Sara Valdeolivas Rojas IBRO InEurope Short Stay Grants Program

IBRO InEurope Short Stay Final Report

This is a brief report to summarize my short stay at the University of East Anglia,
between November 23 and December 22 2015.
At my home laboratory in the Complutense University, we study the
neuroprotective potential of cannabinoids in various models of neurodegenerative
diseases, and it has been recently reported that in these disorders, the
heteromerization of cannabinoid receptors can be altered and thus play a role in the
pathology [1]. Therefore, the first aim of this stay was to learn new skills to study
receptor heteromerization, which are not available at my home laboratory. For this
purpose, we chose the laboratory leaded by Dr. Peter McCormick at the University
of East Anglia. Dr. McCormick has a wide experience in the study of heteromers, and
has also worked before in the cannabinoids research field [2,3]. Hence, the second
aim was to initiate a long term collaboration between the two groups.
As the stay was intended to extent only for one month, we decided to focus firstly on
one of the diseases we study in my home laboratory, which is amyotrophic lateral
sclerosis (ALS). This is a degenerative disease produced by the damage of the upper
and lower motor neurons leading to muscle denervation, atrophy and paralysis [4],
and in which changes in the endocannabinoid signaling in patients have been
reported previously [5]. We used spinal cord slices of two genetic mouse models of
the disease, TDP-43 and SOD1G93A. In both models, our group has found previously a
huge increase in cannabinoid CB2 receptor expression [6, 7]. This receptor is
normally not expressed in the central nervous system, but it turns highly expressed
in reactive microglia in the case of damage such as inflammatory processes. The
P2X7 ion channel expression is also increased in the microglia of mouse models of
ALS [8]. According to that, our first approach was to look for colocalization of these
two molecules in the microglia of our samples by immunofluorescence, and then to
look for heteromerization using the Proximity Ligation Assay (PLA) technique.
The PLA principle is, briefly, the following: two primary antibodies raised in
different species recognize the target antigen or antigens of interest. Speciesspecific secondary antibodies, called PLA probes, each with a unique short DNA
strand attached to it, bind to the primary antibodies. When the PLA probes are in
close proximity, the DNA strands can interact through a subsequent addition of two
other circle-forming DNA oligonucleotides. After joining of the two added
oligonucleotides by enzymatic ligation, they are amplified via rolling circle
amplification using a polymerase. After the amplification reaction, severalhundredfold replication of the DNA circle has occurred, and labeled complementary
oligonucleotide probes highlight the product. The resulting high concentration of
fluorescence in each single-molecule amplification product is easily visible as a
distinct bright spot when viewed with a fluorescence microscope.

Sara Valdeolivas Rojas IBRO InEurope Short Stay Grants Program

During these four weeks I have been able to improve my knowledge and expertise
in immunofluorescence and cell imaging, and also to learn the PLA assay and apply
it to my samples. Once the technique is stablished, our purpose is to increase the
experimental number and, once we see if the heteromer is expressed in wild type
samples, to check if there are changes in the transgenic animals. We will also search
for the presence of other heteromers of interest, such as CB1-A2A or CB1-A1. After
that, we will test the same in human samples of patients with ALS.
This short stay has just been a beginning of what we expect to be a long term
collaboration between the two groups. We are also interested in studying deeply the
heteromerization of cannabinoid receptors in other models of diseases, such as
Parkinsons disease and Dravet syndrome. Therefore, the knowledge acquired
during this fellowship will benefit the whole home laboratory, but also help
maintaining the collaboration with the host group for future projects.
Lastly, I would like to acknowledge all the people from the host group for their
kindness and support, and to Dr. McCormick for accepting me in this short stay, they
all made me feel part of the team. I hope I can have the chance to continue
collaborating with them. And of course, my deepest acknowledge to the IBRO for
giving me this grant, which has meant a great opportunity not only for my scientific
career but also my personal development.

From left to right: Joaquim Botta (PhD student), Sara Valdeolivas (PhD student and IBRO awardee),
Peter McCormick (lecturer and PI), Julia Appelhans (PhD student), Mar Puigdellivol (Postdoc
researcher), Luka Bibi (PhD student)

Sara Valdeolivas Rojas IBRO InEurope Short Stay Grants Program

References
1.

2.
3.

4.
5.
6.

7.

8.

Pinna, A., et al., L-DOPA disrupts adenosine A(2A)-cannabinoid CB(1)-dopamine D(2)

receptor heteromer cross-talk in the striatum of hemiparkinsonian rats: biochemical
and behavioral studies. Exp Neurol, 2014. 253: p. 180-91.
Callen, L., et al., Cannabinoid receptors CB1 and CB2 form functional heteromers in
brain. J Biol Chem, 2012. 287(25): p. 20851-65.
Moreno, E., et al., Cocaine disrupts histamine H3 receptor modulation of dopamine D1
receptor signaling: sigma1-D1-H3 receptor complexes as key targets for reducing
cocaine's effects. J Neurosci, 2014. 34(10): p. 3545-58.
Zarei, S., et al., A comprehensive review of amyotrophic lateral sclerosis. Surg Neurol
Int, 2015. 6: p. 171.
Pryce, G. and D. Baker, Endocannabinoids in Multiple Sclerosis and Amyotrophic
Lateral Sclerosis. Handb Exp Pharmacol, 2015. 231: p. 213-31.
Moreno-Martet, M., et al., Changes in endocannabinoid receptors and enzymes in the
spinal cord of SOD1(G93A) transgenic mice and evaluation of a Sativex((R)) -like
combination of phytocannabinoids: interest for future therapies in amyotrophic
lateral sclerosis. CNS Neurosci Ther, 2014. 20(9): p. 809-15.
Espejo-Porras, F., et al., Changes in the endocannabinoid signaling system in CNS
structures of TDP-43 transgenic mice: relevance for a neuroprotective therapy in TDP43-related disorders. J Neuroimmune Pharmacol, 2015. 10(2): p. 233-44.
Yiangou, Y., et al., COX-2, CB2 and P2X7-immunoreactivities are increased in activated
microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis
spinal cord. BMC Neurol, 2006. 6: p. 12.