Objective/Rationale:
The choreic and dystonic movements, or dyskinesia, which develop in response to prolonged treatment with levodopa, represent one of the major limitations to the current therapy for PD. Using a pre-clinical model of levodopa-induced dyskinesia (LID), we found that this condition is associated with increased activation of the mammalian target of rapamycin complex 1 (mTORC1), which affects neurotransmission by regulating protein synthesis. In this project, we will test the anti-dyskinetic efficacy of interventions aimed at reducing the activity of mTORC1 in specific groups of neurons.
Project Description:
We will use a pre-clinical model of LID generated by reproducing, in the brain, the same type of dopamine deficiency that occurs in PD. To reduce the activity of mTORC1 we will inhibit the synthesis of an important component of the complex, called Raptor. This will be achieved by means of viruses able to produce short sequences of mRNA that interfere with the translation of Raptor. These viruses will be injected in the striatum, which is the region of the brain where mTORC1 is abnormally activated during dyskinesia. This approach will reduce possible side effects caused by a more general blockade of mTORC1. We also plan to use a genetically altered pre-clinical model, in which specially constructed viruses can suppress Raptor expression and mTORC1 function selectively in a specific group of neurons, which we believe are particularly important for the development of LID.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Current pharmacological treatments for LID are only partially effective and rely on the use of drugs, such as amantadine, which produce negative side effects, including confusion, worsening of hallucination and edema. Moreover, dyskinesia has been shown to occur even after transplantation of embryonic dopamine neurons. The validation of mTORC1 as a target for the treatment of LID will provide information for the development of more effective anti-dyskinetic drugs, with lower liability for negative side effects, to be used in combination with different types of anti-parkinsonian therapies.
Anticipated Outcome:
In this study, we will assess the effect on levodopa-induced dyskinesia of inhibition of mTORC1 restricted to a specific brain region (i.e. the striatum) or a specific group of neurons. This approach has the advantage of reducing potential side-effects exerted by systemic inhibition of mTORC1. In addition, the selective knock down of Raptor will be a more specific intervention compared to pharmacological blockade of mTORC1 (which may affect other molecules involved in neurotransmission). Overall, these studies will provide conclusive evidence concerning the involvement of mTORC1 in LID and will prompt the search and development of novel therapies in this direction.
Progress Report
In this first year, we have generated viral vectors able to decrease the production of a protein called Raptor, which is an important regulator of protein synthesis. Decreased Raptor would counteract the activation of the mammalian target of rapamycin, which has been involved in the development of levodopa-induced dyskinesia. We have generated viruses able to nearly abolish the expression of Raptor in isolated cell cultures. These viruses have then been examined for their ability to reduce Raptor in intact pre-clinical models. In a series of initial experiments, we found that injection of the viruses in the striatum, which is the main brain region targeted by levodopa, reduced Raptor only partially. We are currently working at the optimization of the experimental procedure, in order to increase the efficiency of the viral injections and obtain a larger reduction of Raptor.
Final Outcome
Transfection with short hairpin RNA (shRNA) directed against the expression of Raptor, an essential component of the mammalian target of rapamycin complex 1 (mTORC1), produced a very large decrease in the levels of the protein, when tested in cultured cells. Lentiviral vectors expressing the same anti-Raptor shRNA were then examined for their ability to reduce Raptor in intact mice. We tested two protocols based on multiple (2 to 5) stereotaxic injections of increasing volumes of lentiviral vectors in the dorso-lateral striatum. Following this optimization, we examined the effect produced by lentiviral driven expression of anti-Raptor shRNA on the abnormal involuntary movements induced by L-DOPA in a mouse model of Parkinson’s disease. The effect of shRNA on the expression of Raptor in the striatum was modest (20-25% in average). Nevertheless, we observed a small reduction of abnormal involuntary movements (a marker of dyskinesia) in response to a low dose of L-DOPA. However, this effect disappeared when dyskinesia was enhanced with higher doses of L-DOPA. The decrease produced by anti-Raptor shRNA on the dyskinetic response induced by low dose L-DOPA correlated with reduced expression of Raptor and decreased mTORC1 signaling. These data suggest the requirement of Raptor in L-DOPA-induced dyskinesia. In order to corroborate these findings, we made an attempt to improve the downregulation of Raptor in vivo, using miRNA. Even in this case, we failed to observe a robust reduction in the levels of Raptor.
May 2014