Cannabinoid type 2 receptors regulate striatal inflammation following alpha synuclein-induced nigral degeneration in rat model of Parkinson’s disease Open Access

McLaughlin, Caroline (Spring 2020)

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Parkinson’s disease (PD) is characterized by a loss of dopamine neurons in the nigrostriatal pathway of the brain and by the formation of intracellular inclusions or Lewy bodies composed largely of alpha-synuclein (Asyn). Neuroinflammation accompanies the progression of the disease and plays a key role in Asyn-induced degeneration. There is a growing interest in new therapeutic approaches that aim to reduce these inflammatory responses in an effort to slow the progression of the disease. Specifically, targeting cannabinoid type 2 (CB2) receptors has shown to modulate cytokine release and diminish microglia neurotoxicity.  However, it is still not fully understood how pharmacologically targeting CB2 will alter Asyn aggregation and the Asyn-induced inflammatory state. In order to better understand the inflammatory mechanisms of CB2 selective modulation in the striatum, we evaluated the striatal hemispheres of rats unilaterally injected into the substantia nigra with AAV2/5-human wild-type Asyn (2.9E+11 vg/mL) and treated with either systemic SMM-189 (6 mg/kg, ip) or vehicle. SMM-189 is a novel CB2 inverse agonist that has been shown to reduce levels of cytokine and chemokine expression associated with pro-inflammatory microglia. Western blot analyses were conducted on protein extracted from both striatal hemispheres and stained for neuronal markers such as tyrosine hydroxylase (TH), phosphorylated TH, dopamine transporter (DAT), Asyn, and phosphorylated Asyn at serine 129. Additional western blots were performed to evaluate inflammatory markers including GFAP, IBA1, and NfkB. Results show that SMM-189 significantly reduced phosphorylated Asyn and TH expression compared to controls and increased expression of DAT and phosphorylated TH compared to vehicle-treated rats. In addition, inverse and significant correlations were observed between DAT and pSer129 Asyn in both SMM-189 treated rats and control rats; a significant and inverse correlation was seen between TH and Asyn in vehicle treated rats but not in SMM-189 treated rats; and a direct correlation between DAT and Phosphorylated TH, and NFkB and TH was seen in SMM-189-treated rats but not controls.  SMM-189 had no effect on microglia IBA1 expression, but does appear to suppress the up-regulation of GFAP expression seen in the ipsilateral side of vehicle-treated subjects. Our findings suggest that CB2 modulation using the inverse agonist SMM-189 decreases striatal toxic forms of Asyn aggregation, but has no effect on inflammatory markers evaluated, and results in enhanced dopaminergic degeneration as indicated by TH, Phospho TH an DAT expression changes.

Table of Contents

Introduction and Background pg. 9-17

- Parkinson’s Disease and neuroinflammation pg.9

- Peripheral Inflammation in PD pg. 11

- Therapeutic Approaches pg. 12

- Cannabinoid Type 2 Receptors pg. 14

- SMM-189 pg. 15 

- AVV2/5 Mediated Overexpression of Alpha Synuclein pg. 16

- Immunomodulation of SMM-189 pg.17

Hypothesis and Objective pg. 19

Materials and Methods pg.20 -22

- Subjects pg. 20

- Protein Extraction and Sample Preparation pg. 20

- Immuoblot Assays pg. 21

- Stripping pg. 22

- Quantification and Statistical Analysis pg. 22

Results pg. 23- 26

 - Asyn Aggregation pg. 23

- Dopaminergic Markers pg. 24

- Inflammation pg. 25

- Correlations pg. 26

Discussion pg. 27- 36

- Asyn Aggregation pg. 27

- Dopaminergic pg. 28

- Inflammation pg. 29

- Correlations pg. 33

- Limitations pg. 34

- Future Directions pg. 35

Conclusion pg. 37

Figures and Graphs pg. 38- 48

- Table 1: Antibody information pg. 38

- Figure 1: Preliminary data pg. 39

- Figure 2: Successful surgery postmortem pg. 40

-Figure 3: Immunoblot signal intensities pg. 41

- Figure 4: Asyn aggregation pg. 42

- Figure 5: Dopaminergic expression pg. 43

- Figure 6: Inflammatory markers pg. 44

- Figure 7: Ipsi:contra paried t-tests pg. 45

- Figure 8: Vehicle-treated animal correlations pg. 42

-Figure 9: SMM-189-treated animal correlations pg. 43

-Figure 10: Visual Representation of Methodology pg. 48

Works Cited pg. 49-56

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