Josef T. Kittler

Miro1 Is a Calcium Sensor for Glutamate Receptor-Dependent Localization of Mitochondria at Synapses

Summary Energy use, mainly to reverse ion movements in neurons, is a fundamental constraint on brain information processing. Trafficking of mitochondria to locations in neurons where there are large ion fluxes is essential for powering neural function. Mitochondrial trafficking is regulated by Ca2+ entry through ionotropic glutamate receptors, but the underlying mechanism is unknown. We show that the protein Miro1 links mitochondria to KIF5 motor proteins, allowing mitochondria to move along microtubules. This linkage is inhibited by micromolar levels of Ca2+ binding to Miro1. With the EF hand domains of Miro1 mutated to prevent Ca2+ binding, Miro1 could still facilitate mitochondrial motility, but mitochondrial stopping induced by glutamate or neuronal activity was blocked. Activating neuronal NMDA receptors with exogenous or synaptically released glutamate led to Miro1 positioning mitochondria at the postsynaptic side of synapses. Thus, Miro1 is a key determinant of how energy supply is matched to energy usage in neurons.

Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition.

Fast synaptic inhibition in the brain is largely mediated by GABA(A) receptors. These ligand-gated ion channels are crucial in the control of cell and network activity. Therefore, modulating their function or cell surface stability will have major consequences for neuronal excitation. It has become clear that the stability and activity of GABA(A) receptors at synapses can be dynamically modulated by receptor trafficking and phosphorylation. Here, we discuss these regulatory mechanisms, and their consequences for the efficacy of GABA(A) receptor mediated synaptic inhibition.

Brain-derived neurotrophic factor modulates fast synaptic inhibition by regulating GABA(A) receptor phosphorylation, activity, and cell-surface stability.

The efficacy of GABAergic synaptic inhibition is a principal factor in controlling neuronal activity. We demonstrate here that brain-derived neurotrophic factor modulates the activity of GABAA receptors, the main sites of fast synaptic inhibition in the brain, within minutes of application. Temporally, this comprised an early enhancement in the miniature IPSC amplitude, followed by a prolonged depression. This modulation was concurrent with enhanced PKC-mediated phosphorylation, followed by protein phosphatase 2A (PP2A)-mediated dephosphorylation of the GABAA receptor. Mechanistically, these events were facilitated by differential recruitment of PKC, receptor for activated C-kinase, and PP2A to GABAA receptors, depending on the phosphorylation state of the receptor β3-subunit. Thus, transient formation of GABAA receptor signaling complexes has the potential to provide a basis for acute changes in receptor function underlying GABAergic synaptic plasticity.

GABA A receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1

Controlling the number of functional γ-aminobutyric acid A (GABAA) receptors in neuronal membranes is a crucial factor for the efficacy of inhibitory neurotransmission. Here we describe the direct interaction of GABAA receptors with the ubiquitin-like protein Plic-1. Furthermore, Plic-1 is enriched at inhibitory synapses and is associated with subsynaptic membranes. Functionally, Plic-1 facilitates GABAA receptor cell surface expression without affecting the rate of receptor internalization. Plic-1 also enhances the stability of intracellular GABAA receptor subunits, increasing the number of receptors available for insertion into the plasma membrane. Our study identifies a previously unknown role for Plic-1, a modulation of GABAA receptor cell surface number, which suggests that Plic-1 facilitates accumulation of these receptors in dendritic membranes.

Control of mitochondrial transport and localization in neurons

Mitochondria play an essential role in ATP generation, calcium buffering and apoptotic signalling. In neurons, the transport of mitochondria to specific locations where they are needed has emerged as an important process for correct nerve cell function. Recent studies have shed light on the mechanisms that control mitochondrial transport and localization in neurons. We describe the machinery that is important for constitutive transport of mitochondria throughout the cell, and highlight recent advances in our understanding of how signalling pathways can converge on this machinery and allow for rapid activity-dependent control of mitochondrial trafficking and localization. Regulation of mitochondrial trafficking might work in concert with mitochondrial tethering systems to give precise control of mitochondrial delivery and localization to regions of high energy and calcium buffering requirements within neurons.

The Subcellular Distribution of GABARAP and Its Ability to Interact with NSF Suggest a Role for This Protein in the Intracellular Transport of GABAA Receptors

GABA(A) receptors the major sites of fast synaptic inhibition in the brain are composed predominately of alpha, beta, and gamma2 subunits. The receptor gamma2 subunit interacts with a 17-kDa microtubule associated protein GABARAP, but the significance of this interaction remains unknown. Here we demonstrate that GABARAP, which immunoprecipitates with GABA(A) receptors, is not found at significant levels within inhibitory synapses, but is enriched within the Golgi apparatus and postsynaptic cisternae. We also demonstrate that GABARAP binds directly to N-ethylmaleimide-sensitive factor (NSF), a protein critical for intracellular membrane trafficking events. NSF and GABARAP complexes could be detected in neurons and these two proteins also colocalize within intracellular membrane compartments. Together our observations suggest that GABARAP may play a role in intracellular GABA(A) receptor transport but not synaptic anchoring, via its ability to interact with NSF. GABARAP may therefore have an important role in the production of GABAergic synapses.

Cell surface stability of gamma-aminobutyric acid type A receptors. Dependence on protein kinase C activity and subunit composition.

Type A γ-aminobutyric acid receptors (GABAA), the major sites of fast synaptic inhibition in the brain, are believed to be composed predominantly of α, β, and γ subunits. Although cell surface expression is essential for GABAA receptor function, little is known regarding its regulation. To address this issue, the membrane stability of recombinant α1β2 or α1β2γ2 receptors was analyzed in human embryonic kidney cells. α1β2γ2 but not α1β2 receptors were found to recycle constitutively between the cell surface and a microtubule-dependent, perinuclear endosomal compartment. Similar GABAA receptor endocytosis was also seen in cultured hippocampal and cortical neurons. GABAA receptor surface levels were reduced upon protein kinase C (PKC) activation. Like basal endocytosis, this response required the γ2subunit but not receptor phosphorylation. Although inhibiting PKC activity did not block α1β2γ2receptor endocytosis, it did prevent receptor down-regulation, suggesting that PKC activity may block α1β2γ2 receptor recycling to the cell surface. In agreement with this observation, blocking recycling from endosomes with wortmannin selectively reduced surface levels of γ2-containing receptors. Together, our results demonstrate that the surface stability of GABAA receptors can be dynamically and specifically regulated, enabling neurons to modulate cell surface receptor number upon the appropriate cues.

Delivery of GABAARs to Synapses Is Mediated by HAP1-KIF5 and Disrupted by Mutant Huntingtin

The density of GABA(A) receptors (GABA(A)Rs) at synapses regulates brain excitability, and altered inhibition may contribute to Huntingtons disease, which is caused by a polyglutamine repeat in the protein huntingtin. However, the machinery that delivers GABA(A)Rs to synapses is unknown. We demonstrate that GABA(A)Rs are trafficked to synapses by the kinesin family motor protein 5 (KIF5). We identify the adaptor linking the receptors to KIF5 as the huntingtin-associated protein 1 (HAP1). Disrupting the HAP1-KIF5 complex decreases synaptic GABA(A)R number and reduces the amplitude of inhibitory postsynaptic currents. When huntingtin is mutated, as in Huntingtons disease, GABA(A)R transport and inhibitory synaptic currents are reduced. Thus, HAP1-KIF5-dependent GABA(A)R trafficking is a fundamental mechanism controlling the strength of synaptic inhibition in the brain. Its disruption by mutant huntingtin may explain some of the defects in brain information processing occurring in Huntingtons disease and provides a molecular target for therapeutic approaches.

Association of GABAB Receptors and Members of the 14-3-3 Family of Signaling Proteins

Two GABA(B) receptors, GABA(B)R1 and GABA(B)R2, have been cloned recently. Unlike other G protein-coupled receptors, the formation of a heterodimer between GABA(B)R1 and GABA(B)R2 is required for functional expression. We have used the yeast two hybrid system to identify proteins that interact with the C-terminus of GABA(B)R1. We report a direct association between GABA(B) receptors and two members of the 14-3-3 protein family, 14-3-3eta and 14-3-3zeta. We demonstrate that the C-terminus of GABA(B)R1 associates with 14-3-3zeta in rat brain preparations and tissue cultured cells, that they codistribute after rat brain fractionation, colocalize in neurons, and that the binding site overlaps partially with the coiled-coil domain of GABA(B)R1. Furthermore we show a reduced interaction between the C-terminal domains of GABA(B)R1 and GABA(B)R2 in the presence of 14-3-3. The results strongly suggest that GABA(B)R1 and 14-3-3 associate in the nervous system and begin to reveal the signaling complexities of the GABA(B)R1/GABA(B)R2 receptor heterodimer.

GTPase dependent recruitment of Grif-1 by Miro1 regulates mitochondrial trafficking in hippocampal neurons

The transport of mitochondria to specific neuronal locations is critical to meet local cellular energy demands and for buffering intracellular calcium. A critical role for kinesin motor proteins in mitochondrial transport in neurons has been demonstrated. Currently however the molecular mechanisms that underlie the recruitment of motor proteins to mitochondria, and how this recruitment is regulated remain unclear. Here we show that a protein trafficking complex comprising the adaptor protein Grif-1 and the atypical GTPase Miro1 can be detected in mammalian brain where it is localised to neuronal mitochondria. Increasing Miro1 expression levels recruits Grif-1 to mitochondria. This results in an enhanced transport of mitochondria towards the distal ends of neuronal processes. Uncoupling Grif-1 recruitment to mitochondria by expressing a Grif-1/Miro1 binding fragment dramatically reduces mitochondrial transport into neuronal processes. Altering Miro1 function by mutating its first GTPase domain affects Miros ability to recruit Grif-1 to mitochondria and in addition alters mitochondrial distribution and shape along neuronal processes. These data suggest that Miro1 and the kinesin adaptor Grif-1 play an important role in regulating mitochondrial transport in neurons.