Gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, has recently been shown to play an inhibitory role in the immune system. GAT-1, the major high-affinity plasma membrane transporter for GABA, is localized widely in neurons (Itouji et al., 1996; Morara et al., 1996; Conti et al., 2004; Fattorini et al., 2017), particularly on axon terminal (E. Siucinska, A. Hamed, M. Jasinska 2014). GAT-1 is also expressed on astrocytes (Vas et al., 2011; Yadav et al., 2015; Ghirardini et al., 2018) as well as on oligodendrocytes (Fattorini et al., 2017). Regarding its expression on microglial cells, the only evidence comes from a work were the authors showed the enhancement of GABAergic transmission, with specific inhibitor of GAT1 (tiagabine), attenuated LPS-mediated microglial activation (Liu et al., 2015). The goal of this project is to verify the expression and function of GATs, particullarly GAT1, on microglial cells and its involvement in neuroinflammation.
We would perform experiments of fluorescence microscopy on CX3CR1+/gfp mice to reveal the presence of GATs positive microglial cells, by taking advantage of GFP expression exclusively on microglia. Then we will analyze size, morphology and phenotype of GATs positive microglia. Finally, we will perform in vitro functional assays to analyze Na+-dependent GABA uptake on microglial cells. Selective GATs inhibitors (NNC-711 for GAT-1 and SNAP-5114 for GAT-2/3) will be used to verify specific involvement of GATs subunits on microglial cells. Moreover, in vitro, we will analyze if pro-inflammatory stimuli could modify the expression of GATs on microglial cells. We will also confirm these data in vivo, on mice treated with LPS.
The results could suggest that microglial cells may contribute to pathophysiology of several neuroinflammatory diseases through the implication of GATs.
The results we hypothesize to obtain with this study will be important to better define the interaction between neurons and microglia in the field of neurotransmitters. Microglia. the resident immune-related cells located in brain parenchyma, are a double-edged sword, exerting toxic and beneficial roles depending on their polarisation phenotype, activation status and proximity to other cell types. Microglia player of the tripartite synapse or far away from the active synapse represent a target of neuro-signals that could diffused also over a considerable distance. This raises the possibility that microglia could communicate back to neurons by responding to neurotransmitters, collecting and/or synthesising them for a functional feedback loop. And, as well known, neurotransmitters are the main actors in the maintenance the proper firing of neurons that allow the cerebral homeostasis. Neurotransmitter stimulation of microglial cells induce release of many different molecules such as free radicals (superoxide and nitric oxide), chemokines and cytokines (interleukins and growth factors), able to modulate neuronal activity (Mead EL et al. 2012). Among them there is GABA, that reprents the major inhibitory transmitter in the central nervous system and binds to two types of receptors, GABA-A (that are ionotropic receptors) and GABA-B (metabotropic receptors). Microglia express GABA receptors in culture and in vivo (Kuhn SA et al. 2004; Mead EL et al. 2012), as demonstration that these type of cells are targets for the GABA neurotransmitter. Interestingly, recent evidence report microglia as a key regulator of the balance between excitatory and inhibitory inputs. In particular, electrophysiological recordings demonstrated that the reduction in inhibitory GABAergic synapses increased synchronized firing of cortical neurons in gamma-frequency band (Chen et al., 2014). Moreover, one of the principal enzymes involved in the production of GABA is the glutamate decarboxylase (GAD67), has been reported to be consistently inhibited in schizophrenia subjects (Curley AA et al. 2011). Nevertheless, the enhancement of GABAergic transmission, with specific inhibitor of a specific subunit of GABA transporters attenuated LPS-mediated microglial activation (Liu et al., 2015). All these findings prompt us to investigate the presence and functionality of GABA transporters on microglial cells that in the brain represent a central node of connection between immune activation and excitatory/inhibitory neuronal inputs. The advancement of knowledge in this fiels could lead to the definition of new pathophysiological pathways of several neurological diseases in which GABA transporters have been implicated to date.