Activation of WNT and CREB signaling pathways in human neuronal cells in response to the Omega-3 fatty acid docosahexaenoic acid (DHA)

Highlights

• Human iPSC-derived neuronal cells were used for mechanistic studies of DHA's effect on neuroplasticity

• DHA activated WNT and CREB signaling pathways in human neural progenitor cells

• DHA enhanced the survival of iPSC-derived NPCs and differentiating post-mitotic neurons

• DHA increased neurite outgrowth monitored by real-time live-cell imaging

Abstract

A subset of individuals with major depressive disorder (MDD) elects treatment with complementary and alternative medicines (CAMs), including the omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Previous studies in rodents suggest that DHA modulates neurodevelopmental processes, including adult neurogenesis and neuroplasticity, but the molecular and cellular mechanisms of DHA's potential therapeutic effect in the context of human neurobiology have not been well established. Here we sought to address this knowledge gap by investigating the effects of DHA using human iPSC-derived neural progenitor cells (NPCs) and post-mitotic neurons using pathway-selective reporter genes, multiplexed mRNA expression profiling, and a panel of metabolism-based viability assays. Finally, real-time, live-cell imaging was employed to monitor neurite outgrowth upon DHA treatment. Overall, these studies showed that DHA treatment (0–50 μM) significantly upregulated both WNT and CREB signaling pathways in human neuronal cells in a dose-dependent manner with 2- to 3-fold increases in pathway activation. Additionally, we observed that DHA treatment enhanced survival of iPSC-derived NPCs and differentiation of post-mitotic neurons with live-cell imaging, revealing increased neurite outgrowth with DHA treatment within 24 h. Taken together, this study provides evidence that DHA treatment activates critical pathways regulating neuroplasticity, which may contribute to enhanced neuronal cell viability and neuronal connectivity. The extent to which these pathways represent molecular mechanisms underlying the potential beneficial effects of omega-3 fatty acids in MDD and other brain disorders merits further investigation.

Introduction

Major depressive disorder (MDD) is a common and debilitating mental disorder that affects 10–15% of the population worldwide (Al-Harbi, 2012). According to the DSM-V (Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition), MDD is marked by a period of at least two weeks of depressed mood, irritability, fatigue, significant weight change, feelings of worthlessness, and possibly suicidal thoughts. With >300 million people suffering from depression globally (WHO, 2018), the burden of depressive disorders has increasingly become a public health concern (Murray and Lopez, 2013; WHO, 2008). While selective serotonin reuptake inhibitors (SSRIs) are widely recognized as the first-line pharmacologic treatment of depression (NIMH, 2016), not all patients reach remission and some experience adverse side effects (Dunn et al., 1999; Ferguson, 2001; Kostev et al., 2014; Crawford et al., 2014; Uher et al., 2009; Warden et al., 2007).

In addition to possible alterations in serotonergic neurotransmission in depression, accumulating evidence for stress-related abnormalities in MDD patients has led to a ‘stress hypothesis’ for depression (Duman and Monteggia, 2006; Liu et al., 2017; Maes et al., 2011). The associations between depression, oxidative stress, and antioxidant status have been identified in post-mortem samples, together with elevated peripheral markers of oxidative damage in patients' blood samples (Shelton et al., 2011). Since neurons are especially vulnerable to stress, dysregulation of stress pathways results in decreased neurogenesis, synaptogenesis and dendritic spine formation, and increased apoptosis of neurons; the structural abnormalities further lead to deficits of neuronal functions (Holsboer and Barden, 1996; Holsboer, 2000; de Kloet et al., 2005). Indeed, multiple reports note decreased hippocampal volume among patients with depression, regardless of age of onset (Sheline, 2000; Videbech and Ravnkilde, 2004; Neumeister et al., 2005; Frodl et al., 2006; Hoptman et al., 2006; McKinnon et al., 2009; Sapolsky, 2000). These findings indicate the potential for a pharmacological intervention that specifically targets stress-neuroplasticity.

As an alternative or in addition to FDA-approved pharmacologic treatment, complementary and alternative medicines (CAMs), including the omega-3 fatty acids such as docosahexaenoic acid (DHA; 22:6(n-3)) and eicosapentaenoic acid (EPA; 20:5(n-3)), and S-adenosyl methionine (SAMe), a major methyl donor in the brain, have emerged as promising candidates for controlling depressive symptoms (Settle, 2007). Several clinical trials of DHA/EPA report their ability to alleviate MDD symptoms and increase remission rates (Su et al., 2003; Grosso et al., 2014; Smith et al., 2017; Jazayeri et al., 2008), though the effectiveness of DHA as compared to EPA may require more confirmative trials (Martins, 2009; Martins et al., 2012; Sublette et al., 2011; Lin and Su, 2007). In a combination therapy study, Gertsik et al. (2012) found citalopram plus EPA/DHA/other poly-unsaturated fatty acids (PUFAs) co-treatment significantly decreased the symptoms of MDD in comparison to monotherapy. A recent meta-analysis concluded that supplementation with omega-3 PUFAs provides benefits for reducing MDD symptoms, especially when taken as an adjunct treatment with antidepressants (Mocking et al., 2016), although effect sizes may be modest (Appleton et al., 2016). Outside of controlled clinical trials in MDD, omega-3 PUFAs remain popular as dietary supplements and have become of growing interest in the potential treatment of age-related cognitive decline, cardiovascular disease, and cancer (Ajith, 2018; Manson et al., 2019). In all of these contexts, the relative contribution of DHA versus EPA remains important to understand given growing recognition that different omega-3 PUFAs may function separately with varying potencies, as well as function synergistically. This issue has further relevance for efforts to develop omega-3 PUFA-based pharmaceutical products that contain highly purified preparations such as the ethyl ester derivative of EPA, icosapent ethyl, that is being explored for reduction of cardiovascular risk (Bhatt et al., 2019).

Of the omega-3 PUFAs, DHA is the most abundant in the brain and is essential for fetal brain development (Rajarethnem et al., 2017). Literature on DHA reports a diverse role for the compound, including antioxidant and anti-inflammatory properties (Kidd, 2007), as well as modulation of cell membrane fluidity (Tisza et al., 2016; Czysz and Rasenick, 2013). DHA further influences multiple brain development processes, such as neurotransmitter release, gene expression, myelination, neuroinflammation, and neuronal differentiation (Uauy and Dangour, 2006; Haubner et al., 2007; Orr and Bazinet, 2008). While the mechanisms of DHA action remain unclear, growing evidence indicates that it plays a role in regulating neurogenesis and cell survivability. DHA has been shown to increase neurite outgrowth and dendritic branching in rat embryonic hippocampal primary cultures (Calderon and Kim, 2004), as well as to promote axon outgrowth in rat cortical neurons (Cao et al., 2005; Mita et al., 2016). To date, there has been little effort to characterize the mechanism of action of DHA in human rather than rodent cells. Understanding its effects could not only inform us of the compound's potential benefits, but also facilitate in the identification of more targeted treatments.

In the present study, we employ human induced pluripotent stem cell (iPSC)-derived neuronal cells to investigate the impact of DHA at a molecular and cellular level. Our study provides potential insight into the molecular and cellular mechanisms of DHA's antidepressant effect, and provides evidence supporting the stress-neuroplasticity hypothesis of MDD. In particular, our studies demonstrate DHA's effect on the canonical WNT/β-catenin pathway, an essential pathway involved in cell fate, cell proliferation, and cell migration that has been implicated in diverse aspects of the pathophysiology of many neuropsychiatric disorders (Okerlund and Cheyette, 2011; Varela-Nallar and Inestrosa, 2013; Haggarty et al., 2014a; Haggarty et al., 2014b). Additionally, we demonstrate that DHA modulates CREB (cAMP response element-binding protein)-mediated transcription, which also plays an essential role in numerous neuronal biological processes, including cell survival, synaptic structure, and synaptic plasticity (Shaywitz and Greenberg, 1999). The crosstalk between these two signaling pathways has been reported (Grimes and Jope, 2001) but is not well understood in the context of human neuroplasticity. By demonstrating that DHA exposure regulates these pathways and by elucidating functional changes at the level of the transcriptome and neural development, we provide new insight into how DHA may affect the neural substrates critical for neuroplasticity.