Exosomal microRNAs as a Biomarker in Panic Disorder and in Response to CBT

Searching for Exosomal microRNAs and Cellular Biomarkers and Mechanisms Underlying the Differences Between Panic Disorder Patients Who Are Responders and Non-responders to Cognitive Behavior Therapy

Cognitive behavior therapy (CBT) has long been known as an effective treatment for anxiety disorders, either when given by a therapist or when self-administered through a computer program. However, the biological effect of CBT remained largely unexplored. Most studies focused on genomic differences and pursued differences in methylation patterns following CBT, but the findings were very limited in scope, especially when comparing responders and non-responders to CBT. In the currently proposed study, the investigators plan to go one step further and look for changes in exosomal microRNAs (miRs) from serum samples taken before and after CBT from Panic Disorder (PD) patients. Notably, miR changes show a much faster biological response than methylation patterns yet had never been used before in PD research. The primary benefit of this work will be in providing biological validation to psychological treatments. PD is a heavy public health burden, associated with significant market potential for both therapeutic and diagnostic uses.

Cognitive Behavior Therapy (CBT) is an evidence-based treatment, commonly used for treating anxiety disorders. Patients with anxiety disorders are characterized by having cognitive distortions regarding evaluations of threat, which results in safety behaviors to avoid them thereby causing impairment in the patient's quality of life. CBT focuses on changing those "false beliefs" and "maladaptive behaviors" by exposing the patients to their anxiety source in a gradual way, supervised by the therapist. Anxiety disorders are relatively common, and many people have difficulty accessing treatment due to a variety of obstacles. Researchers have therefore explored the possibility of using different methods to administer CBT, resulting in the making of internet-delivered CBT (iCBT). iCBT programs can involve therapist guidance through emails or can be entirely unguided. These programs are typically comprised of 6-15 modules, which are text chapters corresponding to sessions in face-to-face therapy. These programs require little therapist involvement other than guidance and feedback on homework assignments. Current meta-analyses suggest that iCBT does not differ from regular CBT in its efficacy, conducted through a guided program. In this study, the investigators plan to examine whether iCBT and regular CBT can cause biological changes as well as cognitive ones. Few previous studies have shown epigenetic changes in different directions for CBT responders and non-responders. Those studies used methylation patterns as biomarkers, showing that Panic Disorder (PD) patients had lower methylation in specific genes than the control group at the baseline point. After going through CBT the responders showed higher methylation and the non-responders showed even lower methylation than at the baseline point. In addition, a more recent study showed that in comparison to healthy controls, PD patients showed changes in immune system activities. Microglial acid sensing G-protein coupled receptor and T cell death-associated gene-8 (TDAG8), which was found was found higher in PD patients. The investigators wish to take these findings one step forward by using micro-RNAs (miRs) content of circulation exosomes as our biomarker. Exosomes are a type of extracellular vesicles of endocytic origin, used in signaling and cell to cell communication, by transferring proteins, lipids, and variety of RNAs between cells. miRs are short single-stranded RNA molecules that bind to complementary sequences of target mRNAs, causing inhibition of their translation and/or inducing target degradation and affecting brain functioning and mental processes. The investigators will base their work on the hypothesis that the brain's state is reflected, to a certain extent in the miR contents of circulating exosomes. Therefore, the aim of the study will be to test the difference between patients and controls as well as between responders and non-responders by profiling miRs content of circulation exosomes from their plasma samples and seeking association with their clinical data. Whole blood samples will also be collected, in order to serve as secondary outcome measurement, checking for changes in expression of other small non-coding circulating RNAs as well. The investigators will use a cohort of 40 panic disorder patients, comparing them with matching 40 mentally healthy controls. This approach has never been used before in the field of anxiety biomarkers, although miR changes show a much faster biological response than methylation patterns. Therefore, the investigators believe it will provide many new insights on the biological changes following PD and its treatment using CBT/iCBT, especially regarding the biological difference between respondents and non-respondents. The investigators anticipate big benefits for psychologists and psychiatrists who could in the future use simple blood samples to determine which kind of therapy will be most suitable for their patients, or rather to check if the therapy has given the desirable outcomes. Biotech firms may be interested in developing this method as a commercial diagnostic tool that could easily be used by health maintenance organizations just as a simple blood test. PD is a big public health burden, leading the market potential both for therapeutics as well as diagnostics to be large in dollar terms. This study received two ethics approvals that act together. The mentioned ethics approval given by the Hebrew university ethics committee was given for the psychological part of the experiment and include the CBT, using a psychologist or computer program. A second approval, Helsinki approval by the Hadassah hospital committee, covers the biological part of the experiment - including the blood drawing and further analysis.

All patients will be treated with either internet or face to face CBT according to their preference. Circulating exosomal microRNAs will be measured pretreatment and post-treatment.

40 Healthy controls matched on gender and age with no current psychopathology will have exosomal microRNAs measured twice over a 3 month period.

40 adult patients diagnosed with primary panic disorder will have their exosomal microRNAs measured 2x over 3 months.

There are a few common psychotherapies for treating PD, with Cognitive Behavioral Therapy (CBT) as the most common. The most known type of CBT for treating PD consists of two major strategies: cognitive restructuring, and interoceptive and structured exposure to bodily sensations that have become associated with panic attacks (D H Barlow, 1997). The ICBT therapy is based on Barlow and Craske's (2007) protocol for treating PD with elaborations (Huppert & Baker-Morissette, 2003). It includes six modules containing psychoeducation, cognitive restructuring, exposures, acceptance, and consolidation of gains and relapse prevention. The online modules include reading passages, worksheets, videos, and homework assignments. After completing each module, participants practice related skills and complete homework assignments. The treatment is up to 16 weeks long; participants are encouraged to complete the treatment within this time period, and reminders are sent to monitor their progress.

Exosomes are a type of extracellular vesicles of endocytic origin, used in signalling and cell to cell communication, by transferring proteins, lipids, and variety of RNAs between cells. miRs are short single-stranded RNA molecules that bind to complementary sequences of target mRNAs, causing inhibition of their translation and/or inducing target degradation and affecting brain functioning and mental processes. Will be measured twice: pre and post treatment (post or 6 months, whichever is later, to the maximum of 6 months).

Independent evaluators will conduct pre and post treatment evaluations and self report will be administered weekly. The PDSS can be administered as an interview or self-report measure. It is comprised of 7 items that are scored from 0-4, with a total scale score ranging from 0-28. Higher scores mean more severe panic disorder symptoms. Will be administered weekly during treatment, plus at pre and post treatment evaluations.

The ASI-3 is an 18-item self-report measure that measures beliefs regarding panic related bodily sensations. It will be used as a total score, which ranges from 0-72. The ASI-3 will be administered weekly. Higher scores mean more severe anxiety sensitivity.

The Mobility Inventory is a self report measure of agoraphobic avoidance in which patients are asked to rate how much they avoid 26 situations on a 1-5 scale twice, once when alone and a second time when accompanied. The average score of all times will be used, which will range from 1-5. The MI will be administered weekly.

We will also collect whole blood samples that will serve as a secondary outcome measurement, checking for changes in expression of small non-coding circulating RNAs as well. This measurement will act as a more clinically accessible balance for the exosomes approach. Will be measured twice: pre and post treatment (post or 3 months, whichever is later, to the maximum of 6 months).

The DCS-5 is a self-report measure that measures 5 dimensions of curiosity via 25 items which are rated on a 1-7 scale. The total score of all 25 (ranging from 25-175) items and 5 subscales (each ranging from 5-35) will be used for this study. Will be measured twice: pre and post treatment (post or 6 months, whichever is later, to the maximum of 6 months).

The ECR-s is a brief self report instrument that measures both Anxious and avoidance adult attachment styles. The two scales each contain 6 items rated on a 1-7 scale that are summed for each subscale, ranging from 6-42. Will be measured twice: pre and post treatment (post or 6 months, whichever is later, to the maximum of 6 months).

this is a self-report measure of sexual functioning that is comprised of 14 items that are rated on a 1-5 scale and scored by a sum score that ranges between 14-70. Higher scores mean better sexual functioning. Will be measured twice: pre and post treatment (post or 6 months, whichever is later, to the maximum of 6 months).

Inclusion Criteria: Principal DSM-5 primary diagnosis of panic disorder and/or agoraphobia. Aged 18 years or older. PD duration of at least 3 months. If participant is on on medications for PD, the dosage has to remain constant for 3 months prior to the start of treatment and cannot be increased during treatment. The participant must have access to the internet and be willing to use it. Exclusion Criteria: Substance abuse or dependence within the last 6 months. Active suicide potential within the last 6 months. Any current or history of psychosis or bipolar I disorder. Currently in weekly or biweekly psychotherapy. History of a complete course of panic focused CBT

All IPD that underlie results in a publication will be placed on a public website after the manuscript is submitted for publication.

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