GABAA receptors (GABAARs) are ligand‐gated Cl− channels that mediate most of the fast inhibitory neurotransmission in the central nervous system (CNS). Multiple GABAAR subtypes are assembled from a ...family of 19 subunit genes, raising the question of the significance of this heterogeneity. In this review, we discuss the evidence that GABAAR subtypes represent distinct receptor populations with a specific spatio‐temporal expression pattern in the developing and adult CNS, being endowed with unique functional and pharmacological properties, as well as being differentially regulated at the transcriptional, post‐transcriptional and translational levels. GABAAR subtypes are targeted to specific subcellular domains to mediate either synaptic or extrasynaptic transmission, and their action is dynamically regulated by a vast array of molecular mechanisms to adjust the strength of inhibition to the changing needs of neuronal networks. These adaptations involve not only changing the gating or kinetic properties of GABAARs, but also modifying the postsynaptic scaffold organised by gephyrin to anchor specific receptor subtypes at postsynaptic sites. The significance of GABAAR heterogeneity is particularly evident during CNS development and adult neurogenesis, with different receptor subtypes fulfilling distinct steps of neuronal differentiation and maturation. Finally, analysis of the specific roles of GABAAR subtypes reveals their involvement in the pathophysiology of major CNS disorders, and opens novel perspectives for therapeutic intervention. In conclusion, GABAAR subtypes represent the substrate of a multifaceted inhibitory neurotransmission system that is dynamically regulated and performs multiple operations, contributing globally to the proper development, function and plasticity of the CNS.
GABAA receptor heterogeneity arises through combinatorial assembly of a large family of subunits to generate multiple receptor subtypes. It is an important facet of the variety of GABAergic signaling in adult and developing CNS, and a key factor underlying GABAergic synaptic plasticity underlying excitatory/inhibitory balance in neuronal circuits. This review presents and discusses recent progress in elucidating the relevance of GABAA receptor heterogeneity for CNS function in health and disease.
Microglia are highly motile phagocytic cells that infiltrate and take up residence in the developing brain, where they are thought to provide a surveillance and scavenging function. However, although ...microglia have been shown to engulf and clear damaged cellular debris after brain insult, it remains less clear what role microglia play in the uninjured brain. Here, we show that microglia actively engulf synaptic material and play a major role in synaptic pruning during postnatal development in mice. These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.
Distinct types of GABAergic interneurons target different subcellular domains of pyramidal cells, thereby shaping pyramidal cell activity patterns. Whether the presynaptic heterogeneity of GABAergic ...innervation is mirrored by specific postsynaptic factors is largely unexplored. Here we show that dystroglycan, a protein responsible for the majority of congenital muscular dystrophies when dysfunctional, has a function at postsynaptic sites restricted to a subset of GABAergic interneurons. Conditional deletion of Dag1, encoding dystroglycan, in pyramidal cells caused loss of CCK-positive basket cell terminals in hippocampus and neocortex. PV-positive basket cell terminals were unaffected in mutant mice, demonstrating interneuron subtype-specific function of dystroglycan. Loss of dystroglycan in pyramidal cells had little influence on clustering of other GABAergic postsynaptic proteins and of glutamatergic synaptic proteins. CCK-positive terminals were not established at P21 in the absence of dystroglycan and were markedly reduced when dystroglycan was ablated in adult mice, suggesting a role for dystroglycan in both formation and maintenance of CCK-positive terminals. The necessity of neuronal dystroglycan for functional innervation by CCK-positive basket cell axon terminals was confirmed by reduced frequency of inhibitory events in pyramidal cells of dystroglycan-deficient mice and further corroborated by the inefficiency of carbachol to increase IPSC frequency in these cells. Finally, neurexin binding seems dispensable for dystroglycan function because knock-in mice expressing binding-deficient T190M dystroglycan displayed normal CCK-positive terminals. Together, we describe a novel function of dystroglycan in interneuron subtype-specific trans-synaptic signaling, revealing correlation of presynaptic and postsynaptic molecular diversity.
Dystroglycan, an extracellular and transmembrane protein of the dystrophin-glycoprotein complex, is at the center of molecular studies of muscular dystrophies. Although its synaptic distribution in cortical brain regions is long established, function of dystroglycan in the synapse remained obscure. Using mice that selectively lack neuronal dystroglycan, we provide evidence that a subset of GABAergic interneurons requires dystroglycan for formation and maintenance of axonal terminals on pyramidal cells. As such, dystroglycan is the first postsynaptic GABAergic protein for which an interneuron terminal-specific function could be shown. Our findings also offer a new perspective on the mechanisms that lead to intellectual disability in muscular dystrophies without associated brain malformations.
Abstract Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABA A Rs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. ...Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABA A Rs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region ( Gabra2 -1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2 –1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2 -1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.
The use of ultrasound (US) in combination with a responsive chemical agent (sonosensitizer) can selectively trigger the agent's anticancer activity in a process called sonodynamic therapy (SDT). SDT ...shares some properties with photodynamic therapy (PDT), which has been clinically approved, but sets itself apart because of its use of US rather than light to achieve better tissue penetration. SDT provides anticancer effects mainly via the sonosensitizer-mediated generation of reactive oxygen species (ROS), although the precise nature of the underpinning mechanism is still under debate. This work investigates the SDT anticancer activity of hypericin (Hyp) in vitro in two- (2D) and three-dimensional (3D) HT-29 colon cancer models, and uses PDT as a yardstick due to its well-known Hyp phototoxicity. The cancer cell uptake and cellular localization of Hyp were investigated first to determine the proper noncytotoxic concentration and incubation time of Hyp for SDT. Furthermore, ROS production, cell proliferation, and cell death were evaluated after Hyp was exposed to US. Since cancer relapse and transporter-mediated multidrug resistance (MDR) are important causes of cancer treatment failure, the US-mediated ability of Hyp to elicit immunogenic cell death (ICD) and overcome MDR was also investigated. SDT showed strong ROS-mediated anticancer activity 48 h after treatment in both the HT-29 models. Specific damage-associated molecular patterns that are consistent with ICD, such as calreticulin (CRT) exposure and high-mobility group box 1 protein (HMGB1) release, were observed after SDT with Hyp. Moreover, the expression of the ABC transporter, P-glycoprotein (P-gp), in HT-29/MDR cells was not able to hinder cancer cell responsiveness to SDT with Hyp. This work reveals, for the first time, the US responsiveness of Hyp with significant anticancer activity being displayed, making it a full-fledged sonosensitizer for the SDT of cancer.
Sonodynamic therapy (SDT) is a noninvasive method for cancer treatment based on selective activation of a sonosensitiser by ultrasound (US), which results in the generation of reactive oxygen species ...(ROS) and cancer cell death. SDT uses a similar approach to photodynamic therapy (PDT), but can overcome the main drawback of PDT, i.e., poor tissue penetration of light. This research work investigated the anticancer effect of SDT on various two- (2D) and three-dimensional (3D) in vitro tumour models, using PDT as a reference treatment. Sonodynamic experiments were performed with pulsed US, specifically with shock waves (SW) and the prodrug 5-aminolevulinic acid (Ala), which is converted-at the mitochondrial level-into the sonosensitiser protoporphyrin IX (PPIX). SW-mediated PPIX sonodynamic activation resulted in a significant decrease in cell proliferation, especially on human fibrosarcoma (HT-1080) cells, where PPIX accumulation was higher compared to human melanoma (A2058) and neuroblastoma (SH-SY5 Y) cells. Moreover, SW-mediated SDT showed significant ROS generation, cell line-dependent in its amount, probably due to differences in Ala-induced PPIX synthesis. In all cancer cell lines, apoptosis was highlighted as the main cancer cell death pathway determined by SW-mediated SDT, along with significant cytochrome c release, and a consequent increase in DNA damage. The efficacy of SDT with SW and Ala in halting cancer cell proliferation was also confirmed in 3D cancer spheroids. The present study suggests that SW-mediated SDT is a valuable approach to slow down tumour proliferation, thus opening an innovative scenario in cancer treatment.
Gephyrin is the main scaffolding protein at inhibitory postsynaptic sites, and its clusters are the signaling hubs where several molecular pathways converge. Post-translational modifications (PTMs) ...of gephyrin alter GABA
receptor clustering at the synapse, but it is unclear how this affects neuronal activity at the circuit level. We assessed the contribution of gephyrin PTMs to microcircuit activity in the mouse barrel cortex by slice electrophysiology and in vivo two-photon calcium imaging of layer 2/3 (L2/3) pyramidal cells during single-whisker stimulation. Our results suggest that, depending on the type of gephyrin PTM, the neuronal activities of L2/3 pyramidal neurons can be differentially modulated, leading to changes in the size of the neuronal population responding to the single-whisker stimulation. Furthermore, we show that gephyrin PTMs have their preference for selecting synaptic GABA
receptor subunits. Our results identify an important role of gephyrin and GABAergic postsynaptic sites for cortical microcircuit function during sensory stimulation.
Sonodynamic Therapy (SDT) is a new anticancer strategy based on ultrasound (US) technique and is derived from photodynamic therapy (PDT); SDT is still, however, far from clinical application. In ...order to move this therapy forward from bench to bedside, investigations have been focused on treatment selectivity between cancer cells and normal cells. As a result, the effects of the porphyrin activation by SDT on cancer (HT-29) and normal (HDF 106-05) cells were studied in a co-culture evaluating cell cytotoxicity, reactive oxygen species (ROS) production, mitochondrial function and plasma membrane fluidity according to the bilayer sonophore (BLS) theory. While PDT induced similar effects on both HT-29 and HDF 106-05 cells in co-culture, SDT elicited significant cytotoxicity, ROS production and mitochondrial impairment on HT-29 cells only, whereas HDF 106-05 cells were unaffected. Notably, HT-29 and HDF 106-05 showed different cell membrane fluidity during US exposure. In conclusion, our data demonstrate a marked difference between cancer cells and normal cells in co-culture in term of responsiveness to SDT, suggesting that this different behavior can be ascribed to diversity in plasma membrane properties, such as membrane fluidity, according to the BLS theory.
The molecular code that controls synapse formation and maintenance in vivo has remained quite sparse. Here, we identify that the secreted protein Adamtsl3 functions as critical hippocampal synapse ...organizer acting through the transmembrane receptor DCC (deleted in colorectal cancer). Traditionally, DCC function has been associated with glutamatergic synaptogenesis and plasticity in response to Netrin-1 signaling. We demonstrate that early post-natal deletion of Adamtsl3 in neurons impairs DCC protein expression, causing reduced density of both glutamatergic and GABAergic synapses. Adult deletion of Adamtsl3 in either GABAergic or glutamatergic neurons does not interfere with DCC-Netrin-1 function at glutamatergic synapses but controls DCC signaling at GABAergic synapses. The Adamtsl3-DCC signaling unit is further essential for activity-dependent adaptations at GABAergic synapses, involving DCC phosphorylation and Src kinase activation. These findings might be particularly relevant for schizophrenia because genetic variants in Adamtsl3 and DCC have been independently linked with schizophrenia in patients.
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•Adamtsl3 is widely expressed at hippocampal synapses•Adamtsl3 is a ligand for DCC during synaptogenesis and in the mature brain•Presynaptic and postsynaptic Adamtsl3 contribute toward GABAergic synapse maintenance via DCC•Autocrine Adamtsl3-DCC signaling mediates plasticity adaptations at GABAergic postsynapse
The secreted glycoprotein Ce-Punctin clusters neurotransmitter receptors at the C. elegans NMJ. Cramer et al. reveal that Ce-Punctin ortholog Adamtsl3 is a synaptic protein influencing synapse formation via the DCC receptor in the mouse brain. In the adult hippocampus, Adamtsl3-DCC signaling specializes toward GABAergic synapse function with behavioral consequences.
Abstract
Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABA
A
receptor subunit specificity of inhibitory ...synaptic plasticity by studying cerebellar interneuron–Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABA
A
receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABA
A
receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABA
A
receptors and is abolished by preventing CaMKII phosphorylation of GABA
A
receptors. Our results reveal a novel GABA
A
receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.
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