Interaction networks of well-selected molecular targets of lithium and valproate, revealed some striking similarities and clear differences in the biological processes perturbed by these mood stabilizers. Valproate but not lithium networks were highly enriched for nodes associated with the nuclear lumen functional cluster, cell cycle, nucleotide excision repair and DNA replication, representing a major distinction in the cellular effects of these medications. Clearly, the wide array of edges formed by nuclear proteins (including the cyclins, cell division proteins, excision repair proteins, HDACs and transcription factors) suggests that valproate triggers an extensive perturbation of functions of the nucleus. It is not apparent however whether these protein clusters and pathways are the main determinants of valproate’s effect on mania.
We explored the coincident mechanisms involved in lithium and valproate actions to illuminate a molecular repertoire that might be paramount in stabilizing mood. Remarkably, the lithium-valproate intersecting nodes largely reproduced the enriched functional clusters and canonical pathways associated with lithium. The striking enrichment of apoptosis functional clusters, neurotrophin signaling and a range of signaling pathways points to potential core mechanisms involved in the therapeutic effect of these drugs. The interaction networks thus revealed a shared functional signature for lithium and valproate.
Whether lithium or valproate acts to restore the normal kinetics of programmed cell death in brains of bipolar disorder patients is not known. Nevertheless, these medications seem to provoke a yin-yang effect on apoptosis. Molecules differentially regulated by both drugs including AKT1, BCL2 and BDNF and many recruited nodes are known to exert anti-apoptotic properties (Supplementary Figure 1
). In contrast, various pro-apoptotic nodes, including caspases and cyclin-dependent kinase inhibitors are also well represented in the networks (Supplementary Figure 1
). Conceivably, lithium and valproate help fine-tune the apoptotic switch, but the precise timing of this switch in specific cells during the course of treatment remains to be determined.
Neurotrophin signaling was the most enriched pathway identified by identical nodes in lithium and valproate networks. The neurotrophin-signaling cascade transduces signals from BDNF and other neurotrophic factors leading to neuronal cell growth, proliferation and survival. Neurotrophin signaling interacts with downstream pathways including MAPK, which in turn, maintains crosstalk with other significantly enriched pathways, including Wnt, apoptosis, ErbB, and insulin (KEGG Pathways). Stimulation MAPK pathway in rat hippocampus and frontal cortex by mood stabilizers has been reported.31
Regulation of neurotrophin signaling, its interactions with other pathways, and regulation of apoptosis may be the basis for the observed neurotrophic and neuroprotective effects of lithium and valproate.
It is noteworthy that some of the most prominent mood stabilizer biological pathway responses we found in this study are consistent with the major pathway categories recruited by differentially expressed genes in a transcriptome analysis of pairs of bipolar I disorder and control postmortem dorsolateral prefrontal cortex (BA46), that included cellular growth and proliferation, nervous system development and function, and cell death.75
Uncovering key molecules that mediate pathway interactions helps simplify an otherwise overwhelmingly complex molecular response to drug treatment. To isolate these factors we ascertained nodes that recurred frequently across diverse enriched pathways. AKT1/2 and MAPK1/3 were the most preponderant, exposing potential pleiotropy and a major role in coordinating cellular response following lithium and valproate treatment. However the spatial and temporal regulation of the balance between repression and activation of diverse pathways has yet to be established.
AKT1 phosphorylates multiple substrates involved in various cellular processes including regulation of neurogenesis, neuroprotection, cell growth, cell proliferation, cell survival and anti-apoptotic processes.76–78
AKT1 is a vital component of the PI3K growth pathway and its inhibitory effect on GSK3B leads to the activation of Wnt signaling.
Animal models for AKT1, MAPK1 (ERK2) and MAPK3 (ERK1) have exhibited characteristics relevant to brain function. Akt1
knockout mice treated with dopaminergic, adrenergic and cholinergic agents displayed altered working memory.79
Homozygous deletion of MAPK1 is embryonic lethal in mice but mice in which MAPK1 expression was knocked down to ≤ 40% exhibited long-term memory deficits.80
MAPK3 null mice displayed behavioral changes such as hyperactivity, region-specific altered synaptic function and increased brain expression of MAPK1.81
It would be interesting to determine whether these animal models recover wild-type behavior and characteristics following either lithium or valproate treatment.
The lingering question of whether molecular targets for mood stabilizers hold any relevance to the genetic etiology of psychiatric disorders remains to be addressed comprehensively.82–86
For lithium, the newly formed international Consortium on Lithium Genetics (ConLiGen) is assembling and conducting genome-wide association analysis on the largest sample scored for lithium response.87
This study would allow us to determine whether variation in node constituents of enriched pathways and functional clusters is associated with lithium response. Could any combination of node variation help predict response and/or clinical outcome? If so, could nodes in the implicated pathway (s) be used as targets to develop new, effective and faster-acting mood stabilizers with lower toxicity?
Association with gene variants disclosed in small sample sizes must be assessed in the context of recent genome-wide association studies in large schizophrenia and bipolar disorder samples that detected significantly associated genetic variations.88–90
The proposed association of AKT1
variants with schizophrenia in a relatively small sample has not been consistently supported.91
A much weaker association with AKT1
variants has been reported in bipolar disorder and selected phenotypes.92,93
Recently, AKT1 activation has been shown to provoke defects in neuronal development that parallels the effect of DISC1 suppression in newborn neurons.94
In turn, GSK3B has been shown to interact with DISC1, suggesting a possible mechanism by which DISC1 promotes proliferation of adult neural progenitor cells.95
Compelling support for DISC1 as a schizophrenia risk gene has accumulated96
since it was found to be disrupted in an extended Scottish pedigree affected with psychiatric disorders including schizophrenia and bipolar disorder.97–99
ErbB signaling, another highly represented pathway in this study, mediates the action of neuregulin1 (NRG1
), a proposed schizophrenia gene.100
Also, modest support for association of NRG1
variants with psychotic bipolar disorder has been presented.101
Interestingly, AKT1 and GSK3B are downstream effectors of the ErbB signaling cascade that maintains crosstalk with cell cycle progression, MAPK, mTOR and calcium signaling pathways (KEGG pathways).
As we have indicated we assembled genes that show drug-induced differential expression in diverse studies. Clearly, changes in gene expression would have been influenced by dissimilar experimental designs and conditions across studies performed on diverse organisms and cell lines from various origins. Despite these confounding factors, a limited number of differentially expressed genes survived our selection criteria, which thus provide some validity for the inclusion of those genes in the network seeds.
Finally, we need to point out that the extensive network interactions presented here could include false positives. False negatives are also possible because of the paucity of published studies on some differentially regulated molecules, e.g. IMPA1, the lack of functional data on many of the network nodes, and the lack of supporting experimental data for potential drug targets thus were not included in the network seeds. Understudied nodes would fail to establish substantial connections within the networks and/or would not be included in established canonical pathways. Conversely, the comparatively extensive literature on certain genes/proteins may bias the representation of their interactions within the network. Therefore these expansive, multi-directional matrices may actually represent a constrained view of lithium and valproate actions in the cell. Future advances in functional genomics coupled with improvements in network building tools allowing more precise modeling of reactions and interactions within the cell would permit creation of more robust networks.
In summary, biological interaction networks generated in this study offer a glimpse of the molecular repertoire that underlies the global cellular effect of lithium and valproate. Consistent with their divergent chemical structures, valproate but not lithium induced a highly enhanced recruitment of nuclear lumen functional clusters and nodes enriched for the cell cycle, nucleotide excision repair and DNA replication pathways. Conversely, lithium and valproate intersecting network nodes perturbed convergent cellular functions embodied by a striking enrichment of the regulation of apoptosis clusters, neurotrophin signaling and a series of diverse signaling pathways. This shared effect hints at a unique functional footprint for lithium and valproate that may be salient to their effectiveness as mood stabilizers. The emergence of recurrent pathway constituents implies functional pleiotropy, an idea that may harmonize the complexity of intracellular responses to lithium and valproate. AKT and MAPK seem to be central to the execution of these responses, but a more complete picture will require additional experimental work.