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Cardiac disease remains a significant cause of death among humans and therapies to treat the disease are lacking. Importantly, heart failure has also been linked to factors including endoplasmic reticulum stress, mitochondrial bioenergetics, insulin signaling, autophagy, and oxidative stress 1, which are all factors the mitochondria plays a role in. Critically, mitochondria break down across aging and heart also decrease in efficiency in aging. Key factors implicated in mitochondria morphology, such as the mitochondrial contact site and cristae organizing system (MICOS), and its role across aging remains to be seen in cardiac muscle. To better understand the relationship between mitochondria in cardiac muscle, we used transmission electron microscopy (TEM) and serial block facing-scanning electron microscopy (SBF-SEM) to quantitatively analyze the 3D networks in cardiac muscle samples of mice at aging intervals. In studying cristae, the inner folds of mitochondria, we observed a loss of morphology across aging. This mimicked what was observed upon CRISPR/Cas9 knockdown of Mitofilin, Chchd3, Chchd6 (some members of the MICOS complex) and Opa1 which showed poorer quality cristae and fragmented mitochondria, while mitochondria length and volume decreased. We are the first to examine mitochondria changes in cardiac muscle across aging. In combination, these data suggest that, in the heart, loss of the MICOS complex may be implicated in the loss of function in mitochondria that is seen across aging.

Synaptotagmin 9 (SYT9) is a tandem C2 domain Ca2+sensor for exocytosis in neuroendocrine cells; its function in neurons remains unclear. Here, we show that, in mixed-sex cultures, SYT9 does not trigger rapid synaptic vesicle exocytosis in mouse cortical, hippocampal, or striatal neurons, unless it is massively overexpressed. In striatal neurons, loss of SYT9 reduced the frequency of spontaneous neurotransmitter release events (minis). We delved into the underlying mechanism and discovered that SYT9 was localized to dense-core vesicles that contain substance P (SP). Loss of SYT9 impaired SP release, causing the observed decrease in mini frequency. This model is further supported by loss of function mutants. Namely, Ca2+binding to the C2A domain of SYT9 triggered membrane fusionin vitro, and mutations that disrupted this activity abolished the ability of SYT9 to regulate both SP release and mini frequency. We conclude that SYT9 indirectly regulates synaptic transmission in striatal neurons by controlling SP release.SIGNIFICANCE STATEMENTSynaptotagmin 9 (SYT9) has been described as a Ca2+sensor for dense-core vesicle (DCV) exocytosis in neuroendocrine cells, but its role in neurons remains unclear, despite widespread expression in the brain. This article examines the role of SYT9 in synaptic transmission across cultured cortical, hippocampal, and striatal neuronal preparations. We found that SYT9 regulates spontaneous neurotransmitter release in striatal neurons by serving as a Ca2+sensor for the release of the neuromodulator substance P from DCVs. This demonstrates a novel role for SYT9 in neurons and uncovers a new field of study into neuromodulation by SYT9, a protein that is widely expressed in the brain.

While it is commonly thought that microaggressions are isolated incidents, microaggressions are ingrained throughout the academic research institution (Young, Anderson and Stewart 2015; Lee et al. 2020). Persons Excluded from science because of Ethnicity and Race (PEERs) frequently experience microaggressions from various academicians, including graduate students, postdocs and faculty (Asai 2020; Lee et al. 2020). Here, we elaborate on a rationale for concrete actions to cope with and diminish acts of microaggressions that may otherwise hinder the inclusion of PEERs. We encourage Science, Technology, Engineering and Mathematics (STEM) departments and leadership to affirm PEER scholar identities and promote allyship by infusing sensitivity, responsiveness and anti-bias awareness., This article focuses on how mentors can be allies against microaggressions in STEM.

Damaged mitochondria are selectively removed from the cell in a process termed mitophagy. This mitochondrial quality control mechanism is important for neuronal homeostasis, and mutations in pathway components are causative for Parkinson disease and amyotrophic lateral sclerosis (ALS). Here, we discuss our recent work using a novel mild induction paradigm to investigate the spatiotemporal dynamics of mitophagy in primary neurons. Using live-cell imaging, we find that mitophagy-associated proteins translocate to depolarized mitochondrial fragments. These mitophagic events were primarily localized to somatodendritic compartments, suggesting neuronal mitophagy is primarily a somal quality control mechanism. Damaged mitochondria were efficiently sequestered within autophagosomes, but lysosomal fusion or acidification was significantly delayed. Surprisingly, engulfed mitochondria persisted in non-acidified vesicular compartments for hours to days after initial damage. Expression of an ALS-associated mutation disrupted the membrane potential of the mitochondrial network, and oxidative stress exacerbated this effect. Importantly, our results highlight the slow kinetics of mitophagy and suggest that slow turnover of damaged mitochondria may increase neuronal susceptibility to neurodegeneration.

Mitophagy, the selective removal of damaged mitochondria, is thought to be critical to maintain neuronal homeostasis. Mutations of proteins in the pathway cause neurodegenerative diseases, suggesting defective mitochondrial turnover contributes to neurodegeneration. In primary rat hippocampal neurons, we developed a mitophagy induction paradigm where mild oxidative stress induced low levels of mitochondrial damage. Mitophagy-associated proteins were sequentially recruited to depolarized mitochondria followed by sequestration into autophagosomes. The localization of these mitophagy events had a robust somal bias. In basal and induced conditions, engulfed mitochondria remained in non-acidified organelles for hours to days, illustrating efficient autophagosome sequestration but delayed lysosomal fusion or acidification. Furthermore, expression of an ALS-linked mutation in the pathway disrupted mitochondrial network integrity and this effect was exacerbated by oxidative stress. Thus, age-related decline in neuronal health or expression of disease-associated mutations in the pathway may exacerbate the slow kinetics of neuronal mitophagy, leading to neurodegeneration.

The cargo-specific removal of organelles via selective autophagy is important to maintain neuronal homeostasis. Genetic studies indicate that deficits in these pathways are implicated in neurodegenerative diseases, including Parkinson's and amyotrophic lateral sclerosis. Here, we review our current understanding of the pathways that regulate mitochondrial quality control, and compare these mechanisms to those regulating turnover of the endoplasmic reticulum and the clearance of protein aggregates. Research suggests that there are multiple mechanisms regulating the degradation of specific cargos, such as dysfunctional organelles and protein aggregates. These mechanisms are critical for neuronal health, as neurons are uniquely vulnerable to impairment in organelle quality control pathways due to their morphology, size, polarity, and postmitotic nature. We highlight the consequences of dysregulation of selective autophagy in neurons and discuss current challenges in correlating noncongruent findings from in vitro and in vivo systems.

Mitochondrial homeostasis vitally depends on mitophagy, the programmed degradation of mitochondria. The roster of proteins known to participate in mitophagy remains small. We devised here a multidimensional CRISPR/Cas9 genetic screen, using multiple mitophagy reporter systems and pro-mitophagy triggers, and uncover numerous new components of Parkin-dependent mitophagy1. Unexpectedly, we identify the adenine nucleotide translocator (ANT) complex as required for mitophagy in multiple cell types. While pharmacological inhibition of ANT-mediated ADP/ATP exchange promotes mitophagy, genetic ablation of ANT paradoxically suppresses mitophagy. Importantly, ANT promotes mitophagy independently of its nucleotide translocase catalytic activity. Instead, the ANT complex is required for inhibition of the presequence translocase TIM23, leading to PINK1 stabilization, in response to bioenergetic collapse. ANT modulates TIM23 indirectly via interaction with TIM44, known to regulate peptide import through TIM232. Mice lacking ANT1 reveal blunted mitophagy and consequent profound accumulation of aberrant mitochondria. Disease-causing human mutations in ANT1 abrogate binding to TIM44 and TIM23 and inhibit mitophagy. Together, these data identify a novel and essential function for ANT as a fundamental mediator of mitophagy in health and disease.

Synaptotagmin senses fluctuations in the Ca2+environment of neurons near active zones and transduces a signal to the SNARE complex to initiate exocytosis at the presynaptic terminus. The 3D structures of the two tandem C2 domains of synaptotagmin have been determined to high resolution; however, it is currently unclear how each domain dynamically interacts with Ca2+at the atomic level. To study the mechanistic consequences of the lethal mutations at the AD3 locus, we introduced tyrosine to asparagine point mutations in both the C2A and C2B domains of synaptotagmin 1, and we have constructed a model that describes the relationship between Ca2+-binding and the structural changes within each C2 domain. We show that the mobility of loop 3 in the Ca2+binding pocket increases markedly in C2A, while the mobility of loop 1 changes in C2B with the AD3 mutation. This increase in loop mobility results in an increase in the average volume and variance of the Ca2+-binding pockets of C2A and C2B. The volume of the unbound Ca2+-binding pocket in C2A is usually restrained by intra-domain interactions between the tyrosine residue at the AD3 locus and residues on loop 3; however, the AD3 mutation decouples the restraint and results in a larger, more variable Ca2+-binding pocket in C2A. C2B maintains a more compact Ca2+-binding pocket; however, its volume also fluctuates significantly with the AD3 mutation. Changes in binding pocket volume that involve more variable Ca2+binding loops would likely affect Ca2+affinity in the neurons of the affected organism. Using molecular-dynamics simulations, we show that mutations at the AD3 locus alter the mobility of the Ca2+-binding loops by removing a key stabilization mechanism that is normally present in C2 domains. The lack of loop stabilization results in a net increase in the volume of the Ca2+-binding pocket and provides an explanation for the observed lethal phenotype.

Synaptotagmin (syt)-1 is a Ca2+ sensor that triggers rapid synaptic vesicle exocytosis. Mutations that disrupt physical interactions between the tandem Ca2+-sensing modules (C2 domains) of syt-1 disrupt regulated membrane fusion in reconstituted fusion reactions and in neurons. Hence contacts between these domains are important for function., C2 domains are widespread motifs that often serve as Ca2+-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca2+ sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca2+, and shifted the Ca2+ dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation–secretion coupling.

Synaptotagmin-1 is a Ca(2+) sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of synaptotagmin-1 was its ability to aggregate membranes in response to Ca(2+). However, the mechanism and function of this process were poorly understood. Here we show that synaptotagmin-1-mediated vesicle aggregation is driven by trans interactions between synaptotagmin-1 molecules bound to different membranes. We found a strong correlation between the ability of Ca(2+)-bound synaptotagmin-1 to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes-using non-synaptotagmin proteins-also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that synaptotagmin-1 drove the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion-competent heterodimers, independently of aggregation. Thus, membrane aggregation and t-SNARE assembly appear to be two key aspects of fusion reactions that are regulated by Ca(2+)-bound synaptotagmin-1 and catalyzed by SNAREs.

The anterior pituitary releases six different hormones that control virtually all aspects of vertebrate physiology, yet the molecular mechanisms underlying their Ca2+-triggered release remain unknown. A subset of the synaptotagmin (syt) family of proteins serve as Ca2+ sensors for exocytosis in neurons and neuroendocrine cells, and are thus likely to regulate pituitary hormone secretion. Here we show that numerous syt isoforms are highly expressed in the pituitary gland in a lobe, and sex-specific manner. We further investigated a Ca2+-activated isoform, syt-9, and found that it is expressed in a subpopulation of anterior pituitary cells, the gonadotropes. Follicle-stimulating hormone (FSH) and syt-9 are highly co-localized in female, but not male, mice. Loss of syt-9 results in diminished basal and stimulated FSH secretion only in females, resulting in alterations in the oestrus cycle. This work uncovers a new function for syt-9 and reveals a novel sex difference in reproductive hormone secretion., Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) in both male and female mice. Roper et al. show that the synaptotagmin isoform syt-9 is specifically required for FSH secretion, but only in females, revealing a mechanism by which specificity can be encoded in complex secretory cells.

The Ca 2+ sensor synaptotagmin-1 (syt-1) regulates neurotransmitter release by interacting with anionic phospholipids. Here we test the idea that the intrinsic kinetics of syt–membrane interactions determine, in part, the time course of synaptic transmission. To tune the kinetics of this interaction, we grafted structural elements from the slowest isoform, syt-7, onto the fastest isoform, syt-1, resulting in a chimera with intermediate kinetic properties. Moreover, the chimera coupled a physiologically irrelevant metal, Sr 2+ , to membrane fusion in vitro . When substituted for syt-1 in mouse hippocampal neurons, the chimera slowed the kinetics of synaptic transmission. Neurons expressing the chimera also evinced rapid and efficient Sr 2+ triggered release, in contrast to the weak response of neurons expressing syt-1. These findings reveal presynaptic sensor–membrane interactions as a major factor regulating the speed of the release machinery. Finally, the chimera failed to clamp the elevated spontaneous fusion rate exhibited by syt-1 KO neurons, indicating that the metal binding loops of syt-1 regulate the two modes of release by distinct mechanisms. SIGNIFICANCE STATEMENT In calcium, synaptotagmin-1 triggers neurotransmitter release by interacting with membranes. Here, we demonstrate that intrinsic properties of this interaction control the time course of synaptic transmission. We engineered a “chimera” using synaptotagmin-1 and elements of a slower isoform, synaptotagmin-7. When expressed in neurons, the chimera slowed the rate of neurotransmitter release. Furthermore, unlike native synaptotagmin-1, the chimera was able to function robustly in the presence of strontium–a metal not present in cells. We exploited this ability to show that a key function of synaptotagmin-1 is to penetrate cell membranes. This work sheds light on fundamental mechanisms of neurotransmitter release.

Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals. DOI: http://dx.doi.org/10.7554/eLife.01524.001, eLife digest Neurons communicate with one another at junctions called synapses. The arrival of an electrical signal called an action potential at the first neuron triggers the release of chemicals called neurotransmitters into the synapse. These chemicals then diffuse across the gap between the neurons and bind to receptors on the second cell. The neurotransmitter molecules are stored in the first cell in packages known as vesicles, which release their contents by fusing with the cell membrane. Following a fusion event, neurons must replenish their vesicle stocks to ensure that they are ready for the arrival of the next action potential. This replenishment process is known to involve a calcium-dependent pathway and a calcium-independent pathway. A protein called calmodulin, that binds calcium ions, has an important role in the first of these pathways. Now, Liu et al. have shown that another protein, synaptotagmin 7, also has a key role in the replenishment of synaptic vesicles, possibly as a sensor for calcium ions. Moreover, Liu et al. found that synaptotagmin 7 and calmodulin bind to each other to form a complex, which suggests that the calcium-dependent replenishment pathway is regulated by this complex. The synaptotagmins are a family of 17 proteins, three of which are present in all animals. Two of these were known to have roles in synapses, but the role of the third—synaptotagmin 7—had been unclear. In addition to providing a more complete understanding of the replenishment of synaptic vesicles, the work of Liu et al. also supplies the final piece of the jigsaw regarding the role of the synaptotagmins that are present in all animals. DOI: http://dx.doi.org/10.7554/eLife.01524.002

Synaptotagmin is the major calcium sensor for fast synaptic transmission that requires the synchronous fusion of synaptic vesicles. Synaptotagmin contains two calcium-binding domains: C2A and C2B. Mutation of a positively charged residue (R233Q in rat) showed that Ca2+-dependent interactions between the C2A domain and membranes play a role in the electrostatic switch that initiates fusion. Surprisingly, aspartate-to-asparagine mutations in C2A that inhibit Ca2+binding support efficient synaptic transmission, suggesting that Ca2+binding by C2A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca2+-binding residue in C2A (D229E inDrosophila) that inhibited Ca2+binding but maintained the negative charge of the pocket. This C2A aspartate-to-glutamate mutation resulted in ∼80% decrease in synchronous transmitter release and a decrease in the apparent Ca2+affinity of release. Previous aspartate-to-asparagine mutations in C2A partially mimicked Ca2+binding by decreasing the negative charge of the pocket. We now show that the major function of Ca2+binding to C2A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca2+binding by C2A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca2+binding by C2B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca2+influx, but Ca2+binding by both C2domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.