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

Abstract

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.

Keywords: C. elegans; antidepressants; clozapine; diminished motivation; lithium; suicide.

Figure 1

Characterization of suicide risk genes. (A) We compared the frequency of finding counterparts of human genomic genes in the C. elegans genome (gray bar) with the frequency of finding counterparts of the PSRGs in C. elegans (black bar). When analyzed separately, 87.3% of genes derived from GWAS were conserved in C. elegans, whereas that number was essentially the same – 90% – from candidate gene analysis. Significant differences (**p < 0.01) between these comparisons have been indicated by asterisks. (B) The phenotypes associated with PSRG counterparts in C. elegans were tabulated (black bars) and compared with the frequency of phenotypes observed in genome-wide analyses of gene function (gray bars). For the chi-square tests, the Genome control data from whole or partial genome analyses were: 4645/19727 (23.5%) essential genes, 264/2445 (10.8%) lethal genes, 1876/18496 (10.1%) genes that affect lifespan and 185/2072 (8.9%) genes with aldicarb sensitivity. Comparable genomic analysis is not available for Locomotion or Development phenotypes, so only the raw data are shown. Phenotypes are labelled and asterisks indicate p values < 0.05* and < 0.01**.

Figure 2

Evaluation of mutant animals in the immobility assay. (A) Wild-type N2 animals were assessed for movement (foraging) after removal from food on plates suffused with control (CON) buffer or with the same buffer containing a final concentration of 1% DMSO and were compared with various mutants (labelled on the x-axis). The mgl-2;mgl-1 notation refers to a double mutant. Each bar in the figure represents at least 3 separate replicates of the experiment and the error bars depict the standard deviations (A-C). Additional mutants were compared with N2 animals on plates suffused with DMSO or (B) clozapine (CLOZ) or (C) lithium at final concentrations of 160 μM or 6.7 mM, respectively. Significant differences from the N2 group are indicated by hash marks, ^##p < 0.01. Ten of the 16 mutants tested showed immobility (diminished motivation) in the assay. For comparison, only 2 of 25 mutants displayed this phenotype in a separate study of insulin-related effects on recovery of pharyngeal pumping (58). A chi-square test revealed that the difference in the occurrence of the phenotype between the two studies was highly significant, p < 0.0002. For the mutants that showed immobility (B, C), the level of movement on DMSO (baseline) was compared with movement on drug. Significant improvement of movement with a drug is indicated by asterisks: *p < 0.05, **p < 0.01.

Figure 3

Comparison of responses to antidepressant drugs. The data are presented in the same way as in Figure 2 . However, statistical comparisons between the mutants and the N2 group for DMSO conditions have not been indicated with hash marks. (A) Amitriptyline (AMI) was evaluated in the top panel, amoxapine (AMOX) in the middle (B) and trazodone (TRAZ) in panel (C). These drugs were assayed at the same 160 μM concentration. All of the psychotropic drugs (antidepressants, antipsychotics, etc.) used in these studies have been tested with wild-type and other strains and none produce detectable changes in locomotion by themselves. Significant differences between DMSO and drug groups have been indicated with asterisks as before.

Figure 4

Evaluation of (A) imipramine (IMP) and (B) loxapine (LOX) for their ability to restore foraging (motivated) behavior. Both drugs were tested at 160 μM and significant differences have been indicated as before.

Figure 5

Assessment of serotonergic and muscarinic cholinergic antagonists for their ability to restore normal foraging behavior. Mutants were evaluated as described previously in the immobility assay in the presence of (A) cyproheptadine (CYPRO), (B) methiothepin (METH) or (C) atropine (ATRO). CYPRO and METH were tested at 160 μM, whereas atropine was used at a final concentration of 2 mM. Significant differences between DMSO and drug groups have been indicated with asterisks as before.