Evolutionary conservation of putative suicidality-related risk genes that produce diminished motivation corrected by clozapine, lithium and antidepressants.

Background: Genome wide association studies (GWAS) and candidate gene analyses have identified genetic variants and genes that may increase the risk for suicidal thoughts and behaviors (STBs). Important unresolved issues surround these tentative risk variants such as the characteristics of the associated genes and how they might elicit STBs. Methods: Putative suicidality-related risk genes (PSRGs) were identified by comprehensive literature search and were characterized with respect to evolutionary conservation, participation in gene interaction networks and associated phenotypes. Evolutionary conservation was established with database searches and BLASTP queries, whereas gene-gene interactions were ascertained with GeneMANIA. We then examined whether mutations in risk-gene counterparts in C. elegans produced a diminished motivation phenotype previously connected to suicide risk factors. Results and conclusions: From the analysis, 105 risk-gene candidates were identified and found to be: 1) highly conserved during evolution, 2) enriched for essential genes, 3) involved in significant gene-gene interactions, and 4) associated with psychiatric disorders, metabolic disturbances and asthma/allergy. Evaluation of 17 mutant strains with loss-of-function/deletion mutations in PSRG orthologs revealed that 11 mutants showed significant evidence of diminished motivation that manifested as immobility in a foraging assay. Immobility was corrected in some or all of the mutants with clozapine, lithium and tricyclic antidepressant drugs. In addition, 5-HT2 receptor and muscarinic receptor antagonists restored goal-directed behavior in most or all of the mutants. These studies increase confidence in the validity of the PSRGs and provide initial clues about possible mechanisms that mediate STBs.

How Variation in Risk Allele Output and Gene Interactions Shape the Genetic Architecture of Schizophrenia.

Schizophrenia is a highly heritable polygenic psychiatric disorder. Characterization of its genetic architecture may lead to a better understanding of the overall burden of risk variants and how they determine susceptibility to disease. A major goal of this project is to develop a modeling approach to compare and quantify the relative effects of single nucleotide polymorphisms (SNPs), copy number variants (CNVs) and other factors. We derived a mathematical model for the various genetic contributions based on the probability of expressing a combination of risk variants at a frequency that matched disease prevalence. The model included estimated risk variant allele outputs (VAOs) adjusted for population allele frequency. We hypothesized that schizophrenia risk genes would be more interactive than random genes and we confirmed this relationship. Gene-gene interactions may cause network ripple effects that spread and amplify small individual effects of risk variants. The modeling revealed that the number of risk alleles required to achieve the threshold for susceptibility will be determined by the average functional locus output (FLO) associated with a risk allele, the risk allele frequency (RAF), the number of protective variants present and the extent of gene interactions within and between risk loci. The model can account for the quantitative impact of protective variants as well as CNVs on disease susceptibility. The fact that non-affected individuals must carry a non-trivial burden of risk alleles suggests that genetic susceptibility will inevitably reach the threshold for schizophrenia at a recurring frequency in the population.

Catalytic Reaction Model of Suicide.

Suicide is a devastating outcome of unresolved issues that affect mental health, general wellbeing and socioeconomic stress. The biology of suicidal behavior is still poorly understood, although progress has been made. Suicidal behavior runs in families and genetic studies have provided initial glimpses into potential genes that contribute to suicide risk. Here, we attempt to unify the biology and behavioral dimensions into a model that can guide research in this area. The proposed model envisions suicidal behavior as a catalytic reaction that may result in suicide depending on the conditions, analogously to enzyme catalysis of chemical reactions. A wide array of substrates or reactants, such as hopelessness, depression, debilitating illnesses and diminished motivation can mobilize suicidal thoughts and behaviors (STBs), which can then catalyze the final step/act of suicide. Here, we focus on three biological substrates in particular: threat assessment, motivation to engage in life and impulsivity. Genetic risk factors can affect each of these processes and tilt the balance toward suicidal behavior when existential crises (real or perceived) emerge such as loss of a loved one, sudden changes in social status or serious health issues. Although suicide is a uniquely human behavior, many of the fundamental biological processes are evolutionarily conserved. Insights from animal models may help to shape our understanding of suicidal behavior in man. By examining counterparts of the major biological processes in other organisms, new ideas about the role of genetic risk factors may emerge along with possible therapeutic interventions or preventive measures.

Protein Receptors Evolved from Homologous Cohesion Modules That Self-Associated and Are Encoded by Interactive Networked Genes.

Previously, it was proposed that protein receptors evolved from self-binding peptides that were encoded by self-interacting gene segments (inverted repeats) widely dispersed in the genome. In addition, self-association of the peptides was thought to be mediated by regions of amino acid sequence similarity. To extend these ideas, special features of receptors have been explored, such as their degree of homology to other proteins, and the arrangement of their genes for clues about their evolutionary origins and dynamics in the genome. As predicted, BLASTP searches for homologous proteins detected a greater number of unique hits for queries with receptor sequences than for sequences of randomly-selected, non-receptor proteins. This suggested that the building blocks (cohesion modules) for receptors were duplicated, dispersed, and maintained in the genome, due to structure/function relationships discussed here. Furthermore, the genes coding for a representative panel of receptors participated in a larger number of gene-gene interactions than for randomly-selected genes. This could conceivably reflect a greater evolutionary conservation of the receptor genes, with their more extensive integration into networks along with inherent properties of the genes themselves. In support of the latter possibility, some receptor genes were located in active areas of adaptive gene relocation/amalgamation to form functional blocks of related genes. It is suggested that adaptive relocation might allow for their joint regulation by common promoters and enhancers, and affect local chromatin structural domains to facilitate or repress gene expression. Speculation is included about the nature of the coordinated communication between receptors and the genes that encode them.

Analysis of Major Depression Risk Genes Reveals Evolutionary Conservation, Shared Phenotypes, and Extensive Genetic Interactions.

Major depressive disorder (MDD) affects around 15% of the population at some stage in their lifetime. It can be gravely disabling and it is associated with increased risk of suicide. Genetics play an important role; however, there are additional environmental contributions to the pathogenesis. A number of possible risk genes that increase liability for developing symptoms of MDD have been identified in genome-wide association studies (GWAS). The goal of this study was to characterize the MDD risk genes with respect to the degree of evolutionary conservation in simpler model organisms such as Caenorhabditis elegans and zebrafish, the phenotypes associated with variation in these genes and the extent of network connectivity. The MDD risk genes showed higher conservation in C. elegans and zebrafish than genome-to-genome comparisons. In addition, there were recurring themes among the phenotypes associated with variation of these risk genes in C. elegans. The phenotype analysis revealed enrichment for essential genes with pleiotropic effects. Moreover, the MDD risk genes participated in more interactions with each other than did randomly-selected genes from similar-sized gene sets. Syntenic blocks of risk genes with common functional activities were also identified. By characterizing evolutionarily-conserved counterparts to the MDD risk genes, we have gained new insights into pathogenetic processes relevant to the emergence of depressive symptoms in man.

Na+ leak-current channel (NALCN) at the junction of motor and neuropsychiatric symptoms in Parkinson's disease.

Parkinson's disease (PD) is a debilitating movement disorder often accompanied by neuropsychiatric symptoms that stem from the loss of dopaminergic function in the basal ganglia and altered neurotransmission more generally. Akinesia, postural instability, tremors and frozen gait constitute the major motor disturbances, whereas neuropsychiatric symptoms include altered circadian rhythms, disordered sleep, depression, psychosis and cognitive impairment. Evidence is emerging that the motor and neuropsychiatric symptoms may share etiologic factors. Calcium/ion channels (CACNA1C, NALCN), synaptic proteins (SYNJ1) and neuronal RNA-binding proteins (RBFOX1) are among the risk genes that are common to PD and various psychiatric disorders. The Na+ leak-current channel (NALCN) is the focus of this review because it has been implicated in dystonia, regulation of movement, cognitive impairment, sleep and circadian rhythms. It regulates the resting membrane potential in neurons, mediates pace-making activity, participates in synaptic vesicle recycling and is functionally co-localized to the endoplasmic reticulum (ER)-several of the major processes adversely affected in PD. Here, we summarize the literature on mechanisms and pathways that connect the motor and neuropsychiatric symptoms of PD with a focus on recurring relationships to the NALCN. It is hoped that the various connections outlined here will stimulate further discussion, suggest additional areas for exploration and ultimately inspire novel treatment strategies.

Clozapine, nimodipine and endosulfan differentially suppress behavioral defects caused by gain-of-function mutations in a two-pore domain K+ channel (UNC-58).

Two-pore domain K+ channels (K2Ps) regulate the resting membrane potential in excitable cells and determine ease of depolarization. Gain-of-function (gf) mutations in one of these channels (unc-58) in C. elegans switch it to a Na+ conductance channel and cause tremors, paralysis and other defects. We hypothesized that it should be possible to identify drugs that corrected these defects in unc-58(gf) mutant animals by blocking or modulating the over-active channels. We examined dispersal of animals on food because the absence of effective forward locomotion is the most obvious defect. In addition, we quantified egg release over 24 h. Starting with a known inhibitor of mammalian K2Ps and directed structure-based screening, we evaluated numerous drugs in these assays. Loratadine, which inhibits human KCNK18, significantly improved movement as did methiothepin. We confirmed that endosulfan, a GABA-A receptor antagonist, corrected locomotion in the unc-58(gf) strains. Based on structural similarities to other hits, we found that clozapine, loxapine and amoxapine potently suppressed abnormal phenotypes. Curiously, nimodipine, a Ca++-channel blocker, dramatically improved movement and egg laying in unc-58(e665), but not unc-58(n495) animals. Molecular modeling provided initial insights into a possible basis for this difference based on the location of the e665 and n495 mutations. This research may lead to identification of novel K2P modulators and potential leads for drug discovery.

Candidate risk genes for bipolar disorder are highly conserved during evolution and highly interconnected.

OBJECTIVES: Bipolar disorder (BPD) is a highly heritable psychiatric disorder whose genetic complexity and pathogenetic mechanisms are still being unraveled. The main goal of this work was to characterize BPD risk-gene candidates (identified by Nurnberger et al., JAMA Psychiatry 71:657, 2014, and Stahl et al., Nat. Genet. 51:793, 2019) with respect to their evolutionary conservation, associated phenotypes, and extent of gene-gene interactions. METHODS: Database searches and BLAST were used to identify homologous counterparts of human BPD risk genes in C. elegans, zebrafish, and Drosophila. Phenotypes associated with the C. elegans genes were annotated and searched. With GeneMANIA, we characterized and quantified gene-gene interactions among members of the BPD gene set in comparison to randomly chosen gene sets of the same size. RESULTS: BPD risk genes are highly conserved across species and are enriched for essential genes and genes associated with lethality and altered life span. They are significantly more interactive with each other in comparison to random genes. We identified syntenic blocks of risk genes, which provided potential insights into molecular pathways and co-morbidities associated with BPD including coronary disease, obesity, and decreased life expectancy. CONCLUSIONS: BPD risk genes appear to be special in terms of their degree of conservation, interconnectedness, and pleiotropic effects that extend beyond a role in brain function. Key hub genes or pleiotropic regulatory components may represent attractive targets for future drug discovery.

Genomic Chaos Begets Psychiatric Disorder.

The processes that created the primordial genome are inextricably linked to current day vulnerability to developing a psychiatric disorder as summarized in this review article. Chaos and dynamic forces including duplication, transposition, and recombination generated the protogenome. To survive early stages of genome evolution, self-organization emerged to curb chaos. Eventually, the human genome evolved through a delicate balance of chaos/instability and organization/stability. However, recombination coldspots, silencing of transposable elements, and other measures to limit chaos also led to retention of variants that increase risk for disease. Moreover, ongoing dynamics in the genome creates various new mutations that determine liability for psychiatric disorders. Homologous recombination, long-range gene regulation, and gene interactions were all guided by spooky action-at-a-distance, which increased variability in the system. A probabilistic system of life was required to deal with a changing environment. This ensured the generation of outliers in the population, which enhanced the probability that some members would survive unfavorable environmental impacts. Some of the outliers produced through this process in man are ill suited to cope with the complex demands of modern life. Genomic chaos and mental distress from the psychological challenges of modern living will inevitably converge to produce psychiatric disorders in man.

Novel pharmacological modulation of dystonic phenotypes caused by a gain-of-function mutation in the Na+ leak-current channel.

The Na leak-current channel (NALCN) regulates the resting membrane potential in excitable cells, thus determining the likelihood of depolarization in response to incoming signals. Gain-of-function (gf) mutations in this channel are associated with severe dystonic movement disorders in man. Currently, there are no known pharmacological antagonists or selective modulators of this important channel. A gain-of-function mutation in NALCN of C. elegans [known as unc-77(e625)] causes uncoordinated, hyperactive locomotion. We hypothesized that this hyperactive phenotype can be rescued with pharmacological modulators. Here, we summarize the results of targeted drug screening aimed at identification of drugs that corrected locomotion deficits in unc-77(e625) animals. To assay hyperactive locomotion, animals were acutely removed from food and characteristic foraging movements were quantified. Drug screening revealed that 2-aminoethoxydiphenyl borate (2-ABP), nifedipine, nimodipine, flunarizine and ethoxzolamide significantly decreased abnormal movements in unc-77(e625) animals. 2-APB also corrected egg release and coiling deficits in this strain. In addition, serotonin and dopamine both reduced hyperactive locomotion, consistent with regulatory interactions between these systems and the NALCN. 2-APB induced movement phenotypes in wild-type animals that faithfully mimicked those observed in NALCN knockout strains, which suggested that this drug may directly block the channel. Moreover, 2-APB and flunarizine showed significant structural similarities suggestive of overlap in their mode of action. Together, these studies have revealed new insights into regulation of NALCN function and led to the discovery of a potential pharmacological antagonist of the NALCN.

Surprising conservation of schizophrenia risk genes in lower organisms reflects their essential function and the evolution of genetic liability.

Schizophrenia is a devastating psychiatric illness that affects approximately 1% of the population. Genetic variation in multiple genes causes elevated risk for the disorder, but the molecular basis is inadequately understood and it is not clear how risk genes have evolved and persisted in the genome. To address these issues, we have identified orthologs/homologs of 344 schizophrenia risk genes (from the Psychiatric Genomics Consortium dataset) in lower organisms, including C. elegans, Drosophila and zebrafish, along with phenotypes produced by genetic disruption in C. elegans. Schizophrenia risk genes were evolutionarily conserved at significantly higher rates in C. elegans (81%) and zebrafish (88%) than genes in general for these two species (40-70%). The risk-gene equivalents were highly (~3-fold) enriched for essential genes consistent with polygenic mutation threshold models, which propose that genetic susceptibility results from the inevitable expression of harmful combinations of risk variants in the population. Most notably, numerous examples of cross-species synteny revealed how blocks of risk genes geared toward a shared biological purpose coalesced into proximity during evolution. We obtained initial evidence that schizophrenia risk genes affected different stages of development, potentially allowing differential modulation by the environment. Taken together, studies of the conservation of schizophrenia risk genes in simple model organisms provided novel insights into the molecular basis for genetic susceptibility to a complex human psychiatric disorder.

Crossing the Worm-Brain Barrier by Using Caenorhabditis elegans to Explore Fundamentals of Human Psychiatric Illness.

Endophenotypes and Research Domain Criteria (RDoC) represent recent efforts to deconvolute psychiatric illnesses into fundamental symptom clusters or biological markers more closely linked to genetic influences. By taking this one step farther, these biomarkers can be reduced to protophenotypes - endophenotypes conserved during evolution - with counterparts in lower organisms including Caenorhabditis elegans and Drosophila. Striking conservation in C. elegans of genes that increase the risk for mental illness bolsters the relevance of this model system for psychiatric research. Here, I review the characterization of several protophenotypes that are relevant for asociality, avolition/anhedonia, prepulse inhibition, and anorexia. Interestingly, the analogous behavioral defects in C. elegans are also corrected by psychotropic drugs used to treat the corresponding symptoms in man and/or are mediated by the same neurotransmitters. Overall, there is much we can learn about the complex human brain by studying simpler nervous systems directing evolutionarily conserved behaviors. The potential for generating important new insights from model organisms appears limitless when we begin to recognize the vestiges of evolution in ourselves.

Insulin Signaling Deficiency Produces Immobility in Caenorhabditis elegans That Models Diminished Motivation States in Man and Responds to Antidepressants.

Defects in insulin signaling have been reported in schizophrenia and major depressive disorder, which also share certain negative symptoms such as avolition, anhedonia, and apathy. These symptoms reflect diminished motivational states, which have been modeled in rodents as increased immobility in the forced swimming test. We have discovered that loss-of-function mutations in the insulin receptor (daf-2) and syntaxin (unc-64) genes in Caenorhabditis elegans, brief food deprivation, and exposure to DMSO produce immobility and avolition in non-dauer adults. The animals remain responsive to external stimuli; however, they fail to forage and will remain in place for >12 days or until they die. Their immobility can be prevented with drugs used to treat depression and schizophrenia and that reduce immobility in the forced swimming test. This includes amitriptyline, amoxapine, clozapine, and olanzapine, but not benzodiazepines and haloperidol. Recovery experiments confirm that immobility is induced and maintained by excessive signaling via serotonergic and muscarinic cholinergic pathways. The immobility response described here represents a potential protophenotype for avolition/anhedonia in man. This work may provide clues about why there is a significant increase in depression in patients with diabetes and suggest new therapeutic pathways for disorders featuring diminished motivation as a prominent symptom.

Akinesia and freezing caused by Na+ leak-current channel (NALCN) deficiency corrected by pharmacological inhibition of K+ channels and gap junctions.

The Na+ leak-current channel (NALCN) regulates locomotion, respiration, and intellectual development. Previous work highlighted striking similarities between characteristic movement phenotypes of NALCN-deficient animals (Drosophila and Caenorhabditis elegans) and the major symptoms of Parkinson's disease and primary progressive freezing gait. We have discovered novel physiological connections between the NALCN, K+ channels, and gap junctions that mediate regulation of locomotion in C. elegans. Drugs that block K+ channels and gap junctions or that activate Ca++ channels significantly improve movement of NALCN-deficient animals. Loss-of-function of the NALCN creates an imbalance in ions, including K+ and Ca++ , that interferes with normal cycles of depolarization-repolarization. This work suggests new therapeutic strategies for certain human movement disorders. J. Comp. Neurol. 525:1109-1121, 2017. © 2016 Wiley Periodicals, Inc.

Two adjacent phenylalanines in the NMDA receptor GluN2A subunit M3 domain interactively regulate alcohol sensitivity and ion channel gating.

The N-methyl-d-aspartate (NMDA) receptor is a key target of ethanol action in the central nervous system. Alcohol inhibition of NMDA receptor function involves small clusters of residues in the third and fourth membrane-associated (M) domains. Previous results from this laboratory have shown that two adjacent positions in the M3 domain, F636 and F637, can powerfully regulate alcohol sensitivity and ion channel gating. In this study, we report that these positions interact with one another in the regulation of both NMDA receptor gating and alcohol action. Using dual mutant cycle analysis, we detected interactions among various substitution mutants at these positions with respect to regulation of glutamate EC50, steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50. This interaction apparently involves a balancing of forces on the M3 helix, such that the disruption of function due to a substitution at one position can be reversed by a similar substitution at the other position. For example, tryptophan substitution at F636 or F637 increased or decreased channel mean open time, respectively, but tryptophan substitution at both positions did not alter open time. Interestingly, the effects of a number of mutations on receptor kinetics and ethanol sensitivity appeared to depend upon subtle structural differences, such as those between the isomeric amino acids leucine and isoleucine, as they could not be explained on the basis of sidechain molecular volume or hydrophilicity.

Different sites of alcohol action in the NMDA receptor GluN2A and GluN2B subunits.

The NMDA receptor is a major target of alcohol action in the CNS, and recent behavioral and cellular studies have pointed to the importance of the GluN2B subunit in alcohol action. We and others have previously characterized four amino acid positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN2A subunit that influence both ion channel gating and alcohol sensitivity. In this study, we found that substitution mutations at two of the four corresponding positions in the GluN2B subunit, F637 and G826, influence ethanol sensitivity and ion channel gating. Because position 826 contains a glycine residue in the native protein, we focused our attention on GluN2B(F637). Substitution mutations at GluN2B(F637) significantly altered ethanol IC50 values, glutamate EC50 values for peak (Ip) and steady-state (Iss) current, and steady-state to peak current ratios (Iss:Ip). Changes in apparent glutamate affinity were not due to agonist trapping in desensitized states, as glutamate Iss EC50 values were not correlated with Iss:Ip values. Ethanol sensitivity was correlated with values of both Ip and Iss glutamate EC50, but not with Iss:Ip. Values of ethanol IC50, glutamate EC50, and Iss:Ip for mutants at GluN2B(F637) were highly correlated with the corresponding values for mutants at GluN2A(F636), consistent with similar functional roles of this position in both subunits. These results demonstrate that GluN2B(Phe637) regulates ethanol action and ion channel function of NMDA receptors. However, despite highly conserved M domain sequences, ethanol's actions on GluN2A and GluN2B subunits differ.

Social feeding in Caenorhabditis elegans is modulated by antipsychotic drugs and calmodulin and may serve as a protophenotype for asociality.

Here, we define a protophenotype as an endophenotype that has been conserved during evolution. Social feeding in Caenorhabditis elegans may be an example of a protophenotype related to asociality in schizophrenia. It is regulated by the highly conserved neuropeptide Y receptor, NPR-1, and we speculated that social feeding should be affected by antipsychotic drugs. The social feeding strain, npr-1(g320), was exposed to antipsychotic drugs, dopamine or calmodulin antagonists on plates with bacterial lawns, and the number of aggregates on the plates was counted as a measure of social feeding. First-generation antipsychotics, chlorpromazine, trifluoperazine, fluphenazine, and haloperidol, and the second-generation drug, olanzapine, inhibited social feeding. Dopamine accelerated aggregation, whereas selective D2 dopamine receptor antagonists, sulpiride and raclopride, were inhibitory. Calmodulin antagonists effectively inhibited social feeding, as did RNAi knockdown of calmodulin (cmd-1) expression. In addition, gap junction inhibitors prevented aggregation, which is consistent with the hub-and-spoke arrangement of neurons that regulate social feeding via functional gap junctions. The studies described here revealed novel connections between dopaminergic signaling, the NPY receptor, calmodulin, and gap junctions in the regulation of social behavior in C. elegans. These pathways are evolutionarily-conserved, and have also been implicated in the pathogenesis of schizophrenia.

Drug elucidation: invertebrate genetics sheds new light on the molecular targets of CNS drugs.

Many important drugs approved to treat common human diseases were discovered by serendipity, without a firm understanding of their modes of action. As a result, the side effects and interactions of these medications are often unpredictable, and there is limited guidance for improving the design of next-generation drugs. Here, we review the innovative use of simple model organisms, especially Caenorhabditis elegans, to gain fresh insights into the complex biological effects of approved CNS medications. Whereas drug discovery involves the identification of new drug targets and lead compounds/biologics, and drug development spans preclinical testing to FDA approval, drug elucidation refers to the process of understanding the mechanisms of action of marketed drugs by studying their novel effects in model organisms. Drug elucidation studies have revealed new pathways affected by antipsychotic drugs, e.g., the insulin signaling pathway, a trace amine receptor and a nicotinic acetylcholine receptor. Similarly, novel targets of antidepressant drugs and lithium have been identified in C. elegans, including lipid-binding/transport proteins and the SGK-1 signaling pathway, respectively. Elucidation of the mode of action of anesthetic agents has shown that anesthesia can involve mitochondrial targets, leak currents, and gap junctions. The general approach reviewed in this article has advanced our knowledge about important drugs for CNS disorders and can guide future drug discovery efforts.

Insulin/IGF-1 signaling, including class II/III PI3Ks, ß-arrestin and SGK-1, is required in C. elegans to maintain pharyngeal muscle performance during starvation.

In C. elegans, pharyngeal pumping is regulated by the presence of bacteria. In response to food deprivation, the pumping rate rapidly declines by about 50-60%, but then recovers gradually to baseline levels on food after 24 hr. We used this system to study the role of insulin/IGF-1 signaling (IIS) in the recovery of pharyngeal pumping during starvation. Mutant strains with reduced function in the insulin/IGF-1 receptor, DAF-2, various insulins (INS-1 and INS-18), and molecules that regulate insulin release (UNC-64 and NCA-1; NCA-2) failed to recover normal pumping rates after food deprivation. Similarly, reduction or loss of function in downstream signaling molecules (e.g., ARR-1, AKT-1, and SGK-1) and effectors (e.g., CCA-1 and UNC-68) impaired pumping recovery. Pharmacological studies with kinase and metabolic inhibitors implicated class II/III phosphatidylinositol 3-kinases (PI3Ks) and glucose metabolism in the recovery response. Interestingly, both over- and under-activity in IIS was associated with poorer recovery kinetics. Taken together, the data suggest that optimum levels of IIS are required to maintain high levels of pharyngeal pumping during starvation. This work may ultimately provide insights into the connections between IIS, nutritional status and sarcopenia, a hallmark feature of aging in muscle.

Role of the conserved lysine within the Walker A motif of human DMC1.

During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1(K132R)) or alanine (hDMC1(K132A)). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1(K132R) and hDMC1(K132A) variants were devoid of ATP hydrolysis. The hDMC1(K132R) variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1(K132R) variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1(K132A) variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.