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 ...

University of Texas Medical Branch

university of texas medical branch

Altered expression of GABA receptors (GABARs) has been implicated in neurological and psychiatric disorders, but limited information about region-specific GABAR subunit expression in healthy human brains, heteromeric assembly of major isoforms, and their collective organization across healthy individuals, are major roadblocks to understanding their role in non-physiological states. Here, by using microarray and RNA-Seq datasets-from single cell nuclei to global brain expression-from the Allen Institute, we find that transcriptional expression of GABAR subunits is anatomically organized according to their neurodevelopmental origin. The data show a combination of complementary and mutually-exclusive expression patterns that delineate major isoforms, and which is highly stereotypical across brains from control donors. We summarize the region-specific signature of GABR subunits per subject and its variability in a control population sample that can be used as a reference for remodeling changes during homeostatic rearrangements of GABAR subunits after physiological, pharmacological or pathological challenges.

Human brain transcriptome analysis finds region- and subject-specific expression signatures of GABAR subunits.

Extracellular adenosine triphosphate (ATP) participates in maintaining the vascular tone in the CNS, particularly in the retina, via the tonic activity of ligand gated activated P2X1 receptors. P2X1 receptors are characterized by their high affinity for ATP and their strong desensitization to concentrations of ATP that are 200-fold lower than their EC. The mechanism behind P2X1 tonic activity remains unclear. In this study, we expressed human P2X1 (hP2X1) homomeric receptors in Xenopus oocytes to explore the relationship between ATP release from oocytes at rest, hP2X1, and Ca-activated Cl channels. Our results indicate that Xenopus oocytes release ATP at rest via vesicular exocytosis, and this process is a constitutive phenomenon independent of extracellular Ca. Our results also indicate that hP2X1 receptors are able to sustain a tonic activity of Ca-activated Cl channels. In the presence of extracellular Ca the activity of hP2X1 receptors is greatly amplified by its coupling with Ca-activated Cl channels. Future studies addressing the relationship between hP2X1 receptors and Ca-activated Cl channels in vascular smooth muscle cells should provide information about additional mechanisms that regulate the vascular tone and their potential as pharmaceutical targets. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Tonic Calcium-Activated Chloride Current Sustained by ATP Release and Highly Desensitizing Human P2X1 Receptors.

The study of postsynaptic excitation to inhibition (E/I ratio) imbalances in human brain diseases, is a highly relevant functional measurement poorly investigated due to postmortem degradation of synaptic receptors. We show that near-simultaneous recording of microtransplanted synaptic receptors after simulated morgue conditions allows the determination of the postsynaptic E/I ratio for at least 120 h after death, expanding the availability and use of human diseased tissue stored in brain banks.

Preservation of global synaptic excitatory to inhibitory ratio during long postmortem intervals.

Clinical and preclinical studies suggest that some of the behavioral alterations observed in schizophrenia (SZ) may be mechanistically linked to synaptic dysfunction of glutamatergic signaling. Recent genetic and proteomic studies suggest alterations of cortical glutamate receptors of the AMPA-type (AMPARs), which are the predominant ligand-gated ionic channels of fast transmission at excitatory synapses. The impact of gene and protein alterations on the electrophysiological activity of AMPARs is not known in SZ. In this proof of principle work, using human postmortem brain synaptic membranes isolated from the dorsolateral prefrontal cortex (DLPFC), we combined electrophysiological analysis from microtransplanted synaptic membranes (MSM) with transcriptomic (RNA-Seq) and label-free proteomics data in 10 control and 10 subjects diagnosed with SZ. We observed in SZ a reduction in the amplitude of AMPARs currents elicited by kainate, an agonist of AMPARs that blocks the desensitization of the receptor. This reduction was not associated with protein abundance but with a reduction in kainate's potency to activate AMPARs. Electrophysiologically-anchored dataset analysis (EDA) was used to identify synaptosomal proteins that linearly correlate with the amplitude of the AMPARs responses, gene ontology functional annotations were then used to determine protein-protein interactions. Protein modules associated with positive AMPARs current increases were downregulated in SZ, while protein modules that were upregulated in SZ were associated with decreased AMPARs currents. Our results indicate that transcriptomic and proteomic alterations, frequently observed in the DLPFC in SZ, converge at the synaptic level producing a functional electrophysiological impairment of AMPARs.

Functional impairment of cortical AMPA receptors in schizophrenia.

Theoretical and experimental work has demonstrated that excitatory (E) and inhibitory (I) currents within cortical circuits stabilize to a balanced state. This E/I balance, observed from single neuron to network levels, has a fundamental role in proper brain function and its impairment has been linked to numerous brain disorders. Over recent years, large amount of microarray and RNA-Sequencing datasets have been collected, however few studies have made use of these resources for exploring the balance of global gene expression levels between excitatory AMPA receptors (AMPARs) and inhibitory GABA receptors. Here, we analyzed the relative relationships between these receptors to generate a basic transcriptional marker of E/I ratio. Using publicly available data from the Allen Brain Institute, we generated whole brain and regional signatures of AMPAR subunit gene expression in healthy human brains as well as the transcriptional E/I (tE/I) ratio. Then we refined the tE/I ratio to cell-type signatures in the mouse brain using data from the Gene Expression Omnibus. Lastly, we applied our workflow to developmental data from the Allen Brain Institute and revealed spatially and temporally controlled changes in the tE/I ratio during the embryonic and early postnatal stages that ultimately lead to the tE/I balance in adults.

Regional transcriptome analysis of AMPA and GABA receptor subunit expression generates E/I signatures of the human brain.

Myelin facilitates the fast transmission of nerve impulses and provides metabolic support to axons. Differentiation of oligodendrocyte progenitor cells (OPCs) and Schwann cell (SC) precursors is critical for myelination during development and myelin repair in demyelinating disorders. Myelination is tightly controlled by neuron-glia communication and requires the participation of a wide repertoire of signals, including neurotransmitters such as glutamate, ATP, adenosine, or γ-aminobutyric acid (GABA). GABA is the main inhibitory neurotransmitter in the central nervous system (CNS) and it is also present in the peripheral nervous system (PNS). The composition and function of GABA receptors (GABARs) are well studied in neurons, while their nature and role in glial cells are still incipient. Recent studies demonstrate that GABA-mediated signaling mechanisms play relevant roles in OPC and SC precursor development and function, and stand out the implication of GABARs in oligodendrocyte (OL) and SC maturation and myelination. In this review, we highlight the evidence supporting the novel role of GABA with an emphasis on the molecular identity of the receptors expressed in these glial cells and the possible signaling pathways involved in their actions. GABAergic signaling in myelinating cells may have potential implications for developing novel reparative therapies in demyelinating diseases.

Expression and Function of GABA Receptors in Myelinating Cells.

Oligodendrocytes (OLs) express functional GABA receptors (GABARs) that are activated by GABA released at synaptic contacts with axons or by ambient GABA in extrasynaptic domains. In both instances, the receptors' molecular identity has not been fully defined. Furthermore, data on their structural diversity in different brain regions and information on age-dependent changes in their molecular composition are scant. This lack of knowledge has delayed access to a better understanding of the role of GABAergic signaling between neurons and OLs. Here, we used functional, and pharmacological analyses, as well as gene and protein expression of GABAR subunits, to explore the subunit combination that could explain the receptor functional profile expressed in OLs from the neonate rat. We found that GABAR composed of 321 subunits mimicked the characteristics of the endogenous receptor when expressed heterologously in  oocytes. Either 3 or 1 subunit silencing by small interfering RNA transfection changed the GABA-response characteristics in oligodendrocyte precursor cells, indicating their participation in the endogenous receptor conformation. Thus, 3 subunit silencing shifted the mean EC for GABA from 75.1 to 46.6 µM, whereas 1 silencing reduced the current amplitude response by 55%. We also observed that -carbolines differentially enhance GABA responses in oligodendroglia as compared with those in neurons. These results contribute to defining the molecular and pharmacological properties of GABARs in OLs. Additionally, the identification of -carbolines as selective enhancers of GABARs in OLs may help to study the role of GABAergic signaling during myelination. SIGNIFICANCE STATEMENT: GABAergic signaling through GABA receptors (GABARs) expressed in the oligodendroglial lineage contributes to the myelination control. Determining the molecular identity and the pharmacology of these receptors is essential to define their specific roles in myelination. Using GABAR subunit expression and silencing, we identified that the GABAR subunit combination α3β2γ1 conforms the bulk of GABARs in oligodendrocytes from rat neonates. Furthermore, we found that these receptors have differential pharmacological properties that allow specific positive modulation by β-carbolines.

GABA Receptors Expressed in Oligodendrocytes Cultured from the Neonatal Rat Contain 3 and 1 Subunits and Present Differential Functional and Pharmacological Properties.

Receptors to glutamate of the AMPA type (AMPARs) serve as the major gates of excitation in the human brain, where they participate in fundamental processes underlying perception, cognition and movement. Due to their central role in brain function, dysregulation of these receptors has been implicated in neuropathological states associated with a large variety of diseases that manifest with abnormal behaviors. The participation of functional abnormalities of AMPARs in brain disorders is strongly supported by genomic, transcriptomic and proteomic studies. Most of these studies have focused on the expression and function of the subunits that make up the channel and define AMPARs (GRIA1-GRIA4), as well of some accessory proteins. However, it is increasingly evident that native AMPARs are composed of a complex array of accessory proteins that regulate their trafficking, localization, kinetics and pharmacology, and a better understanding of the diversity and regional expression of these accessory proteins is largely needed. In this review we will provide an update on the state of current knowledge of AMPA receptors subunits in the context of their accessory proteins at the transcriptome level. We also summarize the regional expression in the human brain and its correlation with the channel forming subunits. Finally, we discuss some of the current limitations of transcriptomic analysis and propose potential ways to overcome them.

Transcriptomic expression of AMPA receptor subunits and their auxiliary proteins in the human brain.

Synaptic disturbances in excitatory to inhibitory (E/I) balance in forebrain circuits are thought to contribute to the progression of Alzheimer's disease (AD) and dementia, although direct evidence for such imbalance in humans is lacking. We assessed anatomical and electrophysiological synaptic E/I ratios in post-mortem parietal cortex samples from middle-aged individuals with AD (early-onset) or Down syndrome (DS) by fluorescence deconvolution tomography and microtransplantation of synaptic membranes. Both approaches revealed significantly elevated E/I ratios for AD, but not DS, versus controls. Gene expression studies in an independent AD cohort also demonstrated elevated E/I ratios in individuals with AD as compared to controls. These findings provide evidence of a marked pro-excitatory perturbation of synaptic E/I balance in AD parietal cortex, a region within the default mode network that is overly active in the disorder, and support the hypothesis that E/I imbalances disrupt cognition-related shifts in cortical activity which contribute to the intellectual decline in AD.

Agenor Limon

The Mitchell Center for Neurodegenerative Diseases,
Department of Neurology,
The Mitchell Center for Neurodegenerative Diseases, Department of Neurology

University of Texas Medical Branch

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

Agenor Limon

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Mitchell Center for Neurodegenerative Diseases,Department of Neurology,The Mitchell Center for Neurodegenerative Diseases, Department of Neurology

University of Texas Medical Branch

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

The Mitchell Center for Neurodegenerative Diseases,
Department of Neurology,
The Mitchell Center for Neurodegenerative Diseases, Department of Neurology