1. Motor Response Complications and Cycline-Dependent Kinase 5 (Cdk5) Neuroadaptations in an Animal Model of Parkinson’s Disease Heather Minkel Dr. Justin Oh-Lee Neuroscience

2. Parkinson’s Disease  Parkinson’s disease (PD) is a neurodegenerative disease affecting over one million Americans.  It is caused by dopamine depletion.  PD is characterized by such symptoms as akinesia, bradykinesia, resting tremors, and muscular rigidity.

3. Trouble With Treatment  Traditionally treated with twice-daily oral intake of levodopa.  After two to five years of chronic treatment, the drug becomes less effective and severe disabling motor complications appear.  These are termed motor response complications (MRCs) and include: shortening of the effects of levodopa treatment and abnormal involuntary movements (AIMs) or dyskinesias.

4. Cellular Mechanisms  Much of the mechanisms are not clearly understood.  However, we do know: Higher levels of striatal kinases are present after chronic levodopa treatment. Striatal kinase inhibition has lessened motor response complications (MRCs). Cycline-dependent kinase 5 (CDK5) is an inhibitory kinase that regulates striatal kinases.

5. Normal Function Levodopa Treatment INTRASYNAPTIC DOPAMINE LEVELS 0 3 6 0 3 6 0 3 6 Hours Dose Untreated Parkinsonism Dose Hours 11/02

6. Research Question  Because CDK5 has an compensatory neuroadaptive role in striatal neuronal signaling, removing it should worsen levodopa-induced motor response complications.  Using silencing RNA techniques, CDK5 inhibition by SI RNA allows for assessment of its role in the development of MRCs.

7. Methodology  Unilateral lesioning caused hemi- parkinsons.  Deprivation of dopamine results in striatal supersensitization.  Rotation occurs with levodopa treatment.

8. Methodology  We assessed shortened duration of treatment effect (wearing-off effects) and AIMS (dyskinesias).  We performed intracranial injections of flourescence dyed dendrimers conjugated with silencing CDK5 RNA.  12 hours after surgery, following final levodopa injection, behavioral assessments were made.

9. Methodology  Sacrificing, brain harvesting, brain slicing, tissue staining.  Tissue staining allows for CDK5 levels to be compared between the two groups.  The levels of CDK5 and the behavioral assessments for the final treatment were compared for correlation.

10. Exacerbating Effects of Cdk5 SiRNA on Chronic Levodopa-Induced AIMs Severity  Levodopa treatment for 21 days increased both the AIMs amplitude and severity in hemi-parkinsonian rats.  Both amplitude and severity of AIMs was significantly greater in the Cdk5 SiRNA group than the control group on treatment day 22.

11. Effects of Cdk5 SiRNA on duration of levodopa-induced contralateral rotations following chronic levodopa treatment  The duration of the rotational response is compared on levodopa day 1(baseline), day 7, day 14, day 21, and day 22 + local injections (control, SiRNA). On day 22, Cdk5 SiRNA was given 12 hours prior to levodopa challenge. On day 22, shortened response duration was worsened by Cdk5 SiRNA treatment. *p<0.05 #p<0.05 compared with day 21 and control SiRNA, respectively. *#*#

12. Worsening effects of Cdk5 SiRNA + Dendrimer (NC) on chronic levodopa- induced AIMs severity in hemiparkinsonian rats: A treatment with SiRNA after 3 weeks of twice-daily levodopa treatment exacerbated levodopa-induced contralateral AIMs severity on 6-OHDA rats. FITC-labeled dendrimer plus Cdk5 siRNA (FITC green: Panel A) co- stained with propidium iodide nuclear staining (Red; Panel B[Co-label]) in dyskinetic hemiparkinsonian rats 12 Hours after siRNA gene silencing with dendrimer conjugated with Cdk5 SiRNA. Arrows indicate striatal neuronal profile co-labeled with Cdk5 siRNA in cytoplasm (green) and propidium staining in nuleus (red) produced by dendrimer-mediated siRNA gene transfer.

13.  Three weeks of twice-daily levodopa treatment increased the AIMs severity and amplitude, and shortened the duration of the rotational response to acute levodopa challenge.  Both the shortened turning response and augmented AIMs were worsened by Cdk5 removal.  This suggests that Cdk5 activation could attenuate the phosphorylation of striatal glutamatergic receptors and thus ameliorate the severity of dyskinesias. Results

14.  Our results suggest that Cdk5 may be involved in neural adaptive mechanisms to antagonize the effects of repeated levodopa administration.  Findings from the present study provide more accurate understanding of molecular mechanisms underlying striatal neuroadaptive plasticity contributing to the pathogensis of levodopa induce motor complications in advanced PD.  Results from the present study also suggest novel approaches to safer and more effective therapy of motor dysfunction in PD patients and other related dopamine disease states. Conclusion and Discussion

15. Motor Response Complications and Cycline-Dependent Kinase 5 (Cdk5) Neuroadaptations in an Animal Model of Parkinson’s Disease Questions?