Post-Traumatic Stress Disorder and RNA Interference

A micro-review of the role of RNA interference in PTSD.

Post-traumatic stress disorder (PTSD) is a disorder characterized by persistent changes in stress reactivity induced by a traumatic event. Approximately 30-40% of the risk of PTSD is heritable (Ressler et al. 2022). Those with a PTSD diagnosis experience hypervigilance, exaggerated fight-or-flight responses, intrusive memories, and sleep disturbances that persist for at least 30 days following a traumatic experience. There is no cure for PTSD, and the most effective treatments primarily involve counseling; medications are not very effective. The biological foundations of PTSD are unclear; however, canonical correlates involve dysregulations of stress-responsive systems. 

These dysregulations include augmented cortisol dynamics, where the data are mixed, but it appears those with PTSD show lower basal cortisol levels with a protracted time course of cortisol clearance once it’s elevated—they show more significant delta in elevations of cortisol from baseline. PTSD patients exhibit consistently higher corticotropin-releasing factor levels in cerebrospinal fluid (CSF) and blood, and in neuroimaging, have hyperactive amygdalae as well as reduced hippocampal and medial prefrontal cortex size and activity.

Outside of these more commonly understood differences, a body of literature exists that suggests there is a suite of immunological changes that appear to be related to PTSD (e.g., a review from Sun, Qu, & Zhu, 2021). These findings mesh with data suggesting that inflammation is causally related to anxiety-related “sickness behaviors” (e.g., Muscatell et al. 2016).

Before we go further, we need to talk about the “central dogma” of genetics. This dogma maintains that “DNA makes RNA, and RNA makes protein,” in a unidirectional process. However, of course, this is not the case. The central dogma is upended here by a process called RNA interference, which is a process by which nucleic acid translation into protein is interfered with by other nucleic acids.

MicroRNAs (miRNAs) are one such class of nucleic acid involved RNA interference. These are short RNA sequences that allow for dynamic control of messenger RNA’s translation into protein through specific, competitive binding. Physically blocking the cellular machinery needed for translation, these miRNAs allow for what amounts to fine-tuning of the expression of mRNA into protein. This little detail will be relevant in just a moment: miRNAs are expressed as longer, double-stranded RNAs that are then cleaved into smaller, 21-25 nucleotide sections, by an enzyme called Dicer. This then allows them to go and bind their complementary domains on their target mRNAs.

This inflammatory difference in PTSD patients is underemphasized in comparison to the more ‘canonical’ changes associated with PTSD diagnoses, despite high comorbidity of PTSD with disorders of immunity and inflammation. One hypothesis for explaining this phenomenon is that the higher level of inflammatory cytokines is due to an insufficient regulation by cortisol, which is typically an anti-inflammatory hormone, though this must be an incomplete explanation because data regarding cortisol levels are inconsistent (Gill et al., 2009). 

An initial study shining further light on this understudied underside of PTSD was published in 2014. The authors analyzed the blood of combat veterans with PTSD diagnoses and found a significant elevation in some inflammatory cytokines and T-helper cells (CD4+ cells) among peripheral blood mononuclear cells (PBMCs, inclusive of T cells, B cells, and monocytes); the authors followed up on this change by assessing, using high-throughput microRNA (miRNA) microarray hybridization analyses (Zhou et al., 2014), fing 7 miRNAs upregulated and 64 downregulated 2.5-fold or more. Zhou and colleagues linked the downregulation of miR-125a for the observed increase in interferon gamma (IFNG). This group then followed up on these findings and showed that PTSD patients had significant differential expression of 326 genes and 190 miRNAs compared to healthy controls. The primary functional consequence of alterations in miRNA was increased expression of pro-inflammatory cytokines due to reduced expression of miRNAs that regulate their expression, e.g., IF-γ and Interleukin-12B (IL12B), hsa-miR-125a & hsa-miR-193a-5p, respectively, replicating their original finding and expanding out their list of miRNA targets (Bam et al., 2016). 

A subsequent paper found that blood DICER1 levels, an enzyme involved in the processing of miRNAs into their mature forms, were reduced in patients with comorbid PTSD & depression diagnoses (Wingo et al. 2015). (Though the assays were not conducted on PTSD-exclusive patients, there is significant comorbidity between the two disorders.) The functional consequences of reduced blood DICER1 levels was enhanced amygdalar activation, heightened symptom severity, and, as assessed using gene set enrichment analysis (GSEA), elevations of miRNAs related to cytokine signaling and the innate immune system, as well as “genes upregulated in polymorphonuclear leukocytes after Francisella tularensis vaccination.”

However, it is not the case that the entirety of the miRNA-level changes in PTSD patients are directly related to immune function. An examination of the blood miRNA profiles of combat veterans with a PTSD diagnosis revealed 8 miRNAs significantly differentially expressed compared to healthy combat-veteran controls (Martin et al. 2017). Using target prediction and analysis via miRWalk2.0 database, many of the genes these miRNAs targeted were classified as transcription factors. Others were identified as being involved in “axon guidance,” “Wnt signaling pathways,” “adherens junctions,” “MAPK signaling,” “Prostate cancer,” “endocytosis,” and “long-term potentiation” (the last of which is a neuronal phenomenon involved in the formation of memories), and while some of these targets may be related to the integrity of the blood brain barrier, which might be inflammation-associated, others are evidently not constrained to inflammatory processes. 

The reach of this new area of research has not been constrained to the territory of merely correlating blood miRNA levels with the incidence or symptoms of PTSD. A more recent paper, published in 2019, employed a translational rodent model of PTSD wherein the overexpression of a target miRNA identified via sequencing, mir-135b-5p, in the basolateral amygdala was associated with an enhancement of PTSD-like behaviors. The basolateral amygdala is a region of the brain thought to be central to the acquisition and expression of conditioned fear responses, and is, as mentioned previously, a region that is more active in PTSD patients (e.g., Fanselow & LeDoux, 1999). Conversely, the inhibition of mir-135b-5p in animals deemed “stress susceptible” promoted a resilient phenotype, reducing the expression of fear learning in the animals. Centrally, mir-135b-5p is expressed in human amygdalae and its passenger strand, mir-135b-3p is elevated in the blood of PTSD patients. Similar research has been conducted more recently examining other known targets in PTSD risk, such as FKBP5—a recruiter of a co-chaperone protein involved in glucocorticoid-receptor mediated transcriptional responses to stress (Kang et al., 2020).

All said, the role of miRNAs in psychiatric disorders is promising, just beginning, and fascinating.