Artifact 1: ASPET Grant Report

Identification of intramolecular signals controlling PDE11A4

subcellular compartmentalization

Abstract

Phosphodiesterases (PDEs) play an important role in preserving cyclic nucleotide microdomains.

PDE11A4 in brain is expressed almost exclusively in the hippocampal formation, and is enriched
in the cytosol versus soluble membrane fractions of hippocampal neurons. Here, we identify
intramolecular signals that control the subcellular trafficking of PDE11A4 in hopes of identifying
novel ways to therapeutically target this enzyme in a compartment-specific manner. When
expressed in heterologous cells, WT PDE11A4-GFP is distributed throughout the cytoplasm and
in puncta representative of organelles, as indicated by transmission electron microscopy. While
these puncta are not golgi, a subset colocalizes with a lysosomal marker, suggesting that at least
some reflect organelles along the proteolytic pathway. Indeed, live- imaging shows smaller
PDE11A4 puncta moving within the cell and fusing with larger puncta. Phosphomimic mutations
of serine 117 (S117D), S124D, and threonine 499 (T499D) increase trafficking of PDE11A4 into
puncta, while S162D and disruption of homodimerization block this trafficking. Interestingly,
both S117D and S124D appear to synergize, as S117D/S124D potentiates PDE11A4 aggregation
relative to S117D or S124D; whereas S162D is able to completely block the effect of
S117D/S124D. Similarly, we show that PDE11A4 phosphorylated at S162 (pS162) is found
exclusively in cytosol; whereas, pS117p/S124 is in both cytosol and membrane. In addition to
trafficking effects, we find phosphorylation interactions among these residues. pS117 is
decreased in phosphoresistant S124A; whereas, pS117 is increased in phosphomimic S124D.
pS124 is similarly changed in response to the phosphoresistant/mimic status of S117. We believe
pS117/pS124 may drive pS162, because pS162 is significantly decreased in 117A/S124A
mutants relative to WT. Importantly, phosphorylation of these residues is observed in vivo in
mouse hippocampus. These studies are the first to identify intramolecular signals that control
PDE11A4 compartmentalization, and may point to novel mechanisms by which we can
therapeutically target this enzyme in a compartment-specific manner.

Personal Reflection

When describing my involvement in Dr. Kellys lab, many words come to mind: thrilling,
arduous, humbling, motivating, and eye opening are only a few examples. Specifically, working
on my project this summer was an unparalleled learning experience that shaped me as a student,
and as an aspiring physician/scientist. If I could summarize what I gleaned, it would be that
science is full of both ups and downs. From presenting my research at an international
conference in Spain, to discovering groundbreaking results, to learning intricate scientific
techniques, to witnessing my work publish in two peer-reviewed journals, I have felt the raw
exhilaration science has to offer. Simultaneously, enduring through multiple failed hypotheses,
facing the consequences of careless mistakes, struggling to replicate results, and sacrificing
countless hours in lab taught me that research is neither glamorous nor straightforward. In the
end, the ups outweigh the downs; otherwise, no one would venture down the career path. In
a sense, the downs give the ups a profound distinctiveness. They add value to the honor of
contributing towards something greater than any one person or any one lab. Further, I believe
that the downs shaped my scientific understanding just as much as the ups, amounting to an
overall experience that I will never forget.

In addition, my research involvement has led me to pursue an MD/PhD degree. Now that I have
experienced the dynamic link between healthcare and academia, I want to contribute toward the
body of knowledge that drives medicine forward. Specifically, I hope to teach at a leading
medical school and publish clinical data from my practice. I am extremely grateful for the
funding opportunity that ASPET has provided through their SURF grant, and I know that my
future will be much brighter because of what this award allowed me to accomplish.

Contact Information

Telephone: 803-445-7789
Email: wcapell@email.sc.edu or wcapell2@gmail.com Address: 84 Stockman Rd.

Prosperity, SC 29127