ABSTRACT
Members of the synaptophysin and synaptogyrin family are vesicle proteins with four transmembrane domains. In spite of their abundance in synaptic vesicle (SV) membranes, their role remains elusive and only mild defects at the cellular and organismal level are observed in mice lacking one or more family members. Here, we show that coexpression with synapsin in fibroblasts of each of the four brain-enriched members of this family-synaptophysin, synaptoporin, synaptogyrin 1, and synaptogyrin 3-is sufficient to generate clusters of small vesicles in the same size range of SVs. Moreover, mice lacking all these four proteins have larger SVs. We conclude that synaptophysin and synaptogyrin family proteins play an overlapping function in the biogenesis of SVs and in determining their small size.
Subject(s)
Synaptic Vesicles , Synaptogyrins , Synaptophysin , Animals , Synaptophysin/metabolism , Synaptophysin/genetics , Synaptic Vesicles/metabolism , Mice , Synaptogyrins/metabolism , Synaptogyrins/genetics , Synapsins/metabolism , Synapsins/genetics , Mice, Knockout , Fibroblasts/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , Rats , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/geneticsABSTRACT
Voltage-gated CaV2.1 (P/Q-type) Ca2+ channels play a crucial role in regulating neurotransmitter release, thus contributing to synaptic plasticity and to processes such as learning and memory. Despite their recognized importance in neural function, there is limited information on their potential involvement in neurodegenerative conditions such as Alzheimer's disease (AD). Here, we aimed to explore the impact of AD pathology on the density and nanoscale compartmentalization of CaV2.1 channels in the hippocampus in association with GABAB receptors. Histoblotting experiments showed that the density of CaV2.1 channel was significantly reduced in the hippocampus of APP/PS1 mice in a laminar-dependent manner. CaV2.1 channel was enriched in the active zone of the axon terminals and was present at a very low density over the surface of dendritic tree of the CA1 pyramidal cells, as shown by quantitative SDS-digested freeze-fracture replica labelling (SDS-FRL). In APP/PS1 mice, the density of CaV2.1 channel in the active zone was significantly reduced in the strata radiatum and lacunosum-moleculare, while it remained unaltered in the stratum oriens. The decline in Cav2.1 channel density was found to be associated with a corresponding impairment in the GABAergic synaptic function, as evidenced by electrophysiological experiments carried out in the hippocampus of APP/PS1 mice. Remarkably, double SDS-FRL showed a co-clustering of CaV2.1 channel and GABAB1 receptor in nanodomains (~40-50 nm) in wild type mice, while in APP/PS1 mice this nanoarchitecture was absent. Together, these findings suggest that the AD pathology-induced reduction in CaV2.1 channel density and CaV2.1-GABAB1 de-clustering may play a role in the synaptic transmission alterations shown in the AD hippocampus. Therefore, uncovering these layer-dependent changes in P/Q calcium currents associated with AD pathology can benefit the development of future strategies for AD management.
ABSTRACT
Retrograde signaling at the synapse is a fundamental way by which neurons communicate and neuronal circuit function is fine-tuned upon activity. While long-term changes in neurotransmitter release commonly rely on retrograde signaling, the mechanisms remain poorly understood. Here, we identified adenosine/A2A receptor (A2AR) as a retrograde signaling pathway underlying presynaptic long-term potentiation (LTP) at a hippocampal excitatory circuit critically involved in memory and epilepsy. Transient burst activity of a single dentate granule cell induced LTP of mossy cell synaptic inputs, a BDNF/TrkB-dependent form of plasticity that facilitates seizures. Postsynaptic TrkB activation released adenosine from granule cells, uncovering a non-conventional BDNF/TrkB signaling mechanism. Moreover, presynaptic A2ARs were necessary and sufficient for LTP. Lastly, seizure induction released adenosine in a TrkB-dependent manner, while removing A2ARs or TrkB from the dentate gyrus had anti-convulsant effects. By mediating presynaptic LTP, adenosine/A2AR retrograde signaling may modulate dentate gyrus-dependent learning and promote epileptic activity.
Subject(s)
Adenosine , Long-Term Potentiation , Receptor, Adenosine A2A , Seizures , Signal Transduction , Synaptic Transmission , Animals , Seizures/metabolism , Seizures/physiopathology , Receptor, Adenosine A2A/metabolism , Adenosine/metabolism , Synaptic Transmission/physiology , Long-Term Potentiation/physiology , Mice , Dentate Gyrus/metabolism , Male , Receptor, trkB/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Mice, Inbred C57BL , Hippocampus/metabolismABSTRACT
Myelination is the terminal step in a complex and precisely timed program that orchestrates the proliferation, migration and differentiation of oligodendroglial cells. It is thought that Sonic Hedgehog (Shh) acting on Smoothened (Smo) participates in regulating this process, but that these effects are highly context dependent. Here, we investigate oligodendroglial development and remyelination from three specific transgenic lines: NG2-CreERT2 (control), Smofl/fl/NG2-CreERT2 (loss of function), and SmoM2/NG2-CreERT2 (gain of function), as well as pharmacological manipulation that enhance or inhibit the Smo pathway (Smoothened Agonist (SAG) or cyclopamine treatment, respectively). To explore the effects of Shh/Smo on differentiation and myelination in vivo, we developed a highly quantifiable model by transplanting oligodendrocyte precursor cells (OPCs) in the retina. We find that myelination is greatly enhanced upon cyclopamine treatment and hypothesize that Shh/Smo could promote OPC proliferation to subsequently inhibit differentiation. Consistent with this hypothesis, we find that the genetic activation of Smo significantly increased numbers of OPCs and decreased oligodendrocyte differentiation when we examined the corpus callosum during development and after cuprizone demyelination and remyelination. However, upon loss of function with the conditional ablation of Smo, myelination in the same scenarios are unchanged. Taken together, our present findings suggest that the Shh pathway is sufficient to maintain OPCs in an undifferentiated state, but is not necessary for myelination and remyelination.
Subject(s)
Cell Differentiation , Hedgehog Proteins , Mice, Transgenic , Myelin Sheath , Oligodendrocyte Precursor Cells , Smoothened Receptor , Animals , Hedgehog Proteins/metabolism , Oligodendrocyte Precursor Cells/metabolism , Oligodendrocyte Precursor Cells/drug effects , Smoothened Receptor/metabolism , Smoothened Receptor/genetics , Myelin Sheath/metabolism , Cell Differentiation/physiology , Cell Differentiation/drug effects , Veratrum Alkaloids/pharmacology , Mice , Remyelination/physiology , Remyelination/drug effects , Oligodendroglia/metabolism , Oligodendroglia/drug effects , Oligodendroglia/physiology , Mice, Inbred C57BL , Signal Transduction/physiology , Signal Transduction/drug effectsABSTRACT
JOURNAL/nrgr/04.03/01300535-202409000-00040/figure1/v/2024-01-16T170235Z/r/image-tiff Plaques of amyloid-ß (Aß) and neurofibrillary tangles are the main pathological characteristics of Alzheimer's disease (AD). However, some older adult people with AD pathological hallmarks can retain cognitive function. Unraveling the factors that lead to this cognitive resilience to AD offers promising prospects for identifying new therapeutic targets. Our hypothesis focuses on the contribution of resilience to changes in excitatory synapses at the structural and molecular levels, which may underlie healthy cognitive performance in aged AD animals. Utilizing the Morris Water Maze test, we selected resilient (asymptomatic) and cognitively impaired aged Tg2576 mice. While the enzyme-linked immunosorbent assay showed similar levels of Aß42 in both experimental groups, western blot analysis revealed differences in tau pathology in the pre-synaptic supernatant fraction. To further investigate the density of synapses in the hippocampus of 16-18 month-old Tg2576 mice, we employed stereological and electron microscopic methods. Our findings indicated a decrease in the density of excitatory synapses in the stratum radiatum of the hippocampal CA1 in cognitively impaired Tg2576 mice compared with age-matched resilient Tg2576 and non-transgenic controls. Intriguingly, through quantitative immunoelectron microscopy in the hippocampus of impaired and resilient Tg2576 transgenic AD mice, we uncovered differences in the subcellular localization of glutamate receptors. Specifically, the density of GluA1, GluA2/3, and mGlu5 in spines and dendritic shafts of CA1 pyramidal cells in impaired Tg2576 mice was significantly reduced compared with age-matched resilient Tg2576 and non-transgenic controls. Notably, the density of GluA2/3 in resilient Tg2576 mice was significantly increased in spines but not in dendritic shafts compared with impaired Tg2576 and non-transgenic mice. These subcellular findings strongly support the hypothesis that dendritic spine plasticity and synaptic machinery in the hippocampus play crucial roles in the mechanisms of cognitive resilience in Tg2576 mice.
ABSTRACT
Staphylococcus aureus is one of the species with the greatest clinical importance and greatest impact on public health. In fact, methicillin-resistant S. aureus (MRSA) is considered a pandemic pathogen, being essential to develop effective medicines and combat its rapid spread. This study aimed to foster the translation of clinical research outcomes based on metallodrugs into clinical practice for the treatment of MRSA. Bearing in mind the promising anti-Gram-positive effect of the heteroscorpionate ligand 1,1'-(2-(4-isopropylphenyl)ethane-1,1-diyl)bis(3,5-dimethyl-1H-pyrazole) (2P), we propose the coordination of this compound to platinum as a clinical strategy with the ultimate aim of overcoming resistance in the treatment of MRSA. Therefore, the novel metallodrug 2P-Pt were synthetized, fully characterized and its antibacterial effect against the planktonic and biofilm state of S. aureus evaluated. In this sense, three different strains of S. aureus were studied, one collection strain of S. aureus sensitive to methicillin and two clinical MRSA strains. To appraise the antibacterial activity, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) were determined. Moreover, successful outcomes on the development of biofilm in a wound-like medium were obtained. The mechanism of action for 2P-Pt was proposed by measuring the MIC and MBC with EDTA (cation mediated mechanism) and DMSO (exogenous oxidative stress mechanism). Moreover, to shed light on the plausible antistaphylococcal mechanism of this novel platinum agent, additional experiments using transmission electron microscopy were carried out. 2P-Pt inhibited the growth and eradicated the three strains evaluated in the planktonic state. Another point worth stressing is the inhibition in the growth of MRSA biofilm even in a wounded medium. The results of this work support this novel agent as a promising therapeutic alternative for preventing infections caused by MRSA.
Subject(s)
Methicillin-Resistant Staphylococcus aureus , Staphylococcus aureus , Platinum/pharmacology , Anti-Bacterial Agents/pharmacology , Methicillin/pharmacology , Microbial Sensitivity Tests , BiofilmsABSTRACT
BACKGROUND: The basolateral amygdala (BLA) regulates mood and associative learning and has been linked to the development and persistence of alcohol use disorder. The GABABR (gamma-aminobutyric acid B receptor) is a promising therapeutic target for alcohol use disorder, and previous work suggests that exposure to ethanol and other drugs can alter neuronal GABABR-dependent signaling. The effect of ethanol on GABABR-dependent signaling in the BLA is unknown. METHODS: GABABR-dependent signaling in the mouse BLA was examined using slice electrophysiology following repeated ethanol exposure. Neuron-specific viral genetic manipulations were then used to understand the relevance of ethanol-induced neuroadaptations in the basal amygdala subregion (BA) to mood-related behavior. RESULTS: The somatodendritic inhibitory effect of GABABR activation on principal neurons in the basal but not the lateral subregion of the BLA was diminished following ethanol exposure. This adaptation was attributable to the suppression of GIRK (G protein-gated inwardly rectifying K+) channel activity and was mirrored by a redistribution of GABABR and GIRK channels from the surface membrane to internal sites. While GIRK1 and GIRK2 subunits are critical for GIRK channel formation in BA principal neurons, GIRK3 is necessary for the ethanol-induced neuroadaptation. Viral suppression of GIRK channel activity in BA principal neurons from ethanol-naïve mice recapitulated some mood-related behaviors observed in C57BL/6J mice during ethanol withdrawal. CONCLUSIONS: The ethanol-induced suppression of GIRK-dependent signaling in BA principal neurons contributes to some of the mood-related behaviors associated with ethanol withdrawal in mice. Approaches designed to prevent this neuroadaptation and/or strengthen GIRK-dependent signaling may prove useful for the treatment of alcohol use disorder.
Subject(s)
Alcoholism , Basolateral Nuclear Complex , Mice , Animals , Basolateral Nuclear Complex/metabolism , Ethanol/pharmacology , G Protein-Coupled Inwardly-Rectifying Potassium Channels/genetics , G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , Mice, Inbred C57BL , GTP-Binding Proteins , gamma-Aminobutyric AcidABSTRACT
The histoblot (in situ immunoblotting) technique is a simple, reproducible, and sensitive method for protein detection that allows both protein quantitation and analysis of tissue distribution. This easy and fast method allows the direct transfer of native proteins from unfixed frozen tissue sections by mechanical pressure to an immobilizing matrix. Proteins are directly blotted onto nitrocellulose membranes that are then immunolabelled similar to a Western blot, but the result is an immunohistochemical imprint of the section retaining all proteins. The histoblot combines advantages of western blot and immunohistochemical methods and yields optimal accessibility of proteins blotted on membranes whilst also preserving anatomical resolution. In addition, it avoids chemical modifications, crosslinking, or semi-denaturation of proteins, which can alter the access of antibody to epitopes, as introduced by conventional immunohistochemistry. Therefore, the histoblot often enables the use of antibodies that do not recognise the target protein in fixed tissue samples. This method has become a trusted alternative to reveal and compare the regional distribution and expression profile of different proteins in the brain in physiological and pathological conditions. In addition, the technique exhibits a high subregional resolution, although is not suitable to unravel protein distribution at the cellular and subcellular levels. In this review, we introduce the histoblot procedure used in our laboratory on brain sections for the identification of quantitative changes of neurotransmitter receptors, ion channels and other signalling molecules in the brain. We also discuss the potentialities, limitations, and fundamental principles of this technique.
Subject(s)
Brain , Proteins , Blotting, Western , Immunoblotting , ImmunohistochemistryABSTRACT
N-methyl-d-aspartate receptors (NMDARs) are pivotal players in the synaptic transmission and synaptic plasticity underlying learning and memory. Accordingly, dysfunction of NMDARs has been implicated in the pathophysiology of Alzheimer disease (AD). Here, we used histoblot and sodium dodecylsulphate-digested freeze-fracture replica labelling (SDS-FRL) techniques to investigate the expression and subcellular localisation of GluN1, the obligatory subunit of NMDARs, in the hippocampus of P301S mice. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered at 3 months. Using the SDS-FRL technique, excitatory synapses and extrasynaptic sites on spines of pyramidal cells and interneuron dendrites were analysed throughout all dendritic layers in the CA1 field. Our ultrastructural approach revealed a high density of GluN1 in synaptic sites and a substantially lower density at extrasynaptic sites. Labelling density for GluN1 in excitatory synapses established on spines was significantly reduced in P301S mice, compared with age-matched wild-type mice, in the stratum oriens (so), stratum radiatum (sr) and stratum lacunosum-moleculare (slm). Density for synaptic GluN1 on interneuron dendrites was significantly reduced in P301S mice in the so and sr but unaltered in the slm. Labelling density for GluN1 at extrasynaptic sites showed no significant differences in pyramidal cells, and only increased density in the interneuron dendrites of the sr. This differential alteration of synaptic versus extrasynaptic NMDARs supports the notion that the progressive accumulation of phospho-tau is associated with changes in NMDARs, in the absence of amyloid-ß pathology, and may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit.
Subject(s)
Hippocampus , Receptors, N-Methyl-D-Aspartate , Mice , Animals , Mice, Transgenic , Hippocampus/metabolism , CA1 Region, Hippocampal , Dendrites , Synapses/metabolismABSTRACT
The localization and density of any plasma membrane or intracellular protein in chromaffin cells are prerequisites for those studies designed to elucidate their contribution to cellular function within the adrenal gland and can be achieved only by immunoelectron microscopy. The most popular immunoelectron microscopic techniques involved gold particles conjugated to secondary antibodies, leading to electron-dense markers and the so-called immunogold EM method. Two main immunogold electron microscopic techniques exist: the pre-embedding immunogold, whereby the immunolabeling steps take place before samples are embedded, and the post-embedding immunogold, where the immunolabeling steps take place on embedded and sectioned samples. Pre-embedding immunogold is a very sensitive technique useful for simultaneous observation of labeled tissue at the light and electron microscopic levels. Post-embedding immunogold enables the simultaneous localization of different molecules in the cell using secondary antibodies conjugated with gold particles of different size. In this chapter, we introduce pre-embedding and post-embedding immunogold procedures used for the identification of quantitative changes in a wide range of signaling molecules in different tissues and also discuss the limitations inherent to these approaches.
Subject(s)
Chromaffin Cells , Gold , Antibodies , Immunohistochemistry , Microscopy, Electron , Microscopy, ImmunoelectronABSTRACT
Carotenoids are C40 isoprenoids with well-established roles in photosynthesis, pollination, photoprotection, and hormone biosynthesis. The enzymatic or ROS-induced cleavage of carotenoids generates a group of compounds named apocarotenoids, with an increasing interest by virtue of their metabolic, physiological, and ecological activities. Both classes are used industrially in a variety of fields as colorants, supplements, and bio-actives. Crocins and picrocrocin, two saffron apocarotenoids, are examples of high-value pigments utilized in the food, feed, and pharmaceutical industries. In this study, a unique construct was achieved, namely O6, which contains CsCCD2L, UGT74AD1, and UGT709G1 genes responsible for the biosynthesis of saffron apocarotenoids driven by a patatin promoter for the generation of potato tubers producing crocins and picrocrocin. Different tuber potatoes accumulated crocins and picrocrocin ranging from 19.41-360 to 105-800 µg/g DW, respectively, with crocetin, crocin 1 [(crocetin-(ß-D-glucosyl)-ester)] and crocin 2 [(crocetin)-(ß-D-glucosyl)-(ß-D-glucosyl)-ester)] being the main compounds detected. The pattern of carotenoids and apocarotenoids were distinct between wild type and transgenic tubers and were related to changes in the expression of the pathway genes, especially from PSY2, CCD1, and CCD4. In addition, the engineered tubers showed higher antioxidant capacity, up to almost 4-fold more than the wild type, which is a promising sign for the potential health advantages of these lines. In order to better investigate these aspects, different cooking methods were applied, and each process displayed a significant impact on the retention of apocarotenoids. More in detail, the in vitro bioaccessibility of these metabolites was found to be higher in boiled potatoes (97.23%) compared to raw, baked, and fried ones (80.97, 78.96, and 76.18%, respectively). Overall, this work shows that potatoes can be engineered to accumulate saffron apocarotenoids that, when consumed, can potentially offer better health benefits. Moreover, the high bioaccessibility of these compounds revealed that potato is an excellent way to deliver crocins and picrocrocin, while also helping to improve its nutritional value.
ABSTRACT
Tau pathology is a hallmark of Alzheimer's disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD.
Subject(s)
Hippocampus , Receptors, AMPA , Mice , Animals , Receptors, AMPA/genetics , Receptors, AMPA/metabolism , Mice, Transgenic , Hippocampus/metabolism , Synapses/metabolism , Dendrites/metabolismABSTRACT
Alzheimer's disease (AD) is characterized by a reorganization of brain activity determining network hyperexcitability and loss of synaptic plasticity. Precisely, a dysfunction in metabotropic GABAB receptor signalling through G protein-gated inwardly rectifying K+ (GIRK or Kir3) channels on the hippocampus has been postulated. Thus, we determined the impact of amyloid-ß (Aß) pathology in GIRK channel density, subcellular distribution, and its association with GABAB receptors in hippocampal CA1 pyramidal neurons from the APP/PS1 mouse model using quantitative SDS-digested freeze-fracture replica labelling (SDS-FRL) and proximity ligation in situ assay (P-LISA). In wild type mice, single SDS-FRL detection revealed a similar dendritic gradient for GIRK1 and GIRK2 in CA1 pyramidal cells, with higher densities in spines, and GIRK3 showed a lower and uniform distribution. Double SDS-FRL showed a co-clustering of GIRK2 and GIRK1 in post- and presynaptic compartments, but not for GIRK2 and GIRK3. Likewise, double GABAB1 and GIRK2 SDS-FRL detection displayed a high degree of co-clustering in nanodomains (40-50 nm) mostly in spines and axon terminals. In APP/PS1 mice, the density of GIRK2 and GIRK1, but not for GIRK3, was significantly reduced along the neuronal surface of CA1 pyramidal cells and in axon terminals contacting them. Importantly, GABAB1 and GIRK2 co-clustering was not present in APP/PS1 mice. Similarly, P-LISA experiments revealed a significant reduction in GABAB1 and GIRK2 interaction on the hippocampus of this animal model. Overall, our results provide compelling evidence showing a significant reduction on the cell surface density of pre- and postsynaptic GIRK1 and GIRK2, but not GIRK3, and a decline in GABAB receptors and GIRK2 channels co-clustering in hippocampal pyramidal neurons from APP/PS1 mice, thus suggesting that a disruption in the GABAB receptor-GIRK channel membrane assembly causes dysregulation in the GABAB signalling via GIRK channels in this AD animal model.
Subject(s)
G Protein-Coupled Inwardly-Rectifying Potassium Channels , Receptors, GABA-B , Animals , G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , G Protein-Coupled Inwardly-Rectifying Potassium Channels/ultrastructure , Hippocampus/metabolism , Mice , Neuronal Plasticity , Receptors, GABA-B/metabolism , gamma-Aminobutyric AcidABSTRACT
GluN3A is an atypical glycine-binding subunit of NMDA receptors (NMDARs) whose actions in the brain are mostly unknown. Here, we show that the expression of GluN3A subunits controls the excitability of mouse adult cortical and amygdalar circuits via an unusual signaling mechanism involving the formation of excitatory glycine GluN1/GluN3A receptors (eGlyRs) and their tonic activation by extracellular glycine. eGlyRs are mostly extrasynaptic and reside in specific neuronal populations, including the principal cells of the basolateral amygdala (BLA) and SST-positive interneurons (SST-INs) of the neocortex. In the BLA, tonic eGlyR currents are sensitive to fear-conditioning protocols, are subject to neuromodulation by the dopaminergic system, and control the stability of fear memories. In the neocortex, eGlyRs control the in vivo spiking of SST-INs and the behavior-dependent modulation of cortical activity. GluN3A-containing eGlyRs thus represent a novel and widespread signaling modality in the adult brain, with attributes that strikingly depart from those of conventional NMDARs.
Subject(s)
Amygdala , Neocortex , Receptors, Glycine , Receptors, N-Methyl-D-Aspartate , Amygdala/metabolism , Animals , Cerebral Cortex/metabolism , Glycine/metabolism , Interneurons/metabolism , Mice , Neocortex/metabolism , Neurons/metabolism , Receptors, Glycine/genetics , Receptors, Glycine/metabolism , Receptors, N-Methyl-D-Aspartate/genetics , Receptors, N-Methyl-D-Aspartate/metabolismABSTRACT
G protein-coupled receptors (GPCRs) constitute the largest group of membrane receptor proteins controlling brain activity. Accordingly, GPCRs are the main target of commercial drugs for most neurological and neuropsychiatric disorders. One of the mechanisms by which GPCRs regulate neuronal function is by homo- and heteromerization, with the establishment of direct protein-protein interactions between the same and different GPCRs. The occurrence of GPCR homo- and heteromers in artificial systems is generally well accepted, but more specific methods are necessary to address GPCR oligomerization in the brain. Here, we revise some of the techniques that have mostly contributed to reveal GPCR oligomers in native tissue, which include immunogold electron microscopy, proximity ligation assay (PLA), resonance energy transfer (RET) between fluorescent ligands and the Amplified Luminescent Proximity Homogeneous Assay (ALPHA). Of note, we use the archetypical GPCR oligomer, the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer as an example to illustrate the implementation of these techniques, which can allow visualizing GPCR oligomers in the human brain under normal and pathological conditions. Indeed, GPCR oligomerization may be involved in the pathophysiology of neurological and neuropsychiatric disorders.
Subject(s)
Receptors, Dopamine , Receptors, G-Protein-Coupled , Adenosine , Brain/metabolism , Humans , Ligands , Receptors, Dopamine/metabolism , Receptors, G-Protein-Coupled/metabolismABSTRACT
During development, neural circuit formation requires the stabilization of active γ-aminobutyric acidmediated (GABAergic) synapses and the elimination of inactive ones. Here, we demonstrate that, although the activation of postsynaptic GABA type A receptors (GABAARs) and adenosine A2A receptors (A2ARs) stabilizes GABAergic synapses, only A2AR activation is sufficient. Both GABAAR- and A2AR-dependent signaling pathways act synergistically to produce adenosine 3',5'-monophosphate through the recruitment of the calciumcalmodulinadenylyl cyclase pathway. Protein kinase A, thus activated, phosphorylates gephyrin on serine residue 303, which is required for GABAAR stabilization. Finally, the stabilization of pre- and postsynaptic GABAergic elements involves the interaction between gephyrin and the synaptogenic membrane protein Slitrk3. We propose that A2ARs act as detectors of active GABAergic synapses releasing GABA, adenosine triphosphate, and adenosine to regulate their fate toward stabilization or elimination.
Subject(s)
Adenosine/metabolism , Hippocampus/growth & development , Neurons/physiology , Receptor, Adenosine A2A/metabolism , Signal Transduction , Synapses/physiology , gamma-Aminobutyric Acid/metabolism , Adenosine A2 Receptor Antagonists , Adenosine Triphosphate/metabolism , Animals , Calcium/metabolism , Cognition , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Hippocampus/metabolism , Male , Membrane Proteins/metabolism , Mice , Nerve Tissue Proteins , Phosphorylation , Receptor, Adenosine A2A/genetics , Receptors, GABA-A/metabolismABSTRACT
Despite the well-accepted role of the two main neuropathological markers (ß-amyloid and tau) in the progression of Alzheimer's disease, the interaction and specific contribution of each of them is not fully elucidated. To address this question, in the present study, an adeno-associated virus (AAV9) carrying the mutant P301L form of human tau, was injected into the dorsal hippocampi of APP/PS1 transgenic mice or wild type mice (WT). Three months after injections, memory tasks, biochemical and immunohistochemical analysis were performed. We found that the overexpression of hTauP301L accelerates memory deficits in APP/PS1 mice, but it did not affect memory function of WT mice. Likewise, biochemical assays showed that only in the case of APP/PS1-hTauP301L injected mice, an important accumulation of tau was observed in the insoluble urea fraction. Similarly, electron microscopy images revealed that numerous clusters of tau immunoparticles appear at the dendrites of APP/PS1 injected mice and not in WT animals, suggesting that the presence of amyloid is necessary to induce tau aggregation. Interestingly, these tau immunoparticles accumulate in dendritic mitochondria in the APP/PS1 mice, whereas most of mitochondria in WT injected mice remain free of tau immunoparticles. Taken together, it seems that amyloid induces tau aggregation and accumulation in the dendritic mitochondria and subsequently may alter synapse function, thus, contributing to accelerate cognitive decline in APP/PS1 mice.
Subject(s)
Alzheimer Disease/complications , Amyloid beta-Peptides/adverse effects , Cognition Disorders/pathology , Disease Models, Animal , Mitochondria/pathology , tau Proteins/metabolism , Amyloid beta-Protein Precursor/physiology , Animals , Cognition Disorders/etiology , Cognition Disorders/metabolism , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/metabolism , Phosphorylation , Presenilin-1/physiology , Synapses , tau Proteins/geneticsABSTRACT
G protein-gated inwardly rectifying K+ (GIRK) channels are the main targets controlling excitability and synaptic plasticity on hippocampal neurons. Consequently, dysfunction of GIRK-mediated signalling has been implicated in the pathophysiology of Alzheimer´s disease (AD). Here, we provide a quantitative description on the expression and localisation patterns of GIRK2 in two transgenic mice models of AD (P301S and APP/PS1 mice), combining histoblots and immunoelectron microscopic approaches. The histoblot technique revealed differences in the expression of GIRK2 in the two transgenic mice models. The expression of GIRK2 was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered in APP/PS1 mice at 12 months compared to age-matched wild type mice. Ultrastructural approaches using the pre-embedding immunogold technique, demonstrated that the subcellular localisation of GIRK2 was significantly reduced along the neuronal surface of CA1 pyramidal cells, but increased in its frequency at cytoplasmic sites, in both P301S and APP/PS1 mice. We also found a decrease in plasma membrane GIRK2 channels in axon terminals contacting dendritic spines of CA1 pyramidal cells in P301S and APP/PS1 mice. These data demonstrate for the first time a redistribution of GIRK channels from the plasma membrane to intracellular sites in different compartments of CA1 pyramidal cells. Altogether, the pre- and post-synaptic reduction of GIRK2 channels suggest that GIRK-mediated alteration of the excitability in pyramidal cells could contribute to the cognitive dysfunctions as described in the two AD animal models.