressant-like effects. In this study, we sought to study the role of SR58611A in behavioral changes and its potential cellular and molecular mechanism in the prefrontal cortex (PFC). We found that rats with SR58611A (1 mg/kg) enhanced PFC-mediated recognition memory, whereas administration of higher dosage of SR58611A (20 mg/kg) caused hyperlocomotion, and exhibited an impairment effect on recognition memory. Electrophysiological data also indicated that SR58611A (1 mg/kg) selectively enhanced NMDA receptor-mediated excitatory postsynaptic currents (EPSC) through interacting with norepinephrine (NE) system and activating ß3-AR, whereas higher dosage of SR58611A (20 mg/kg) reduced both AMPA receptor- and NMDA receptor-mediated EPSC. SR58611A-induced different effects on EPSC linked with the change of the surface expression quantity of NMDA receptor and/or AMPA receptor subunits. Synaptosomal-associated protein 25 (SNAP-25), which is a key soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein involved in incorporation of NMDA receptor to postsynaptic membrane, contributed to SR58611A (1 mg/kg)-induced enhancement of recognition memory and NMDA receptor function. Moreover, SR58611A (1 mg/kg) could rescue repeated stress-induced defect of both recognition memory and NMDA receptor function through a SNAP-25-dependent mechanism. These results provide a potential mechanism underlying the cognitive-enhancing effects of SR58611A (1 mg/kg).

An overview of the synaptic vesicle lipid composition.

Chemical neurotransmission is the major mechanism of neuronal communication. Neurotransmitters are released from secretory organelles, the synaptic vesicles (SVs) via exocytosis into the synaptic cleft. Fusion of SVs with the presynaptic plasma membrane is balanced by endocytosis, thus maintaining the presynaptic membrane at steady-state levels. The protein machineries responsible for exo- and endocytosis have been extensively investigated. In contrast, less is known about the role of lipids in synaptic transmission and how the lipid composition of SVs is affected by dynamic exo-endocytotic cycling. Here we summarize the current knowledge about the composition, organization, and function of SV membrane lipids. We also cover lipid biogenesis and maintenance during the synaptic vesicle cycle.