HRB SUMMER STUDENT SCHOLARSHIP 2015

Scholars’ Report

(Please complete this form and email as an attachment to summerstudents@hrb.ie by Friday, 16 October 2015)

1. Name of student: Kelly Moore

2. Name of University/ Higher Education Institute where student is studying:National University of Ireland, Galway

3. Degree course and stage reached: 4. 3rd year Undeminominated Science

5. Name and address of University/Higher Education Institute where research was undertaken:National University of Ireland, Galway

6. Country in which research was undertaken: 7. Ireland 8. Name and address of supervisor (in Ireland):

Dr. Eilís Dowd, Pharmacology & TherapeuticsNational University of Ireland, Galway

7. Project Title:The potential of the CB2 subtype of cannabinoid receptor as pharmacological target for disease modification and neuroprotection in Parkinson's disease

8. Project aims/Hypothesis: (maximum 100 words)The aim of my project was to validate the CB2 subtype of cannabinoid receptor as a potential pharmacological target for disease modification and neuroprotection in Parkinson's disease. The specific objectives were 1) to determine if pharmacological targeting of the CB2 receptor would prevent nigrostriatal neurodegeneration in a rat model of Parkinson’s disease, and 2) to determine if the CB2 receptor is expressed in the substantia nigra of post mortem human patients' brains. The hypothesis being tested in this study is that activation of the CB 2 receptor will prevent Parkinsonian neurodegeneration thus highlighting its potential as a target for disease modification in this disease.

HRB Summer Student Scholarships 2015Student Report Form

9. Project Description: (maximum 1,000 words) to include abstract (summary); aims and objectives; materials and methods; results; discussion; conclusion. Use tables, diagrams or photographs where appropriate (these are not included in the word limit)

Background: The endocannabinoid system has recently emerged as a potential target to curb the chronic self-sustaining cycle of neuroinflammation and neurodegeneration observed in Parkinson’s disease (Ashton & Glass, 2007; Benito et al., 2008; Ehrhart et al., 2005; Kannarkat et al., 2013; Tansey & Goldberg, 2010). Moreover, the Dowd group has recently shown that this receptor significantly upregulated in an inflammation-driven rat model of this condition (Concannon et al., 2015).

Aim: Therefore, the aim of this study was to determine if pharmacological activation of the CB2

receptor in this inflammation-driven model could prevent nigrostriatal neurodegeneration. We also completed some preliminary studies to determine if the CB2 receptor is expressed in the substantia nigra of post mortem human patients' brains in order to maximise the translational relevance of our study.

Methods: Male Sprague Dawley rats were assigned to four groups: [1] Naive & Vehicle (n=4), [2] Naive & CB2 agonist (n=4), [3] LPS lesion & Vehicle (n=10), [4] LPS lesion & CB 2 agonist (n=10). Animals in the lesion groups received a unilateral, intra-nigral LPS lesion (10 µg in 2 µl). Animals were injected with either CB2 agonist (JWH-133, 1 mg/kg i.p.) or vehicle (sterile saline), 2 hr prior to surgery, and daily for a further 13 days, and were then sacrificed 14 days post-surgery. Quantitative immunohistochemical analysis was performed to assess neuroinflammation and neurodegeneration in the nigrostriatal pathway. For the human brains, preliminary immunohistochemical staining for microglia and the CB2 receptor was carried out on brain sections procured from the Dublin Brain Bank.

Results: Injection of LPS into the rat substantia nigra caused significant nigral microgliosis (Fig. 1) and nigrostriatal neurodegeneration (Fig. 3) but no astrocytic scarring (Fig. 2). Chronic JWH-133 attenuated LPS-induced dopaminergic neurodegeneration at the level of the cell bodies in the substantia nigra. Interestingly, the CB2 agonist did not reduce microglial infiltration/proliferation at the lesion site, indicating that the protective effects of JWH-133 treatment may involve other anti-inflammatory mechanisms such as reduction in release of pro-inflammatory mediators from these cells. In the human substantia nigra, clear microglial staining was observed but the CB2

staining was not conclusive (Fig. 4).

Conclusion: Overall, this study has shown that pharmacological targeting of the CB2 receptor protects the nigrostriatal pathway from inflammation-driven dopaminergic neurodegeneration via a mechanism that does not involve a reduction in microgliosis. This study indicates that targeting the CB2 receptor may represent a viable target for anti-inflammatory disease modification in Parkinson’s disease.

HRB Summer Student Scholarships 2015Student Report Form

Fig. 1 Immunohistochemical analysis for microglia. OX-42 immunohistochemistry revealed that LPS induced nigral microgliosis but this was not reduced by the CB2 agonist JWH-133.

HRB Summer Student Scholarships 2015Student Report Form

LPS & JWHLPS & Veh

JWHVeh

LPS & JWHLPS & Veh

JWHVeh

Fig. 2 Immunohistochemical analysis for astrocytes. GFAP immunohistochemistry revealed that LPS did not induce any nigral astrocytosis.

HRB Summer Student Scholarships 2015Student Report Form

LPS & JWHLPS & Veh

JWHVeh

LPS & JWHLPS & Veh

JWHVeh

Fig. 3 Immunohistochemical analysis for nigrostriatal degeneration. Tyrosine hydroxylase immunohistochemistry revealed that LPS caused significant nigrostriatal neurodegeneration, and importantly, this was prevented by the CB2 agonist JWH-133.

HRB Summer Student Scholarships 2015Student Report Form

LPS & JWHLPS & Veh

JWHVeh

LPS & JWHLPS & Veh

JWHVeh

Fig. 4 Immunohistochemical staining of human brain sections. Iba1 staining revealed clear microgliosis in the human substantia nigra (left photomicrograph). However, as no clear cellular morphology was observed after CB2 receptor staining (right photomicrograph), we could not definitively conclude that the CB2 receptor was expressed in the human Parkinsonian brain.

HRB Summer Student Scholarships 2015Student Report Form

10. Overview of Research Experience: My overall experience as a HRB Summer Scholar was extremely positive. I gained invaluable experience while working in the Dowd laboratory. In my first week, I got an excellent introduction to the lab - I got to see how a lab is run efficiently and I met all the brilliant people who work and/or are studying there. I got a broad exposure to various aspects of lab work and research, and I now have a major appreciation for all the research carried out with a laboratory setting, the time put in, and the cost of each project. I found the lab a very friendly environment, and staff and students were more than willing to help when I had any difficulty. If I encountered a problem that I couldn’t resolve myself, the other lab members were always very helpful and informative. However, they also encouraged me to figure out some problems on my own, this helped with my own self confidence and competence in the lab.

I also got great experience of working in a team and I got an insight into the importance of teamwork. I got run my own immunohistochemistry assay to obtain new data and also got the opportunity to analyse existing immunohistochemical slides already obtained. This gave me a greater understanding of the bigger picture of the project. My project was part of an existing project (funded by the HRB (HRA_POR/2012/12)) and will contribute significantly to the future direction of the Dowd team. As my research showed that activation of the CB 2 receptor could prevent LPS-induced nigrostriatal neurodegeneration, this could have important implications for the development of new drugs for Parkinson’s disease in the future.

By chatting to the research students and staff I was able to gain a greater understanding of the world of research and I know have better knowledge of the work it entails. This gives me the ability to make more educated decision on my own future career. From my eight weeks working in the lab, I can now say with certainty that I would be interested in a career in research.

11. Describe the role played by you in the project:I played a big role in project and gained significant new skills that will be invaluable to me in my future career such as laboratory animal maintenance, brain sectioning, immunohistochemistry, immunofluorescence, microscopy, Image J analysis, data presentation/statistical analysis, and report writing. Specifically:

1. I got the opportunity also to observe and help with the maintenance of live animals in the lab. 2. I got to use the microtome to section rat brain samples at 30 mm. 3. I preformed the GFAP immunohistochemistry for astrocytes from start to finish

independently. Moreover, I also captured the photomicrographs, used Image J to graph and analyse the data, and I drew my own conclusions based on these findings.

4. I also got substantially more experience in data analysis and presentation as I reanalysed the previously generated photomicrographs of the OX42 and tyrpsine hydroxylase immunostaining.

5. I also worked with the human brain sections which I found particularly interesting. Specifically, I completed the microglial and CB2 staining independently from start to finish.

12. References (maximum 8):

1. Ashton JC, Glass M.

HRB Summer Student Scholarships 2015Student Report Form

The cannabinoid CB2 receptor as a target for inflammation-dependent neurodegeneration.Curr Neuropharmacol. 2007;5(2):73-80.PMID:18615177

2. Benito C, Tolón RM, Pazos MR, Núñez E, Castillo AI, Romero J.Cannabinoid CB2 receptors in human brain inflammation.Br J Pharmacol. 2008 Jan;153(2):277-85. Epub 2007 Oct 15. Review.PMID:17934510

3. Concannon RM, Okine BN, Finn DP, Dowd E.Differential upregulation of the cannabinoid CB₂ receptor in neurotoxic and inflammation-driven rat models of Parkinson's disease.Exp Neurol. 2015 Jul;269:133-41. doi: 10.1016/j.expneurol.2015.04.007. Epub 2015 Apr 18.PMID:25895887

4. Ehrhart J, Obregon D, Mori T, Hou H, Sun N, Bai Y, Klein T, Fernandez F, Tan J, Shytle RD.Stimulation of cannabinoid receptor 2 (CB2) suppresses microglial activation.J Neuroinflammation. 2005 Dec 12;2:29.PMID:16343349

5. Kannarkat GT, Boss JM, Tansey MG.The role of innate and adaptive immunity in Parkinson's disease.J Parkinsons Dis. 2013;3(4):493-514. doi: 10.3233/JPD-130250. Review.PMID:24275605

6. Tansey MG, Goldberg MS.Neuroinflammation in Parkinson's disease: its role in neuronal death and implications for therapeutic intervention.Neurobiol Dis. 2010 Mar;37(3):510-8. doi: 10.1016/j.nbd.2009.11.004. Epub 2009 Nov 10. Review.PMID:19913097

13. Please list any new skills acquired: Brain sectioning Immunohistochemistry Immunofluorence Photomicroscopy Image J analysis Data Analysis

14. Presentations, posters, conference abstracts, or publications arising from the research (if any):n/a

HRB Summer Student Scholarships 2015Student Report Form