Fast synaptic inhibition is largely mediated by GABAA receptors (GABAA Rs), ligand-gated chloride channels that play an essential role in the control of cell and network activity in the brain. Recent ...work has demonstrated that the delivery, number and stability of GABAA Rs at inhibitory synapses play a key role in the dynamic regulation of inhibitory synaptic efficacy and plasticity. The regulatory pathways essential for the fine-tuning of synaptic inhibition have also emerged as key sites of vulnerability during pathological changes in cell excitability in disease states.
Effective inhibitory synaptic transmission requires efficient stabilization of GABA(A) receptors (GABA(A)Rs) at synapses, which is essential for maintaining the correct excitatory-inhibitory balance ...in the brain. However, the signaling mechanisms that locally regulate synaptic GABA(A)R membrane dynamics remain poorly understood. Using a combination of molecular, imaging, and electrophysiological approaches, we delineate a GIT1/βPIX/Rac1/PAK signaling pathway that modulates F-actin and is important for maintaining surface GABA(A)R levels, inhibitory synapse integrity, and synapse strength. We show that GIT1 and βPIX are required for synaptic GABA(A)R surface stability through the activity of the GTPase Rac1 and downstream effector PAK. Manipulating this pathway using RNAi, dominant-negative and pharmacological approaches leads to a disruption of GABA(A)R clustering and decrease in the strength of synaptic inhibition. Thus, the GIT1/βPIX/Rac1/PAK pathway plays a crucial role in regulating GABA(A)R synaptic stability and hence inhibitory synaptic transmission with important implications for inhibitory plasticity and information processing in the brain.
The spatiotemporal distribution of mitochondria is crucial for precise ATP provision and calcium buffering required to support neuronal signaling. Fast-spiking GABAergic interneurons expressing ...parvalbumin (PV+) have a high mitochondrial content reflecting their large energy utilization. The importance for correct trafficking and precise mitochondrial positioning remains poorly elucidated in inhibitory neurons. Miro1 is a Ca²
+
-sensing adaptor protein that links mitochondria to the trafficking apparatus, for their microtubule-dependent transport along axons and dendrites, in order to meet the metabolic and Ca
2+
-buffering requirements of the cell. Here, we explore the role of Miro1 in PV+ interneurons and how changes in mitochondrial trafficking could alter network activity in the mouse brain. By employing live and fixed imaging, we found that the impairments in Miro1-directed trafficking in PV+ interneurons altered their mitochondrial distribution and axonal arborization, while PV+ interneuron-mediated inhibition remained intact. These changes were accompanied by an increase in the ex vivo hippocampal γ-oscillation (30–80 Hz) frequency and promoted anxiolysis. Our findings show that precise regulation of mitochondrial dynamics in PV+ interneurons is crucial for proper neuronal signaling and network synchronization.
Effective inhibitory synaptic transmission requires efficient stabilization of GABA
A
receptors (GABA
A
Rs) at synapses, which is essential for maintaining the correct excitatory-inhibitory balance ...in the brain. However, the signaling mechanisms that locally regulate synaptic GABA
A
R membrane dynamics remain poorly understood. Using a combination of molecular, imaging, and electrophysiological approaches, we delineate a GIT1/βPIX/Rac1/PAK signaling pathway that modulates F-actin and is important for maintaining surface GABA
A
R levels, inhibitory synapse integrity, and synapse strength. We show that GIT1 and βPIX are required for synaptic GABA
A
R surface stability through the activity of the GTPase Rac1 and downstream effector PAK. Manipulating this pathway using RNAi, dominant-negative and pharmacological approaches leads to a disruption of GABA
A
R clustering and decrease in the strength of synaptic inhibition. Thus, the GIT1/βPIX/Rac1/PAK pathway plays a crucial role in regulating GABA
A
R synaptic stability and hence inhibitory synaptic transmission with important implications for inhibitory plasticity and information processing in the brain.
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GIT1 and βPIX are present at inhibitory synapses and complex with GABA
A
Rs
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GIT1 and βPIX are important for GABA
A
R clustering and inhibitory transmission
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Rac1 and PAK activity is required for stabilization of GABA
A
Rs at synapses
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A GIT1/βPIX/Rac1/PAK pathway is required for inhibitory synaptic transmission
Clustering of GABA
A
receptors at inhibitory synapses is important for maintaining the correct balance of excitation and inhibition in the brain. Smith et al. reveal a signaling mechanism at inhibitory synapses involving the scaffold GIT1, which anchors βPIX to the inhibitory synaptic site and activates Rac1 and PAK, thereby stabilizing F-actin. This signaling pathway underlies the stabilization of synaptic GABA
A
receptors and therefore contributes to efficient inhibitory synaptic transmission in the brain.
γ-Aminobutyric acid type A receptors ( GABAARs) are the major sites of fast synaptic inhibition in the brain. An essential determinant for the efficacy of synaptic inhibition is the regulation of ...GABAAR cell surface stability. Here, we have examined the regulation of GABAAR endocytic sorting, a critical regulator of cell surface receptor number. In neurons, rapid constitutive endocytosis of GABAARs was evident. Internalized receptors were then either rapidly recycled back to the cell surface, or on a slower time scale, targeted for lysosomal degradation. This sorting decision was regulated by a direct interaction of GABAARs with Huntingtin-associated protein 1 (HAP1). HAP1 modulated synaptic GABAAR number by inhibiting receptor degradation and facilitating receptor recycling. Together these observations have identified a role for HAP1 in regulating GABAAR sorting, suggesting an important role for this protein in the construction and maintenance of inhibitory synapses.
NMDA receptors have been shown to contribute to glutamate‐evoked currents in oligodendrocytes. Activation of these receptors damages myelin in ischaemia, in part because they are more weakly blocked ...by Mg2+ than are most neuronal NMDA receptors. This weak Mg2+ block was suggested to reflect an unusual subunit composition including the NR2C and NR3A subunits. Here we expressed NR1/NR2C and triplet NR1/NR2C/NR3A recombinant receptors in HEK cells and compared their currents with those of NMDA‐evoked currents in rat cerebellar oligodendrocytes. NR1/NR2C/3A receptors were less blocked by 2 mm Mg2+ than were NR1/NR2C receptors (the remaining current was 30% and 18%, respectively, of that seen without added Mg2+) and showed less channel noise, suggesting a smaller single channel conductance. NMDA‐evoked currents in oligodendrocytes showed a Mg2+ block (to 32%) similar to that observed for NR1/NR2C/NR3A and significantly different from that for NR1/NR2C receptors. Co‐immunoprecipitation revealed interactions between NR1, NR2C and NR3A subunits in a purified myelin preparation from rat brain. These data are consistent with NMDA‐evoked currents in oligodendrocytes reflecting the activation of receptors containing NR1, NR2C and NR3A subunits.
To speed the transmission of information in the brain, a fatty substance called myelin is wrapped around nerve cells by another cell type called oligodendrocytes. Oligodendrocytes, and the cells they develop from, can become damaged in diseases such as cerebral palsy, stroke, multiple sclerosis and spinal cord injury. This damage is often caused by the release of glutamate. Glutamate acts on different types of receptor protein, including NMDA receptors. The NMDA receptors in oligodendrocytes have been suggested to have properties different from those in nerve cells, as a result of the protein subcomponents they are constructed from, making it easier for them to damage the cells. This paper examines the subcomponents contributing to oligodendrocyte NMDA receptors and concludes that they are likely to be made of three proteins called NR1, NR2C and NR3A. This may help the design of drugs that specifically target oligodendrocyte NMDA receptors.
Astrocytic GLT-1 is the main glutamate transporter involved in glutamate buffering in the brain, pivotal for glutamate removal at excitatory synapses to terminate neurotransmission and for preventing ...excitotoxicity. We show here that the surface expression and function of GLT-1 can be rapidly modulated through the interaction of its N-terminus with the nonadrenergic imidazoline-1 receptor protein, Nischarin. The phox domain of Nischarin is critical for interaction and internalization of surface GLT-1. Using live super-resolution imaging, we found that glutamate accelerated Nischarin-GLT-1 internalization into endosomal structures. The surface GLT-1 level increased in Nischarin knockout astrocytes, and this correlated with a significant increase in transporter uptake current. In addition, Nischarin knockout in astrocytes is neuroprotective against glutamate excitotoxicity. These data provide new molecular insights into regulation of GLT-1 surface level and function and suggest new drug targets for the treatment of neurological disorders.
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•Nischarin phox domain interacts with the N-terminus of the glutamate transporter, GLT-1•Nischarin promotes internalization of GLT-1 to endosomes•Glutamate modulates GLT-1 surface levels by regulating the Nischarin-GLT-1 interaction•Nischarin loss enhances GLT-1 surface levels, transport currents, and neuroprotection
Molecular biology; Molecular neuroscience; Cellular neuroscience; Cell biology
Brain computation is metabolically expensive and requires the supply of significant amounts of energy. Mitochondria are highly specialized organelles whose main function is to generate cellular ...energy. Due to their complex morphologies, neurons are especially dependent on a set of tools necessary to regulate mitochondrial function locally in order to match energy provision with local demands. By regulating mitochondrial transport, neurons control the local availability of mitochondrial mass in response to changes in synaptic activity. Neurons also modulate mitochondrial dynamics locally to adjust metabolic efficiency with energetic demand. Additionally, neurons remove inefficient mitochondria through mitophagy. Neurons coordinate these processes through signalling pathways that couple energetic expenditure with energy availability. When these mechanisms fail, neurons can no longer support brain function giving rise to neuropathological states like metabolic syndromes or neurodegeneration.
The strength of synaptic inhibition depends partly on the number of GABAA receptors (GABAARs) found at synaptic sites. The trafficking of GABAARs within the endocytic pathway is a key determinant of ...surface GABAAR number and is altered in neuropathologies, such as cerebral ischemia. However, the molecular mechanisms and signaling pathways that regulate this trafficking are poorly understood. Here, we report the subunit specific lysosomal targeting of synaptic GABAARs. We demonstrate that the targeting of synaptic GABAARs into the degradation pathway is facilitated by ubiquitination of a motif within the intracellular domain of the γ2 subunit. Blockade of lysosomal activity or disruption of the trafficking of ubiquitinated cargo to lysosomes specifically increases the efficacy of synaptic inhibition without altering excitatory currents. Moreover, mutation of the ubiquitination site within the γ2 subunit retards the lysosomal targeting of GABAARs and is sufficient to block the loss of synaptic GABAARs after anoxic insult. Together, our results establish a previously unknown mechanism for influencing inhibitory transmission under normal and pathological conditions.
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