In an effort to confirm and characterize possible suicide risk genes, we compiled a comprehensive list of implicated genes and determined that they are evolutionarily conserved, enriched for genes that are essential for life and are highly networked with each other. Additionally, we identified common themes among the phenotypes associated with the risk genes including psychiatric symptoms, metabolic outcomes and asthma/allergy. Moreover, these studies revealed that mutation of the suicide risk genes caused a relevant phenotype in C. elegans that was corrected by drugs shown clinically to reduce suicidal behavior. It is remarkable that so many of the genes examined were associated with immobility/diminished motivation in our model system – this did not have to be so. These findings are significant because they increase our confidence that these genes should qualify as bona fide suicide risk genes. Furthermore, they implicate certain signaling pathways (serotonin and insulin) and fundamental behaviors such as diminished goal-directed motivation in the emergence of STBs.
The immobility assay is a measure of drive or motivation to search for food in response to food deprivation. The absence of goal-directed behavior in animals fully capable of locomotion has been interpreted to reflect diminished motivation [41]. Diminished motivation to engage in life activities (known as anhedonia) is a hallmark feature of major depression and other psychiatric disorders and is also akin to the hopelessness or despair underlying suicidal ideation and behavior [57]. Elsewhere, we have discussed in detail how diminished motivation contributes to STBs [43].
Foraging or area restricted search has been characterized previously and is mediated neurochemically by serotonin [58], dopamine/glutamate [59], acetylcholine and insulin [41]. Cyproheptadine and methiothepin effectively corrected immobility in all of the mutants tested, whereas atropine was effective in 7 of 10 mutant strains. The pharmacology of the response in animals with mutations in suicide risk genes is consistent with previous studies [41] and implicates an excess of serotonin and acetylcholine in diminished motivation. The role of serotonin in depression has always been complicated. Early studies found that individuals with depression tended to show either very low or high levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in CSF that also predicted suicide attempts [60]. Furthermore, 5-HT2 receptor antagonists also treat depressive symptoms [61, 62]. This complicated biology of serotonin is consistent when we recognize its major role as a mediator of homeostasis [63]. Importantly, the adverse effects of elevated serotonin might explain the observation that SSRIs can increase STBs [64, 65].
Another striking feature of the data is the apparent interaction between drug effects and genetic mutations. Consequently, certain drugs did not correct immobility in all of the mutants despite the strains being essentially identical genetically outside of the primary mutant allele. In particular, lithium, atropine, imipramine and trazodone were effective at improving motivation in only some of the mutants tested. The diversity of responses by the mutants to the panel of drugs and the comparable effects of different serotonergic antagonists strongly argue against non-specific effects in our system. The variability in responsiveness could occur because some of the mutations affect pathways that the drug can effectively address, whereas other mutations affect different pathways that the drug cannot correct. Alternatively, a mutant may not respond to a drug because the mutation directly affects the receptor, pathway, etc. targeted by that drug. For example, the failure of lithium to correct immobility in the pdk-1 mutants may be due to the fact that lithium can act via a signaling pathway that requires the insulin receptor and pdk-1 [66]. Future studies will be needed to distinguish between these alternatives or to discover others.
The broad effects of psychotropic drugs may complicate interpretation of the findings, although this may ultimately allow greater understanding of their mechanism of action in our system and in patients treated with them. Clozapine and loxapine – two of the most effective drugs – inhibit an array of monoaminergic and cholinergic receptors in addition to promoting Akt activation [67, 68]. Older tricyclic antidepressants such as amitriptyline and amoxapine also block muscarinic cholinergic receptors, which is similar to atropine, an active drug in our assay. The anticholinergic drug scopolamine shows rapid antidepressant effects [69]. Similarly, when imipramine was first introduced, it treated anhedonia and suicidal tendencies within a few days [70]. At that time, it was used at higher doses that routinely produced anticholinergic effects such as dry mouth, etc. [70], which might explain the quicker onset of therapeutic benefit.
Taken together, analysis of the phenotypes and pathways implicated by the mutants suggests relevant functional connections to STBs. Multiple suicide risk genes were associated with diabetes (17) and asthma (15). Diabetes has been linked to increased apathy (diminished motivation [44]), depression [45, 71] and suicidal ideation and attempts [46, 72]. Likewise, there is a 2.5-4-fold increased risk of suicide attempts in patients with asthma [73, 74]. The present studies confirm that motivation to forage is regulated by insulin, serotonin and acetylcholine. Furthermore, these three signaling molecules are linked to asthma [75, 76], so the overlap is likely to be meaningful.
Several mutants clearly fit this story because they affect insulin signaling, dauer or life span in C. elegans, including pdk-1, tbx-2, sad-1, and hpk-1. Previously, we [36] identified among major depression risk genes a block of genes brought together via adaptive relocation that includes insulin-IGF2, tyrosine hydroxylase, D2 dopamine receptor-4 and brain-specific kinase 2 (BRSK2, a sad-1 counterpart). Interestingly, human BRSK2 and sad-1, and human HIPK4 and hpk-1 contain consensus phosphorylation sites for Akt (Supplementary Fig. 2), the major kinase of the insulin signaling pathway downstream of PDK-1. We speculate that these kinases are targets of regulation by Akt activated in response to insulin and other growth factors such as BDNF. Other suicide risk genes have been implicated in regulating synaptic responses and neuronal excitability including egl-10 (RGS6), cpx-1 (CPLX1), and fox-1 (RBFOX1) [77–80], which is consistent with the findings of Sokolowski et al. [26]. C. elegans risk-gene counterparts, unc-40 and ttx-3, both regulate neuronal development [81, 82], whereas unc-40 has also been reported to promote assembly of synaptic terminals [81]. Loss of additional synaptic components, namely AMPA receptors, mediates the delayed immobility phenotype displayed by glr-1 mutants, which might serve as a useful model for chronic stress/exhaustion [83]. Finally, Mealer et al. [84] linked GALNT10 (gly-10) to defects in glycosylation in schizophrenia – it may be needed to glycosylate key neurotransmitter receptors and/or ion channels to regulate their function. Thus, a picture begins to emerge of how the suicide risk genes act at the molecular level to potentially affect neuronal mediation of motivated behavior.
Additional findings revealed that suicide risk genes participated in more gene-gene interactions than randomly-selected genes. Suicide risk genes may be more interactive because they are evolutionarily-conserved, which means a longer residence time in the genome, with the potential for more extensive integration into networks [36]. This observation has previously been cited as providing additional validation for the authenticity of risk genes for bipolar disorder [37].
Limitations of this study include the lack of independent GWAS or other confirmation of many of the genetic variants as bona fide risk factors for suicide. Of course, the studies reported here were aimed at addressing this issue by providing functional validation of a selection of risk genes. Some of the differences in responsiveness to drugs between mutants could have emerged because not all of the strains have been backcrossed allowing for contributions by unknown mutations in other genes. However, consistent patterns observed when comparing mutant strains and their responses to drugs, together with the low odds of random mutations affecting motivation in multiple mutants, argue against this possibility as a major factor. Furthermore, diminished motivation, as measured in the immobility assay, could be interpreted in different ways than our view. Nevertheless, it is striking that so many of the selected risk genes caused a potentially relevant phenotype at a rate that appears to defy coincidence.
Future studies will need to evaluate additional suicide risk-gene candidates for their effects on motivation as well as other suitable phenotypes both in C. elegans and other model systems. The possibility that diminished motivation as measured in the immobility assay constitutes a protophenotype [40] for aspects of suicidal behavior in man merits serious consideration. Finally, the risk genes characterized here should be considered meaningful leads for learning more about the genetic and mechanistic contributions to suicide.