Protein interactome and cell-type expression analyses reveal that cytoplasmic FMR1-interacting protein 1 (CYFIP1), but not CYFIP2, associates with astrocytic focal adhesion.

The two members of the cytoplasmic FMR1-interacting protein family, CYFIP1 and CYFIP2, are evolutionarily conserved multifunctional proteins whose defects are associated with distinct types of brain disorders. Even with high sequence homology between CYFIP1 and CYFIP2, several lines of evidence indicate their different functions in the brain; however, the underlying mechanisms remain largely unknown. Here, we performed reciprocal immunoprecipitation experiments using CYFIP1-2 × Myc and CYFIP2-3 × Flag knock-in mice and found that CYFIP1 and CYFIP2 are not significantly co-immunoprecipitated with each other in the knock-in brains compared with negative control wild-type (WT) brains. Moreover, CYFIP1 and CYFIP2 showed different size distributions by size-exclusion chromatography of WT mouse brains. Specifically, mass spectrometry-based analysis of CYFIP1-2 × Myc knock-in brains identified 131 proteins in the CYFIP1 interactome. Comparison of the CYFIP1 interactome with the previously identified brain region- and age-matched CYFIP2 interactome, consisting of 140 proteins, revealed only eight common proteins. Investigations using single-cell RNA-sequencing databases suggested non-neuronal cell- and neuron-enriched expression of Cyfip1 and Cyfip2, respectively. At the protein level, CYFIP1 was detected in both neurons and astrocytes, while CYFIP2 was detected only in neurons, suggesting the predominant expression of CYFIP1 in astrocytes. Bioinformatic characterization of the CYFIP1 interactome, and co-expression analysis of Cyfip1 with astrocytic genes, commonly linked CYFIP1 with focal adhesion proteins. Immunocytochemical analysis and proximity ligation assay suggested partial co-localization of CYFIP1 and focal adhesion proteins in cultured astrocytes. Together, these results suggest a CYFIP1-specific association with astrocytic focal adhesion, which may contribute to the different brain functions and dysfunctions of CYFIP1 and CYFIP2. Cover Image for this issue: https://doi.org/10.1111/jnc.15410.

PSMD14 Targeting Triggers Paraptosis in Breast Cancer Cells by Inducing Proteasome Inhibition and Ca2+ Imbalance.

PSMD14, a subunit of the 19S regulatory particles of the 26S proteasome, was recently identified as a potential prognostic marker and therapeutic target in diverse human cancers. Here, we show that the silencing and pharmacological blockade of PSMD14 in MDA-MB 435S breast cancer cells induce paraptosis, a non-apoptotic cell death mode characterized by extensive vacuolation derived from the endoplasmic reticulum (ER) and mitochondria. The PSMD14 inhibitor, capzimin (CZM), inhibits proteasome activity but differs from the 20S proteasome subunit-inhibiting bortezomib (Bz) in that it does not induce aggresome formation or Nrf1 upregulation, which underlie Bz resistance in cancer cells. In addition to proteasome inhibition, the release of Ca2+ from the ER into the cytosol critically contributes to CZM-induced paraptosis. Induction of paraptosis by targeting PSMD14 may provide an attractive therapeutic strategy against cancer cells resistant to proteasome inhibitors or pro-apoptotic drugs.

Correction: LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

[This corrects the article DOI: 10.1371/journal.pbio.1002516.].

Pleiotropic Mitochondria: The Influence of Mitochondria on Neuronal Development and Disease.

Mitochondria play many important biological roles, including ATP production, lipid biogenesis, ROS regulation, and calcium clearance. In neurons, the mitochondrion is an essential organelle for metabolism and calcium homeostasis. Moreover, mitochondria are extremely dynamic and able to divide, fuse, and move along microtubule tracks to ensure their distribution to the neuronal periphery. Mitochondrial dysfunction and altered mitochondrial dynamics are observed in a wide range of conditions, from impaired neuronal development to various neurodegenerative diseases. Novel imaging techniques and genetic tools provide unprecedented access to the physiological roles of mitochondria by visualizing mitochondrial trafficking, morphological dynamics, ATP generation, and ultrastructure. Recent studies using these new techniques have unveiled the influence of mitochondria on axon branching, synaptic function, calcium regulation with the ER, glial cell function, neurogenesis, and neuronal repair. This review provides an overview of the crucial roles played by mitochondria in the CNS in physiological and pathophysiological conditions.

LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

Individual synapses vary significantly in their neurotransmitter release properties, which underlie complex information processing in neural circuits. Presynaptic Ca2+ homeostasis plays a critical role in specifying neurotransmitter release properties, but the mechanisms regulating synapse-specific Ca2+ homeostasis in the mammalian brain are still poorly understood. Using electrophysiology and genetically encoded Ca2+ sensors targeted to the mitochondrial matrix or to presynaptic boutons of cortical pyramidal neurons, we demonstrate that the presence or absence of mitochondria at presynaptic boutons dictates neurotransmitter release properties through Mitochondrial Calcium Uniporter (MCU)-dependent Ca2+ clearance. We demonstrate that the serine/threonine kinase LKB1 regulates MCU expression, mitochondria-dependent Ca2+ clearance, and thereby, presynaptic release properties. Re-establishment of MCU-dependent mitochondrial Ca2+ uptake at glutamatergic synapses rescues the altered neurotransmitter release properties characterizing LKB1-null cortical axons. Our results provide novel insights into the cellular and molecular mechanisms whereby mitochondria control neurotransmitter release properties in a bouton-specific way through presynaptic Ca2+ clearance.

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn.

The receptor-type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPrho is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto-domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto-domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain-specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.

Neuronal mitochondrial morphology is significantly affected by both fixative and oxygen level during perfusion.

Neurons in the brain have a uniquely polarized structure consisting of multiple dendrites and a single axon generated from a cell body. Interestingly, intracellular mitochondria also show strikingly polarized morphologies along the dendrites and axons: in cortical pyramidal neurons (PNs), dendritic mitochondria have a long and tubular shape, while axonal mitochondria are small and circular. Mitochondria play important roles in each compartment of the neuron by generating adenosine triphosphate (ATP) and buffering calcium, thereby affecting synaptic transmission and neuronal development. In addition, mitochondrial shape, and thereby function, is dynamically altered by environmental stressors such as oxidative stress or in various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Although the importance of altered mitochondrial shape has been claimed by multiple studies, methods for studying this stress-sensitive organelle have not been standardized. Here we address pertinent steps that influence mitochondrial morphology during experimental processes. We demonstrate that fixative solutions containing only paraformaldehyde (PFA), or that introduce hypoxic conditions during the procedure, induce dramatic fragmentation of mitochondria both in vitro and in vivo. This disruption was not observed following the use of glutaraldehyde (GA) addition or oxygen supplementation, respectively. Finally, using pre-formed fibril α-synuclein treated neurons, we show fixative choice can alter experimental outcomes. Specifically, α-synuclein-induced mitochondrial remodeling could not be observed with PFA only fixation as fixation itself caused mitochondrial fragmentation. Our study provides optimized methods for examining mitochondrial morphology in neurons and demonstrates that fixation conditions are critical when investigating the underlying cellular mechanisms involving mitochondria in physiological and neurodegenerative disease models.

Trans-synaptic adhesions between netrin-G ligand-3 (NGL-3) and receptor tyrosine phosphatases LAR, protein-tyrosine phosphatase delta (PTPdelta), and PTPsigma via specific domains regulate excitatory synapse formation.

Synaptic cell adhesion molecules regulate various steps of synapse formation. The trans-synaptic adhesion between postsynaptic NGL-3 (for netrin-G ligand-3) and presynaptic LAR (for leukocyte antigen-related) regulates excitatory synapse formation in a bidirectional manner. However, little is known about the molecular details of the NGL-3-LAR adhesion and whether two additional LAR family proteins, protein-tyrosine phosphatase delta (PTPdelta), and PTPsigma, also interact with NGL-3 and are involved in synapse formation. We report here that the leucine-rich repeat (LRR) domain of NGL-3, containing nine LRRs, interacts with the first two fibronectin III (FNIII) domains of LAR to induce bidirectional synapse formation. Moreover, Gln-96 in the first LRR motif of NGL-3 is critical for LAR binding and induction of presynaptic differentiation. PTPdelta and PTPsigma also interact with NGL-3 via their first two FNIII domains. These two interactions promote synapse formation in a different manner; the PTPsigma-NGL-3 interaction promotes synapse formation in a bidirectional manner, whereas the PTPdelta-NGL-3 interaction instructs only presynaptic differentiation in a unidirectional manner. mRNAs encoding LAR family proteins display overlapping and differential expression patterns in various brain regions. These results suggest that trans-synaptic adhesion between NGL-3 and the three LAR family proteins regulates excitatory synapse formation in shared and distinct neural circuits.

The NGL family of leucine-rich repeat-containing synaptic adhesion molecules.

Cell adhesion molecules at neuronal synapses regulate diverse aspects of synaptic development, including axo-dendritic contact establishment, early synapse formation, and synaptic maturation. Recent studies have identified several synaptogenic adhesion molecules. The NGL (netrin-G ligand; LRRC4) family of synaptic cell adhesion molecules belongs to the superfamily of leucine-rich repeat (LRR) proteins. The three known members of the NGL family, NGL-1, NGL-2, and NGL-3, are mainly localized to the postsynaptic side of excitatory synapses, and interact with the presynaptic ligands, netrin-G1, netrin-G2, and LAR, respectively. NGLs interact with the abundant postsynaptic density (PSD) protein, PSD-95, and other postsynaptic proteins, including NMDA receptors. These interactions are thought to couple synaptic adhesion events to the assembly of synaptic proteins. In addition, NGL proteins regulate axonal outgrowth and lamina-specific dendritic segmentation, suggesting that the NGL-dependent adhesion system is important for the development of axons, dendrites, and synapses. Consistent with these functions, defects in NGLs and their ligands are associated with impaired learning and memory, hyperactivity, and an abnormal acoustic startle response in transgenic mice, and schizophrenia, bipolar disorder, and Rett syndrome in human patients.

NGL family PSD-95-interacting adhesion molecules regulate excitatory synapse formation.

Synaptic cell adhesion molecules (CAMs) regulate synapse formation through their trans-synaptic and heterophilic adhesion. Here we show that postsynaptic netrin-G ligand (NGL) CAMs associate with netrin-G CAMs in an isoform-specific manner and, through their cytosolic tail, with the abundant postsynaptic scaffold postsynaptic density-95 (PSD-95). Overexpression of NGL-2 in cultured rat neurons increased the number of PSD-95-positive dendritic protrusions. NGL-2 located on heterologous cells or beads induced functional presynaptic differentiation in contacting neurites. Direct aggregation of NGL-2 on the surface membrane of dendrites induced the clustering of excitatory postsynaptic proteins. Competitive inhibition by soluble NGL-2 reduced the number of excitatory synapses. NGL-2 knockdown reduced excitatory, but not inhibitory, synapse numbers and currents. These results suggest that NGL regulates the formation of excitatory synapses.

Dysfunction of Mitochondrial Ca2+ Regulatory Machineries in Brain Aging and Neurodegenerative Diseases.

Calcium ions (Ca2+) play critical roles in neuronal processes, such as signaling pathway activation, transcriptional regulation, and synaptic transmission initiation. Therefore, the regulation of Ca2+ homeostasis is one of the most important processes underlying the basic cellular viability and function of the neuron. Multiple components, including intracellular organelles and plasma membrane Ca2+-ATPase, are involved in neuronal Ca2+ control, and recent studies have focused on investigating the roles of mitochondria in synaptic function. Numerous mitochondrial Ca2+ regulatory proteins have been identified in the past decade, with studies demonstrating the tissue- or cell-type-specific function of each component. The mitochondrial calcium uniporter and its binding subunits are major inner mitochondrial membrane proteins contributing to mitochondrial Ca2+ uptake, whereas the mitochondrial Na+/Ca2+ exchanger (NCLX) and mitochondrial permeability transition pore (mPTP) are well-studied proteins involved in Ca2+ extrusion. The level of cytosolic Ca2+ and the resulting characteristics of synaptic vesicle release properties are controlled via mitochondrial Ca2+ uptake and release at presynaptic sites, while in dendrites, mitochondrial Ca2+ regulation affects synaptic plasticity. During brain aging and the progress of neurodegenerative disease, mitochondrial Ca2+ mishandling has been observed using various techniques, including live imaging of Ca2+ dynamics. Furthermore, Ca2+ dysregulation not only disrupts synaptic transmission but also causes neuronal cell death. Therefore, understanding the detailed pathophysiological mechanisms affecting the recently discovered mitochondrial Ca2+ regulatory machineries will help to identify novel therapeutic targets. Here, we discuss current research into mitochondrial Ca2+ regulatory machineries and how mitochondrial Ca2+ dysregulation contributes to brain aging and neurodegenerative disease.

The small GTPase ARF6 regulates GABAergic synapse development.

ADP ribosylation factors (ARFs) are a family of small GTPases composed of six members (ARF1-6) that control various cellular functions, including membrane trafficking and actin cytoskeletal rearrangement, in eukaryotic cells. Among them, ARF1 and ARF6 are the most studied in neurons, particularly at glutamatergic synapses, but their roles at GABAergic synapses have not been investigated. Here, we show that a subset of ARF6 protein is localized at GABAergic synapses in cultured hippocampal neurons. In addition, we found that knockdown (KD) of ARF6, but not ARF1, triggered a reduction in the number of GABAergic synaptic puncta in mature cultured neurons in an ARF activity-dependent manner. ARF6 KD also reduced GABAergic synaptic density in the mouse hippocampal dentate gyrus (DG) region. Furthermore, ARF6 KD in the DG increased seizure susceptibility in an induced epilepsy model. Viewed together, our results suggest that modulating ARF6 and its regulators could be a therapeutic strategy against brain pathologies involving hippocampal network dysfunction, such as epilepsy.

Super-Resolution Three-Dimensional Imaging of Actin Filaments in Cultured Cells and the Brain via Expansion Microscopy.

Actin is an essential protein in almost all life forms. It mediates diverse biological functions, ranging from controlling the shape of cells and cell movements to cargo transport and the formation of synaptic connections. Multiple diseases are closely related to the dysfunction of actin or actin-related proteins. Despite the biological importance of actin, super-resolution imaging of it in tissue is still challenging, as it forms very dense networks in almost all cells inside the tissue. In this work, we demonstrate multiplexed super-resolution volumetric imaging of actin in both cultured cells and mouse brain slices via expansion microscopy (ExM). By introducing a simple labeling process, which enables the anchoring of an actin probe, phalloidin, to a swellable hydrogel, the multiplexed ExM imaging of actin filaments was achieved. We first showed that this technique could visualize the nanoscale details of actin filament organizations in cultured cells. Then, we applied this technique to mouse brain slices and visualized diverse actin organizations, such as the parallel actin filaments along the long axis of dendrites and dense actin structures in postsynaptic spines. We examined the postsynaptic spines in the mouse brain and showed that the organizations of actin filaments are highly diverse. This technique, which enables the high-throughput 60 nm resolution imaging of actin filaments and other proteins in cultured cells and thick tissue slices, would be a useful tool to study the organization of actin filaments in diverse biological circumstances and how they change under pathological conditions.

Altered presynaptic function and number of mitochondria in the medial prefrontal cortex of adult Cyfip2 heterozygous mice.

Variants of the cytoplasmic FMR1-interacting protein (CYFIP) gene family, CYFIP1 and CYFIP2, are associated with numerous neurodevelopmental and neuropsychiatric disorders. According to several studies, CYFIP1 regulates the development and function of both pre- and post-synapses in neurons. Furthermore, various studies have evaluated CYFIP2 functions in the postsynaptic compartment, such as regulating dendritic spine morphology; however, no study has evaluated whether and how CYFIP2 affects presynaptic functions. To address this issue, in this study, we have focused on the presynapses of layer 5 neurons of the medial prefrontal cortex (mPFC) in adult Cyfip2 heterozygous (Cyfip2+/-) mice. Electrophysiological analyses revealed an enhancement in the presynaptic short-term plasticity induced by high-frequency stimuli in Cyfip2+/- neurons compared with wild-type neurons. Since presynaptic mitochondria play an important role in buffering presynaptic Ca2+, which is directly associated with the short-term plasticity, we analyzed presynaptic mitochondria using electron microscopic images of the mPFC. Compared with wild-type mice, the number, but not the volume or cristae density, of mitochondria in both presynaptic boutons and axonal processes in the mPFC layer 5 of Cyfip2+/- mice was reduced. Consistent with an identification of mitochondrial proteins in a previously established CYFIP2 interactome, CYFIP2 was detected in a biochemically enriched mitochondrial fraction of the mouse mPFC. Collectively, these results suggest roles for CYFIP2 in regulating presynaptic functions, which may involve presynaptic mitochondrial changes.

Enhanced Prefrontal Neuronal Activity and Social Dominance Behavior in Postnatal Forebrain Excitatory Neuron-Specific Cyfip2 Knock-Out Mice.

The cytoplasmic fragile X mental retardation 1 (FMR1)-interacting protein 2 (CYFIP2) gene is associated with epilepsy, intellectual disability (ID), and developmental delay, suggesting its critical role in proper neuronal development and function. CYFIP2 is involved in regulating cellular actin dynamics and also interacts with RNA-binding proteins. However, the adult brain function of CYFIP2 remains unclear because investigations thus far are limited to Cyfip2 heterozygous (Cyfip2+/- ) mice owing to the perinatal lethality of Cyfip2-null mice. Therefore, we generated Cyfip2 conditional knock-out (cKO) mice with reduced CYFIP2 expression in postnatal forebrain excitatory neurons (CaMKIIα-Cre). We found that in the medial prefrontal cortex (mPFC) of adult Cyfip2 cKO mice, CYFIP2 expression was decreased in both layer 2/3 (L2/3) and layer 5 (L5) neurons, unlike the L5-specific CYFIP2 reduction observed in adult Cyfip2+/- mice. Nevertheless, filamentous actin (F-actin) levels were increased only in L5 of Cyfip2 cKO mPFC possibly because of a compensatory increase in CYFIP1, the other member of CYFIP family, in L2/3 neurons. Abnormal dendritic spines on basal, but not on apical, dendrites were consistently observed in L5 neurons of Cyfip2 cKO mPFC. Meanwhile, neuronal excitability and activity were enhanced in both L2/3 and L5 neurons of Cyfip2 cKO mPFC, suggesting that CYFIP2 functions of regulating F-actin and excitability/activity may be mediated through independent mechanisms. Unexpectedly, adult Cyfip2 cKO mice did not display locomotor hyperactivity or reduced anxiety observed in Cyfip2+/- mice. Instead, both exhibited enhanced social dominance accessed by the tube test. Together, these results provide additional insights into the functions of CYFIP2 in the adult brain.

The Neomycin Resistance Cassette in the Targeted Allele of Shank3B Knock-Out Mice Has Potential Off-Target Effects to Produce an Unusual Shank3 Isoform.

Variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3), which encodes postsynaptic scaffolds, are associated with brain disorders. The targeted alleles in a few Shank3 knock-out (KO) lines contain a neomycin resistance (Neo) cassette, which may perturb the normal expression of neighboring genes; however, this has not been investigated in detail. We previously reported an unexpected increase in the mRNA expression of Shank3 exons 1-12 in the brains of Shank3B KO mice generated by replacing Shank3 exons 13-16 with the Neo cassette. In this study, we confirmed that the increased Shank3 mRNA in Shank3B KO brains produced an unusual ∼60 kDa Shank3 isoform (Shank3-N), which did not properly localize to the synaptic compartment. Functionally, Shank3-N overexpression altered the dendritic spine morphology in cultured neurons. Importantly, Shank3-N expression in Shank3B KO mice was not a compensatory response to a reduction of full-length Shank3 because expression was still detected in the brain after normalizing the level of full-length Shank3. Moreover, in another Shank3 KO line (Shank3 gKO) with a similar Shank3 exonal deletion as that in Shank3B KO mice but without a Neo cassette, the mRNA expression levels of Shank3 exons 1-12 were lower than those of wild-type mice and Shank3-N was not detected in the brain. In addition, the expression levels of genes neighboring Shank3 on chromosome 15 were altered in the striatum of Shank3B KO but not Shank3 gKO mice. These results suggest that the Neo cassette has potential off-target effects in Shank3B KO mice.

PINK1 controls mitochondrial localization of Parkin through direct phosphorylation.

PTEN-induced putative kinase 1 (PINK1) and Parkin, encoded by their respective genes associated with Parkinson's disease (PD), are linked in a common pathway involved in the protection of mitochondrial integrity and function. However, the mechanism of their interaction at the biochemical level has not been investigated yet. Using both mammalian and Drosophila systems, we here demonstrate that the PINK1 kinase activity is required for its function in mitochondria. PINK1 regulates the localization of Parkin to the mitochondria in its kinase activity-dependent manner. In detail, Parkin phosphorylation by PINK1 on its linker region promotes its mitochondrial translocation, and the RING1 domain of Parkin is critical for this occurrence. These results demonstrate the biochemical relationship between PINK1, Parkin, and the mitochondria and thereby suggest the possible mechanism of PINK-Parkin-associated PD pathogenesis.

Emerging roles of mitochondria in synaptic transmission and neurodegeneration.

Mitochondria play numerous critical physiological functions in neurons including ATP production, Ca2+ regulation, lipid synthesis, ROS signaling, and the ability to trigger apoptosis. Recently developed technologies, including in vivo 2-photon imaging in awake behaving mice revealed that unlike in the peripheral nervous system (PNS), mitochondrial transport decreases strikingly along the axons of adult neurons of the central nervous system (CNS). Furthermore, the improvements of genetically-encoded biosensors have enabled precise monitoring of the spatial and temporal impact of mitochondria on Ca2+, ATP and ROS homeostasis in a compartment-specific manner. Here, we discuss recent findings that begin to unravel novel physiological and pathophysiological properties of neuronal mitochondria at synapses. We also suggest new directions in the exploration of mitochondrial function in synaptic transmission, plasticity and neurodegeneration.

MFF-dependent mitochondrial fission regulates presynaptic release and axon branching by limiting axonal mitochondria size.

Neurons display extreme degrees of polarization, including compartment-specific organelle morphology. In cortical, long-range projecting, pyramidal neurons (PNs), dendritic mitochondria are long and tubular whereas axonal mitochondria display uniformly short length. Here we explored the functional significance of maintaining small mitochondria for axonal development in vitro and in vivo. We report that the Drp1 'receptor' Mitochondrial fission factor (MFF) is required for determining the size of mitochondria entering the axon and then for maintenance of their size along the distal portions of the axon without affecting their trafficking properties, presynaptic capture, membrane potential or ability to generate ATP. Strikingly, this increase in presynaptic mitochondrial size upon MFF downregulation augments their capacity for Ca2+ ([Ca2+]m) uptake during neurotransmission, leading to reduced presynaptic [Ca2+]c accumulation, decreased presynaptic release and terminal axon branching. Our results uncover a novel mechanism controlling neurotransmitter release and axon branching through fission-dependent regulation of presynaptic mitochondrial size.

ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons.

Interfaces between organelles are emerging as critical platforms for many biological responses in eukaryotic cells. In yeast, the ERMES complex is an endoplasmic reticulum (ER)-mitochondria tether composed of four proteins, three of which contain a SMP (synaptotagmin-like mitochondrial-lipid binding protein) domain. No functional ortholog for any ERMES protein has been identified in metazoans. Here, we identified PDZD8 as an ER protein present at ER-mitochondria contacts. The SMP domain of PDZD8 is functionally orthologous to the SMP domain found in yeast Mmm1. PDZD8 was necessary for the formation of ER-mitochondria contacts in mammalian cells. In neurons, PDZD8 was required for calcium ion (Ca2+) uptake by mitochondria after synaptically induced Ca2+-release from ER and thereby regulated cytoplasmic Ca2+ dynamics. Thus, PDZD8 represents a critical ER-mitochondria tethering protein in metazoans. We suggest that ER-mitochondria coupling is involved in the regulation of dendritic Ca2+ dynamics in mammalian neurons.