Shared structural features of Miro binding control mitochondrial homeostasis.

Miro proteins are universally conserved mitochondrial calcium-binding GTPases that regulate a multitude of mitochondrial processes, including transport, clearance, and lipid trafficking. The exact role of Miro in these functions is unclear but involves binding to a variety of client proteins. How this binding is operated at the molecular level and whether and how it is important for mitochondrial health, however, remains unknown. Here, we show that known Miro interactors-namely, CENPF, Trak, and MYO19-all use a similar short motif to bind the same structural element: a highly conserved hydrophobic pocket in the first calcium-binding domain of Miro. Using these Miro-binding motifs, we identified direct interactors de novo, including MTFR1/2/1L, the lipid transporters Mdm34 and VPS13D, and the ubiquitin E3-ligase Parkin. Given the shared binding mechanism of these functionally diverse clients and its conservation across eukaryotes, we propose that Miro is a universal mitochondrial adaptor coordinating mitochondrial health.

Mitochondrial regulation of local supply of energy in neurons.

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.

SARS-CoV-2 triggers pericyte-mediated cerebral capillary constriction.

The SARS-CoV-2 receptor, ACE2, is found on pericytes, contractile cells enwrapping capillaries that regulate brain, heart and kidney blood flow. ACE2 converts vasoconstricting angiotensin II into vasodilating angiotensin-(1-7). In brain slices from hamster, which has an ACE2 sequence similar to human ACE2, angiotensin II evoked a small pericyte-mediated capillary constriction via AT1 receptors, but evoked a large constriction when the SARS-CoV-2 receptor binding domain (RBD, original Wuhan variant) was present. A mutated non-binding RBD did not potentiate constriction. A similar RBD-potentiated capillary constriction occurred in human cortical slices, and was evoked in hamster brain slices by pseudotyped virions expressing SARS-CoV-2 spike protein. This constriction reflects an RBD-induced decrease in the conversion of angiotensin II to angiotensin-(1-7) mediated by removal of ACE2 from the cell surface membrane and was mimicked by blocking ACE2. The clinically used drug losartan inhibited the RBD-potentiated constriction. Thus, AT1 receptor blockers could be protective in COVID-19 by preventing pericyte-mediated blood flow reductions in the brain, and perhaps the heart and kidney.

Mitochondrial Ca2+ uniporter haploinsufficiency enhances long-term potentiation at hippocampal mossy fibre synapses.

Long-term changes in synaptic strength form the basis of learning and memory. These changes rely upon energy-demanding mechanisms, which are regulated by local Ca2+ signalling. Mitochondria are optimised for providing energy and buffering Ca2+. However, our understanding of the role of mitochondria in regulating synaptic plasticity is incomplete. Here, we have used optical and electrophysiological techniques in cultured hippocampal neurons and ex vivo hippocampal slices from mice with haploinsufficiency of the mitochondrial Ca2+ uniporter (MCU+/-) to address whether reducing mitochondrial Ca2+ uptake alters synaptic transmission and plasticity. We found that cultured MCU+/- hippocampal neurons have impaired Ca2+ clearance, and consequently enhanced synaptic vesicle fusion at presynapses occupied by mitochondria. Furthermore, long-term potentiation (LTP) at mossy fibre (MF) synapses, a process which is dependent on presynaptic Ca2+ accumulation, is enhanced in MCU+/- slices. Our results reveal a previously unrecognised role for mitochondria in regulating presynaptic plasticity of a major excitatory pathway involved in learning and memory.

Phosphorylation of neuroligin-2 by PKA regulates its cell surface abundance and synaptic stabilization.

The trans-synaptic adhesion molecule neuroligin-2 (NL2) is essential for the development and function of inhibitory synapses. NL2 recruits the postsynaptic scaffold protein gephyrin, which, in turn, stabilizes γ-aminobutyric acid type A receptors (GABAARs) in the postsynaptic domain. Thus, the amount of NL2 at the synapse can control synaptic GABAAR concentration to tune inhibitory neurotransmission efficacy. Here, using biochemistry, imaging, single-particle tracking, and electrophysiology, we uncovered a key role for cAMP-dependent protein kinase (PKA) in the synaptic stabilization of NL2. We found that PKA-mediated phosphorylation of NL2 at Ser714 caused its dispersal from the synapse and reduced NL2 surface amounts, leading to a loss of synaptic GABAARs. Conversely, enhancing the stability of NL2 at synapses by abolishing PKA-mediated phosphorylation led to increased inhibitory signaling. Thus, PKA plays a key role in regulating NL2 function and GABA-mediated synaptic inhibition.

The non-adrenergic imidazoline-1 receptor protein nischarin is a key regulator of astrocyte glutamate uptake.

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.

Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response.

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post-natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.

Miro1-dependent mitochondrial dynamics in parvalbumin interneurons.

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 Ca2+-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.

Cdk5 and GSK3ß inhibit fast endophilin-mediated endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Fast Endophilin-mediated endocytosis, FEME, is not constitutively active but triggered upon receptor activation. High levels of growth factors induce spontaneous FEME, which can be suppressed upon serum starvation. This suggested a role for protein kinases in this growth factor receptor-mediated regulation. Using chemical and genetic inhibition, we find that Cdk5 and GSK3ß are negative regulators of FEME. They antagonize the binding of Endophilin to Dynamin-1 and to CRMP4, a Plexin A1 adaptor. This control is required for proper axon elongation, branching and growth cone formation in hippocampal neurons. The kinases also block the recruitment of Dynein onto FEME carriers by Bin1. As GSK3ß binds to Endophilin, it imposes a local regulation of FEME. Thus, Cdk5 and GSK3ß are key regulators of FEME, licensing cells for rapid uptake by the pathway only when their activity is low.

KCC2 is required for the survival of mature neurons but not for their development.

The K+/Cl- cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl- levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.

Regulation of peroxisomal trafficking and distribution.

Peroxisomes are organelles that perform a wide range of essential metabolic processes. To ensure that peroxisomes are optimally positioned in the cell, they must be transported by both long- and short-range trafficking events in response to cellular needs. Here, we review our current understanding of the mechanisms by which the cytoskeleton and organelle contact sites alter peroxisomal distribution. Though the focus of the review is peroxisomal transport in mammalian cells, findings from flies and fungi are used for comparison and to inform the gaps in our understanding. Attention is given to the apparent overlap in regulatory mechanisms for mitochondrial and peroxisomal trafficking, along with the recently discovered role of the mitochondrial Rho-GTPases, Miro, in peroxisomal dynamics. Moreover, we outline and discuss the known pathological and pharmacological conditions that perturb peroxisomal positioning. We conclude by highlighting several gaps in our current knowledge and suggest future directions that require attention.

DISC1 Regulates Mitochondrial Trafficking in a Miro1-GTP-Dependent Manner.

The disrupted in schizophrenia 1 (DISC1) protein is implicated in major mental illnesses including schizophrenia and bipolar disorder. A key feature of psychiatric disease is aberrant synaptic communication. Correct synaptic transmission is dependent on spatiotemporally regulated energy provision and calcium buffering. This can be achieved by precise distribution of mitochondria throughout the elaborate architecture of the neuron. Central to this process is the calcium sensor and GTPase Miro1, which allows mitochondrial trafficking by molecular motors. While the role of Miro1-calcium binding in mitochondrial transport is well described, far less is known regarding the functions of the two GTPase domains. Here, we investigate the effects of a psychiatric disease-associated mutation in DISC1 on mitochondrial trafficking. We show that this DISC1 mutation impairs Miro1's ability to transport mitochondria. We also demonstrate the necessity of the first Miro1 GTPase domain in determining direction of mitochondrial transport and the involvement of DISC1 in this process. Finally, we describe the effects of mutant DISC1 on positioning of mitochondria at synapses.

Peroxisomal fission is modulated by the mitochondrial Rho-GTPases, Miro1 and Miro2.

Peroxisomes are essential for a number of cellular functions, including reactive oxygen species metabolism, fatty acid ß-oxidation and lipid synthesis. To ensure optimal functionality, peroxisomal size, shape and number must be dynamically maintained; however, many aspects of how this is regulated remain poorly characterised. Here, we show that the localisation of Miro1 and Miro2-outer mitochondrial membrane proteins essential for mitochondrial trafficking-to peroxisomes is not required for basal peroxisomal distribution and long-range trafficking, but rather for the maintenance of peroxisomal size and morphology through peroxisomal fission. Mechanistically, this is achieved by Miro negatively regulating Drp1-dependent fission, a function that is shared with the mitochondria. We further find that the peroxisomal localisation of Miro is regulated by its first GTPase domain and is mediated by an interaction through its transmembrane domain with the peroxisomal-membrane protein chaperone, Pex19. Our work highlights a shared regulatory role of Miro in maintaining the morphology of both peroxisomes and mitochondria, supporting a crosstalk between peroxisomal and mitochondrial biology.

Axonal autophagosome maturation defect through failure of ATG9A sorting underpins pathology in AP-4 deficiency syndrome.

Adaptor protein (AP) complexes mediate key sorting decisions in the cell through selective incorporation of transmembrane proteins into vesicles. Little is known of the roles of AP-4, despite its loss of function leading to a severe early onset neurological disorder, AP-4 deficiency syndrome. Here we demonstrate an AP-4 epsilon subunit knockout mouse model that recapitulates characteristic neuroanatomical phenotypes of AP-4 deficiency patients. We show that ATG9A, critical for autophagosome biogenesis, is an AP-4 cargo, which is retained within the trans-Golgi network (TGN) in vivo and in culture when AP-4 function is lost. TGN retention results in depletion of axonal ATG9A, leading to defective autophagosome generation and aberrant expansions of the distal axon. The reduction in the capacity to generate axonal autophagosomes leads to defective axonal extension and de novo generation of distal axonal swellings containing accumulated ER, underlying the impaired axonal integrity in AP-4 deficiency syndrome.Abbreviations: AP: adaptor protein; AP4B1: adaptor-related protein complex AP-4, beta 1; AP4E1: adaptor-related protein complex AP-4, epsilon 1; ATG: autophagy-related; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GFAP: glial fibrillary acidic protein; GOLGA1/Golgin-97/GOLG97: golgi autoantigen, golgin subfamily a, 1; GOLGA2/GM130: golgi autoantigen, golgin subfamily a, 2; HSP: hereditary spastic paraplegia; LC3/MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MAPK8IP1/JIP1: mitogen-acitvated protein kinase 8 interacting protein 1; NEFH/NF200: neurofilament, heavy polypeptide; RBFOX3/NeuN (RNA binding protein, fox-1 homolog [C. elegans] 3); SQSTM1/p62: sequestosome 1; TGN: trans-Golgi network; WIPI2: WD repeat domain, phosphoinositide interacting protein 2.

The adaptor proteins HAP1a and GRIP1 collaborate to activate the kinesin-1 isoform KIF5C.

Binding of motor proteins to cellular cargoes is regulated by adaptor proteins. HAP1 and GRIP1 are kinesin-1 adaptors that have been implicated individually in the transport of vesicular cargoes in the dendrites of neurons. We find that HAP1a and GRIP1 form a protein complex in the brain, and co-operate to activate the kinesin-1 subunit KIF5C in vitro Based upon this co-operative activation of kinesin-1, we propose a modification to the kinesin activation model that incorporates stabilisation of the central hinge region known to be critical to autoinhibition of kinesin-1.

SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling.

GABAA receptors mediate fast inhibitory transmission in the brain, and their number can be rapidly up- or downregulated to alter synaptic strength. Neuroligin-2 plays a critical role in the stabilization of synaptic GABAA receptors and the development and maintenance of inhibitory synapses. To date, little is known about how the amount of neuroligin-2 at the synapse is regulated and whether neuroligin-2 trafficking affects inhibitory signaling. Here, we show that neuroligin-2, when internalized to endosomes, co-localizes with SNX27, a brain-enriched cargo-adaptor protein that facilitates membrane protein recycling. Direct interaction between the PDZ domain of SNX27 and PDZ-binding motif in neuroligin-2 enables membrane retrieval of neuroligin-2, thus enhancing synaptic neuroligin-2 clusters. Furthermore, SNX27 knockdown has the opposite effect. SNX27-mediated up- and downregulation of neuroligin-2 surface levels affects inhibitory synapse composition and signaling strength. Taken together, we show a role for SNX27-mediated recycling of neuroligin-2 in maintenance and signaling of the GABAergic synapse.

Miro clusters regulate ER-mitochondria contact sites and link cristae organization to the mitochondrial transport machinery.

Mitochondrial Rho (Miro) GTPases localize to the outer mitochondrial membrane and are essential machinery for the regulated trafficking of mitochondria to defined subcellular locations. However, their sub-mitochondrial localization and relationship with other critical mitochondrial complexes remains poorly understood. Here, using super-resolution fluorescence microscopy, we report that Miro proteins form nanometer-sized clusters along the mitochondrial outer membrane in association with the Mitochondrial Contact Site and Cristae Organizing System (MICOS). Using knockout mouse embryonic fibroblasts we show that Miro1 and Miro2 are required for normal mitochondrial cristae architecture and Endoplasmic Reticulum-Mitochondria Contacts Sites (ERMCS). Further, we show that Miro couples MICOS to TRAK motor protein adaptors to ensure the concerted transport of the two mitochondrial membranes and the correct distribution of cristae on the mitochondrial membrane. The Miro nanoscale organization, association with MICOS complex and regulation of ERMCS reveal new levels of control of the Miro GTPases on mitochondrial functionality.

Autism and Schizophrenia-Associated CYFIP1 Regulates the Balance of Synaptic Excitation and Inhibition.

Altered excitatory/inhibitory (E/I) balance is implicated in neuropsychiatric and neurodevelopmental disorders, but the underlying genetic etiology remains poorly understood. Copy number variations in CYFIP1 are associated with autism, schizophrenia, and intellectual disability, but its role in regulating synaptic inhibition or E/I balance remains unclear. We show that CYFIP1, and the paralog CYFIP2, are enriched at inhibitory postsynaptic sites. While CYFIP1 or CYFIP2 upregulation increases excitatory synapse number and the frequency of miniature excitatory postsynaptic currents (mEPSCs), it has the opposite effect at inhibitory synapses, decreasing their size and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Contrary to CYFIP1 upregulation, its loss in vivo, upon conditional knockout in neocortical principal cells, increases expression of postsynaptic GABAA receptor ß2/3-subunits and neuroligin 3, enhancing synaptic inhibition. Thus, CYFIP1 dosage can bi-directionally impact inhibitory synaptic structure and function, potentially leading to altered E/I balance and circuit dysfunction in CYFIP1-associated neurological disorders.

Nanoscale tweezers for single-cell biopsies.

Much of the functionality of multicellular systems arises from the spatial organization and dynamic behaviours within and between cells. Current single-cell genomic methods only provide a transcriptional 'snapshot' of individual cells. The real-time analysis and perturbation of living cells would generate a step change in single-cell analysis. Here we describe minimally invasive nanotweezers that can be spatially controlled to extract samples from living cells with single-molecule precision. They consist of two closely spaced electrodes with gaps as small as 10-20 nm, which can be used for the dielectrophoretic trapping of DNA and proteins. Aside from trapping single molecules, we also extract nucleic acids for gene expression analysis from living cells without affecting their viability. Finally, we report on the trapping and extraction of a single mitochondrion. This work bridges the gap between single-molecule/organelle manipulation and cell biology and can ultimately enable a better understanding of living cells.

Quantum dot conjugated nanobodies for multiplex imaging of protein dynamics at synapses.

Neurons communicate with each other through synapses, which show enrichment for specialized receptors. Although many studies have explored spatial enrichment and diffusion of these receptors in dissociated neurons using single particle tracking, much less is known about their dynamic properties at synapses in complex tissue like brain slices. Here we report the use of smaller and highly specific quantum dots conjugated with a recombinant single domain antibody fragment (VHH fragment) against green fluorescent protein to provide information on diffusion of adhesion molecules at the growth cone and neurotransmitter receptors at synapses. Our data reveals that QD-nanobodies can measure neurotransmitter receptor dynamics at both excitatory and inhibitory synapses in primary neuronal cultures as well as in ex vivo rat brain slices. We also demonstrate that this approach can be applied to tagging multiple proteins to simultaneously monitor their behavior. Thus, we provide a strategy for multiplex imaging of tagged membrane proteins to study their clustering, diffusion and transport both in vitro as well as in native tissue environments such as brain slices.