RTP801/REDD1: a stress coping regulator that turns into a troublemaker in neurodegenerative disorders

Mechanistic target of Rapamycin (mTOR) pathway regulates essential processes directed to preserve cellular homeostasis, such as cell growth, proliferation, survival, protein synthesis and autophagy. Importantly, mTOR pathway deregulation has been related to many diseases. Indeed, it has become a hallmark in neurodegenerative disorders, since a fine-tuned regulation of mTOR activities is crucial for neuron function and survival. RTP801/REDD1/Dig2 has become one of the most puzzling regulators of mTOR. Although the mechanism is not completely understood, RTP801 inactivates mTOR and Akt via the tuberous sclerosis complex (TSC1/TSC2) in many cellular contexts. Intriguingly, RTP801 protects dividing cells from hypoxia or H2O2-induced apoptosis, while it sensitizes differentiated cells to stress. Based on experimental models of Parkinson's disease (PD), it has been proposed that at early stages of the disease, stress-induced RTP801 upregulation contributes to mTOR repression, in an attempt to maintain cell function and viability. However, if RTP801 elevation is sustained, it leads to neuron cell death by a sequential inhibition of mTOR and Akt. Here, we will review RTP801 deregulation of mTOR in a context of PD and other neurodegenerative disorders

RTP801 is involved in mutant huntingtin-induced cell death

RTP801 expression is induced by cellular stress and has a pro-apoptotic function in non-proliferating differentiated cells such as neurons. In several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease, elevated levels of RTP801 have been observed, which suggests a role for RTP801 in neuronal death. Neuronal death is also a pathological hallmark in Huntington's disease (HD), an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Currently, the exact mechanisms underlying mutant huntingtin (mhtt)-induced toxicity are still unclear. Here, we investigated whether RTP801 is involved in (mhtt)-induced cell death. Ectopic exon-1 mhtt elevated RTP801 mRNA and protein levels in nerve growth factor (NGF)-differentiated PC12 cells and in rat primary cortical neurons. In neuronal PC12 cells, mhtt also contributed to RTP801 protein elevation by reducing its proteasomal degradation rate, in addition to promoting RTP801 gene expression. Interestingly, silencing RTP801 expression with short hairpin RNAs (shRNAs) blocked mhtt-induced cell death in NGF-differentiated PC12 cells. However, RTP801 protein levels were not altered in the striatum of Hdh(Q7/Q111) and R6/1 mice, two HD models that display motor deficits but not neuronal death. Importantly, RTP801 protein levels were elevated in both neural telencephalic progenitors differentiated from HD patient-derived induced pluripotent stem cells and in the putamen and cerebellum of human HD postmortem brains. Taken together, our results suggest that RTP801 is a novel downstream effector of mhtt-induced toxicity and that it may be relevant to the human disease

Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

Parkinson's disease (PD) is a disorder characterized by the degeneration of certain neuronal populations in the central and peripheral nervous system. One of the hallmarks of the disease is the toxic accumulation of proteins within susceptible neurons due to major impairment in the degradation/clearance protein systems. RTP801 is a pro-apoptotic protein that is sufficient and necessary to induce neuronal death in cellular and animal models of PD. RTP801 is also upregulated in sporadic and parkin mutant PD brains. Here, we report the role of NEDD4, an E3 ligase involved in α-synuclein degradation and PD pathogenesis, in the regulation of RTP801 protein levels and toxicity. NEDD4 polyubiquitinates RTP801 in a cell-free system and in cellular cultures, and they interact physically. NEDD4 conjugates K63-ubiquitin chains to RTP801 and targets it for degradation. NEDD4 regulates RTP801 protein levels in both cultured cells and in the brain tissue. NEDD4 levels are diminished in nigral neurons from human PD brains. Interestingly, neurotoxin 6-OHDA decreases dramatically NEDD4 protein expression but elevates RTP801 protein levels. Moreover, NEDD4 protects neuronal PC12 cells from both 6-OHDA and RTP801-induced toxicity. In primary cortical neurons, NEDD4 knockdown toxicity is mediated by RTP801 since the double knockdown of RTP801 and NEDD4 abrogates the loss of phospho Ser473-Akt and the appearance of caspase-cleaved spectrin fragments. Thus, NEDD4 ligase regulates RTP801 and is sensitive to PD-associated oxidative stress. This suggests that NEDD4 loss of function in PD could contribute importantly into neuronal death by elevating RTP801

Parkin loss of function contributes to RTP801 elevation and neurodegeneration in Parkinson"s disease

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

Study of pro-apoptotic protein RTP801 homeostasis and its regulation by NEDD4 in Parkinson's disease

[eng] Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons of the Substantia Nigra pars compacta (SNpc) and the presence of cytoplasmic protein inclusions named Lewy Bodies. Current treatments are directed principally to ameliorate the clinical manifestations of the disease rather than suppressing the underlying neuron degeneration and death. This is due, in part, to an incomplete understanding of the pathways that lead to neurodegeneration in PD. RTP801 is induced in cellular and animal models of Parkinson's disease (PD) and is elevated in neuromelanin positive neurons in the Substantia Nigra (SN) of PD patients. In a variety of neuronal systems, RTP801 overexpression or upregulation is sufficient to trigger cell death. This involves a sequential mechanism in which it sequentially inactivates mTOR and survival kinase Akt. RTP801 is a protein with a very short half-life (2-7 minutes), so it should be strictly and dynamically controlled at post-translational level to precisely modulate mTOR pathway. Hence, elucidating which proteins mediate RTP801 degradation would be a stepping-stone to design new therapies to block neurodegeneration. NEDD4, is an E3 ubiquitin ligase highly expressed in mammalian neurons that has been directly linked to PD pathogenesis since it has been reported to promote α-synuclein degradation and to be protective against its toxicity. In this work, we report that NEDD4 contributes to RTP801 protein degradation. We show in neuronal cells that there is a pool of RTP801 degraded via lysosomal pathway. We also demonstrate that both proteins interact and that NEDD4 enhances RTP801 polyubiquitination by preferentially conjugating K63 ubiquitin chains. Importantly, NEDD4 regulates RTP801 protein levels in cultured cells and in a conditional knockout mouse model. We also provide evidence that NEDD4 protects against RTP801 toxicity by mediating its polyubiquitination. Furthermore, NEDD4 levels are decreased in the 6-OHDA PD cellular model and its restoration protects against 6-OHDA-induced cell death by reducing RTP801 protein levels. Moreover, NEDD4 loss of function is toxic in neurons due to RTP801 elevation and the subsequent mTOR/Akt inactivation. A role for NEDD4 in PD is supported by observation of diminished NEDD4 in nigral neurons from postmortem sporadic PD brains. In line with these findings, the NEDD4 signaling inducer NAB2 reduces RTP801 protein levels in control and in 6-OHDA-treated cortical neurons. However, NAB2 is not sufficient to confer protection against 6-OHDA toxicity, because the compound itself represses mTOR signaling pathway compromising cell survival.[spa] RTP801/REDD1 es una proteína pro-apoptótica que se encuentra aumentada en modelos celulares y animales y en muestras humanas de la enfermedad de Parkinson. RTP801 es suficiente y necesaria para promover muerte celular mediante un mecanismo de inhibición de la quinasas de supervivencia mTOR y Akt. NEDD4 es una E3 ubiquitina ligasa que ubiquitiniza proteínas señalizándolas para su posterior degradación. Esta proteína se ha relacionado recientemente con la enfermedad de Parkinson, ya que se ha visto que ubiquitiniza la proteína alfa-sinucleína. En este trabajo, se ha descrito que la RTP801 es un sustrato de la ubiquitina ligasa NEDD4. Se ha demostrado que ambas proteínas interaccionan y que NEDD4 ubiquitiniza RTP801 con cadenas de ubiquitina del tipo K63, señalizándola para su posterior degradación lisosomal. También, se ha observado que NEDD4 regula los niveles de RTP801 en sistemas neuronales, y que la pérdida de función de NEDD4 es tóxica debido al incremento de RTP801 y la consecuente inhibición de las quinasas mTOR/Akt. De forma interesante, la NEDD4 ectópica es capaz de proteger ante la toxicidad provocada por la sobreexpresión de RTP801 y ante la toxina parkinsoniana 6-OHDA. Además, la proteína NEDD4 se encuentra disminuida en neuronas nigrales de la SNpc de cerebros post mortem con la enfermedad de Parkinson. De forma consistente, el compuesto NAB2, un activador de NEDD4, disminuye los niveles de la RTP801 en neuronas. No obstante, la acción de este compuesto no es suficiente para proteger ante la toxicidad de la 6-OHDA. También, se ha identificado una posible regulación entre las E3 ligasas parkina y NEDD4, que conferiría un grado de complejidad mayor a la regulación de la RTP801. Diferentes mutantes de la proteína RTP801 han sido generados para estudiar cómo afecta la ubiquitinización a su estabilidad y función. De forma interesante, el mutante RTP801-K185R pierde la función pro-apoptótica de la RTP801, poniendo en relieve que la ubiquitinización en este residuo es crítica para su función. Finalmente, mediante estudios proteómicos se han identificado numerosos interactores de RTP801 putativos, entre los cuáles se encuentra la clatrina y la proteína adaptadora AP-2, sugiriendo un posible papel de la RTP801 en procesos relacionados con la endocitosis mediada por clatrina

RTP801/REDD1: a stress coping regulator that turns into a troublemaker in neurodegenerative disorders

Mechanistic target of Rapamycin (mTOR) pathway regulates essential processes directed to preserve cellular homeostasis, such as cell growth, proliferation, survival, protein synthesis and autophagy. Importantly, mTOR pathway deregulation has been related to many diseases. Indeed, it has become a hallmark in neurodegenerative disorders, since a fine-tuned regulation of mTOR activities is crucial for neuron function and survival. RTP801/REDD1/Dig2 has become one of the most puzzling regulators of mTOR. Although the mechanism is not completely understood, RTP801 inactivates mTOR and Akt via the tuberous sclerosis complex (TSC1/TSC2) in many cellular contexts. Intriguingly, RTP801 protects dividing cells from hypoxia or H2O2-induced apoptosis, while it sensitizes differentiated cells to stress. Based on experimental models of Parkinson's disease (PD), it has been proposed that at early stages of the disease, stress-induced RTP801 upregulation contributes to mTOR repression, in an attempt to maintain cell function and viability. However, if RTP801 elevation is sustained, it leads to neuron cell death by a sequential inhibition of mTOR and Akt. Here, we will review RTP801 deregulation of mTOR in a context of PD and other neurodegenerative disorders

Loss of NEDD4 contributes to RTP801 elevation and neuron toxicity: implications for Parkinson's disease

Parkinson's disease (PD) is a disorder characterized by the degeneration of certain neuronal populations in the central and peripheral nervous system. One of the hallmarks of the disease is the toxic accumulation of proteins within susceptible neurons due to major impairment in the degradation/clearance protein systems. RTP801 is a pro-apoptotic protein that is sufficient and necessary to induce neuronal death in cellular and animal models of PD. RTP801 is also upregulated in sporadic and parkin mutant PD brains. Here, we report the role of NEDD4, an E3 ligase involved in α-synuclein degradation and PD pathogenesis, in the regulation of RTP801 protein levels and toxicity. NEDD4 polyubiquitinates RTP801 in a cell-free system and in cellular cultures, and they interact physically. NEDD4 conjugates K63-ubiquitin chains to RTP801 and targets it for degradation. NEDD4 regulates RTP801 protein levels in both cultured cells and in the brain tissue. NEDD4 levels are diminished in nigral neurons from human PD brains. Interestingly, neurotoxin 6-OHDA decreases dramatically NEDD4 protein expression but elevates RTP801 protein levels. Moreover, NEDD4 protects neuronal PC12 cells from both 6-OHDA and RTP801-induced toxicity. In primary cortical neurons, NEDD4 knockdown toxicity is mediated by RTP801 since the double knockdown of RTP801 and NEDD4 abrogates the loss of phospho Ser473-Akt and the appearance of caspase-cleaved spectrin fragments. Thus, NEDD4 ligase regulates RTP801 and is sensitive to PD-associated oxidative stress. This suggests that NEDD4 loss of function in PD could contribute importantly into neuronal death by elevating RTP801

RTP801 regulates motor cortex synaptic transmission and learning

Background: RTP801/REDD1 is a stress-regulated protein whose upregulation is necessary and sufficient to trigger neuronal death in in vitro and in vivo models of Parkinson's and Huntington's diseases and is up regulated in compromised neurons in human postmortem brains of both neurodegenerative disorders. Indeed, in both Parkinson's and Huntington's disease mouse models, RTP801 knockdown alleviates motor-learning deficits. Results: We investigated the physiological role of RTP801 in neuronal plasticity and we found RTP801 in rat, mouse and human synapses. The absence of RTP801 enhanced excitatory synaptic transmission in both neuronal cultures and brain slices from RTP801 knock-out (KO) mice. Indeed, RTP801 KO mice showed improved motor learning, which correlated with lower spine density but increased basal filopodia and mushroom spines in the motor cortex layer V. This paralleled with higher levels of synaptosomal GluA1 and TrkB receptors in homogenates derived from KO mice motor cortex, proteins that are associated with synaptic strengthening.Conclusions: Altogether, these results indicate that RTP801 has an important role modulating neuronal plasticity and motor learning. They will help to understand its role in neurodegenerative disorders where RTP801 levels are detrimentally upregulated

Parkin loss of function contributes to RTP801 elevation and neurodegeneration in Parkinson"s disease

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