1. Knock Out Mice in the iron pathway as a potential model of RLS Three Models: Thy 1 knockout mice H-ferritin deficient mice Iron regulatory protein 1 knockout mice James R Connor, Ph.D. University Distinguished Professor Vice-Chair of Neurosurgery

2. Summary of RLS and Iron Status of Neuromelanin cells: l protein profile expression of iron deficiency with a major exception l Transferrin receptor expression is decreased l Iron Regulatory Protein 1 (but not IRP2) expression is decreased in RLS l Hypothesis: A decrease in IRP1 expression results in a destablization of Transferrin receptor mRNA leading to cellular iron deficiency.

3. What are the Consequences of Iron deficiency in Neuromelanin Cells: l Effect on Dopamine? n no decrease in Tyrosine Hydroxlyase in RLS phosphorylated Tyrosine hydroxylase is elevated n Dopamine levels in the Substantia Nigra are elevated in RLS D2 is decreased in putamen in RLS l Effect on Thy1? Gene profiling study identified Thy1 as an iron responsive gene Ye and Connor (2000), Nucleic Acids Res. 28(8), 1802-1807

4. Thy1 cell adhesion molecule that influences T-cell activation accounts for 7.5% of the surface protein on neurons stabilizes synapses involved in regulated vesicular release of neurotransmitters at the synapse expression related temporally and spatially to development of dopaminergic axons and synapses

6. Thy1 expression in total brain homogenates of mildly iron deficient rats and substantia nigra of human brains (control and RLS)

7. Dose Response of Thy1 and Transferrin receptors to iron chelation in PC 12 cells

8. D2 receptors increase with loss of Thy1 D2 receptors are decreased in the RLS brains: Absence of Thy1 is associated with increased D2 perhaps to compensate for structurally compromised synapse Does the failure of D2 increase in RLS indicate a loss of compensatory capacity because of iron deficiency :

9. The active form of TH is increased by ID In RLS pTH increases: this effect is consistent with ID

10. Synaptic efficacy and response to DA agent is altered in Thy1 KO mice Figure 1. A and B show population spikes from exemplar Thy-1 and Control slices, respectively. Raclopride reduces the population spike for the Control but not the Thy-1 slice. Bars: 5ms, 0.5 mV. C and D show the mean input-output curves of the Thy-1 and Control slices treated with raclopride, respectively. Raclopride, a D 2 -receptor antagonist reduces synaptic transmission in the Controls but not the Thy-1 slices. Washout is not shown but is identical to the before condition for both groups. E shows input output-curves for separate groups of Thy-1 and Control slices. Thy-1 synaptic transmission is slightly elevated compared to the Controls. Th, threshold stimulation intensity; Max, maximum stimulation intensity.

11. Gabapentin and Thy 1 A: Low dose Gabapentin (300 μM) reduced synaptic responses in young (~6 mo) Thy-1 mice slices but WT-control mice slices were not affected. B: High dose Gabapentin (600 μM) reduced synaptic response in both young (~6 mo) Thy-1 knockout and control mice slices.

12. Thy1 knockout mice and RLS l Compensatory response to the absence of Thy1 in the synapse is to have greater response to stimulation (loss of inhibitory influence) and increase D2 receptors n In RLS D2 receptors are decreased u Failure to increase due to iron deficiency? u Female Thy1 KO mice who are also iron deficient do not have the compensatory increase in D2 receptors, males do –Women have more RLS than men n If there is heightened response to stimuli with Thy1 deficiency in RLS with a decrease in D2 receptors would this result in abnormally low transmission u suboptimal stimulation of inhibtory pathway drives the urge to move??? n Is heightened synaptic intensity combined with a exogenous source of dopamine agonist a recipe for augmentation

13. Thy1 knockout mice and RLS l Potential for pharmacologic model of RLS n There is a drug effect on dopaminergic synapses u Poor response to D2 agonist suggests decrease D2 receptors at synaptic surface n Evaluate potential for augmentation n Behavioral model to study startle response n Gabapentin is effective in dampening the increased intensity of stimulation response in Thy1 defective synapses u Mechanism of gabapentin action is different from D2 receptor u Maybe it is better to decrease the synaptic response then try to stimulate the receptors in this model? –Underlie a difference in susceptibility to augmentation

14. Building A Story: The biological basis of RLS symptoms involves a compromise in the dopaminergic system at the level of the synapse.  The dopaminergic alterations could stem from loss of structural (loss of Thy1) and biochemical (decreased D2) modalities and both of these can be attributed to insufficient iron levels.  Studies on Thy1 knockout mice favor a DA profile that supports an iron deficiency model for RLS  Expected compensatory response to Thy1 deficit not seen in RLS---effect of iron deficiency?

15. H-ferritin deficient mice as a model for the decrease in H-ferritin seen in RLS brains l Mice Homozygotic for mutation are embryonic lethal l Heterozygotes are viable n More realistic model for RLS

16. Altered ferritin levels in the brain (Thompson et al., 2003) ~80% decrease ~70% increase * * H-Ferritin mostly found in neurons. Functionally L-ferritin cannot compensate for H-ferritin loss Neurons did not express L-ferritin

17. Establish a neurochemical profile of the brain in the H-ferritin deficient mouse High-Resolution Magic Angle Spin Proton Magnetic Resonance Spectroscopy (MR-MAS 1 H-MRS).

18. H-ferritin deficiency alters the regional neurochemical profile in the hippocampus and cortex. H-ferritin deficient animals had increased hippocampal choline and increased cortical and hippocampal N-Acetyl-Aspartyl-Glutamate (NAAG), a storage molecule for glutamate. Bars are mean (n=4)±SEM, ∗ indicates p < 0.05, t-test.

19. p<0.05 DAT increased due to neuronal iron deficiency? DAT unchanged in Putamen in RLS autopsy

20. Remember Decreased D2 in RLS Putamen and Decreased IRP1 activity in RLS Is Loss of IRP1 associated with A Decrease in D2 expression

21. Animal Model conclusions l D2 receptor loss is due to iron and not a compromise of the structural integrity of the synapse n D2 should increase with loss of Thy1 but is prevented by ID—relevant to RLS l Loss of H-ferritin shows compromise in energy n Look beyond the DA profile in RLS l The loss of IRP1 activity in RLS is not responsible for the decrease in D2 n Impact on IRP1 is possibly a direct metabolic effect

22. Connor Laboratory 2008 Matt McErchon Cezar Goletani

23. Thank you!