STUDYING THE RAS-ERK PATHWAY THROUGH SINGLE-CELL ANALYSIS IN ACUTE STRIATAL SLICES
Creators: name: MORELLA, ILARIA MARIA
Publisher: name: Universit� degli Studi di Milano
Year: 2013

description: In the central nervous system, the Ras-ERK signaling module, triggered by the small GTPases of the Ras family, plays a key role in different forms of behavioural plasticity, including learning and consolidation of long-term memory. In particular, in the striatum the combined engagement of dopaminergic and glutamatergic pathways leads to a sustained activation of the Ras-ERK pathway, necessary for the normal striatal functions, including learning of procedural actions and habit formation, but also for the behavioural responses to the rewarding properties of drugs of abuse. In addition, an aberrant ERK hyperactivation is implicated in the development of L-DOPA- induced dyskinesia (LID), a severe and disabling pathology characterized by involuntary movements that develop as consequence of L-DOPA treatment in parkinsonian patients.
In this work, I showed the development of a new experimental approach in which striatal slices acutely prepared from adult mice can be incubated in a perfusing chamber and challenged with appropriate agonists and antagonists. First, I validated our system by Western blot analysis performed on striatal slices stimulated for 10 min with glutamate and I confirmed a significant ERK1/2 activation. Next, I developed a system to study ERK activation at a single cell definition monitoring by immunofluorescence analysis the phosphorylation state of two proteins downstream of ERK1/2: S6 ribosomal protein (cytoplasmic target) and histone H3 (nuclear target). I demonstrated that both these proteins are robustly activated upon the application of glutamate, the D1Rs agonist SKF38393 or the neurotrophin BDNF 100 ?M and that this event is specifically regulated by the ERK cascade, thus demonstrating that these two proteins are reliable indicators of ERK induction. 
Having validated this experimental setting, I next investigated the integration between dopaminergic and glutamatergic signaling specifically in the D1Rs-expressing neurons (direct pathway) and in the D2Rs-expressing neurons (indirect pathway) in adult striatal slices obtained from M4-EGFP mice, expressing EGFP in the direct pathway. My results indicate that an additive integration between glutamate and SKF38393 is necessary to elicit S6, but not histone H3 phosphorylation, and this event is restricted to the direct pathway. Unexpectedly, the application of the D1Rs agonist activates S6 ribosomal protein also in the indirect pathway in a D1Rs-dependent manner. Further experiments will be necessary to clarify the exact mechanism, either direct or indirect, through which D1Rs stimulation mediates S6 activation in the indirect pathway. In addition, I demonstrated that, whereas D1Rs-mediated signalling depends on glutamatergic activity, glutamatergic activity does not entirely depend on dopaminergic signalling since glutamate-mediated S6 phosphorylation is only partially inhibited (48% of inhibition) following D1Rs blockade. In addition, in the direct pathway, S6 activation seems to be more inhibited (55%) by D1Rs antagonist with respect to the indirect pathway (41%). Further experiments will be performed also in A2A-EGFP mice, expressing EGFP in the indirect pathway, in order to confirm the results concerning the indirect pathway.
In the last part of the project, I used the technique I developed to investigate the role of the Ras-ERK signalling in LID. In particular, I demonstrated that the antidyskinetic property of the recent discovered NOP receptor agonist (N/OFQ) correlates with a negative modulation of D1Rs-mediated signalling in striatal slices obtained from na�ve mice, highlighting NOP receptor as a possible drug target. Successively, since recent evidence has demostrated that ERK hyperactivation in dyskinetic mice is reduced by the loss of Ras-GRF1, a neuronal specific Ras exchange factor, ameliorating the dyskinetic symptoms, I wanted to further investigate the effect of Ras-GRF1 ablation on either S6 and histone H3 activation in dyskinetic mice; my results have demonstrated that histone H3 phosphorylation as well as ERK phosphorylation are reduced in dyskinetic Ras-GRF1 KO mice, whereas the level of S6 phosphorylation does not change in comparison to wild-type mice. These findings may indicate that the chronic treatment with L-DOPA produces some adaptive processes that bypass Ras-GRF1 and possibly ERK requirement. For instance, lack of Ras-GRF1 may be compensated by other calcium-sensitive Ras-GEFs, including the striatal-enriched members of Ras-GRP/CalDAG GEFs family, although I cannot exclude that other pathways, including mTOR, converge to activate S6 in dyskinetic mice effectively bypassing ERK. In order to test our hypothesis, further experiments will be performed, for instance by treating dyskinetic wild-type and Ras-GRF1 KO mice or striatal slices obtained from these animals with a MEK inhibitor, to investigate the effect of ERK blockade on L-DOPA induced S6 hyperactivation.