Mitochondrial defects triggered by amg-1 mutation elicit UPRmt and phagocytic clearance during spermatogenesis in C. elegans.

Mitochondria are the powerhouses of many biological processes. During spermatogenesis, post-transcriptional regulation of mitochondrial gene expression is mediated by nuclear-encoded mitochondrial RNA-binding proteins (mtRBPs). We identified AMG-1 as an mtRBP required for reproductive success in Caenorhabditis elegans. amg-1 mutation led to defects in mitochondrial structure and sperm budding, resulting in mitochondria being discarded into residual bodies, which ultimately delayed spermatogenesis in the proximal gonad. In addition, mitochondrial defects triggered the gonadal mitochondrial unfolded protein response and phagocytic clearance to ensure spermatogenesis but ultimately failed to rescue hermaphroditic fertility. These findings reveal a previously undiscovered role for AMG-1 in regulating C. elegans spermatogenesis, in which mitochondrial-damaged sperm prevented the transmission of defective mitochondria to mature sperm by budding and phagocytic clearance, a process which may also exist in the reproductive systems of higher organisms.

Protocol for CRISPR-Cas9-mediated genome editing to study spermatogenesis in Caenorhabditis elegans.

Gene silencing by P-element-induced wimpy testis-interacting RNAs is a mechanism to maintain genome integrity in germ cells. Here, we present a protocol for knockin or knockout editing of male germline genome mediated by CRISPR-Cas9 technology in Caenorhabditis elegans. We describe steps for constructing edited plasmids, microinjecting worms with these plasmids, and screening edited worms. We then detail procedures for dissecting released sperm and their observation with fluorescence microscopy. Engineered worms provide a model for studying hermaphrodite/male fertility or protein localization in vivo. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).1.

RNA-binding protein complex AMG-1/SLRP-1 mediates germline development and spermatogenesis by maintaining mitochondrial homeostasis in Caenorhabditis elegans.

The mechanisms of RNA-binding proteins (RBPs)-mediated post-transcriptional regulation of pre-existing mRNAs, which is essential for spermatogenesis, remain poorly understood. In this study, we identify that a germline-specific mitochondrial RBP AMG-1(abnormal mitochondria in germline 1), a homolog of mammalian leucine-rich PPR motif-containing protein (LRPPRC), is required for spermatogenesis in Caenorhabditis elegans. The amg-1 mutation hinders germline development without affecting somatic development and leads to the aberrant mitochondrial morphology and structure associated with mitochondrial dysfunctions specifically in the germline. We demonstrate that AMG-1 is most frequently bound to mtDNA-encoded 12S and 16S ribosomal RNA, the essential components of mitochondrial ribosomes, and that 12S rRNA expression mediated by AMG-1 is crucial for germline mitochondrial protein homeostasis. Furthermore, steroid receptor RNA activator (SRA) stem loop interacting RNA binding protein (SLRP-1), a homolog of mammalian SRA stem loop interacting RNA binding protein (SLIRP) in C. elegans, interacts with AMG-1 genetically to regulate germline development and reproductive success in C. elegans. Overall, these findings reveal the novel function of mtRBP, specifically in regulating germline development.

The mechanism of STING autoinhibition and activation.

2',3'-cGAMP, produced by the DNA sensor cGAS, activates stimulator of interferon genes (STING) and triggers immune response during infection. Tremendous effort has been placed on unraveling the mechanism of STING activation. However, little is known about STING inhibition. Here, we found that apo-STING exhibits a bilayer with head-to-head as well as side-by-side packing, mediated by its ligand-binding domain (LBD). This type of assembly holds two endoplasmic reticulum (ER) membranes together not only to prevent STING ER exit but also to eliminate the recruitment of TBK1, representing the autoinhibited state of STING. Additionally, we obtained the filament structure of the STING/2',3'-cGAMP complex, which adopts a bent monolayer assembly mediated by LBD and transmembrane domain (TMD). The active, curved STING polymer could deform ER membrane to support its ER exit and anterograde transportation. Our data together provide a panoramic vision regarding STING autoinhibition and activation, which adds substantially to current understanding of the cGAS-STING pathway.

MIG-23 is involved in sperm migration by modulating extracellular ATP levels in Ascaris suum.

In nematodes, spermiogenesis is a process of sperm activation in which nonmotile spermatids are transformed into crawling spermatozoa. Sperm motility acquisition during this process is essential for successful fertilization, but the underlying mechanisms remain to be clarified. Herein, we have found that extracellular adenosine-5'-triphosphate (ATP) level regulation by MIG-23, which is a homolog of human ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), was required for major sperm protein (MSP) filament dynamics and sperm motility in the nematode Ascaris suum. During sperm activation, a large amount of ATP was produced in mitochondria and was stored in refringent granules (RGs). Some of the produced ATP was released to the extracellular space through innexin channels. MIG-23 was localized in the sperm plasma membrane and contributed to the ecto-ATPase activity of spermatozoa. Blocking MIG-23 activity resulted in a decrease in the ATP hydrolysis activity of spermatozoa and an increase in the depolymerization rate of MSP filaments in pseudopodia, which eventually affected sperm migration. Overall, our data suggest that MIG-23, which contributes to the ecto-ATPase activity of spermatozoa, regulates sperm migration by modulating extracellular ATP levels.

Membrane contact site-dependent cholesterol transport regulates Na+/K+-ATPase polarization and spermiogenesis in Caenorhabditis elegans.

Spermiogenesis in nematodes is a process whereby round and quiescent spermatids differentiate into asymmetric and crawling spermatozoa. The molecular mechanism underlying this symmetry breaking remains uncharacterized. In this study, we revealed that sperm-specific Na+/K+-ATPase (NKA) is evenly distributed on the plasma membrane (PM) of Caenorhabditis elegans spermatids but is translocated to and subsequently enters the invaginated membrane of the spermatozoa cell body during sperm activation. The polarization of NKA depends on the transport of cholesterol from the PM to membranous organelles (MOs) via membrane contact sites (MCSs). The inositol 5-phosphatase CIL-1 and the MO-localized PI4P phosphatase SAC-1 may mediate PI4P metabolism to drive cholesterol countertransport via sterol/lipid transport proteins through MCSs. Furthermore, the NKA function is required for C. elegans sperm motility and reproductive success. Our data imply that the lipid dynamics mediated by MCSs might play crucial roles in the establishment of cell polarity. eGraphical abstract.

Astrocytic Epoxyeicosatrienoic Acid Signaling in the Medial Prefrontal Cortex Modulates Depressive-like Behaviors.

Major depressive disorder is the most common mental illness. Mounting evidence indicates that astrocytes play a crucial role in the pathophysiology of depression; however, the underlying molecular mechanisms remain elusive. Compared with other neuronal cell types, astrocytes are enriched for arachidonic acid metabolism. Herein, we observed brain-region-specific alterations of epoxyeicosatrienoic acid (EET) signaling, which is an arachidonic acid metabolic pathway, in both a mouse model of depression and postmortem samples from patients with depression. The enzymatic activity of soluble epoxide hydrolase (sEH), the key enzyme in EET signaling, was selectively increased in the mPFC of susceptible mice after chronic social defeated stress and was negatively correlated with the social interaction ratio, which is an indicator of depressive-like behavior. The specific deletion of Ephx2 (encode sEH) in adult astrocytes induced resilience to stress, whereas the impaired EET signaling in the mPFC evoked depressive-like behaviors in response to stress. sEH was mainly expressed on lysosomes of astrocytes. Using pharmacological and genetic approaches performed on C57BL/6J background adult male mice, we found that EET signaling modulated astrocytic ATP release in vitro and in vivo Moreover, astrocytic ATP release was required for the antidepressant-like effect of Ephx2 deletion in adult astrocytes. In addition, sEH inhibitors produced rapid antidepressant-like effects in multiple animal models of depression, including chronic social defeated stress and chronic mild stress. Together, our results highlight that EET signaling in astrocytes in the mPFC is essential for behavioral adaptation in response to psychiatric stress.SIGNIFICANCE STATEMENT Astrocytes, the most abundant glial cells of the brain, play a vital role in the pathophysiology of depression. Astrocytes secrete adenosine ATP, which modulates depressive-like behaviors. Notably, astrocytes are enriched for arachidonic acid metabolism. In the present study, we explored the hypothesis that epoxyeicosatrienoic acid signaling, an arachidonic acid metabolic pathway, modulates astrocytic ATP release and the expression of depressive-like behaviors. Our work demonstrated that epoxyeicosatrienoic acid signaling in astrocytes in the mPFC is essential for behavioral homeostatic adaptation in response to stress, and the extent of astrocyte functioning is greater than expected based on earlier reports.

The lysosomal membrane protein SCAV-3 maintains lysosome integrity and adult longevity.

Lysosomes degrade macromolecules and recycle metabolites as well as being involved in diverse processes that regulate cellular homeostasis. The lysosome is limited by a single phospholipid bilayer that forms a barrier to separate the potent luminal hydrolases from other cellular constituents, thus protecting the latter from unwanted degradation. The mechanisms that maintain lysosomal membrane integrity remain unknown. Here, we identified SCAV-3, the Caenorhabditis elegans homologue of human LIMP-2, as a key regulator of lysosome integrity, motility, and dynamics. Loss of scav-3 caused rupture of lysosome membranes and significantly shortened lifespan. Both of these phenotypes were suppressed by reinforced expression of LMP-1 or LMP-2, the C. elegans LAMPs, indicating that longevity requires maintenance of lysosome integrity. Remarkably, reduction in insulin/insulin-like growth factor 1 (IGF-1) signaling suppressed lysosomal damage and extended the lifespan in scav-3(lf) animals in a DAF-16-dependent manner. Our data reveal that SCAV-3 is essential for preserving lysosomal membrane stability and that modulation of lysosome integrity by the insulin/IGF-1 signaling pathway affects longevity.

Protein kinase C controls lysosome biogenesis independently of mTORC1.

Lysosomes respond to environmental cues by controlling their own biogenesis, but the underlying mechanisms are poorly understood. Here we describe a protein kinase C (PKC)-dependent and mTORC1-independent mechanism for regulating lysosome biogenesis, which provides insights into previously reported effects of PKC on lysosomes. By identifying lysosome-inducing compounds we show that PKC couples activation of the TFEB transcription factor with inactivation of the ZKSCAN3 transcriptional repressor through two parallel signalling cascades. Activated PKC inactivates GSK3ß, leading to reduced phosphorylation, nuclear translocation and activation of TFEB, while PKC activates JNK and p38 MAPK, which phosphorylate ZKSCAN3, leading to its inactivation by translocation out of the nucleus. PKC activation may therefore mediate lysosomal adaptation to many extracellular cues. PKC activators facilitate clearance of aggregated proteins and lipid droplets in cell models and ameliorate amyloid ß plaque formation in APP/PS1 mouse brains. Thus, PKC activators are viable treatment options for lysosome-related disorders.

Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans.

In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but how paternal mitochondria, but not maternal mitochondria, are selectively targeted for degradation is poorly understood. Here we report that mitochondrial dynamics have a profound effect on PME. A defect in fission of paternal mitochondria delays PME, whereas a defect in fusion of paternal mitochondria accelerates PME. Surprisingly, a defect in maternal mitochondrial fusion delays PME, which is reversed by a fission defect in maternal mitochondria or by increasing maternal mitochondrial membrane potential using oligomycin. Electron microscopy and tomography analyses reveal that a proportion of maternal mitochondria are compromised when they fail to fuse normally, leading to their competition for the autophagy machinery with damaged paternal mitochondria and delayed PME. Our study indicates that mitochondrial dynamics play a critical role in regulating both the kinetics and the specificity of PME.

Clathrin and AP2 are required for phagocytic receptor-mediated apoptotic cell clearance in Caenorhabditis elegans.

Clathrin and the multi-subunit adaptor protein complex AP2 are central players in clathrin-mediated endocytosis by which the cell selectively internalizes surface materials. Here, we report the essential role of clathrin and AP2 in phagocytosis of apoptotic cells. In Caenorhabditis elegans, depletion of the clathrin heavy chain CHC-1 and individual components of AP2 led to a significant accumulation of germ cell corpses, which resulted from defects in both cell corpse engulfment and phagosome maturation required for corpse removal. CHC-1 and AP2 components associate with phagosomes in an inter-dependent manner. Importantly, we found that the phagocytic receptor CED-1 interacts with the α subunit of AP2, while the CED-6/Gulp adaptor forms a complex with both CHC-1 and the AP2 complex, which likely mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment triggered by the CED-1 signaling pathway. In addition, CHC-1 and AP2 promote the phagosomal association of LST-4/Snx9/18/33 and DYN-1/dynamin by forming a complex with them, thereby facilitating the maturation of phagosomes necessary for corpse degradation. These findings reveal a non-classical role of clathrin and AP2 and establish them as indispensable regulators in phagocytic receptor-mediated apoptotic cell clearance.

Cytosolic Ca(2+) as a multifunctional modulator is required for spermiogenesis in Ascaris suum.

The dynamic polar polymers actin filaments and microtubules are usually employed to provide the structural basis for establishing cell polarity in most eukaryotic cells. Radially round and immotile spermatids from nematodes contain almost no actin or tubulin, but still have the ability to break symmetry to extend a pseudopod and initiate the acquisition of motility powered by the dynamics of cytoskeleton composed of major sperm protein (MSP) during spermiogenesis (sperm activation). However, the signal transduction mechanism of nematode sperm activation and motility acquisition remains poorly understood. Here we show that Ca(2+) oscillations induced by the Ca(2+) release from intracellular Ca(2+) store through inositol (1,4,5)-trisphosphate receptor are required for Ascaris suum sperm activation. The chelation of cytosolic Ca(2+) suppresses the generation of a functional pseudopod, and this suppression can be relieved by introducing exogenous Ca(2+) into sperm cells. Ca(2+) promotes MSP-based sperm motility by increasing mitochondrial membrane potential and thus the energy supply required for MSP cytoskeleton assembly. On the other hand, Ca(2+) promotes MSP disassembly by activating Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase calcineurin. In addition, Ca(2+)/camodulin activity is required for the fusion of sperm-specifi c membranous organelle with the plasma membrane, a regulated exocytosis required for sperm motility. Thus, Ca(2+) plays multifunctional roles during sperm activation in Ascaris suum.

The micronutrient element zinc modulates sperm activation through the SPE-8 pathway in Caenorhabditis elegans.

Immotile spermatids produced in the testis must undergo a series of poorly understood morphological, physiological and biochemical processes called sperm activation to become motile, fertilization-competent spermatozoa. In Caenorhabditis elegans, the spe-8 group contains sperm-specific genes active in both males and hermaphrodites, although their activity is required only for hermaphrodite self-sperm activation. The activating signal upstream of the SPE-8 signaling cascade remains unknown. Here, we show that the micronutrient zinc is sufficient to trigger sperm activation in vitro, and that extracellular zinc induces the intracellular redistribution of labile zinc. We demonstrate that other activating signals promote the similar redistribution of labile zinc, indicating that zinc might have first and/or second messenger roles during sperm activation. Moreover, zinc-induced sperm activation is SPE-8 pathway dependent. Labile zinc was enriched in the spermatheca, the normal site for self-sperm activation in hermaphrodites. High levels of zinc were also found in the secretory cells in the male gonad, suggesting that zinc might be secreted from these cells during copulation and become a component of seminal fluid, to modulate sperm activation post-copulation. These data indicate that zinc regulates sperm activation in both male and hermaphrodite C. elegans, a finding with important implications for understanding hermaphroditic evolution.

Cholesterol and the biosynthesis of glycosphingolipids are required for sperm activation in Caenorhabditis elegans.

Ejaculated mammalian sperm must acquire fertilization capacity after residing into the female reproductive tract, a process collectively known as capacitation. Cholesterol efflux was required for sperm maturation. Different from flagellated sperm, C. elegans sperm are crawling cells. C. elegans sperm are highly enriched with cholesterol though this animal species lacks biosynthetic pathway for cholesterol and its survival requires an exogenous cholesterol supply. The low abundance of cholesterol in C. elegans lipid extract is thought insufficient to form lipid microdomains ubiquitously in this organism. We present evidence that cholesterol is enriched in the plasma membrane of C. elegans spermatids and that cholesterol- and glycosphingolipids (GSLs)-enriched membrane microdomains (lipid microdomains) mediate sperm activation. Disruption of sperm lipid microdomains by acute manipulation of cholesterol in vitro blocks the sperm activation. Restriction of cholesterol uptake also results in the abnormal sperm activation in both males and hermaphrodites. Manipulation of the integrity of lipid microdomains by targeting the biosynthesis of GSLs inhibits sperm activation and the inhibition can be rescued by the addition of exogenous GSLs. The cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, which are exclusively found in lipid microdomains, also affects sperm activation. We conclude that localized signaling mediated by lipid microdomains is critical for worm sperm activation. Lipid microdomains composed of cholesterol and GSLs have been observed in flagellated sperm of several animal species, thus cholesterol, before its efflux from the plasma membrane, might be needed to assemble into a platform for some more important upstream signal sorting during spermatogenesis than was previously thought.

BK channels reveal novel phosphate sensitivity in SNr neurons.

Whether large conductance Ca(2+)-activated potassium (BK) channels are present in the substantia nigra pars reticulata (SNr) is a matter of debate. Using the patch-clamp technique, we examined the functional expression of BK channels in neurons of the SNr and showed that the channels were activated or inhibited by internal high-energy phosphates (IHEPs) at positive and negative membrane potentials, respectively. SNr neurons showed membrane potential hyperpolarization under glucose-deprivation conditions which was attenuated by paxilline, a specific BK channel blocker. In addition, Fluo-3 fluorescence recording detected an increase in the level of internal free calcium ([Ca(2+)](i)) during ischemic hyperpolarization. These results confirm that BK channels are present in SNr neurons and indicate that their unique IHEP sensitivity is requisite in neuronal ischemic responses. Bearing in mind that the K(ATP) channel blocker tolbutamide also attenuated the hyperpolarization, we suggest that BK channels may play a protective role in the basal ganglia by modulating the excitability of SNr neurons along with K(ATP) channels under ischemic stresses.

Transient oxygen-glucose deprivation causes immediate changes in redox activity in mouse brain tissue.

Redox activity is an important property of living cells, and decreases in redox activity are likely to be an upstream event in ischemic brain injuries. In this study, immediate changes in redox activity caused by ischemic injury were investigated in oxygen-glucose deprivation (OGD) treated mouse brain tissue. Adult mouse brain slices were subjected to 10 min or 15 min OGD treatments and were immediately stained with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) staining procedure. After 10 min OGD, the redox activity decreased in the lateral globus pallidus (LGP), medial globus pallidus (MGP), pyramidal cell layer of hippocampus CA1 (CA1(PL)) and the granular layer of the cerebellum (cereb(GL)). After 15 min OGD, decreases also occurred in the substantia nigra (SN) and several other areas of the brain stem. Hoechst 33342 was used to confirm that changes in redox activity occurred before morphological alterations in the cellular nuclei--morphological changes were not observed even after a 60 min OGD. The results presented here indicate that functional ischemic vulnerability exists in several brain regions, and will be helpful for systematic research on mammalian brain injury caused by transient metabolic stress.