RTP801 PARKIN · RTP801, A NEGATIVE REGULATOR OF mTOR AND AKT, AS A TARGET OF PARKIN""

RTP801, A NEGATIVE REGULATOR OF mTOR AND AKT, AS A TARGET OF PARKIN

SCIENTIFIC OBJECTIVES The goals of this study are to determine whether levels of RTP801 are regulated by parkin, whether parkin mutants cause cell death and if so, whether this death is associated to an accumulation of RTP801. If this is correct, then we will determine the mechanisms by which death occurs and we will compare them with our current working model in which RTP801 is sufficient to induce cell death by sequentially inactivating mTOR and Akt. Such information may lead to a better understanding of PD-associated neurodegeneration and therefore, to PD treatments aimed at RTP801 in both genetic and idiopathic cases. Using cellular models and animal models, our specific aims are: 1. To investigate whether RTP801 is a substrate and/or transcriptional target for parkin. 2. To determine whether loss of parkin activity results in accumulation of RTP801 and neuron cell death. 3. To investigate whether mutant parkin, via RTP801, triggers cell death, and if so, whether it is done by down-regulating mTOR and Akt activity. 4. To determine whether RTP801 is elevated in SN dopaminergic neurons of post-mortem brains of patients with parkin-associated PD and in mutated parkin animal models of PD (if and when available). SUMMARY OVERVIEW OF RESULTS: Mutations in the PARK2 gene are associated with an autosomal recessive form of juvenile parkinsonism (AR-JP). These mutations affect parkin solubility and impair its E3 ligase activity, leading to a toxic accumulation of proteins within susceptible neurons that results in a slow but progressive neuronal degeneration and cell death. Here, we report that RTP801/ REDD1, a pro-apoptotic negative regulator of survival kinases mTOR and Akt, is one of such parkin substrates. We observed that parkin knockdown elevated RTP801 in sympathetic neurons and neuronal PC12 cells, whereas ectopic parkin enhanced RTP801 poly-ubiquitination and proteasomal degradation. In parkin knockout mouse brains and in human fibroblasts from AR-JP patients with parkin mutations, RTP801 levels were elevated. Moreover, in human postmortem PD brains with mutated parkin, nigral neurons were highly positive for RTP801. Further consistent with the idea that RTP801 is a substrate for parkin, the two endogenous proteins interacted in reciprocal co-immunoprecipitates of cell lysates. A potential physiological role for parkin-mediated RTP801 degradation is indicated by observations that parkin protects neuronal cells from death caused by RTP801 overexpression by mediating its degradation, whereas parkin knockdown exacerbates such death. Similarly, parkin knockdown enhanced RTP801 induction in neuronal cells exposed to the Parkinson’s disease mimetic 6-hydroxydopamine and increased sensitivity to this toxin. This response to parkin loss of function appeared to be mediated by RTP801 as it was abolished by RTP801 knockdown. Taken together these results indicate that RTP801 is a novel parkin substrate that may contribute to neurodegeneration caused by loss of parkin expression or activity. CONCLUSIONS: Our work in cellular and animal models and in human samples strongly indicates that RTP801 is a substrate of parkin and that RTP801 elevation due to parkin loss of function in both AR-JP and sporadic PD may contribute to neurodegeneration. Any genetic conditions or stress situa- tions that compromise parkin activity have the potential to produce a toxic accumulation of RTP801. This is relevant to design of therapeutic approaches to promote neuron survival and block neurodegeneration in both sporadic and AR-JP. SOCIO-ECONOMIC IMPACTS: The results that we achieved so far within this project helped to start a whole new line of investigation in the department of Pharmacology. During these years, I started up a research group to investigate the signaling pathways responsible of neurodegeneration in PD, using cellular and animal models. All this work lead to publish two papers related directly to the project, one review and another paper that shows RTP801 protein as a new target for mutated huntingtin in Huntington’s disease. Moreover the project helped to finance three PhD thesis that soon will be defended. Our knowledge of neurodegeneration and signaling helped to establish very productive collaborations with other groups in the University of Barcelona and in the Hospital Clínic: a) We established collaboration with the group of Dr. Eduard Tolosa. He is one of the best neurologists in Barcelona and he treats and monitors all kind of PD patients in the Hospital Clínic. We obtained from them primary human skin fibroblasts from patients carrying parkin mutations. With these cultures we could test our initial hypothesis and we confirmed that RTP801 is more elevated in these parkin mutant patients. b) We established collaboration with the group of Dr. Esther Perez-Navarro and Jordi Alberch. They are studying the molecular basis of neurodegeneration in Huntington’s disease. We are working together to study the role of pro-apoptotic protein RTP801 in HD models and in the disease itself. The paper was accepted in april 2015. c) We also helped the group of Dr. Amalia Lafuente in the department of Pharmacology with their in vitro and biochemical studies, since they only had experience in pharmacogenetics. We also screened for SNPs in the mTOR pathway that could sensitize schizophrenic patients to anti-psychotics adverse effects, like the extrapyramidal syndrome (EPS), and we found that the combination of SNPs in four genes of the mTOR pathway, DDIT4/RTP801, FCHSD1 (that regulates DDIT4/RTP801), raptor and Akt, predicted a higher chance to suffer from EPS, that is a pharmacological form of Parkinson. With this study we submitted a patent (patent number: EP13382027.4) to use these four SNPs as a predictor to EPS in schizophrenic patients and choose the best antipsychotic for them. We are also planning to explore these four SNPs as a combinational early biomarker for PD. All these collaborations and transfer of knowledge within the department and in the university/hospital will help my permanent integration in a near future.

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