CMTR-1 RNA methyltransferase mutations activate widespread expression of a dopaminergic neuron-specific mitochondrial complex I gene.

The mitochondrial proteome is comprised of approximately 1,100 proteins,1 all but 12 of which are encoded by the nuclear genome in C. elegans. The expression of nuclear-encoded mitochondrial proteins varies widely across cell lineages and metabolic states,2,3,4 but the factors that specify these programs are not known. Here, we identify mutations in two nuclear-localized mRNA processing proteins, CMTR1/CMTR-1 and SRRT/ARS2/SRRT-1, which we show act via the same mechanism to rescue the mitochondrial complex I mutant NDUFS2/gas-1(fc21). CMTR-1 is an FtsJ-family RNA methyltransferase that, in mammals, 2'-O-methylates the first nucleotide 3' to the mRNA CAP to promote RNA stability and translation5,6,7,8. The mutations isolated in cmtr-1 are dominant and lie exclusively in the regulatory G-patch domain. SRRT-1 is an RNA binding partner of the nuclear cap-binding complex and determines mRNA transcript fate.9 We show that cmtr-1 and srrt-1 mutations activate embryonic expression of NDUFS2/nduf-2.2, a paralog of NDUFS2/gas-1 normally expressed only in dopaminergic neurons, and that nduf-2.2 is necessary for the complex I rescue by the cmtr-1 G-patch mutant. Additionally, we find that loss of the cmtr-1 G-patch domain cause ectopic localization of CMTR-1 protein to processing bodies (P bodies), phase-separated organelles involved in mRNA storage and decay.10 P-body localization of the G-patch mutant CMTR-1 contributes to the rescue of the hyperoxia sensitivity of the NDUFS2/gas-1 mutant. This study suggests that mRNA methylation at P bodies may control nduf-2.2 gene expression, with broader implications for how the mitochondrial proteome is translationally remodeled in the face of tissue-specific metabolic requirements and stress.

Neuroendocrine gene expression coupling of interoceptive bacterial food cues to foraging behavior of C. elegans.

Animal internal state is modulated by nutrient intake, resulting in behavioral responses to changing food conditions. The neural mechanisms by which internal states are generated and maintained are not well understood. Here, we show that in the nematode Caenorhabditis elegans, distinct cues from bacterial food - interoceptive signals from the ingestion of bacteria and gustatory molecules sensed from nearby bacteria - act antagonistically on the expression of the neuroendocrine TGF-beta ligand DAF-7 from the ASJ pair of sensory neurons to modulate foraging behavior. A positive-feedback loop dependent on the expression of daf-7 from the ASJ neurons acts to promote transitions between roaming and dwelling foraging states and influence the persistence of roaming states. SCD-2, the C. elegans ortholog of mammalian anaplastic lymphoma kinase (ALK), which has been implicated in the central control of metabolism of mammals, functions in the AIA interneurons to regulate foraging behavior and cell-non-autonomously control the expression of DAF-7 from the ASJ neurons. Our data establish how a dynamic neuroendocrine daf-7 expression feedback loop regulated by SCD-2 functions to couple sensing and ingestion of bacterial food to foraging behavior. We further suggest that this neuroendocrine feedback loop underlies previously characterized exploratory behaviors in C. elegans. Our data suggest that the expression of daf-7 from the ASJ neurons contributes to and is correlated with an internal state of 'unmet need' that regulates exploratory foraging behavior in response to bacterial cues in diverse physiological contexts.

Hypoxia and intra-complex genetic suppressors rescue complex I mutants by a shared mechanism.

The electron transport chain (ETC) of mitochondria, bacteria, and archaea couples electron flow to proton pumping and is adapted to diverse oxygen environments. Remarkably, in mice, neurological disease due to ETC complex I dysfunction is rescued by hypoxia through unknown mechanisms. Here, we show that hypoxia rescue and hyperoxia sensitivity of complex I deficiency are evolutionarily conserved to C. elegans and are specific to mutants that compromise the electron-conducting matrix arm. We show that hypoxia rescue does not involve the hypoxia-inducible factor pathway or attenuation of reactive oxygen species. To discover the mechanism, we use C. elegans genetic screens to identify suppressor mutations in the complex I accessory subunit NDUFA6/nuo-3 that phenocopy hypoxia rescue. We show that NDUFA6/nuo-3(G60D) or hypoxia directly restores complex I forward activity, with downstream rescue of ETC flux and, in some cases, complex I levels. Additional screens identify residues within the ubiquinone binding pocket as being required for the rescue by NDUFA6/nuo-3(G60D) or hypoxia. This reveals oxygen-sensitive coupling between an accessory subunit and the quinone binding pocket of complex I that can restore forward activity in the same manner as hypoxia.

Genetic Variants That Modify the Neuroendocrine Regulation of Foraging Behavior in C. elegans.

The molecular mechanisms underlying diversity in animal behavior are not well understood. A major experimental challenge is determining the contribution of genetic variants that affect neuronal gene expression to differences in behavioral traits. The neuroendocrine TGF-beta ligand, DAF-7, regulates diverse behavioral responses of Caenorhabditis elegans to bacterial food and pathogens. The dynamic neuron-specific expression of daf-7 is modulated by environmental and endogenous bacteria-derived cues. Here, we investigated natural variation in the expression of daf-7 from the ASJ pair of chemosensory neurons and identified common variants in gap-2, encoding a GTPase-Activating Protein homologous to mammalian SynGAP proteins, which modify daf-7 expression cell-non-autonomously and promote exploratory foraging behavior in a DAF-7-dependent manner. Our data connect natural variation in neuron-specific gene expression to differences in behavior and suggest that genetic variation in neuroendocrine signaling pathways mediating host-microbe interactions may give rise to diversity in animal behavior.

Neuroendocrine Gene Expression Coupling of Interoceptive Bacterial Food Cues to Foraging Behavior of C. elegans.

Animal internal state is modulated by nutrient intake, resulting in behavioral responses to changing food conditions. The neural mechanisms by which internal states are generated and maintained are not well understood. Here, we show that in the nematode Caenorhabditis elegans, distinct cues from bacterial food - interoceptive signals from the ingestion of bacteria and gustatory molecules sensed from nearby bacteria - act antagonistically on the expression of the neuroendocrine TGF-beta ligand DAF-7 from the ASJ pair of sensory neurons to modulate foraging behavior. A positive-feedback loop dependent on the expression of daf-7 from the ASJ neurons acts to promote transitions between roaming and dwelling foraging states and influence the persistence of roaming states. SCD-2, the C. elegans ortholog of mammalian Anaplastic Lymphoma Kinase (ALK), which has been implicated in the central control of metabolism of mammals, functions in the AIA interneurons to regulate foraging behavior and cell-non-autonomously control the expression of DAF-7 from the ASJ neurons. Our data establish how a dynamic neuroendocrine daf-7 expression feedback loop regulated by SCD-2 functions to couple sensing and ingestion of bacterial food to foraging behavior. We further suggest that this neuroendocrine feedback loop underlies previously characterized exploratory behaviors in C. elegans. Our data suggest that the expression of daf-7 from the ASJ neurons contributes to and is correlated with an internal state of "unmet need" that regulates exploratory foraging behavior in response to bacterial cues in diverse physiological contexts.

MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations.

The mammalian mitochondrial proteome is under dual genomic control, with 99% of proteins encoded by the nuclear genome and 13 originating from the mitochondrial DNA (mtDNA). We previously developed MitoCarta, a catalogue of over 1000 genes encoding the mammalian mitochondrial proteome. This catalogue was compiled using a Bayesian integration of multiple sequence features and experimental datasets, notably protein mass spectrometry of mitochondria isolated from fourteen murine tissues. Here, we introduce MitoCarta3.0. Beginning with the MitoCarta2.0 inventory, we performed manual review to remove 100 genes and introduce 78 additional genes, arriving at an updated inventory of 1136 human genes. We now include manually curated annotations of sub-mitochondrial localization (matrix, inner membrane, intermembrane space, outer membrane) as well as assignment to 149 hierarchical 'MitoPathways' spanning seven broad functional categories relevant to mitochondria. MitoCarta3.0, including sub-mitochondrial localization and MitoPathway annotations, is freely available at http://www.broadinstitute.org/mitocarta and should serve as a continued community resource for mitochondrial biology and medicine.

Immediate activation of chemosensory neuron gene expression by bacterial metabolites is selectively induced by distinct cyclic GMP-dependent pathways in Caenorhabditis elegans.

Dynamic gene expression in neurons shapes fundamental processes in the nervous systems of animals. However, how neuronal activation by different stimuli can lead to distinct transcriptional responses is not well understood. We have been studying how microbial metabolites modulate gene expression in chemosensory neurons of Caenorhabditis elegans. Considering the diverse environmental stimuli that can activate chemosensory neurons of C. elegans, we sought to understand how specific transcriptional responses can be generated in these neurons in response to distinct cues. We have focused on the mechanism of rapid (<6 min) and selective transcriptional induction of daf-7, a gene encoding a TGF-ß ligand, in the ASJ chemosensory neurons in response to the pathogenic bacterium Pseudomonas aeruginosa. DAF-7 is required for the protective behavioral avoidance of P. aeruginosa by C. elegans. Here, we define the involvement of two distinct cyclic GMP (cGMP)-dependent pathways that are required for daf-7 expression in the ASJ neuron pair in response to P. aeruginosa. We show that a calcium-independent pathway dependent on the cGMP-dependent protein kinase G (PKG) EGL-4, and a canonical calcium-dependent signaling pathway dependent on the activity of a cyclic nucleotide-gated channel subunit CNG-2, function in parallel to activate rapid, selective transcription of daf-7 in response to P. aeruginosa metabolites. Our data suggest that fast, selective early transcription of neuronal genes require PKG in shaping responses to distinct microbial stimuli in a pair of C. elegans chemosensory neurons.

Hypoxia Rescues Frataxin Loss by Restoring Iron Sulfur Cluster Biogenesis.

Friedreich's ataxia (FRDA) is a devastating, multisystemic disorder caused by recessive mutations in the mitochondrial protein frataxin (FXN). FXN participates in the biosynthesis of Fe-S clusters and is considered to be essential for viability. Here we report that when grown in 1% ambient O2, FXN null yeast, human cells, and nematodes are fully viable. In human cells, hypoxia restores steady-state levels of Fe-S clusters and normalizes ATF4, NRF2, and IRP2 signaling events associated with FRDA. Cellular studies and in vitro reconstitution indicate that hypoxia acts through HIF-independent mechanisms that increase bioavailable iron as well as directly activate Fe-S synthesis. In a mouse model of FRDA, breathing 11% O2 attenuates the progression of ataxia, whereas breathing 55% O2 hastens it. Our work identifies oxygen as a key environmental variable in the pathogenesis associated with FXN depletion, with important mechanistic and therapeutic implications.

MICU1 imparts the mitochondrial uniporter with the ability to discriminate between Ca2+ and Mn2+.

The mitochondrial uniporter is a Ca2+-activated Ca2+ channel complex that displays exceptionally high conductance and selectivity. Here, we report cellular metal toxicity screens highlighting the uniporter's role in Mn2+ toxicity. Cells lacking the pore-forming uniporter subunit, MCU, are more resistant to Mn2+ toxicity, while cells lacking the Ca2+-sensing inhibitory subunit, MICU1, are more sensitive than the wild type. Consistent with these findings, Caenorhabditis elegans lacking the uniporter's pore have increased resistance to Mn2+ toxicity. The chemical-genetic interaction between uniporter machinery and Mn2+ toxicity prompted us to hypothesize that Mn2+ can indeed be transported by the uniporter's pore, but this transport is prevented by MICU1. To this end, we demonstrate that, in the absence of MICU1, both Mn2+ and Ca2+ can pass through the uniporter, as evidenced by mitochondrial Mn2+ uptake assays, mitochondrial membrane potential measurements, and mitoplast electrophysiology. We show that Mn2+ does not elicit the conformational change in MICU1 that is physiologically elicited by Ca2+, preventing Mn2+ from inducing the pore opening. Our work showcases a mechanism by which a channel's auxiliary subunit can contribute to its apparent selectivity and, furthermore, may have implications for understanding how manganese contributes to neurodegenerative disease.

Inhibition of Lithium-Sensitive Phosphatase BPNT-1 Causes Selective Neuronal Dysfunction in C. elegans.

Lithium has been a mainstay for the treatment of bipolar disorder, yet the molecular mechanisms underlying its action remain enigmatic. Bisphosphate 3'-nucleotidase (BPNT-1) is a lithium-sensitive phosphatase that catalyzes the breakdown of cytosolic 3'-phosphoadenosine 5'-phosphate (PAP), a byproduct of sulfation reactions utilizing the universal sulfate group donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) [1-3]. Loss of BPNT-1 leads to the toxic accumulation of PAP in yeast and non-neuronal cell types in mice [4, 5]. Intriguingly, BPNT-1 is expressed throughout the mammalian brain [4], and it has been hypothesized that inhibition of BPNT-1 could contribute to the effects of lithium on behavior [5]. Here, we show that loss of BPNT-1 in Caenorhabditis elegans results in the selective dysfunction of two neurons, the bilaterally symmetric pair of ASJ chemosensory neurons. As a result, BPNT-1 mutants are defective in behaviors dependent on the ASJ neurons, such as dauer exit and pathogen avoidance. Acute treatment with lithium also causes dysfunction of the ASJ neurons, and we show that this effect is reversible and mediated specifically through inhibition of BPNT-1. Finally, we show that the selective effect of lithium on the nervous system is due in part to the limited expression of the cytosolic sulfotransferase SSU-1 in the ASJ neuron pair. Our data suggest that lithium, through inhibition of BPNT-1 in the nervous system, can cause selective toxicity to specific neurons, resulting in corresponding effects on behavior of C. elegans.

Physiology and evolution of nitrate acquisition in Prochlorococcus.

Prochlorococcus is the numerically dominant phototroph in the oligotrophic subtropical ocean and carries out a significant fraction of marine primary productivity. Although field studies have provided evidence for nitrate uptake by Prochlorococcus, little is known about this trait because axenic cultures capable of growth on nitrate have not been available. Additionally, all previously sequenced genomes lacked the genes necessary for nitrate assimilation. Here we introduce three Prochlorococcus strains capable of growth on nitrate and analyze their physiology and genome architecture. We show that the growth of high-light (HL) adapted strains on nitrate is ∼17% slower than their growth on ammonium. By analyzing 41 Prochlorococcus genomes, we find that genes for nitrate assimilation have been gained multiple times during the evolution of this group, and can be found in at least three lineages. In low-light adapted strains, nitrate assimilation genes are located in the same genomic context as in marine Synechococcus. These genes are located elsewhere in HL adapted strains and may often exist as a stable genetic acquisition as suggested by the striking degree of similarity in the order, phylogeny and location of these genes in one HL adapted strain and a consensus assembly of environmental Prochlorococcus metagenome sequences. In another HL adapted strain, nitrate utilization genes may have been independently acquired as indicated by adjacent phage mobility elements; these genes are also duplicated with each copy detected in separate genomic islands. These results provide direct evidence for nitrate utilization by Prochlorococcus and illuminate the complex evolutionary history of this trait.

Chemosensation of bacterial secondary metabolites modulates neuroendocrine signaling and behavior of C. elegans.

Discrimination between pathogenic and beneficial microbes is essential for host organism immunity and homeostasis. Here, we show that chemosensory detection of two secondary metabolites produced by Pseudomonas aeruginosa modulates a neuroendocrine signaling pathway that promotes avoidance behavior in the simple animal host Caenorhabditis elegans. Secondary metabolites phenazine-1-carboxamide and pyochelin activate a G-protein-signaling pathway in the ASJ chemosensory neuron pair that induces expression of the neuromodulator DAF-7/TGF-ß. DAF-7, in turn, activates a canonical TGF-ß signaling pathway in adjacent interneurons to modulate aerotaxis behavior and promote avoidance of pathogenic P. aeruginosa. Our data provide a chemical, genetic, and neuronal basis for how the behavior and physiology of a simple animal host can be modified by the microbial environment and suggest that secondary metabolites produced by microbes may provide environmental cues that contribute to pathogen recognition and host survival.

Behavioral avoidance of pathogenic bacteria by Caenorhabditis elegans.

The simple animal host Caenorhabditis elegans utilizes its nervous system to respond to diverse microbial cues, and can engage in a protective behavioral avoidance response to environmental pathogens. This behavior bears hallmarks of an immune response, with sensors and recognition systems that trigger a protective response following a learning experience. Neuronal circuits required for aversive learning have been defined, revealing conserved signaling modules with dual roles in immunity and neuronal responses to pathogenic bacteria. Identification of natural polymorphisms that modulate avoidance behavior has enabled an improved understanding of host-microbe interactions at the molecular level. We review here these findings and discuss how the microbial cues and host responses defined in C. elegans may provide insight into evolutionarily diverse host-microbe interactions.