The C. elegans homolog of human panic-disorder risk gene TMEM132D orchestrates neuronal morphogenesis through the WAVE-regulatory complex.

TMEM132D is a human gene identified with multiple risk alleles for panic disorders, anxiety and major depressive disorders. Defining a conserved family of transmembrane proteins, TMEM132D and its homologs are still of unknown molecular functions. By generating loss-of-function mutants of the sole TMEM132 ortholog in C. elegans, we identify abnormal morphologic phenotypes in the dopaminergic PDE neurons. Using a yeast two-hybrid screen, we find that NAP1 directly interacts with the cytoplasmic domain of human TMEM132D, and mutations in C. elegans tmem-132 that disrupt interaction with NAP1 cause similar morphologic defects in the PDE neurons. NAP1 is a component of the WAVE regulatory complex (WRC) that controls F-actin cytoskeletal dynamics. Decreasing activity of WRC rescues the PDE defects in tmem-132 mutants, whereas gain-of-function of TMEM132D in mammalian cells inhibits WRC, leading to decreased abundance of select WRC components, impaired actin nucleation and cell motility. We propose that metazoan TMEM132 family proteins play evolutionarily conserved roles in regulating NAP1 protein homologs to restrict inappropriate WRC activity, cytoskeletal and morphologic changes in the cell.

The conserved autoimmune-disease risk gene TMEM39A regulates lysosome dynamics.

TMEM39A encodes an evolutionarily conserved transmembrane protein and carries single-nucleotide polymorphisms associated with increased risk of major human autoimmune diseases, including multiple sclerosis. The exact cellular function of TMEM39A remains not well understood. Here, we report that TMEM-39, the sole Caenorhabditis elegans (C. elegans) ortholog of TMEM39A, regulates lysosome distribution and accumulation. Elimination of tmem-39 leads to lysosome tubularization and reduced lysosome mobility, as well as accumulation of the lysosome-associated membrane protein LMP-1. In mammalian cells, loss of TMEM39A leads to redistribution of lysosomes from the perinuclear region to cell periphery. Mechanistically, TMEM39A interacts with the dynein intermediate light chain DYNC1I2 to maintain proper lysosome distribution. Deficiency of tmem-39 or the DYNC1I2 homolog in C. elegans impairs mTOR signaling and activates the downstream TFEB-like transcription factor HLH-30. We propose evolutionarily conserved roles of TMEM39 family proteins in regulating lysosome distribution and lysosome-associated signaling, dysfunction of which in humans may underlie aspects of autoimmune diseases.

The conserved transmembrane protein TMEM-39 coordinates with COPII to promote collagen secretion and regulate ER stress response.

Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. Here, we report that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. We identify the C. elegans ortholog TMEM-39 from an unbiased RNAi screen and show that deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. We propose that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved.

Cytoprotection by a naturally occurring variant of ATP5G1 in Arctic ground squirrel neural progenitor cells.

Many organisms in nature have evolved mechanisms to tolerate severe hypoxia or ischemia, including the hibernation-capable Arctic ground squirrel (AGS). Although hypoxic or ischemia tolerance in AGS involves physiological adaptations, little is known about the critical cellular mechanisms underlying intrinsic AGS cell resilience to metabolic stress. Through cell survival-based cDNA expression screens in neural progenitor cells, we identify a genetic variant of AGS Atp5g1 that confers cell resilience to metabolic stress. Atp5g1 encodes a subunit of the mitochondrial ATP synthase. Ectopic expression in mouse cells and CRISPR/Cas9 base editing of endogenous AGS loci revealed causal roles of one AGS-specific amino acid substitution in mediating cytoprotection by AGS ATP5G1. AGS ATP5G1 promotes metabolic stress resilience by modulating mitochondrial morphological change and metabolic functions. Our results identify a naturally occurring variant of ATP5G1 from a mammalian hibernator that critically contributes to intrinsic cytoprotection against metabolic stress.

When animals hibernate, they lower their body temperature and metabolism to conserve the energy they need to withstand cold harsh winters. One such animal is the Arctic ground squirrel, an extreme hibernator that can drop its body temperatures to below 0°C. This hibernation ability means the cells of Arctic ground squirrels can survive severe shortages of blood and oxygen. But, it is unclear how their cells are able to endure this metabolic stress. To answer this question, Singhal, Bai et al. studied the cells of Arctic ground squirrels for unique features that might make them more durable to stress. Examining the genetic code of these resilient cells revealed that Arctic ground squirrels may have a variant form of a protein called ATP5G1. This protein is found in a cellular compartment called the mitochondria, which is responsible for supplying energy to the rest of the cell and therefore plays an important role in metabolic processes. Singhal, Bai et al. found that when this variant form of ATP5G1 was introduced into the cells of mice, their mitochondria was better at coping with stress conditions, such as low oxygen, low temperature and poisoning. Using a gene editing tool to selectively substitute some of the building blocks, also known as amino acids, that make up the ATP5G1 protein revealed that improvements to the mitochondria were caused by switching specific amino acids. However, swapping these amino acids, which presumably affects the role of ATP5G1, did not completely remove the cells' resilience to stress. This suggests that variants of other genes and proteins may also be involved in providing protection. These findings provide the first evidence of a protein variant that is responsible for protecting cells during the metabolic stress conditions caused by hibernation. The approach taken by Singhal, Bai et al. could be used to identify and study other proteins that increase resilience to metabolic stress. These findings could help develop new treatments for diseases caused by a limited blood supply to human organs, such as a stroke or heart attack.

Broadly conserved roles of TMEM131 family proteins in intracellular collagen assembly and secretory cargo trafficking.

Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, we identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. We find that amino termini of human TMEM131 contain bacterial PapD chaperone-like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. We provide evidence that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans.

A genetic program mediates cold-warming response and promotes stress-induced phenoptosis in C. elegans.

How multicellular organisms respond to and are impacted by severe hypothermic stress is largely unknown. From C. elegans screens for mutants abnormally responding to cold-warming stimuli, we identify a molecular genetic pathway comprising ISY-1, a conserved uncharacterized protein, and ZIP-10, a bZIP-type transcription factor. ISY-1 gatekeeps the ZIP-10 transcriptional program by regulating the microRNA mir-60. Downstream of ISY-1 and mir-60, zip-10 levels rapidly and specifically increase upon transient cold-warming exposure. Prolonged zip-10 up-regulation induces several protease-encoding genes and promotes stress-induced organismic death, or phenoptosis, of C. elegans. zip-10 deficiency confers enhanced resistance to prolonged cold-warming stress, more prominently in adults than larvae. We conclude that the ZIP-10 genetic program mediates cold-warming response and may have evolved to promote wild-population kin selection under resource-limiting and thermal stress conditions.

The CDK8 Complex and Proneural Proteins Together Drive Neurogenesis from a Mesodermal Lineage.

At least some animal species can generate neurons from mesoderm or endoderm, but the underlying mechanisms remain unknown. We screened for C. elegans mutants in which the presumptive mesoderm-derived I4 neuron adopts a muscle-like cell fate. From this screen, we identified HLH-3, the C. elegans homolog of a mammalian proneural protein (Ascl1) used for in vitro neuronal reprogramming, as required for efficient I4 neurogenesis. We discovered that the CDK-8 Mediator kinase module acts together with a second proneural protein, HLH-2, and in parallel to HLH-3 to promote I4 neurogenesis. Genetic analysis revealed that CDK-8 most likely promotes I4 neurogenesis by inhibiting the CDK-7/CYH-1 (CDK7/cyclin H) kinase module of the transcription initiation factor TFIIH. Ectopic expression of HLH-2 and HLH-3 together promoted expression of neuronal features in non-neuronal cells. These findings reveal that the Mediator CDK8 kinase module can promote non-ectodermal neurogenesis and suggest that inhibiting CDK7/cyclin H might similarly promote neurogenesis.

The conserved LIM domain-containing focal adhesion protein ZYX-1 regulates synapse maintenance in Caenorhabditis elegans.

We describe the identification of zyxin as a regulator of synapse maintenance in mechanosensory neurons in C. elegans. zyx-1 mutants lacked PLM mechanosensory synapses as adult animals. However, most PLM synapses initially formed during development but were subsequently lost as the animals developed. Vertebrate zyxin regulates cytoskeletal responses to mechanical stress in culture. Our work provides in vivo evidence in support of such a role for zyxin. In particular, zyx-1 mutant synaptogenesis phenotypes were suppressed by disrupting locomotion of the mutant animals, suggesting that zyx-1 protects mechanosensory synapses from locomotion-induced forces. In cultured cells, zyxin is recruited to focal adhesions and stress fibers via C-terminal LIM domains and modulates cytoskeletal organization via the N-terminal domain. The synapse-stabilizing activity was mediated by a short isoform of ZYX-1 containing only the LIM domains. Consistent with this notion, PLM synaptogenesis was independent of α-actinin and ENA-VASP, both of which bind to the N-terminal domain of zyxin. Our results demonstrate that the LIM domain moiety of zyxin functions autonomously to mediate responses to mechanical stress and provide in vivo evidence for a role of zyxin in neuronal development.

Change of glutamic acid decarboxylase antibody and protein tyrosine phosphatase antibody in Chinese patients with acute-onset type 1 diabetes mellitus.

BACKGROUND: Glutamic acid decarboxylase antibody (GADA) and protein tyrosine phosphatase antibody (IA-2A) are two major autoantibodies, which exert important roles in the process of type 1 diabetes mellitus (T1D). Our study aimed to investigate the changes in positivity and titers of GADA and IA-2A during the course of Chinese acute-onset T1D patients and their relationships with clinical features. METHODS: Two hundreds and forty-seven Chinese newly diagnosed acute-onset T1D patients were consecutively recruited. GADA and IA-2A were detected at the time of diagnosis, one year later, 3-5 years later after diagnosis during the follow-up; all the clinical data were recorded and analyzed as well. RESULTS: During the course of acute-onset T1D, the majority of patients remained stable for GADA or IA-2A, however, a few patients changed from positivity to negativity and fewer patients converted from negativity to positivity. The prevalence of GADA was 56.3% at diagnosis, decreasing to 50.5% one year later, and 43.3% 3-5 years later while the corresponding prevalence of IA-2A were 32.8%, 31.0% and 23.3%, respectively. The median GADA titers were 0.0825 at diagnosis, declining to 0.0585 one year later and 0.0383 3-5 years later (P < 0.001), while the corresponding median titers were 0.0016, 0.0010, 0.0014 for IA-2A, respectively. Fasting C-peptide (FCP) and postprandial C-peptide 2 hours (PCP2h) levels of GADA or IA-2A negativity persistence patients were higher than those of positivity persistence and negativity conversion patients (P < 0.05) which indicated GADA or IA-2A negativity persistence T1D patients had a less loss of ß cell function. CONCLUSION: Our data suggest that repeated detection of GADA and IA-2A are necessary for differential diagnosis of autoimmune diabetes and the indirect prediction of the ß cell function in Chinese patients.

HID-1, a new component of the peptidergic signaling pathway.

hid-1 was originally identified as a Caenorhabditis elegans gene encoding a novel conserved protein that regulates the decision to enter into the enduring dauer larval stage. We isolated a novel allele of hid-1 in a forward genetic screen for mutants mislocalizing RBF-1 rabphilin, a RAB-27 effector. Here we demonstrate that HID-1 functions in the nervous system to regulate neuromuscular signaling and in the intestine to regulate the defecation motor program. We further show that a conserved N-terminal myristoylated motif of both invertebrate and vertebrate HID-1 is essential for its association with intracellular membranes in nematodes and PC12 cells. C. elegans neuronal HID-1 resides on intracellular membranes in neuronal cell somas; however, the kinesin UNC-104 also transports HID-1 to synaptic regions. HID-1 accumulates in the axons of unc-13 and unc-31 mutants, suggesting it is associated with neurosecretory vesicles. Consistent with this, genetic studies place HID-1 in a peptidergic signaling pathway. Finally, a hid-1 null mutation reduces the levels of endogenous neuropeptides and alters the secretion of fluorescent-tagged cargos derived from neuronal and intestinal dense core vesicles (DCVs). Taken together, our findings indicate that HID-1 is a novel component of a DCV-based neurosecretory pathway and that it regulates one or more aspects of the biogenesis, maturation, or trafficking of DCVs.

[Value of zinc transporter 8 autoantibody in the diagnostic classification of acute-onset diabetics].

OBJECTIVE: to explore the application significance of zinc transporter 8 autoantibody (ZnT8A) in the diagnostic classification of acute-onset diabetics. METHODS: according to the status of glutamic acid decarboxylase antibody (GADA) and tyrosine phosphatase antibody (IA2-A), 453 acute-onset diabetics were divided into A+ subgroup (any antibody positive) and A- subgroup (both antibodies negative). A total of 555 type 2 diabetics and 405 healthy controls were recruited. The distribution and correlated factors of ZnT8A were analyzed in the acute-onset diabetic group and two subgroups (A+ and A-). The clinical characteristics were compared between the patients with ZnT8A positive alone and patients without any antibody. All these islet antibodies were measured by radioligand assay. RESULTS: the prevalence of ZnT8A in acute-onset diabetics was 24.3% and it was significantly higher than that in type 2 diabetics (1.8%) and healthy controls (1.0%) (both P < 0.01). The frequency of ZnT8A in A+ subgroup was much higher than A- subgroup (29.7% vs 15.8%, P < 0.01). The positive rates of ZnT8A were much higher in all the subgroups with age at onset of < 30 yr than those with ≥ 30 yr (0 - 9, 34.9%; 10 - 19, 26.7%; 20 - 29, 26.3% vs ≥ 30 yr, 18.3%; all P < 0.05); furthermore, the rates were also higher in BMI < 21.0 kg/m(2) and 21.0 - 25.0 kg/m(2) subgroups than in BMI > 25.0 kg/m(2) subgroup (25.5% and 25.9% vs 8.7%, both P < 0.05). The ZnT8A level was only positively correlated with IA2-A titer (r = 0.165, P = 0.01), but not related to such factors as GADA titer, age at onset, duration, body mass index, HbA1c and CP levels (all P > 0.05). As compared with Ab- patients, the patients with ZnT8A positive alone had much higher insulin injection dosage [(35.5 ± 9.3) U/d vs (29.8 ± 14.7) U/d, P < 0.05], and much lower systolic blood pressures [(107 ± 15) mm Hg vs (113 ± 16) mm Hg, P < 0.05] and diastolic blood pressures [(69 ± 12) mm Hg vs (73 ± 12) mm Hg, P < 0.05]. CONCLUSION: ZnT8A testing may be applied in the diagnostic classification of acute-onset diabetics, especially in those without an evidence of GADA and IA2-A since it helps to identify a clinical phenotype which is more similar to the classic type 1 diabetes.

Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans.

The Caenorhabditis elegans defecation motor program (DMP) is a highly coordinated rhythmic behavior that requires two GABAergic neurons that synapse onto the enteric muscles. One class of DMP mutants, called anterior body wall muscle contraction and expulsion defective (aex) mutants, exhibits similar defects to those caused by the loss of these two neurons. Here, we demonstrate that aex-2 encodes a G-protein-coupled receptor (GPCR) and aex-4 encodes an exocytic SNAP25 homologue. We found that aex-2 functions in the nervous system and activates a G(s)alpha signaling pathway to regulate defecation. aex-4, on the other hand, functions in the intestinal epithelial cells. Furthermore, we show that aex-5, which encodes a pro-protein convertase, functions in the intestine to regulate the DMP and that its secretion from the intestine is impaired in aex-4 mutants. Activation of the G(s)alpha GPCR pathway in GABAergic neurons can suppress the defecation defect of the intestinal mutants aex-4 and aex-5. Lastly, we demonstrate that activation of GABAergic neurons using the light-gated cation channel channelrhodopsin-2 is sufficient to suppress the behavioral defects of aex-2, aex-4, and aex-5. These results genetically place intestinal genes aex-4 and aex-5 upstream of GABAergic GPCR signaling. We propose a model whereby the intestinal genes aex-4 and aex-5 control the DMP by regulating the secretion of a signal, which activates the neuronal receptor aex-2.

Regulators of kinesin involved in polarized trafficking and axon outgrowth.

Proteins such as UNC-76 that associate with kinesin motors are important in directing neurite extension. A small Caenorhabditis elegans coiled-coil protein, UNC-69, has now been shown to interact with UNC-76 and to be involved in axonal (but not dendritic) transport and outgrowth, as well as synapse formation.

Regulation of synaptic transmission by RAB-3 and RAB-27 in Caenorhabditis elegans.

Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function.

Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay.

Caenorhabditis elegans has emerged as a powerful model system for studying the biology of the synapse. Here we describe a widely used assay for synaptic transmission at the C. elegans neuromuscular junction. This protocol monitors the sensitivity of C. elegans to the paralyzing affects of an acetylcholinesterase inhibitor, aldicarb. Briefly, adult worms are incubated in the presence of aldicarb and scored for the time-course of aldicarb-induced paralysis. Animals harboring mutations in genes that affect synaptic transmission generally exhibit a change in their sensitivity to aldicarb (either increased sensitivity for enhancements in synaptic transmission or decreased sensitivity for blockage in synaptic transmission). This technique provides a simple assay for the accurate comparative analysis of synaptic transmission in multiple C. elegans strains. The protocol described can be performed relatively quickly and is a practical alternative to other techniques used to study synaptic transmission. This protocol can also be modified to follow the paralytic effects with other pharmacological reagents. The assay can be performed in about 3-6 hours depending on the severity of synaptic transmission defects.

{alpha}-Syntrophin regulates ARMS localization at the neuromuscular junction and enhances EphA4 signaling in an ARMS-dependent manner.

EphA4 signaling has recently been implicated in the regulation of synapse formation and plasticity. In this study, we show that ankyrin repeat-rich membrane spanning (ARMS; also known as a kinase D-interacting substrate of 220 kD), a substrate for ephrin and neurotrophin receptors, was expressed in developing muscle and was concentrated at the neuromuscular junction (NMJ). Using yeast two-hybrid screening, we identified a PDZ (PSD-95, Dlg, ZO-1) domain protein, alpha-syntrophin, as an ARMS-interacting protein in muscle. Overexpression of alpha-syntrophin induced ARMS clustering in a PDZ domain-dependent manner. Coexpression of ARMS enhanced EphA4 signaling, which was further augmented by the presence of alpha-syntrophin. Moreover, the ephrin-A1-induced tyrosine phosphorylation of EphA4 was reduced in C2C12 myotubes after the blockade of ARMS and alpha-syntrophin expression by RNA interference. Finally, alpha-syntrophin-null mice exhibited a disrupted localization of ARMS and EphA4 at the NMJ and a reduced expression of ARMS in muscle. Altogether, our findings suggest that ARMS may play an important role in regulating postsynaptic signal transduction through the syntrophin-mediated localization of receptor tyrosine kinases such as EphA4.

Characterization of methanogenic Archaea in the leachate of a closed municipal solid waste landfill.

Cultivation-independent molecular approaches were used to investigate the phylogenetic composition of Archaea and the relative abundance of phylogenetically defined groups of methanogens in the leachate of a closed municipal solid waste landfill. Cloning and phylogenetic analysis of archaeal 16S rRNA gene sequences (16S rDNA) revealed that the landfill leachate harbored a diverse Archaea community, with sequence types distributed within the two archaeal kingdoms of the Euryarchaeota and the Crenarchaeota. Of the 80 clones examined, 51 were phylogenetically associated with well-defined methanogen lineages covering two major methanogenic phenotypes; 20 were related to Thermoplasma and were grouped with some novel archaeal rRNA gene sequences recently recovered from various anaerobic habitats; finally, five belonged to Crenarchaeota and were not closely related to any hitherto cultivated species. Most of the methanogen-like clones were affiliated with the hydrogenotrophic Methanomicrobiales and the methylotrophic and acetoclastic Methanosarcinales. Quantitative oligonucleotide hybridization experiments showed that methanogens in the leachate accounted for only a very small fraction of the total community (approximately 2%) and that Methanomicrobiales and Methanosarcinales constituted the majority of the total methanogenic population.

Diversity and structure of the archaeal community in the leachate of a full-scale recirculating landfill as examined by direct 16S rRNA gene sequence retrieval.

The diversity and structure of the archaeal community in the effluent leachate from a full-scale recirculating landfill was characterized by direct 16S rRNA gene (16S rDNA) retrieval. Total-community DNA was extracted from the microbial assemblages in the landfill leachate, and archaeal 16S rDNAs were amplified with a universally conserved primer and an Archaea-specific primer. The amplification product was then used to construct a 16S rDNA clone library, and 70 randomly selected archaeal clones in the library were grouped by restriction fragment length polymorphism (RFLP) analysis. Sequencing and phylogenetic analysis of representatives from each unique RFLP type showed that the archaeal library was dominated by methanogen-like rDNAs. Represented in the kingdom of Euryarchaeota were phylotypes highly similar to the methanogenic genera Methanoculleus, Methanosarcina, Methanocorpusculum, Methanospirillum and Methanogenium, where the clone distribution was 48, 11, 3, 1 and 1, respectively. No sequences related to known Methanosaeta spp. were retrieved. Four rDNA clones were not affiliated with the known methanogenic Archaea, but instead, they were clustered with the uncultured archaeal sequences recently recovered from anaerobic habitats. Two chimeric sequences were identified among the clones analyzed.