Individuals affected with PTSD (Post-Traumatic Stress Disorder) demonstrate changes in microRNA (miRNA) molecules associated with gene regulation. A controlled study, involving Dutch military personnel on deployment to a combat zone in Afghanistan, provided evidence for the role of blood-based miRNAs as candidate biomarkers for symptoms of PTSD. This finding may offer an approach towards screening for symptoms of PTSD, and it holds promise for understanding other trauma-related psychiatric disorders. Limitations of the study are that this was a small pilot study, and the findings need to be validated, extended, and confirmed. First results were presented at the 30th conference of the European College of Neuropsychopharmacology (ENCP) in Paris in early September.
PTSD is a psychiatric disorder which can manifest following exposure to a traumatic event, such as combat, assault, or natural disaster. Among individuals exposed to traumatic events, only a minority of individuals will develop PTSD, while others will show resiliency. Little is known of the mechanisms behind these different responses. The last few years have seen much attention given to whether the modification and expression of genes – epigenetic modifications – might be involved. But there are several practical and ethical challenges in designing a research study on humans undergoing such experiences, meaning that designing relevant study approaches is difficult.
A research group from the Netherlands worked with just over 1,000 Dutch soldiers and the Dutch Ministry of Defense to study changes in biology in relation to changes in presentations of symptoms of PTSD in soldiers who were deployed to a combat zone in Afghanistan. In a longitudinal study, they collected blood samples before deployment as well as 6 months after deployment. Most of the soldiers had been exposed to trauma, and some of the soldiers had developed symptoms of PTSD.
For this pilot study, from the initial group, 24 subjects were selected in 3 subgroups of 8. Eight of the soldiers had developed symptoms of PTSD; 8 had endorsed traumatic experiences but had not developed symptoms of PTSD; and another 8 had not been in serious traumatic circumstances and served as a control group. Using modern sequencing techniques, several types of miRNAs for which the blood levels differed between the groups were identified.
MiRNAs (Micro RiboNucleic Acids) are small molecules with chemical building blocks similar to DNA. Unlike the more famous DNA, miRNAs are typically very short – comprising only around 20 to 25 base units (the building blocks of nucleic acids), and they do not code, in other words they do not specify the production of a protein or peptide. However, they have very important roles in biology (every miRNA regulates the expression, and thereby also the activity of several other genes), and they are known to regulate the impact of environmental factors on biology. In addition, brain-derived miRNA can circulate throughout the human body and can be detected in the blood.
Differences in miRNA levels have been associated with certain diseases, such as some cancers, kidney disease, and even alcoholism. This regulatory role makes them also a candidate for investigation in PTSD.
“We discovered that these small molecules, called miRNAs, are present in different amounts in the blood of persons suffering from PTSD compared to trauma-exposed and control subjects without PTSD,” said first author Dr Laurence de Nijs of Maastricht University.
“We identified over 900 different types of these small molecules. 40 of them were regulated differently in people who developed PTSD, whereas there were differences in 27 of the miRNAs in trauma-exposed individuals who did not develop PTSD.”
“Interestingly, previous studies have found circulating miRNA levels to be not only correlated with different types of cancer, but also with certain psychiatric disorders including major depressive disorders. These preliminary results of our pilot study suggest that miRNAs might indeed be candidates as predictive blood markers (biomarkers) to distinguish between persons at high and low risk of developing PTSD. However, several steps need to be performed before such results can really have an impact on the larger field and in clinical practice. In addition to working towards biomarkers, the results may also provide novel information about the biological mechanisms underlying the development of PTSD.”
Dr de Nijs explained:
“Most of our stressful experiences don’t leave a long-lasting psychological scar. However, for some people who experience chronic severe stress or really terrible traumatic events, the stress does not go away. They are stuck with it, and the body’s stress response is stuck in ‘on’ mode. This can lead to the development of mental illness such as PTSD.
These individuals experience symptoms including re-experiencing of the traumatic event through flashbacks or recurrent nightmares, constant avoidance of reminders of the event, negative mood, and extreme arousal. This can manifest itself through insomnia and or hyper-alertness. Individuals with PTSD are six times more at risk of committing suicide and having marital problems, and the annual loss of productivity is estimated to be approximately $3 billion. Currently, there is no definite cure for patients with PTSD, and available treatments often are not effective.”
Commenting, Professor Josef Zohar (Ex-ECNP Chair, Tel Aviv, Israel) said:
“The relevance of a better understanding of stress-related events is unfortunately becoming clearer and clearer after each terror attack. This work points to an innovative avenue regarding the potential identification of risk factors for susceptibility to developing post-traumatic stress disorder.”
Funding: Dr de Nijs was awarded a Marie Curie fellowship grant by the European Union to perform this study, within a network of other expert scientists in PTSD and epigenetics. The Dutch cohort of soldiers (PRISMO) was funded through the Dutch Ministry of Defence.
Glioblastoma multiforme is the most common type of malignant brain tumor. These tumors actively divide and send invasive cells throughout the brain. This migration complicates patient treatment, because simply removing the tumor does not clear the brain of migrating cells, which can initiate new tumors elsewhere. This problem has nearly halted progress in prognosis or treatment of glioblastoma over the past 20 years, and has afflicted many of our most important political figures, including Ted Kennedy and John McCain. This astonishing lack of improvement in treatment has motivated Dr. Xandra Breakefield and her team to explore an alternative pathway for treatment. Dr. Breakefield discussed her work in a recent Wednesday afternoon lecture at NIH.
To start their investigation, Dr. Breakefield gathered a “fresh” sample of glioblastoma multiforme that came straight out of the operating room. The idea of studying a fresh sample was novel; previously, samples were a few hours old or from mouse models. Examining fresh samples in a living condition revealed that glioblastomas are extremely physically active, constantly extending protrusions from the surface of the tumor cells. These protrusions turn into extracellular vesicles (EVs) of many sizes, released at the rate of about 10,000 vesicles per cell per day. When profiled, these EVs were found to contain RNAs, enzymes, transcription factors, other proteins, and many other cellular components. The team hypothesized that the EVs released by the tumor must somehow promote tumor progression.
Using a fluorescent tag that labels both cell and vesicle membranes, they tracked the tumor EVs in the living brain of mice and found that many of them are taken up by surrounding healthy myeloid cells — microglia and macrophages. Macrophages are the primary form of defense in the central nervous system. Normally functioning microglia are sentinels, warriors, and nurturers. As sentinels, they actively scan the brain for damage, then rush in to nurture injured areas to repair the damage. In their warrior role they kill and eat invading cells. Ironically, a higher density of microglia and macrophages in a tumor results in a worse prognosis. They are attracted to but then subjugated by the glioblastoma, and are coerced into an abnormal function of supporting tumor growth. Understanding how glioblastoma sabotages these immune cells and co-opts them to support tumor growth is key to ultimately finding a cure for the disease.
Glioblastomas and their EVs typically have very high levels of miR-21, a microRNA affiliated with various cancer pathways. Dr. Breakefield and her team found that microglia grown near a glioblastoma and exposed to its EVs had higher levels of miR-21, higher proliferation rates, and lower expression of proteins from pathways involved in sensing and attacking invaders. They hypothesized that miR-21 transferred from the tumor to surrounding benign microglia by EVs plays a key role in recruiting and transforming them. Then they asked whether microglia that have taken up many tumor vesicles have a different phenotype than microglia that haven’t taken up as many. To test this question, the Breakefield lab implanted fluorescently (GFP) labelled gliomas into the brains of mice and developed a flow cytometry method to sort cells extracted from that environment based on their level of GFP. The more tumor vesicles the surrounding normal cells have taken up, the more GFP they should have. The researchers used single-cell RNA sequencing to compare the expression profile of mRNAs in microglia and macrophages with high vs. low levels of GFP, indicating uptake of tumor vesicles. They found significant differences in mRNA expression in brain microglia but not in macrophages.
The genetic pathways responsible for microglia’s sentinel role have been called the “microglial sensome” (Hickman et al., 2013). The mRNA for most sensome genes were down-regulated in microglia that had taken up more tumor EVs – thus, the tumor EVs “blinded” the microglia. Pathways for immune suppression were up-regulated – showing that uptake of the tumor EVs compromised the microglias’ warrior function. Finally, pathways for tissue repair were up-regulated – indicating that the tumor EVs co-opted the microglias’ nurturing function to support the tumor instead of normal brain cells.
EVs are a promising biomarker for earlier diagnosis of disease. They are released by all cells and found in all biofluids. They can contain a wide variety of different information, including cell specific proteins and RNAs.
Dr. Breakefield was interested in finding biomarkers for glioblastoma that could be found in the bloodstream or other accessible biofluids. She targeted EGFRvIII, a deletion in the EGFR gene quite common in glioblastomas, and IDH1/2, a single point mutation that results in lower grade tumors with better prognosis. Her team is examining cerebrospinal fluid (CSF) and serum from patients to identify the presence or absence of EGFRvIII with promising success. Examining exRNA and free circulating DNA in plasma, her team was able to differentiate IDH1/2 mutant carriers from healthy volunteers. That work is being developed into a clinical tool for earlier diagnosis of low-grade gliomas which have a better prognosis and different treatment options than non-IDH1/2 gliomas.
Therapy EVs are proven, effective therapies for a variety of diseases. They can be obtained from many cell types and protect their fragile molecular cargo when administered into the body. They can also be efficiently taken up by specific target cells in vivo. EVs have been used therapeutically in immune modulation, tissue repair, and antigen presentation for vaccination.
After working on glioblastomas for an extended period, Dr. Breakefield decided to study schwannomas, a related but benign class of tumor. There are three types of hereditary diseases that stem from schwannomas: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), and schwannomatosis. These diseases cause motor dysfunction, pain, and potential hearing loss.
In NF2, tumors form along nerve fibers, including those at the base of the spine. The current treatment is surgical removal of the tumor, but that can cause irreparable nerve damage. Because these tumors are not malignant, reducing their size could be an effective alternative treatment.
Dr. Breakefield and colleagues made a model system using human Schwann cells from an NF2 patient. They implanted those cells in the sciatic nerve of mice, forming tumors. They then created an adenovirus-associated virus (AAV) vector containing the pro-inflammatory enzyme caspase 1 attached to a promoter (P0) exclusively active in Schwann cells. They then injected this vector into the tumors to try to shrink them.
In this model system, the tumor grew when the AAV-GFP was injected. It regressed when AAV-P0-ICE was injected. P0-ICE creates a “bystander” effect, in which cells surrounding the injected cell are also killed. Surprisingly, Schwann cells appeared completely normal after treatment with P0-ICE, while the tumor (schwannoma) shrunk after injection of the tumor with AAV-P0-ICE. Although the EVs’ role in this finding is not known, Dr. Breakefield theorizes that caspase-1 is incorporated into the EVs and transferred to surrounding schwannoma cells.
There is still much to be discovered about EVs’ roles in tumors, both benign and malignant. We have not yet found glioblastoma’s Achilles’ heel, but further research may yet do so. Dr. Breakefield looks forward to continuing her work and potentially creating better treatment options for those afflicted by these devastating tumors. The previously unrecognized role of the tumor-derived EVs in transferring genetic information and co-opting other cells in their microenviroment may be an important key.
Early detection of viral infections is extremely important for control of disease transmission, prompt initiation of treatment, and prevention of infection-related complications. Because of our hypothesis that viral DNA, messenger RNA, and proteins cannot be detected in all infected individuals, we wanted to determine whether detection of exogenous miRNAs encoded by viruses represents a more sensitive assay of the true prevalence of infection by viruses including latent Kaposi Sarcoma Herpes Virus (KSHV) and Epstein Barr Virus (EBV). Therefore, we measured plasma miRNAs using RT-qPCR and compared it to the current standard method for detection of viral infection, an enzyme-linked immunosorbent assay (ELISA) of blood plasma, which detects antibodies generated by the host against the infecting virus. Our study population was 214 Caucasian patients from the United States and Romania, separated into four independent patient cohorts. We examined a total of 300 plasma samples from this population. This study enabled us to develop an approach to detect infection by KSHV using multiplexed RT-qPCR of multiple viral miRNAs.
We found that our method had clear advantages over the current ELISA-based approach. It detected a significantly higher prevalence of KSHV infection than that determined by seropositivity, with the difference most pronounced in immuno-depressed patients. When applied to EBV, our new method based on plasma viral miRNA quantification proved that EBV infection is ubiquitous. This strategy has the potential to become a gold standard method in clinical practice to detect latency of viruses and viremia — viral infection of the bloodstream — in both general and immune-compromised populations.
Fuentes-Mattei E, Giza DE, Shimizu M, Ivan C, Manning JT, Tudor S, Ciccone M, Kargin OA, Zhang X, Mur P, do Amaral NS, Chen M, Tarrand JJ, Lupu F, Ferrajoli A, Keating MJ, Vasilescu C, Yeung SJ, Calin GA. Plasma viral miRNAs indicate a high prevalence of occult viral infections. EBioMedicine. (2017) 20:182-192. doi: 10.1016/j.ebiom.2017.04.018. Pubmed: 28465156.