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.

Copy number variation (CNV) at the 15q11.2 region has been identified as a significant risk locus for neurological and neuropsychiatric conditions such as schizophrenia (SCZ) and autism spectrum disorder (ASD). However, the individual roles for genes at this locus in nervous system development, function and connectivity remain poorly understood. Haploinsufficiency of one gene in this region, Cyfip1, may provide a model for 15q11.2 CNV-associated neuropsychiatric phenotypes. Here we show that altering CYFIP1 expression levels in neurons both in vitro and in vivo influences dendritic complexity, spine morphology, spine actin dynamics and synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor lateral diffusion. CYFIP1 is highly enriched at synapses and its overexpression in vitro leads to increased dendritic complexity. Neurons derived from Cyfip1 heterozygous animals on the other hand, possess reduced dendritic complexity, increased mobile F-actin and enhanced GluA2-containing AMPA receptor mobility at synapses. Interestingly, Cyfip1 overexpression or haploinsufficiency increased immature spine number, whereas activity-dependent changes in spine volume were occluded in Cyfip1 haploinsufficient neurons. In vivo, Cyfip1 heterozygous animals exhibited deficits in dendritic complexity as well as an altered ratio of immature-to-mature spines in hippocampal CA1 neurons. In summary, we provide evidence that dysregulation of CYFIP1 expression levels leads to pathological changes in CNS maturation and neuronal connectivity, both of which may contribute to the development of the neurological symptoms seen in ASD and SCZ.

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 Ca2+. Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca2+-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 Ca2+in astrocytic processes. Thus, the regulation of intracellular Ca2+signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca2+wave propagation, gliotransmission, and ultimately neuronal function.SIGNIFICANCE STATEMENTMitochondria are key cellular organelles that play important roles in providing cellular energy and buffering intracellular calcium ions. The mechanisms that control mitochondrial distribution within the processes of glial cells called astrocytes and the impact this may have on calcium signaling remains unclear. We show that activation of glutamate receptors or increased neuronal activity leads to the altered transport of mitochondria and their positioning at synapses dependent on a key mitochondrial trafficking protein called Miro1. We also show that, the control of mitochondrial movement and stopping by Miro plays an important role in regulating astrocyte calcium responses. Thus the regulation of intracellular calcium signaling, by Miro-mediated mitochondrial positioning, could have important consequences for astrocyte signaling and neuron–glial interactions.

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. GLIA 2016;64:1252–1264

SummaryInhibitory synaptic transmission requires the targeting and stabilization of GABAA receptors (GABAARs) at synapses. The mechanisms responsible remain poorly understood, and roles for transmembrane accessory proteins have not been established. Using molecular, imaging, and electrophysiological approaches, we identify the tetraspanin LHFPL4 as a critical regulator of postsynaptic GABAAR clustering in hippocampal pyramidal neurons. LHFPL4 interacts tightly with GABAAR subunits and is selectively enriched at inhibitory synapses. In LHFPL4 knockout mice, there is a dramatic cell-type-specific reduction in GABAAR and gephyrin clusters and an accumulation of large intracellular gephyrin aggregates in vivo. While GABAARs are still trafficked to the neuronal surface in pyramidal neurons, they are no longer localized at synapses, resulting in a profound loss of fast inhibitory postsynaptic currents. Hippocampal interneuron currents remain unaffected. Our results establish LHFPL4 as a synapse-specific tetraspanin essential for inhibitory synapse function and provide fresh insights into the molecular make-up of inhibitory synapses.