SummaryEnergy 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.

Synapses enable neurons to communicate with each other and are therefore a prerequisite for normal brain function. Presynaptically, this communication requires energy and generates large fluctuations in calcium concentrations. Mitochondria are optimized for supplying energy and buffering calcium, and they are actively recruited to presynapses. However, not all presynapses contain mitochondria; thus, how might synapses with and without mitochondria differ? Mitochondria are also increasingly recognized to serve additional functions at the presynapse. Here, we discuss the importance of presynaptic mitochondria in maintaining neuronal homeostasis and how dysfunctional presynaptic mitochondria might contribute to the development of disease.

In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho‐GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double‐knockout mouse embryos and single‐ and double‐knockout embryonic fibroblasts, we demonstrate the essential and non‐redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double‐knockout cells. In contrast, we show that TRAK2‐mediated retrograde mitochondrial transport is Miro1‐dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin‐dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double‐knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine‐tune actin‐ and tubulin‐dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.

No abstract

Type A GABA receptors (GABA A ) mediate the majority of fast synaptic inhibition in the brain and are believed to be predominantly composed of ␣, ␤, and ␥ subunits. Although changes in cell surface GABA A receptor number have been postulated to be of importance in modulating inhibitory synaptic transmission, little is currently known on the mechanism used by neurons to modify surface receptor levels at inhibitory synapses. To address this issue, we have studied the cell surface expression and maintenance of GABA A receptors. Here we show that constitutive internalization of GABA A receptors in hippocampal neurons and recombinant receptors expressed in A293 cells is mediated by clathrin-dependent endocytosis. Furthermore, we identify an interaction between the GABA A receptor ␤ and ␥ subunits with the adaptin complex AP2, which is critical for the recruitment of integral membrane proteins into clathrin-coated pits. GABA A receptors also colocalize with AP2 in cultured hippocampal neurons. Finally, blocking clathrin-dependant endocytosis with a peptide that disrupts the association between amphiphysin and dynamin causes a large sustained increase in the amplitude of miniature IPSCs in cultured hippocampal neurons. These results suggest that GABA A receptors cycle between the synaptic membrane and intracellular sites, and their association with AP2 followed by recruitment into clathrin-coated pits represents an important mechanism in the postsynaptic modulation of inhibitory synaptic transmission.

The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABA A receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABA A receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABA A receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABA A receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABA A receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABA A receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABA A receptors, facilitating their accumulation at inhibitory synapses.

The density of GABAA receptors (GABAARs) at synapses regulates brain excitability, and altered inhibition may contribute to Huntington’s disease, which is caused by a polyglutamine repeat in the protein huntingtin. However, the machinery that delivers GABAARs to synapses is unknown. We demonstrate that GABAARs 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 GABAAR number, and reduces the amplitude of inhibitory postsynaptic currents. When huntingtin is mutated as in Huntington’s disease, GABAAR transport and inhibitory synaptic currents are reduced. Thus, HAP1-KIF5 dependent GABAAR 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 Huntington’s disease, and provides a new molecular target for therapeutic approaches.

The efficacy of synaptic inhibition depends on the number of ␥-aminobutyric acid type A receptors (GABA A Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABAAR endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABAAR ␤ subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the ␤3 subunit) incorporates the major sites of serine phosphorylation within receptor ␤ subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pep␤3) corresponding to the AP2 binding motif in the GABAAR ␤3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pep␤3 peptide, but not its phosphorylated equivalent (pep␤3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABAAR current. These effects of pep␤3 on GABAAR current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pep␤3 on GABAAR endocytosis. Therefore phosphodependent regulation of AP2 binding to GABAARs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission. endocytosis ͉ phosphorylation G ABA A receptors (GABA A Rs) are the major sites of fast synaptic inhibition in the brain (1). These pentameric ligandgated ion channels can be constructed from seven subunit classes: ␣1-6, ␤ 1-3, ␥ 1-3, ␦, , , and (2), with the majority of benzodiazepine-sensitive receptor subtypes being assembled from ␣, ␤, and ␥2 subunits (1, 2). A primary determinant for the efficacy of synaptic inhibition and, hence, neuronal excitation is the number of functional GABA A Rs expressed on the surface of neurons (3-10). Therefore, there has been considerable interest in understanding the cellular mechanism that neurons use to regulate GABA A R cell surface stability and activity. Collectively these studies have revealed that neuronal GABA A Rs undergo significant rates of constitutive endocytosis (3,8,(11)(12)(13)(14)(15), a process that has been established to regulate synaptic inhibition (8). GABA A Rs enter the endocytic pathway by a clathrin-mediated dynamindependent mechanism (8,(11)(12)(13)(14), a process that is facilitated by the clathrin adaptor protein 2 (AP2) complex, which is intimately associated with these receptors in neurons (8, 13). Internalized GABA A Rs are then subjected to either rapid recycling or targeted for lysozomal degradation, an endoctytic sorting decision that is regulated by the Huntingtin associated protein-1 (15). Therefore, changes in the rates of GABA A R endocytosis and͞or endocytic sorting represent potentially powerful mechanisms to regulate GABA A R cell surface number and inhibitory synaptic transmission (8,15).A potential mechanism to regulate target protein endocytosis is modulating interaction with the AP2 adaptor protein complex (1...