Coordination of RAB-8 and RAB-11 during unconventional protein secretion.

Multiple physiology-pertinent transmembrane proteins reach the cell surface via the Golgi-bypassing unconventional protein secretion (UcPS) pathway. By employing C. elegans-polarized intestine epithelia, we recently have revealed that the small GTPase RAB-8/Rab8 serves as an important player in the process. Nonetheless, its function and the relevant UcPS itinerary remain poorly understood. Here, we show that deregulated RAB-8 activity resulted in impaired apical UcPS, which increased sensitivity to infection and environmental stress. We also identified the SNARE VTI-1/Vti1a/b as a new RAB-8-interacting factor involved in the apical UcPS. Besides, RAB-11/Rab11 was capable of recruiting RABI-8/Rabin8 to reduce the guanine nucleotide exchange activity of SMGL-1/GEF toward RAB-8, indicating the necessity of a finely tuned RAB-8/RAB-11 network. Populations of RAB-8- and RAB-11-positive endosomal structures containing the apical UcPS cargo moved toward the apical side. In the absence of RAB-11 or its effectors, the cargo was retained in RAB-8- and RAB-11-positive endosomes, respectively, suggesting that these endosomes are utilized as intermediate carriers for the UcPS.

Age-associated decline in RAB-10 efficacy impairs intestinal barrier integrity.

The age-related decline in the ability of the intestinal barrier to maintain selective permeability can lead to various physiological disturbances. Adherens junctions play a vital role in regulating intestinal permeability, and their proper assembly is contingent upon endocytic recycling. However, how aging affects the recycling efficiency and, consequently, the integrity of adherens junctions remains unclear. Here we show that RAB-10/Rab10 functionality is reduced during senescence, leading to impaired adherens junctions in the Caenorhabditis elegans intestine. Mechanistic analysis reveals that SDPN-1/PACSINs is upregulated in aging animals, suppressing RAB-10 activation by competing with DENN-4/GEF. Consistently, SDPN-1 knockdown alleviates age-related abnormalities in adherens junction integrity and intestinal barrier permeability. Of note, the inhibitory effect of SDPN-1 on RAB-10 requires KGB-1/JUN kinase, which presumably enhances the potency of SDPN-1 by altering its oligomerization state. Together, by examining age-associated changes in endocytic recycling, our study sheds light on how aging can impact intestinal barrier permeability.

Oligomeric CHMP7 mediates three-way ER junctions and ER-mitochondria interactions.

In metazoans the endoplasmic reticulum (ER) undergoes extensive remodeling during the cell cycle. The endosomal sorting complexes required for transport (ESCRT) protein CHMP7 coordinates ESCRT-III dependent nuclear envelope reformation during mitotic exit. However, potential roles of ER-associated CHMP7 at non-mitotic stages remain unclear. Here we discovered a new role of CHMP7 in mediating three-way ER and ER-mitochondrial membrane contact sites (MCSs). We showed that CHMP7 localizes to multiple cellular membranes including the ER, mitochondrial-associated membranes (MAMs) and the outer mitochondrial membrane (OMM) via its N-terminal membrane-binding domain. CHMP7 undergoes dynamic assembly at three-way ER junctions and ER-mitochondrial MCSs through hydrophobic interactions among α helix-1 and α helix-2 of the C-terminal CHMP-like domain, which was required for tethering different organelles in vivo. Furthermore, CHMP7 mediates the formation of three-way ER junctions in parallel with Atlastins (ATLs). Importantly, CHMP7 also regulates ER-mitochondrial interactions and its depletion affects mitochondrial division independently of ESCRT complex. Taken together, our results suggest a direct role of CHMP7 in the formation of the ER contacts in interphase.

Rab GTPases: The principal players in crafting the regulatory landscape of endosomal trafficking.

After endocytosis, diverse cargos are sorted into endosomes and directed to various destinations, including extracellular macromolecules, membrane lipids, and membrane proteins. Some cargos are returned to the plasma membrane via endocytic recycling. In contrast, others are delivered to the Golgi apparatus through the retrograde pathway, while the rest are transported to late endosomes and eventually to lysosomes for degradation. Rab GTPases are major regulators that ensure cargos are delivered to their proper destinations. Rabs are localized to distinct endosomes and play predominant roles in membrane budding, vesicle formation and motility, vesicle tethering, and vesicle fusion by recruiting effectors. The cascades between Rabs via shared effectors or the recruitment of Rab activators provide an additional layer of spatiotemporal regulation of endocytic trafficking. Notably, several recent studies have indicated that disorders of Rab-mediated endocytic transports are closely associated with diseases such as immunodeficiency, cancer, and neurological disorders.

A Tau Pathogenesis-Based Network Pharmacology Approach for Exploring the Protections of Chuanxiong Rhizoma in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of neurodegenerative dementia and one of the top medical concerns worldwide. Currently, the approved drugs to treat AD are effective only in treating the symptoms, but do not cure or prevent AD. Although the exact causes of AD are not understood, it is recognized that tau aggregation in neurons plays a key role. Chuanxiong Rhizoma (CR) has been widely reported as effective for brain diseases such as dementia. Thus, we explored the protections of CR in AD by a tau pathogenesis-based network pharmacology approach. According to ultra-HPLC with triple quadrupole mass spectrometry data and Lipinski's rule of five, 18 bioactive phytochemicals of CR were screened out. They were shown corresponding to 127 tau pathogenesis-related targets, among which VEGFA, IL1B, CTNNB1, JUN, ESR1, STAT3, APP, BCL2L1, PTGS2, and PPARG were identified as the core ones. We further analyzed the specific actions of CR-active phytochemicals on tau pathogenesis from the aspects of tau aggregation and tau-mediated toxicities. It was shown that neocnidilide, ferulic acid, coniferyl ferulate, levistilide A, Z-ligustilide, butylidenephthalide, and caffeic acid can be effective in reversing tau hyperphosphorylation. Neocnidilide, senkyunolide A, butylphthalide, butylidenephthalide, Z-ligustilide, and L-tryptophan may be effective in promoting lysosome-associated degradation of tau, and levistilide A, neocnidilide, ferulic acid, L-tryptophan, senkyunolide A, Z-ligustilide, and butylidenephthalide may antagonize tau-mediated impairments of intracellular transport, axon and synaptic damages, and neuron death (especially apoptosis). The present study suggests that acting on tau aggregation and tau-mediated toxicities is part of the therapeutic mechanism of CR against AD.

SMGL-1/NBAS acts as a RAB-8 GEF to regulate unconventional protein secretion.

Unconventional protein secretion (UPS) pathways are conserved across species. However, the underlying mechanisms that regulate Golgi-bypassing UPS of integral proteins remain elusive. In this study, we show that RAB-8 and SMGL-1/NBAS are required for the UPS of integral proteins in C. elegans intestine. SMGL-1 resides in the ER-Golgi intermediate compartment and adjacent RAB-8-positive structures, and NRZ complex component CZW-1/ZW10 is required for this residency. Notably, SMGL-1 acts as a guanine nucleotide exchange factor for RAB-8, ensuring UPS of integral proteins by driving the activation of RAB-8. Furthermore, we show that Pseudomonas aeruginosa infection elevated the expression of SMGL-1 and RAB-8. Loss of SMGL-1 or RAB-8 compromised resistance to environmental colchicine, arsenite, and pathogenic bacteria. These results suggest that the SMGL-1/RAB-8-mediated UPS could integrate environmental signals to serve as a host defense response. Together, by establishing the C. elegans intestine as a multicellular model, our findings provide insights into RAB-8-dependent Golgi-bypassing UPS, especially in the context of epithelia in vivo.

Circadian rhythm modulates endochondral bone formation via MTR1/AMPKß1/BMAL1 signaling axis.

The circadian clock is a master regulator in coordinating daily oscillations of physiology and behaviors. Nevertheless, how the circadian rhythm affects endochondral ossification is poorly understood. Here we showed that endochondral bone formation exhibits circadian rhythms, manifested as fast DNA replication in the daytime, active cell mitosis, and matrix synthesis at night. Circadian rhythm disruption led to endochondral ossification deformities. The mechanistic dissection revealed that melatonin receptor 1 (MTR1) periodically activates the AMPKß1 phosphorylation, which then orchestrates the rhythms of cell proliferation and matrix synthesis via destabilizing the clock component CRY1 and triggering BMAL1 expression. Accordingly, the AMPKß1 agonist is capable of alleviating the abnormity of endochondral ossification caused by circadian dysrhythmias. Taken together, these findings indicated that the central circadian clock could control endochondral bone formation via the MTR1/AMPKß1/BMAL1 signaling axis in chondrocytes. Also, our results suggested that the AMPKß1 signaling activators are promising medications toward endochondral ossification deformities.

AP-1 Recruits SMAP-1/SMAPs to the trans-Golgi Network to Promote Sorting in Polarized Epithelia.

Coordinated AP-1 and clathrin coat assembly mediate secretory sorting on the trans-Golgi network (TGN) during conventional secretion. Here we found that SMAP-1/SMAPs deficiency caused the apical protein ERM-1 to accumulate on the basolateral side of the TGN. In contrast, the basolateral protein SLCF-1 appeared abnormally on the apical membrane. SMAP-1 colocalized with AP-1 on the TGN. The integrity of AP-1 is required for the subcellular presence of SMAP-1. Moreover, we found that the loss of SMAP-1 reduced clathrin-positive structures in the cytosol, suggesting that SMAP-1 has a regulatory role in clathrin assembly on the TGN. Functional experiments showed that overexpressing clathrin effectively alleviated exocytic defects due to the lack of SMAP-1, corroborating the role of SMAP-1 in promoting the assembly of clathrin on the TGN. Together, our results suggested that the AP-1 complex regulates the TGN localization of SMAP-1, promoting clathrin assembly to ensure polarized conventional secretion in C. elegans intestinal epithelia.

WTS-1/LATS regulates endocytic recycling by restraining F-actin assembly in a synergistic manner.

The disruption of endosomal actin architecture negatively affects endocytic recycling. However, the underlying homeostatic mechanisms that regulate actin organization during recycling remain unclear. In this study, we identified a synergistic endosomal actin assembly restricting mechanism in C. elegans involving WTS-1, the homolog of LATS kinases, which is a core component of the Hippo pathway. WTS-1 resides on the sorting endosomes and colocalizes with the actin polymerization regulator PTRN-1 [the homolog of the calmodulin-regulated spectrin-associated proteins (CAMSAPs)]. We observed an increase in PTRN-1-labeled structures in WTS-1-deficient cells, indicating that WTS-1 can limit the endosomal localization of PTRN-1. Accordingly, the actin overaccumulation phenotype in WTS-1-depleted cells was mitigated by the associated PTRN-1 loss. We further demonstrated that recycling defects and actin overaccumulation in WTS-1-deficient cells were reduced by the overexpression of constitutively active UNC-60A(S3A) (a cofilin protein homolog), which aligns with the role of LATS as a positive regulator of cofilin activity. Altogether, our data confirmed previous findings, and we propose an additional model, that WTS-1 acts alongside the UNC-60A-mediated actin disassembly to restrict the assembly of endosomal F-actin by curbing PTRN-1 dwelling on endosomes, preserving recycling transport.

RTKN-1/Rhotekin shields endosome-associated F-actin from disassembly to ensure endocytic recycling.

Cargo sorting and the subsequent membrane carrier formation require a properly organized endosomal actin network. To better understand the actin dynamics during endocytic recycling, we performed a genetic screen in C. elegans and identified RTKN-1/Rhotekin as a requisite to sustain endosome-associated actin integrity. Loss of RTKN-1 led to a prominent decrease in actin structures and basolateral recycling defects. Furthermore, we showed that the presence of RTKN-1 thwarts the actin disassembly competence of UNC-60A/cofilin. Consistently, in RTKN-1-deficient cells, UNC-60A knockdown replenished actin structures and alleviated the recycling defects. Notably, an intramolecular interaction within RTKN-1 could mediate the formation of oligomers. Overexpression of an RTKN-1 mutant form that lacks self-binding capacity failed to restore actin structures and recycling flow in rtkn-1 mutants. Finally, we demonstrated that SDPN-1/Syndapin acts to direct the recycling endosomal dwelling of RTKN-1 and promotes actin integrity there. Taken together, these findings consolidated the role of SDPN-1 in organizing the endosomal actin network architecture and introduced RTKN-1 as a novel regulatory protein involved in this process.

Key Phytochemicals and Biological Functions of Chuanxiong Rhizoma Against Ischemic Stroke: A Network Pharmacology and Experimental Assessment.

Presently, the treatment options for ischemic stroke (IS) are limited due to the complicated pathological process of the disease. Chuanxiong Rhizome (CR), also known as Conioselinum anthriscoides "Chuanxiong" (rhizome), is the most widely used traditional Chinese medicine for treating stroke. This study aimed to uncover the key phytochemicals and biological functions of CR against IS through a network pharmacology approach combining with IS pathophysiology analysis. We employed permanent unilateral common carotid artery ligation to construct a mouse model of global cerebral ischemia and found that cerebral ischemia injuries were improved after 7 days of gavage treatment of CR (1,300 mg/kg/day). CR exerts protective effects on neurons mainly by acting on targets related to synaptic structure, synaptic function, neuronal survival and neuronal growth. A total of 18 phytochemicals from CR based on UHPLC-MS/MS that corresponded to 85 anti-IS targets. Coniferyl ferulate, neocnidilide and ferulic acid were identified as the key phytochemicals of CR against IS. Its brain protective effects involve anti-inflammatory, anti-oxidative stress, and anti-cell death activities and improves blood circulation. Additionally, the two most important synergistic effects of CR phytochemicals in treating IS are prevention of infection and regulation of blood pressure. In brain samples of Sham mice, L-tryptophan and vanillin were detected, while L-tryptophan, gallic acid, vanillin and cryptochlorogenic acid were detected in IS mice by UHPLC-MS/MS. Our findings provide a pathophysiology relevant pharmacological basis for further researches on IS therapeutic drugs.

LET-502/ROCK Regulates Endocytic Recycling by Promoting Activation of RAB-5 in a Distinct Subpopulation of Sorting Endosomes.

To explore the mechanism of Rab5/RAB-5 activation during endocytic recycling, we perform a genome-wide RNAi screen and identify a recycling regulator, LET-502/ROCK. LET-502 preferentially interacts with RAB-5(GDP) and activates RABX-5 GEF activity toward RAB-5, presumably by disrupting the self-inhibiting conformation of RABX-5. Furthermore, we find that the concomitant loss of LET-502 and another CED-10 effector, TBC-2/RAB-5-GAP, results in an endosomal buildup of RAB-5, indicating that CED-10 directs TBC-2-mediated RAB-5 inactivation and re-activates RAB-5 via LET-502 afterward. Then, we compare the functional position of LET-502 with that of RME-6/RAB-5-GEF. Loss of LET-502-RABX-5 module or RME-6 leads to diminished RAB-5 presence in spatially distinct endosome groups. We conclude that in the intestine of C. elegans, RAB-5 resides in discrete endosome subpopulations. Under the oversight of CED-10, LET-502 synergizes with RABX-5 to revitalize RAB-5 on a subset of endosomes in the deep cytosol, ensuring the progress of basolateral recycling.

An EHBP-1-SID-3-DYN-1 axis promotes membranous tubule fission during endocytic recycling.

The ACK family tyrosine kinase SID-3 is involved in the endocytic uptake of double-stranded RNA. Here we identified SID-3 as a previously unappreciated recycling regulator in the Caenorhabditis elegans intestine. The RAB-10 effector EHBP-1 is required for the endosomal localization of SID-3. Accordingly, animals with loss of SID-3 phenocopied the recycling defects observed in ehbp-1 and rab-10 single mutants. Moreover, we detected sequential protein interactions between EHBP-1, SID-3, NCK-1, and DYN-1. In the absence of SID-3, DYN-1 failed to localize at tubular recycling endosomes, and membrane tubules breaking away from endosomes were mostly absent, suggesting that SID-3 acts synergistically with the downstream DYN-1 to promote endosomal tubule fission. In agreement with these observations, overexpression of DYN-1 significantly increased recycling transport in SID-3-deficient cells. Finally, we noticed that loss of RAB-10 or EHBP-1 compromised feeding RNAi efficiency in multiple tissues, implicating basolateral recycling in the transport of RNA silencing signals. Taken together, our study demonstrated that in C. elegans intestinal epithelia, SID-3 acts downstream of EHBP-1 to direct fission of recycling endosomal tubules in concert with NCK-1 and DYN-1.

Revealing Functional Crosstalk between Distinct Bioprocesses through Reciprocal Functional Tests of Genetically Interacting Genes.

To systematically explore the genes mediating functional crosstalk between metazoan biological processes, we apply comparative genetic interaction (GI) mapping in Saccharomyces cerevisiae and Caenorhabditis elegans to generate an inter-bioprocess network consisting of 178 C. elegans GIs. The GI network spans six annotated biological processes including aging, intracellular transport, microtubule-based processes, cytokinesis, lipid metabolic processes, and anatomical structure development. By proposing a strategy called "reciprocal functional test" for interacting gene pairs, we discover a group of genes that mediate crosstalk between distinct biological processes. In particular, we identify the ribosomal S6 Kinase/RSKS-1, previously characterized as an mTOR (mechanistic target of rapamycin) effector, as a regulator of DAF-2 endosomal recycling transport, which traces a functional correlation between endocytic recycling and aging processes. Together, our results provide an alternative and effective strategy for identifying genes and pathways that mediate crosstalk between bioprocesses with little prior knowledge.

A Model of Hereditary Sensory and Autonomic Neuropathy Type 1 Reveals a Role of Glycosphingolipids in Neuronal Polarity.

Hereditary sensory and autonomic neuropathy Type 1 (HSAN1) is a rare autosomal dominantly inherited neuropathy, clinically characterized by a loss of distal peripheral sensory and motoneuronal function. Mutations in subunits of serine palmitoyltransferase (SPT) have been linked to the majority of HSAN1 cases. SPTs catalyze the condensation of l-serine with palmitoyl-CoA, the first committed and rate-limiting step in de novo sphingolipid biosynthesis. Despite extensive investigation, the molecular pathogenesis of HSAN1 remains controversial. Here, we established a Caenorhabditis elegans (C. elegans) model of HSAN1 by generating a sptl-1(c363g) mutation, encoding SPTL-1(C121W) and equivalent to human SPTLC1C133W, at the C. elegans genomic locus through CRISPR. The sptl-1(c363g) homozygous mutants exhibited the same larval lethality and epithelial polarity defect as observed in sptl-1(RNAi) animals, suggesting a loss-of-function effect of the SPTL-1(C121W) mutation. sptl-1(c363g)/+ heterozygous mutants displayed sensory dysfunction with concomitant neuronal morphology and axon-dendrite polarity defects, demonstrating that the C. elegans model recapitulates characteristics of the human disease. sptl-1(c363g)-derived neuronal defects were copied in animals with defective sphingolipid biosynthetic enzymes downstream of SPTL-1, including ceramide glucosyltransferases, suggesting that SPTLC1C133W contributes to the HSAN1 pathogenesis by limiting the production of complex sphingolipids, including glucosylceramide. Overexpression of SPTL-1(C121W) led to similar epithelial and neuronal defects and to reduced levels of complex sphingolipids, specifically glucosylceramide, consistent with a dominant-negative effect of SPTL-1(C121W) that is mediated by loss of this downstream product. Genetic interactions between SPTL-1(C121W) and components of directional trafficking in neurons suggest that the neuronal polarity phenotype could be caused by glycosphingolipid-dependent defects in polarized vesicular trafficking.SIGNIFICANCE STATEMENT The symptoms of inherited metabolic diseases are often attributed to the accumulation of toxic intermediates or byproducts, no matter whether the disease-causing enzyme participates in a biosynthetic or a degradation pathway. By showing that the phenotypes observed in a C. elegans model of HSAN1 disease could be caused by loss of a downstream product (glucosylceramide) rather than the accumulation of a toxic byproduct, our work provides new insights into the origins of the symptoms of inherited metabolic diseases while expanding the repertoire of sphingolipid functions, specifically, of glucosylceramides. These findings not only have their most immediate relevance for neuroprotective treatments for HSAN1, they may also have implications for a much broader range of neurologic conditions.

Sam50 Regulates PINK1-Parkin-Mediated Mitophagy by Controlling PINK1 Stability and Mitochondrial Morphology.

PINK1 and Parkin mediate mitophagy, the cellular process that clears dysfunctional mitochondria. Mitophagy is regulated by mitochondrial dynamics, but the molecules linking these two processes remain poorly understood. Here, we show that Sam50, the core component of the sorting and assembly machinery (SAM), is a critical regulator of mitochondrial dynamics and PINK1-Parkin-mediated mitophagy. In response to Sam50 depletion, normal tubular mitochondria are first fragmented and subsequently merged into large spheres. Sam50 interacts with PINK1 to facilitate its processing and degradation. Depletion of Sam50 results in PINK1 accumulation, Parkin recruitment, and mitophagy. Interestingly, Sam50 deficiency induces a piecemeal mode of mitophagy that eliminates mitochondria "bit by bit" but spares mtDNA. In C. elegans, the Sam50 homolog gop-3 is required for the maintenance of mitochondrial morphology and mass. Our findings reveal that Sam50 directly links mitochondrial dynamics and mitophagy and that Sam50 depletion induces elimination of mitochondria without affecting mtDNA content.

SAC-1 ensures epithelial endocytic recycling by restricting ARF-6 activity.

Arf6/ARF-6 is a crucial regulator of the endosomal phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) pool in endocytic recycling. To further characterize ARF-6 regulation, we performed an ARF-6 interactor screen in Caenorhabditis elegans and identified SAC-1, the homologue of the phosphoinositide phosphatase Sac1p in yeast, as a novel ARF-6 partner. In the absence of ARF-6, basolateral endosomes show a loss of SAC-1 staining in epithelial cells. Steady-state cargo distribution assays revealed that loss of SAC-1 specifically affected apical secretory delivery and basolateral recycling. PI(4,5)P2 levels and the endosomal labeling of the ARF-6 effector UNC-16 were significantly elevated in sac-1 mutants, suggesting that SAC-1 functions as a negative regulator of ARF-6. Further analyses revealed an interaction between SAC-1 and the ARF-6-GEF BRIS-1. This interaction outcompeted ARF-6(guanosine diphosphate [GDP]) for binding to BRIS-1 in a concentration-dependent manner. Consequently, loss of SAC-1 promotes the intracellular overlap between ARF-6 and BRIS-1. BRIS-1 knockdown resulted in a significant reduction in PI(4,5)P2 levels in SAC-1-depleted cells. Interestingly, the action of SAC-1 in sequestering BRIS-1 is independent of SAC-1's catalytic activity. Our results suggest that the interaction of SAC-1 with ARF-6 curbs ARF-6 activity by limiting the access of ARF-6(GDP) to its guanine nucleotide exchange factor, BRIS-1.

PTRN-1/CAMSAP promotes CYK-1/formin-dependent actin polymerization during endocytic recycling.

Cargo sorting and membrane carrier initiation in recycling endosomes require appropriately coordinated actin dynamics. However, the mechanism underlying the regulation of actin organization during recycling transport remains elusive. Here we report that the loss of PTRN-1/CAMSAP stalled actin exchange and diminished the cytosolic actin structures. Furthermore, we found that PTRN-1 is required for the recycling of clathrin-independent cargo hTAC-GFP The N-terminal calponin homology (CH) domain and central coiled-coils (CC) region of PTRN-1 can synergistically sustain the flow of hTAC-GFP We identified CYK-1/formin as a binding partner of PTRN-1. The N-terminal GTPase-binding domain (GBD) of CYK-1 serves as the binding interface for the PTRN-1 CH domain. The presence of the PTRN-1 CH domain promoted CYK-1-mediated actin polymerization, which suggests that the PTRN-1-CH:CYK-1-GBD interaction efficiently relieves autoinhibitory interactions within CYK-1. As expected, the overexpression of the CYK-1 formin homology domain 2 (FH2) substantially restored actin structures and partially suppressed the hTAC-GFP overaccumulation phenotype in ptrn-1 mutants. We conclude that the PTRN-1 CH domain is required to stimulate CYK-1 to facilitate actin dynamics during endocytic recycling.

Melatonin Mitigates Kainic Acid-Induced Neuronal Tau Hyperphosphorylation and Memory Deficits through Alleviating ER Stress.

Kainic acid (KA) exposure causes neuronal degeneration featured by Alzheimer-like tau hyperphosphorylation and memory deficits. Melatonin (Mel) is known to protect hippocampal neurons against KA-induced damage. However, the underlying mechanisms remain elusive. In the current study, we investigated the protective effect of melatonin on KA-induced tau hyperphosphorylation by focusing on endoplasmic reticulum (ER) stress-mediated signaling pathways. By using primary hippocampal neurons and mouse brain, we showed that KA treatment specifically induced ER stress and activated GSK-3ß and CDK5, two major kinases responsible for tau phosphorylation. Inhibition of ER stress efficiently inactivated GSK-3ß and CDK5. Mechanistically, we found that KA-induced ER stress significantly activated calpain, a calcium-dependent protease. Inhibition of ER stress or calpain leads to the reduction in KA-induced GSK-3ß and CDK5 activities and tau phosphorylation. Moreover, GSK-3ß or CDK5 inhibition failed to downregulate ER stress efficiently, suggesting that ER stress functions upstream of GSK-3ß or CDK5. Notably, our results revealed that melatonin acts against KA-induced neuronal degeneration and tau hyperphosphorylation via easing ER stress, further highlighting the protective role of melatonin in the KA-induced neuronal defects.

LET-413/Erbin acts as a RAB-5 effector to promote RAB-10 activation during endocytic recycling.

RAB-10/Rab10 is a master regulator of endocytic recycling in epithelial cells. To better understand the regulation of RAB-10 activity, we sought to identify RAB-10(GDP)-interacting proteins. One novel RAB-10(GDP)-binding partner that we identified, LET-413, is the Caenorhabditis elegans homologue of Scrib/Erbin. Here, we focus on the mechanistic role of LET-413 in the regulation of RAB-10 within the C. elegans intestine. We show that LET-413 is a RAB-5 effector and colocalizes with RAB-10 on endosomes, and the overlap of LET-413 with RAB-10 is RAB-5 dependent. Notably, LET-413 enhances the interaction of DENN-4 with RAB-10(GDP) and promotes DENN-4 guanine nucleotide exchange factor activity toward RAB-10. Loss of LET-413 leads to cytosolic dispersion of the RAB-10 effectors TBC-2 and CNT-1. Finally, we demonstrate that the loss of RAB-10 or LET-413 results in abnormal overextensions of lateral membrane. Hence, our studies indicate that LET-413 is required for DENN-4-mediated RAB-10 activation, and the LET-413-assisted RAB-5 to RAB-10 cascade contributes to the integrity of C. elegans intestinal epithelia.