Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling

It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function

Neuronal activity mediated regulation of glutamate transporter GLT-1 surface diffusion in rat astrocytes in dissociated and slice cultures.

The astrocytic GLT-1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live-cell imaging to study the mechanisms regulating GLT-1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP-time lapse imaging, we show that GLT-1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity-dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT-1 is more stable than diffuse GLT-1 and that glutamate increases GLT-1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT-1 isoforms expressed in the brain, GLT-1a and GLT-1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT-1b more so. GLT-1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT-1 isoforms. Altogether, these data reveal that astrocytic GLT-1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission

GABAA receptor dependent synaptic inhibition rapidly tunes KCC2 activity via the Cl--sensitive WNK1 kinase

This is the final version of the article. Available from the publisher via the DOI in this record.There is another ORE record for this publication: http://hdl.handle.net/10871/33406The K+-Cl-co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAAreceptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl-]i. KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABAAreceptor (GABAAR)-mediated transmission in mature hippocampal neurons. Enhancing GABAAR-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl-as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl--sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl-]ito GABAAR activity.This work was supported in part by Inserm, Sorbonne Université-UPMC, as well as the Fondation pour la Recherche Médicale (Equipe FRM DEQ20140329539 to J.C.P.), the Human Frontier Science Program (RGP0022/2013 to J.C.P.) and the Fondation pour la Recherche sur le Cerveau (to S.L.). Equipment at the IFM was also supported by DIM NeRF from Région Ile-de-France and by the FRC/Rotary ‘Espoir en tête’. M.H. was the recipient of a doctoral fellowship from the Université Pierre and Marie Curie, as well as from Bio-Psy Laboratory of excellence. K.T.K. is supported by the National Institutes of Health, the Simons Foundation, and the March of Dimes Foundation Basil O’Connor Award. The Poncer/Lévi lab is affiliated with the Paris School of Neuroscience (ENP) and the Bio-Psy Laboratory of excellence

KCC2 Regulates Neuronal Excitability and Hippocampal Activity via Interaction with Task-3 Channels

KCC2 regulates neuronal transmembrane chloride gradients and thereby controls GABA signaling in the brain. KCC2 downregulation is observed in numerous neurological and psychiatric disorders. Paradoxical, excitatory GABA signaling is usually assumed to contribute to abnormal network activity underlying the pathology. We tested this hypothesis and explored the functional impact of chronic KCC2 downregulation in the rat dentate gyrus. Although the reversal potential of GABAA receptor currents is depolarized in KCC2 knockdown neurons, this shift is compensated by depolarization of the resting membrane potential. This reflects downregulation of leak potassium currents. We show KCC2 interacts with Task-3 (KCNK9) channels and is required for their membrane expression. Increased neuronal excitability upon KCC2 suppression altered dentate gyrus rhythmogenesis, which could be normalized by chemogenetic hyperpolarization. Our data reveal KCC2 downregulation engages complex synaptic and cellular alterations beyond GABA signaling that perturb network activity thus offering additional targets for therapeutic intervention. Reduced KCC2 expression is associated with numerous neurological and psychiatric disorders and assumed to primarily affect GABA signaling. Goutierre et al. demonstrate chronic KCC2 knockdown in rat hippocampus has little effect on GABA signaling but affects neuronal excitability and network activity by downregulating membrane expression of Task-3 leak potassium channels.We are grateful to Q. Chevy for sharing original observations related to the present work, X. Marques (Imaging Facility, Institut du Fer à Moulin) for assistance with confocal imaging, and M. Heubl for providing WB analysis of RNAi efficiency. We also thank R. Miles and K. Kaila for critical reading of the manuscript. This work was supported in part by INSERM, Sorbonne Université, as well as the Fondation pour la Recherche Médicale (Equipe FRM DEQ20140329539 to J.C.P.), the Human Frontier Science Program (RGP0022/2013 to J.C.P. and L.M.d.l.P.), ERANET-Neuron (funded by ANR to J.C.P. and MINECO to L.M.d.l.P.), and the Fondation Française pour la Recherche sur l’Epilepsie - Fédération pour la Recherche sur le Cerveau (research grant to J.C.P.). M.G. and F.D. were recipients of fellowships from Sorbonne Université, and M.G. was also supported by Fondation pour la Recherche Médicale, as well as an IBRO-InEurope Short Stay Grant. The Poncer lab is affiliated with the Paris School of Neuroscience (ENP) and the Bio-Psy Laboratory of Excellence