No Pain, FAAH-OUT

Researchers from the University College London have brought us one step closer to understanding how we feel pain. Their study, published in Brain, describes a rare genetic mutation found in a patient who can feel no physical pain, anxiety, or fear.

Joanne Cameron is a seventy-two-year-old retired teacher who was first referred to pain geneticists at UCL in 2013 after her doctor noticed that she had no reported symptoms of pain or discomfort after two major surgeries. Since then, scientists have been mystified by her inability to feel physical pain.

In interviews, Cameron recounts how as a child she fell and hurt her arm while roller-skating but had no idea it was broken until her mother noticed the arm hanging strangely. After giving birth to her son, Cameron could only describe the experience of natural childbirth as a tickle rather than anything remotely painful, stating that “it was over before [she] knew it”.

Now, Mikaeili et al. report that the secret to Joanne Cameron’s pain-free life lies in two genetic mutations affecting the genes FAAH and FAAH-OUT. An even bigger surprise is that FAAH-OUT is not a protein-coding gene, but a long non-coding RNA.

What are FAAH and FAAH-OUT?

Both FAAH and FAAH-OUT are part of the endogenous cannabinoid system. The endogenous cannabinoid system is responsible for several important functions including anxiety and stress responses, pain modulation, learning and memory, and wound healing. FAAH is a protein found in several regions of the brain that are responsible for pain signals.

The FAAH protein and its associated gene are known to be involved in pain signaling and have been the target of several pain studies and potential treatments for the past twenty years. However, as of today, no FAAH-inhibiting drugs have been approved after human trials.

While the FAAH gene has been the focus of pain research for the past twenty years, much less is known about FAAH-OUT. FAAH-OUT is a long non-coding RNA. Scientists have long understood that shorter segments of RNA like ribosomal RNA (rRNA) and transfer RNA (tRNA) are essential to produce proteins. However, the function of long non-coding RNA has remained much more cryptic.

Much as its name implies, long non-coding RNA are stretches of more than 200 nucleotides that do not encode any proteins. However, in 2007, a study investigating the role of long non-coding RNA discovered that these seemingly purposeless stretches of genetic material actually contain several characteristics of shorter, protein-producing segments of RNA. Evidence of functions like polyadenylation, splicing, and 5’ capping suggested that long non-coding RNA may be able to directly interact with and alter other genes and proteins. Now, a handful of studies have implicated long non-coding RNAs in the progression of neurological diseases and cancer, suggesting that long non-coding RNAs may play a more significant role in our body than previously realized.

So, how are FAAH and FAAH-OUT involved in pain? After examining Joanne Cameron’s genetic makeup, Mikaeili et al. found that in addition to a slight mutation of the FAAH gene, Cameron was missing an entire section of the FAAH-OUT gene. This prompted the researchers to examine not only how mutations in FAAH may impact pain signaling, but also how a deletion of the FAAH-OUT long non-coding RNA may contribute to the production of pain signals.

To examine this, the researchers cultured human cells in a petri dish and used CRISPR to delete the FAAH-OUT gene within the cells. From there, Mikaeili et al. tracked how the production of FAAH protein was impacted over time.

Interestingly, by deleting the FAAH-OUT gene, Mikaeili et al. found that the cells produced significantly less FAAH protein. Additional experiments showed that when FAAH-OUT was enhanced, the production of FAAH protein increased by almost two-fold. These results suggest that FAAH-OUT may directly regulate the production of FAAH.

To strengthen the association between the FAAH-OUT gene and FAAH protein, the researchers then marked FAAH-OUT and FAAH with fluorescent proteins to assess where both genes were located in the cells. After examining the location of FAAH and FAAH-OUT transcripts in human tissue samples of the brain, Mikaeili et al. found that both transcripts were expressed in the same cells, further supporting the conclusion that FAAH-OUT regulates the production of FAAH protein.

In addition to Jo Cameron’s inability to feel pain, Cameron also reported feeling little to no sense of anxiety and a consistently elevated mood. With this, researchers were interested in investigating how FAAH-OUT impacts pain-related genes as well as how the long non-coding RNA may impact mood-related genes.

One gene of interest was brain-derived neurotrophic factor (BDNF). When BDNF becomes dysfunctional or is decreased, it is known to cause malfunctions in brain cell communication, decreasing the number of excitatory brain cells, and inducing symptoms of depression. Interestingly, after analyzing Jo Cameron’s genetic makeup, researchers found that the expression of BDNF was increased. Further experiments examining the relationship between FAAH protein and BDNF found that inhibiting the production of FAAH protein increased levels of BDNF. These results suggested that Jo Cameron’s deletion of the FAAH-OUT long non-coding RNA may decrease her body’s production of FAAH protein, leading to increased levels of BDNF and an elevated mood.

Overall, the results of this study bring us one step closer to understanding the biological mechanisms that underlie our ability to feel pain. Hopefully, as we continue to delve into the role of FAAH and FAAH-OUT, we will not only develop new targets for pain treatments and medications but will deepen our understanding of long non-coding RNAs and their role in the body.