Organelle communication maintains mitochondrial and endosomal homeostasis during podocyte lipotoxicity.

Organelle stress exacerbates podocyte injury, contributing to perturbed lipid metabolism. Simultaneous organelle stresses occur in kidney tissues; therefore, a thorough analysis of organelle communication is crucial for understanding the progression of kidney diseases. Although organelles closely interact with one another at membrane contact sites, limited studies have explored their involvement in kidney homeostasis. The endoplasmic reticulum (ER) protein, PDZ domain-containing 8 (PDZD8), is implicated in multiple organelle tethering processes and cellular lipid homeostasis. In this study, we aimed to elucidate the role of organelle communication in podocyte injury using podocyte-specific Pdzd8-knockout mice. Our findings demonstrated that Pdzd8 deletion exacerbated podocyte injury in an accelerated obesity-related kidney disease model. Proteomic analysis of isolated glomeruli revealed that Pdzd8 deletion exacerbated mitochondrial and endosomal dysfunction during podocyte lipotoxicity. Additionally, electron microscopy revealed the accumulation of "fatty abnormal endosomes" in Pdzd8-deficient podocytes during obesity-related kidney diseases. Lipidomic analysis indicated that glucosylceramide accumulated in Pdzd8-deficient podocytes, owing to accelerated production and decelerated degradation. Thus, the organelle-tethering factor, PDZD8, plays a crucial role in maintaining mitochondrial and endosomal homeostasis during podocyte lipotoxicity. Collectively, our findings highlight the importance of organelle communication at the three-way junction among the ER, mitochondria, and endosomes in preserving podocyte homeostasis.

Optimization of an adeno-associated viral vector for epidermal keratinocytes in vitro and in vivo.

BACKGROUND: Local gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. OBJECTIVE: To optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. METHODS: AAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. RESULTS: A novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. CONCLUSION: The combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies.

PDZD8-FKBP8 tethering complex at ER-mitochondria contact sites regulates mitochondrial complexity.

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identified the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-Electron Microscopy (Cryo-EM) tomography, and correlative light-EM (CLEM). Single molecule tracking revealed highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and capture at MERCS. Overexpression of FKBP8 was sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrated their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. Here, we demonstrate using multiple, independent, in vitro and in vivo approaches that the majority (~80-90%) of axonal mitochondria in cortical pyramidal neurons (CPNs), lack mitochondrial DNA (mtDNA). Using dynamic, optical imaging analysis of genetically encoded sensors for mitochondrial matrix ATP and pH, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial complex V (ATP synthase) functions in a reverse way, consuming ATP and protruding H+ out of the matrix to maintain mitochondrial membrane potential. Our results demonstrate that in mammalian CPNs, axonal mitochondria do not play a major role in ATP supply, despite playing other functions critical to regulating neurotransmission such as Ca2+ buffering.

An interactive deep learning-based approach reveals mitochondrial cristae topologies.

The convolution of membranes called cristae is a critical structural and functional feature of mitochondria. Crista structure is highly diverse between different cell types, reflecting their role in metabolic adaptation. However, their precise three-dimensional (3D) arrangement requires volumetric analysis of serial electron microscopy and has therefore been limiting for unbiased quantitative assessment. Here, we developed a novel, publicly available, deep learning (DL)-based image analysis platform called Python-based human-in-the-loop workflow (PHILOW) implemented with a human-in-the-loop (HITL) algorithm. Analysis of dense, large, and isotropic volumes of focused ion beam-scanning electron microscopy (FIB-SEM) using PHILOW reveals the complex 3D nanostructure of both inner and outer mitochondrial membranes and provides deep, quantitative, structural features of cristae in a large number of individual mitochondria. This nanometer-scale analysis in micrometer-scale cellular contexts uncovers fundamental parameters of cristae, such as total surface area, orientation, tubular/lamellar cristae ratio, and crista junction density in individual mitochondria. Unbiased clustering analysis of our structural data unraveled a new function for the dynamin-related GTPase Optic Atrophy 1 (OPA1) in regulating the balance between lamellar versus tubular cristae subdomains.

Compartmentalization of soluble endocytic proteins in synaptic vesicle clusters by phase separation.

Synaptic vesicle (SV) clusters, which reportedly result from synapsin's capacity to undergo liquid-liquid phase separation (LLPS), constitute the structural basis for neurotransmission. Although these clusters contain various endocytic accessory proteins, how endocytic proteins accumulate in SV clusters remains unknown. Here, we report that endophilin A1 (EndoA1), the endocytic scaffold protein, undergoes LLPS under physiologically relevant concentrations at presynaptic terminals. On heterologous expression, EndoA1 facilitates the formation of synapsin condensates and accumulates in SV-like vesicle clusters via synapsin. Moreover, EndoA1 condensates recruit endocytic proteins such as dynamin 1, amphiphysin, and intersectin 1, none of which are recruited in vesicle clusters by synapsin. In cultured neurons, like synapsin, EndoA1 is compartmentalized in SV clusters through LLPS, exhibiting activity-dependent dispersion/reassembly cycles. Thus, beyond its essential function in SV endocytosis, EndoA1 serves an additional structural function by undergoing LLPS, thereby accumulating various endocytic proteins in dynamic SV clusters in concert with synapsin.

Compartment-specific tuning of dendritic feature selectivity by intracellular Ca2+ release.

Dendritic calcium signaling is central to neural plasticity mechanisms that allow animals to adapt to the environment. Intracellular calcium release (ICR) from the endoplasmic reticulum has long been thought to shape these mechanisms. However, ICR has not been investigated in mammalian neurons in vivo. We combined electroporation of single CA1 pyramidal neurons, simultaneous imaging of dendritic and somatic activity during spatial navigation, optogenetic place field induction, and acute genetic augmentation of ICR cytosolic impact to reveal that ICR supports the establishment of dendritic feature selectivity and shapes integrative properties determining output-level receptive fields. This role for ICR was more prominent in apical than in basal dendrites. Thus, ICR cooperates with circuit-level architecture in vivo to promote the emergence of behaviorally relevant plasticity in a compartment-specific manner.

New insights into the regulation of synaptic transmission and plasticity by the endoplasmic reticulum and its membrane contacts.

Mammalian neurons are highly compartmentalized yet very large cells. To provide each compartment with its distinct properties, metabolic homeostasis and molecular composition need to be precisely coordinated in a compartment-specific manner. Despite the importance of the endoplasmic reticulum (ER) as a platform for various biochemical reactions, such as protein synthesis, protein trafficking, and intracellular calcium control, the contribution of the ER to neuronal compartment-specific functions and plasticity remains elusive. Recent advances in the development of live imaging and serial scanning electron microscopy (sSEM) analysis have revealed that the neuronal ER is a highly dynamic organelle with compartment-specific structures. sSEM studies also revealed that the ER forms contacts with other membranes, such as the mitochondria and plasma membrane, although little is known about the functions of these ER-membrane contacts. In this review, we discuss the mechanisms and physiological roles of the ER structure and ER-mitochondria contacts in synaptic transmission and plasticity, thereby highlighting a potential link between organelle ultrastructure and neuronal functions.

Endoplasmic Reticulum-Mitochondria Contact Sites-Emerging Intracellular Signaling Hubs.

It has become apparent that our textbook illustration of singular isolated organelles is obsolete. In reality, organelles form complex cooperative networks involving various types of organelles. Light microscopic and ultrastructural studies have revealed that mitochondria-endoplasmic reticulum (ER) contact sites (MERCSs) are abundant in various tissues and cell types. Indeed, MERCSs have been proposed to play critical roles in various biochemical and signaling functions such as Ca2+ homeostasis, lipid transfer, and regulation of organelle dynamics. While numerous proteins involved in these MERCS-dependent functions have been reported, how they coordinate and cooperate with each other has not yet been elucidated. In this review, we summarize the functions of mammalian proteins that localize at MERCSs and regulate their formation. We also discuss potential roles of the MERCS proteins in regulating multiple organelle contacts.

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.

Diverse gene regulatory mechanisms mediated by Polycomb group proteins during neural development.

While all the developmental genes are temporarily repressed for future activation in the pluripotent stem cells, non-neural genes become persistently repressed in the course of commitment to the neuronal lineage. Although Polycomb group proteins (PcG) are key factors for both temporary and persistent repression of the developmental genes, how the same group of proteins can differentially repress target genes remains unclarified. The identification of a variety of PcG complexes and activities sheds light on these issues. In this review, based on the recent findings including those with the use of interactome and Chromosome Conformation Capture (3C)-type analyses, we summarize the molecular mechanisms of PcG-mediated gene regulation and discuss how PcG regulates cell fate specification during neural development.

Ubiquitination-Independent Repression of PRC1 Targets during Neuronal Fate Restriction in the Developing Mouse Neocortex.

Polycomb repressive complex (PRC) 1 maintains developmental genes in a poised state through monoubiquitination (Ub) of histone H2A. Although Ub-independent functions of PRC1 have also been suggested, it has remained unclear whether Ub-dependent and -independent functions of PRC1 operate differentially in a developmental context. Here, we show that the E3 ubiquitin ligase activity of Ring1B, a core component of PRC1, is necessary for the temporary repression of key neuronal genes in neurogenic (early-stage) neural stem or progenitor cells (NPCs) but is dispensable for the persistent repression of these genes associated with the loss of neurogenic potential in astrogliogenic (late-stage) NPCs. Our results also suggest that histone deacetylase (HDAC) activity of the NuRD/MBD3 complex and Phc2-dependent PRC1 clustering are necessary for the transition from the Ub-dependent to -independent function of PRC1. Together, these results indicate that Ub-independent mode of repression by PRC1 plays a key role in mammalian development during cell fate restriction.

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.

Correlated Light-Serial Scanning Electron Microscopy (CoLSSEM) for ultrastructural visualization of single neurons in vivo.

A challenging aspect of neuroscience revolves around mapping the synaptic connections within neural circuits (connectomics) over scales spanning several orders of magnitude (nanometers to meters). Despite significant improvements in serial section electron microscopy (SSEM) technologies, several major roadblocks have impaired its general applicability to mammalian neural circuits. In the present study, we introduce a new approach that circumvents some of these roadblocks by adapting a genetically-encoded ascorbate peroxidase (APEX2) as a fusion protein to a membrane-targeted fluorescent reporter (CAAX-Venus), and introduce it in single pyramidal neurons in vivo using extremely sparse in utero cortical electroporation. This approach allows us to perform Correlated Light-SSEM (CoLSSEM), a variant of Correlated Light-EM (CLEM), on individual neurons, reconstructing their dendritic and axonal arborization in a targeted way via combination of high-resolution confocal microscopy, and subsequent imaging of its ultrastructural features and synaptic connections with ATUM-SEM (automated tape-collecting ultramicrotome - scanning electron microscopy) technology. Our method significantly will improve the feasibility of large-scale reconstructions of neurons within a circuit, and permits the description of some ultrastructural features of identified neurons with their functional and/or structural connectivity, one of the main goal of connectomics.

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.].

Optogenetic Control of Endoplasmic Reticulum-Mitochondria Tethering.

The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER-mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER-mitochondrial contacts.

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.

Multicluster Pcdh diversity is required for mouse olfactory neural circuit assembly.

The vertebrate clustered protocadherin (Pcdh) cell surface proteins are encoded by three closely linked gene clusters (Pcdhα, Pcdhß, and Pcdhγ). Here, we show that all three gene clusters functionally cooperate to provide individual mouse olfactory sensory neurons (OSNs) with the cell surface diversity required for their assembly into distinct glomeruli in the olfactory bulb. Although deletion of individual Pcdh clusters had subtle phenotypic consequences, the loss of all three clusters (tricluster deletion) led to a severe axonal arborization defect and loss of self-avoidance. By contrast, when endogenous Pcdh diversity is overridden by the expression of a single-tricluster gene repertoire (α and ß and γ), OSN axons fail to converge to form glomeruli, likely owing to contact-mediated repulsion between axons expressing identical combinations of Pcdh isoforms.

Organelle-Specific Sensors for Monitoring Ca2+ Dynamics in Neurons.

Calcium (Ca2+) plays innumerable critical functions in neurons ranging from regulation of neurotransmitter release and synaptic plasticity to activity-dependent transcription. Therefore, more than any other cell types, neurons are critically dependent on spatially and temporally controlled Ca2+ dynamics. This is achieved through an exquisite level of compartmentalization of Ca2+ storage and release from various organelles. The function of these organelles in the regulation of Ca2+ dynamics has been studied for decades using electrophysiological and optical methods combined with pharmacological and genetic alterations. Mitochondria and the endoplasmic reticulum (ER) are among the organelles playing the most critical roles in Ca2+ dynamics in neurons. At presynaptic boutons, Ca2+ triggers neurotransmitter release and synaptic plasticity, and postsynaptically, Ca2+ mobilization mediates long-term synaptic plasticity. To explore Ca2+ dynamics in live cells and intact animals, various synthetic and genetically encoded fluorescent Ca2+ sensors were developed, and recently, many groups actively increased the sensitivity and diversity of genetically encoded Ca2+ indicators (GECIs). Following conjugation with various signal peptides, these improved GECIs can be targeted to specific subcellular compartments, allowing monitoring of organelle-specific Ca2+ dynamics. Here, we review recent findings unraveling novel roles for mitochondria- and ER-dependent Ca2+ dynamics in neurons and at synapses.

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.