Mitofusin 2 Sustains the Axonal Mitochondrial Network to Support Presynaptic Ca2+ Homeostasis and the Synaptic Vesicle Cycle in Rat Hippocampal Axons.

Mitochondria exert powerful control over cellular physiology, contributing to ion homeostasis, energy production, and metabolite biosynthesis. The trafficking and function of these organelles are particularly important in neurons, with impaired mitochondrial function or altered morphology observed in every neurodegenerative disorder studied. While mitochondrial biosynthetic products play a crucial role in maintaining cellular function, their resulting byproducts can have negative consequences. Thus, organelle quality control (QC) mechanisms that maintain mitochondrial function are imperative to restrict destructive signaling cascades in the cell. Axons are particularly sensitive to damage, and there is little consensus regarding the mechanisms that mediate mitochondrial QC in this compartment. Here, we first investigated the unstressed behavior of mitochondria in rat hippocampal neurons of mixed sex, focusing on mitochondrial trafficking and fusion to better understand potential QC mechanisms. We observed size and redox asymmetry of mitochondrial traffic in axons, suggesting an active QC mechanism in this compartment. We also document biochemical complementation upon the fusion and fission of axonal mitochondria. Eliminating fusion by knocking down the neuronal mitochondrial fusion protein mitofusin 2 (MFN2) reduced the rates of axonal mitochondrial trafficking and fusion, decreased the levels of synaptic vesicle (SV) proteins, inhibited exocytosis, and impaired SV recruitment from the reserve pool during extended stimulation. MFN2 knockdown also resulted in presynaptic Ca2+ dyshomeostasis. Remarkably, upon MFN2 knockdown, presynaptic mitochondria sequestered Ca2+ more efficiently, effectively limiting presynaptic Ca2+ transients during stimulation. These results support an active mitochondrial trafficking and fusion-related QC process that supports presynaptic Ca2+ handling and the SV cycle.SIGNIFICANCE STATEMENT Decreased or altered mitochondrial function is observed in many disease states. All neurodegenerative diseases co-present with some sort of mitochondrial abnormality. Therefore, identifying quality control mechanisms that sustain the mitochondrial network in neurons, and particularly in axons, is of significant interest. The response of axonal mitochondria to acutely applied toxins or injury has been studied in detail. Although informative, the response of neurons to these insults might not be physiologically relevant, so it is crucial to also study the basal behavior of axonal mitochondria. Here, we use fluorescent biosensors to investigate the mitochondrial network in neurons and examine the role of mitofusin 2 in maintaining the axonal mitochondrial network and in supporting the synaptic vesicle cycle.

Synaptic vesicle proteins are selectively delivered to axons in mammalian neurons.

Neurotransmitter-filled synaptic vesicles (SVs) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading model posits that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH (retention using selective hooks) system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct transmembrane SV proteins, synaptotagmin 1 and synaptobrevin 2, from the soma of mature cultured rat and mouse neurons. For these studies, the SV reporter constructs were expressed at carefully controlled, very low levels. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons with minimal entry into dendrites. However, even moderate overexpression resulted in the spillover of SV proteins into dendrites, potentially explaining the origin of previous non-polarized transport models, revealing the limited, saturable nature of the direct axonal trafficking pathway. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway, for SV proteins, in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis.

Knockoff: Druggable Cleavage of Membrane Proteins.

Comparative cell biology relies on methods that disrupt protein function. Traditional approaches target the gene that encodes the protein of interest via conventional knockout (KO) methodology, conditional Cre-lox system, or recently, flexible protocols based on CRISPR/Cas9. However, these technologies lack precise temporal control (hours), whereby the slow half-lives of proteins may confound measurements, possibly resulting in misleading phenotypes. Targeting the protein itself bypasses issues pertaining to protein half-life, resulting in more acute disruption. An ideal system would enable controllable protein disruption, dependent on the presence or absence of a small molecule, with high temporal control achieved through washout/addition of the small molecule. Here, we outline the use of knockoff, a general method to disrupt membrane proteins based on the NS3/4A protease of the hepatitis C virus. This technique has been used in post-mitotic cells to study the function of long-lived integral membrane proteins and is suitable for the study of other membrane-bound proteins. Graphic abstract: Removal of the protease inhibitor induces cleavage from the membrane. General model of knockoff method. Inh, Inhibitor; POI, Protein of Interest; NS3/4A, Hepatitis C viral protease.

Synaptotagmin 1 oligomerization via the juxtamembrane linker regulates spontaneous and evoked neurotransmitter release.

Synaptotagmin 1 (syt1) is a Ca2+ sensor that regulates synaptic vesicle exocytosis. Cell-based experiments suggest that syt1 functions as a multimer; however, biochemical and electron microscopy studies have yielded contradictory findings regarding putative self-association. Here, we performed dynamic light scattering on syt1 in solution, followed by electron microscopy, and we used atomic force microscopy to study syt1 self-association on supported lipid bilayers under aqueous conditions. Ring-like multimers were clearly observed. Multimerization was enhanced by Ca2+ and required anionic phospholipids. Large ring-like structures (∼180 nm) were reduced to smaller rings (∼30 nm) upon neutralization of a cluster of juxtamembrane lysine residues; further substitution of residues in the second C2-domain completely abolished self-association. When expressed in neurons, syt1 mutants with graded reductions in self-association activity exhibited concomitant reductions in 1) clamping spontaneous release and 2) triggering and synchronizing evoked release. Thus, the juxtamembrane linker of syt1 plays a crucial role in exocytosis by mediating multimerization.

Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons.

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. 'Zap-and-freeze' electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.

Membrane dynamics and protein targets of lipid droplet microautophagy during ER stress-induced proteostasis in the budding yeast, Saccharomyces cerevisiae.

Our previous studies reveal a mechanism for lipid droplet (LD)-mediated proteostasis in the endoplasmic reticulum (ER) whereby unfolded proteins that accumulate in the ER in response to lipid imbalance-induced ER stress are removed by LDs and degraded by microlipophagy (µLP), autophagosome-independent LD uptake into the vacuole (the yeast lysosome). Here, we show that dithiothreitol- or tunicamycin-induced ER stress also induces µLP and identify an unexpected role for vacuolar membrane dynamics in this process. All stressors studied induce vacuolar fragmentation prior to µLP. Moreover, during µLP, fragmented vacuoles fuse to form cup-shaped structures that encapsulate and ultimately take up LDs. Our studies also indicate that proteins of the endosome sorting complexes required for transport (ESCRT) are upregulated, required for µLP, and recruited to LDs, vacuolar membranes, and sites of vacuolar membrane scission during µLP. We identify possible target proteins for LD-mediated ER proteostasis. Our live-cell imaging studies reveal that one potential target (Nup159) localizes to punctate structures that colocalizes with LDs 1) during movement from ER membranes to the cytosol, 2) during microautophagic uptake into vacuoles, and 3) within the vacuolar lumen. Finally, we find that mutations that inhibit LD biogenesis, homotypic vacuolar membrane fusion or ESCRT function inhibit stress-induced autophagy of Nup159 and other ER proteins. Thus, we have obtained the first direct evidence that LDs and µLP can mediate ER stress-induced ER proteostasis, and identified direct roles for ESCRT and vacuolar membrane fusion in that process.

Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons.

The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.

Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration.

Syntaphilin (SNPH) is a major mitochondrial anchoring protein targeted to axons and excluded from dendrites. In this study, we provide in vivo evidence that this spatial specificity is lost in Shiverer (Shi) mice, a model for progressive multiple sclerosis (MS), resulting in inappropriate intrusion of SNPH into dendrites of cerebellar Purkinje cells with neurodegenerative consequences. Thus, reconstituting dendritic SNPH intrusion in SNPH-KO mice by viral transduction greatly sensitizes Purkinje cells to excitotoxicity when the glutamatergic climbing fibers are stimulated. Finally, we demonstrate in vitro that overexpression of SNPH in dendrites compromises neuronal viability by inducing N-methyl-D-aspartate (NMDA) excitotoxicity, reducing mitochondrial calcium uptake, and interfering with quality control of mitochondria by blocking somal mitophagy. Collectively, we propose that inappropriate immobilization of dendritic mitochondria by SNPH intrusion produces excitotoxicity and suggest that interception of dendritic SNPH intrusion is a therapeutic strategy to combat neurodegeneration.

Reduced mitochondrial fusion and Huntingtin levels contribute to impaired dendritic maturation and behavioral deficits in Fmr1-mutant mice.

Fragile X syndrome results from a loss of the RNA-binding protein fragile X mental retardation protein (FMRP). How FMRP regulates neuronal development and function remains unclear. Here we show that FMRP-deficient immature neurons exhibit impaired dendritic maturation, altered expression of mitochondrial genes, fragmented mitochondria, impaired mitochondrial function, and increased oxidative stress. Enhancing mitochondrial fusion partially rescued dendritic abnormalities in FMRP-deficient immature neurons. We show that FMRP deficiency leads to reduced Htt mRNA and protein levels and that HTT mediates FMRP regulation of mitochondrial fusion and dendritic maturation. Mice with hippocampal Htt knockdown and Fmr1-knockout mice showed similar behavioral deficits that could be rescued by treatment with a mitochondrial fusion compound. Our data unveil mitochondrial dysfunction as a contributor to the impaired dendritic maturation of FMRP-deficient neurons and suggest a role for interactions between FMRP and HTT in the pathogenesis of fragile X syndrome.

Lipid droplet autophagy during energy mobilization, lipid homeostasis and protein quality control.

Lipid droplets (LDs) have well-established functions as sites for lipid storage and energy mobilization to meet the metabolic demands of cells. However, recent studies have expanded the roles of LDs to protein quality control. Lipophagy, or LD degradation by autophagy, plays a vital role not only in the mobilization of free fatty acids (FFAs) and lipid homeostasis at LDs, but also in the adaptation of cells to certain forms of stress including lipid imbalance. Recent studies have provided new mechanistic insights about the diverse types of lipophagy, in particular microlipophagy. This review summarizes key findings about the mechanisms and functions of lipophagy and highlights a novel function of LD microlipophagy as a mechanism to maintain endoplasmic reticulum (ER) proteostasis under conditions of lipid imbalance.

Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons.

Recent reports suggest that botulinum neurotoxin (BoNT) A, which is widely used clinically to inhibit neurotransmission, can spread within networks of neurons to have distal effects, but this remains controversial. Moreover, it is not known whether other members of this toxin family are transferred between neurons. Here, we investigate the potential distal effects of BoNT/A, BoNT/D, and tetanus toxin (TeNT), using central neurons grown in microfluidic devices. Toxins acted upon the neurons that mediated initial entry, but all three toxins were also taken up, via an alternative pathway, into non-acidified organelles that mediated retrograde transport to the somato-dendritic compartment. Toxins were then released into the media, where they entered and exerted their effects upon upstream neurons. These findings directly demonstrate that these agents undergo transcytosis and interneuronal transfer in an active form, resulting in long-distance effects.

A role for Mfb1p in region-specific anchorage of high-functioning mitochondria and lifespan in Saccharomyces cerevisiae.

Previous studies indicate that replicative lifespan in daughter cells of Sacchraromyces cerevisiae depends on the preferential inheritance of young, high-functioning mitochondria. We report here that mitochondria are functionally segregated even within single mother cells in S. cerevisiae. A high-functioning population of mitochondria accumulates at the tip of the mother cell distal to the bud. We find that the mitochondrial F-box protein (Mfb1p) localizes to mitochondria in the mother tip and is required for mitochondrial anchorage at that site, independent of the previously identified anchorage protein Num1p. Deletion of MFB1 results in loss of the mother-tip-localized mitochondrial population, defects in mitochondrial function and premature replicative ageing. Inhibiting mitochondrial inheritance to buds, by deletion of MMR1, in mfb1Δ cells restores mitochondrial distribution, promotes mitochondrial function and extends replicative lifespan. Our results identify a mechanism that retains a reservoir of high-functioning mitochondria in mother cells and thereby preserves maternal reproductive capacity.

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae.

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud.

The transcriptional repressor Sum1p counteracts Sir2p in regulation of the actin cytoskeleton, mitochondrial quality control and replicative lifespan in Saccharomyces cerevisiae.

Increasing the stability or dynamics of the actin cytoskeleton can extend lifespan in C. elegans and S. cerevisiae. Actin cables of budding yeast, bundles of actin filaments that mediate cargo transport, affect lifespan control through effects on mitochondrial quality control. Sir2p, the founding member of the Sirtuin family of lifespan regulators, also affects actin cable dynamics, assembly, and function in mitochondrial quality control. Here, we obtained evidence for novel interactions between Sir2p and Sum1p, a transcriptional repressor that was originally identified through mutations that genetically suppress sir2∆ phenotypes unrelated to lifespan. We find that deletion of SUM1 in wild-type cells results in increased mitochondrial function and actin cable abundance. Furthermore, deletion of SUM1 suppresses defects in actin cables and mitochondria of sir2∆ yeast, and extends the replicative lifespan and cellular health span of sir2∆ cells. Thus, Sum1p suppresses Sir2p function in control of specific aging determinants and lifespan in budding yeast.

Role for Lipid Droplet Biogenesis and Microlipophagy in Adaptation to Lipid Imbalance in Yeast.

The immediate responses to inhibition of phosphatidylcholine (PC) biosynthesis in yeast are altered phospholipid levels, slow growth, and defects in the morphology and localization of ER and mitochondria. With chronic lipid imbalance, yeast adapt. Lipid droplet (LD) biogenesis and conversion of phospholipids to triacylglycerol are required for restoring some phospholipids to near-wild-type levels. We confirmed that the unfolded protein response is activated by this lipid stress and find that Hsp104p is recruited to ER aggregates. We also find that LDs form at ER aggregates, contain polyubiquitinated proteins and an ER chaperone, and are degraded in the vacuole by a process resembling microautophagy. This process, microlipophagy, is required for restoration of organelle morphology and cell growth during adaptation to lipid stress. Microlipophagy does not require ATG7 but does requires ESCRT components and a newly identified class E VPS protein that localizes to ER and is upregulated by lipid imbalance.

Role of asymmetric cell division in lifespan control in Saccharomyces cerevisiae.

Aging determinants are asymmetrically distributed during cell division in S. cerevisiae, which leads to production of an immaculate, age-free daughter cell. During this process, damaged components are sequestered and retained in the mother cell, and higher functioning organelles and rejuvenating factors are transported to and/or enriched in the bud. Here, we will describe the key quality control mechanisms in budding yeast that contribute to asymmetric cell division of aging determinants including mitochondria, endoplasmic reticulum (ER), vacuoles, extrachromosomal rDNA circles (ERCs), and protein aggregates.

Inheritance of the fittest mitochondria in yeast.

Eukaryotic cells compartmentalize their biochemical processes within organelles, which have specific functions that must be maintained for overall cellular health. As the site of aerobic energy mobilization and essential biosynthetic activities, mitochondria are critical for cell survival and proliferation. Here, we describe mechanisms to control the quality and quantity of mitochondria within cells with an emphasis on findings from the budding yeast Saccharomyces cerevisiae. We also describe how mitochondrial quality and quantity control systems that operate during cell division affect lifespan and cell cycle progression.

Actin dynamics affect mitochondrial quality control and aging in budding yeast.

Actin cables of budding yeast are bundles of F-actin that extend from the bud tip or neck to the mother cell tip, serve as tracks for bidirectional cargo transport, and undergo continuous movement from buds toward mother cells [1]. This movement, retrograde actin cable flow (RACF), is similar to retrograde actin flow in lamellipodia, growth cones, immunological synapses, dendritic spines, and filopodia [2-5]. In all cases, actin flow is driven by the push of actin polymerization and assembly at the cell cortex, and myosin-driven pulling forces deeper within the cell [6-10]. Therefore, for movement and inheritance from mothers to buds, mitochondria must "swim upstream" against the opposing force of RACF [11]. We find that increasing RACF rates results in increased fitness of mitochondria inherited by buds and that the increase in mitochondrial fitness leads to extended replicative lifespan and increased cellular healthspan. The sirtuin SIR2 is required for normal RACF and mitochondrial fitness, and increasing RACF rates in sir2Δ cells increases mitochondrial fitness and cellular healthspan but does not affect replicative lifespan. These studies support the model that RACF serves as a filter for segregation of fit from less-fit mitochondria during inheritance, which controls cellular lifespan and healthspan. They also support a role for Sir2p in these processes.

Ratiometric biosensors that measure mitochondrial redox state and ATP in living yeast cells.

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.