Study of the mTOR pathway in neurodegenerative diseases: from synapses to genes

Abstract

Huntington's disease (HD) and Parkinson's disease (PD) are devastating neurodegenerative diseases that progress with the death of selective neuronal subpopulations. Neuronal dysfunction and death are consequence of multiple pathogenic processes which alter signalling cascades. The identification of such molecular pathways is crucial to understand the cellular processes that triggers the symptomatology of diseases. One of the common affected pathways in neurodegeneration is the mTOR pathway. It regulates multiple cellular processes to preserve cellular viability and function. Consequently, to maintain a proper function mTOR activity needs to be fine-tuned. RTP801 is an mTOR negative regulator whose action over this pathway plays a significant role in neurodegeneration. RTP801 protein is induced in an attempt to cope cellular stress. However a sustained RTP801 increase leads to neuronal death by sequentially inactivating first mTOR and then Akt pro-survival kinase. RTP801 pathological increase has been involved in neurodegenerative diseases such as PD. Therefore, identifying RTP801 as a possible new therapeutic target in HD would be highly valuable in designing new pharmacological therapies that block, or at least delay, the neurodegeneration and changes in synaptic plasticity associated with it. Our results show that the overexpression of pathogenic N-terminal htt increases RTP801 levels by both lengthening the protein half-life and up-regulating its gene expression. Blockade of RTP801 expression prevents mhtt-induced cell death in HD cellular models. Importantly, RTP801 is elevated in HD-iPSC and putamen, caudate nucleus and cerebellum of human HD post-mortem brain. Although total RTP801 levels in the striatum of HD murine models are not altered, RTP801 is increased in the synaptic compartment which contributes to motor learning deficits in the R6/1 model. Downregulation of striatal RTP801 preserves motor learning skills in R6/1 mice. Hence, RTP801 is identified as a novel downstream effector of mhtt which mediates its toxicity. Recently, exosomes have emerged as a key mechanism to maintain trophic support between neural cells and as vehicle for the clearance of toxic proteins from neurons. Since RTP801 is upregulated under stressful conditions, its propagation by exosomes may allow the neuron-to-neuron spreading of RTP801 toxicity through the modulation of mTOR/Akt pathway. Our results have elucidated a novel function of RTP801 as an exosomal protein. We demonstrate that both ectopic and endogenous RTP801 can be found in exosomes derived from HEK293 cells. In cortical neurons, exosomal RTP801 elevation is sensitive to PD mimetic 6-OHDA (6-hydroxydopamine) but not to potassium depolarization. Consequently, 6-OHDA exposure induces the loading of RTP801 into exosomes released from cortical neurons. Intriguingly, mhtt does not elevate RTP801 in exosomes obtained from a cellular model. In addition, we demonstrate that exosomes have a protective role promoting the activation of both mTOR complex 1 and 2 in recipient neurons, but increased RTP801 counteracts exosomal mTOR pathway activation suggesting that RTP801 negatively modulates pro-survival signals transneuronally. Altered protein functions of the mTOR pathway are a common hallmark in many neurodegenerative diseases. However, little is known about the contribution of genetic variants or single nucleotide polymorphisms (SNPs) that belong to the mTOR pathway. As a multifactorial disease, we studied the association of SNPs in genes encoding mTOR pathway protein components with the susceptibility PD and the response to levodopa (L-DOPA) treatment. The data found indicate that polymorphisms in genetic markers of the mTOR pathway contribute to the susceptibility to PD and the response to L-DOPA treatment in PD patients. We show that these SNPs influence the outcome individually or interacting epistatically with other genetic markers. Taken together, our findings indicate that deregulation of the mTOR signalling pathway plays an important role in the pathogenesis associated with PE and HD and its regulation is crucial to maintain adequate neuronal function and viability.

La enfermedad de Huntington (EH) y la enfermedad de Parkinson (EP) son enfermedades neurodegenerativas devastadoras caracterizadas por la muerte de subpoblaciones neuronales selectivas. La disfunción neuronal y la muerte son consecuencia de múltiples procesos patogénicos que llevan a la alteración de cascadas de señalización. Una de las vías afectadas de forma común en los procesos neurodegenerativos es la vía mTOR. Como modulador de numerosos procesos celulares, la vía de mTOR está regulada para mantener la supervivencia neuronal y la plasticidad sináptica. Una de las proteínas que modula esta cascada de señalización es RTP801. RTP801 se induce en respuesta a factores de estrés celular y su aumento desencadena la muerte neuronal al regular negativamente la vía mTOR/Akt. La implicación de RTP801 en la EP ha sido ampliamente estudiada, sin embargo, su contribución a la patogénesis de la EH nunca antes había sido explorada. Específicamente, nuestros resultados han identificado a RTP801 como un mediador de la toxicidad inducida por huntingtina mutada. El aumento de RTP801 medía la muerte celular inducida por huntingtina mutada y contribuye a la disfunción del aprendizaje motor en el modelo murino R6/1. El silenciamiento de RTP801 en el estriado de los ratones R6/1 contribuye a preservar la plasticidad sináptica de la vía corticoestriatal, y por tanto del aprendizaje motor. Por otra parte, mostramos que los exosomas secretados por neuronas activan la vía de supervivencia mTOR/Akt en neuronas recipientes. Sin embargo, ante un estrés celular, la toxicidad de RTP801 es propagada a través de exosomas que contrarrestan la activación trófica de la vía mTOR/Akt. Finalmente, demostramos que variaciones genéticas en los componentes de la vía de mTOR modulan la susceptibilidad y la edad de inicio de la EP y, contribuyen a la aparición y severidad de la discinesia inducida por levodopa. En conjunto, nuestros hallazgos indican que la desregulación de la vía de mTOR desempeña un papel importante en la patogénesis asociada a la EP y la EH y, su correcta regulación es crucial para mantener la viabilidad y función neuronal.