Proteasome-independent K63 polyubiquitination selectively regulates ATP levels and proteasome activity during fear memory formation in the female amygdala

The National Center for PTSD (post-traumatic stress disorder) reports women are twice as likely to develop PTSD than men…Why? We are wondering the same thing.
Published in Neuroscience
Proteasome-independent K63 polyubiquitination selectively regulates ATP levels and proteasome activity during fear memory formation in the female amygdala

In this blog, we will dive into a specific molecular mechanism involved in fear memory formation and examine how it differs between males and females in an attempt to better understand why females are more likely than males to develop post-traumatic stress disorder (PTSD). Before pursuing this study, we had already observed sex differences in a different form of this specific mechanism, and this led to our passion for studying sex differences in fear memory. At first, we thought it would be impossible due to the complexity of this specific mechanism, but as they say, where there is a will, there is a way.

Setting the scene

It all started in the amygdala, the emotional control center of the brain. It was here that the ubiquitin-proteasome system was working nonstop to label proteins with the small regulatory protein, ubiquitin. This ubiquitin protein has multiple linkage sites on it that can be used to label a protein, with each site having a unique role or function. You might be thinking, this is complicated, and we would have to agree. To keep it simple, we will look at two examples. One protein gains a linkage at lysine-48 (K48), which labels the protein for degradation (destruction). We previously found that K48 labels different proteins for degradation in the amygdala of males and females during fear memory formation. Another protein gains a linkage at lysine-63 (K63), which would not lead to destruction of the protein. While we know that K63 does not typically cause degradation, it has never been studied in the brain and its role in fear memory formation is unknown. The motivation for this study was to understand the role of the K63 ubiquitin linkage site in the amygdala of male and female rats for fear memory.

K63 doesn’t do the same thing in males and females?

            To begin our study, we identified all of the proteins labeled with the K63 linkage in the amygdala of male and female rats one hour following fear conditioning. This time point is widely accepted as a peak time for ubiquitin-proteasome system activity. What we found was exciting (and, admittedly, unexpected)! We identified 22 proteins in females that either lost or gained K63 during fear memory formation, but only one protein in males, which lost the K63 mark, was identified. That means that not a single protein was gaining the K63 mark in the amygdala of males during fear memory formation. This was the first time we had observed this degree of differences between males and females in ubiquitin-proteasome signaling, so we were excited to dig deeper into whether males and females both need the K63 linkage to form a fear memory. However, first we needed to figure out how to remove the K63 linkage in order to see if fear memory would be impacted.

Soo… what are you waiting for?

As we mentioned briefly, the ubiquitin-proteasome system is complicated. Well, really ubiquitin was the complicated player in this game. Instead of having one coding gene, ubiquitin has four AND each ubiquitin linkage site is part of the same protein. To make a long story short, our typical CRISPR-dCas9 editing system was not sophisticated enough to target just K63. To get around this predicament, we decided to use another system called CRISPR-dCas13b-ADAR2DD. This system allowed us to target RNA (instead of DNA) to essentially remove the K63 linkage site on ubiquitin. Since this system had never been used in the brain, we had to be very careful. We did a lot of testing in cell culture to make sure we could change a specific linkage site without changing the neighboring linkage sites AND without killing the cells. Eventually, the system was ready, and we could get back to our goal: Identifying the need for K63 in the amygdala for fear memory formation.

Role of K63 linkage in the amygdala for fear memory. Normal animals of both sexes remember fear memory. Females with K63 knockdown do not remember fear memory, but males with K63 knockdown do remember.

This image depicts the role of the K63 linkage for fear memory in the amygdala of both sexes. The left panel shows animals with a normal amount of K63 linkages in the amygdala. Both sexes are trained using contextual fear conditioning, which causes normal animals to associate the environment with fear. The next day, when animals are placed back into the same environment, both sexes remember the training as indicated by a fear response. The right panel shows K63 knockdown animals with less K63 in the amygdala compared to normal animals. The K63 knockdown animals are trained using contextual fear conditioning. The next day, female K63 knockdown animals do not remember the training, but male K63 knockdown animals do remember the training. Created with

Females need K63 for context fear, males don’t

            We injected the CRISPR-dCas13b-ADAR2DD system into the amygdala of male and female rats. Then animals were trained to contextual fear conditioning and fear memory was evaluated. As if our first finding wasn’t exciting enough, this experiment revealed that males could remember just fine without the K63 linkage, but females could not. We needed to cover all bases, so we did additional experiments in females. First, we examined anxiety-like behavior to make sure removing the K63 linkage did not change this behavior and impact fear memory. Good news, it didn’t. There are different types of fear memories, so we wanted to see if the K63 linkage was important for auditory fear too. Crazy enough, although context fear memory was impaired, removing the K63 linkage in the amygdala of females did not impact auditory fear memory. So, what did we learn? Females, but NOT males, need the K63 linkage in the amygdala for context fear memory.

We were still left wondering, what is the role of the K63 linkage in the amygdala of females during fear memory formation? We noticed that proteins involved in ATP production and proteasome activity were labeled with K63 in females, so we compared data collected from control (they have K63) and K63 knockdown (they have less K63 due to CRISPR-dCas13b-ADAR2DD) animals. ATP levels and proteasome activity both increased in control animals, but the K63 knockdown reduced this increase. While this isn’t the most striking data, we remembered that K63 had 22 different protein targets and that the CRISPR-dCas13b-ADAR2DD cannot target the K63 linkage in every single cell of the amygdala. This makes determining a single role for K63 quite difficult, so these moderate reductions are actually pretty exciting! Considering that this was the FIRST study of the K63 linkage in the brain, our data provide a solid foundation for future studies to build upon. In the future, it will be important to study why this sex difference exists, how K63 may contribute to sex differences in the prevalence of PTSD, and the role of other ubiquitin linkages in fear memory. For now, we hope that these exciting data highlight the importance of studying both sexes and provide a potential mechanism involved in PTSD development. 

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