We report that rapamycin, an allosteric inhibitor of certain but not all actions of the key cellular kinase mammalian target of rapamycin (mTOR), protects neurons from death in both cellular and animal toxin models of Parkinson's disease (PD). This protective action appears to be attributable to blocked translation of RTP801/REDD1/Ddit4, a protein that is induced in cell and animal models of PD and in affected neurons of PD patients and that causes neuron death by leading to dephosphorylation of the survival kinase Akt. In support of this mechanism, in PD models, rapamycin spares phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity. The capacity of rapamycin to provide neuroprotection in PD models appears to arise from its selective suppression of some but not all actions of mTOR, as indicated by the contrasting finding that Torin1, a full catalytic mTOR inhibitor, is not protective and induces Akt dephosphorylation and neuron death.

ER stress–induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress–mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress–induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ) and JNK. Remarkably, CaMKIIγ was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress–induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIγ-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress–induced apoptosis.

Previously, we reported that RTP801, a stress regulated protein, is induced in multiple cellular models of Parkinson's disease (PD), in an animal model of PD and in dopaminergic neurons of PD patients. In cellular PD models, RTP801 is both sufficient and necessary for death. We further showed that RTP801 and PD mimetics such as 6-OHDA trigger neuron death by suppressing activation of the key kinase mammalian target of rapamycin (mTOR). Here, we report that as a consequence of mTOR signaling blockade, 6-OHDA suppresses the phosphorylation and activation of Akt, a major supporter of neuron survival. This effect is mediated by RTP801 and appears to underlie neuron death induced by 6-OHDA. Examination of postmortem dopaminergic neurons reveals a consistent depletion of phospho-Akt, but not of total Akt in PD patients. These observations support a sequential mechanism in which PD-associated stresses induce RTP801, suppress mTOR signaling, deplete phosphorylated/activated Akt and permit neuron degeneration and death.

The molecules underlying neuron loss in Parkinson's disease (PD) are essentially unknown, and current therapies focus on diminishing symptoms rather than preventing neuron death. We identified RTP801 as a gene whose transcripts were highly induced in a cellular model of PD in which death of neuronal catecholaminergic PC12 cells was triggered by the PD mimetic 6-OHDA. Here, we find that RTP801 protein is also induced in this and additional cellular and animal PD models. To assess the relevance of these observations to PD, we used immunohistochemistry to compare RTP801 expression in postmortem brains from PD and control patients. For all PD brains examined, expression was highly elevated within neuromelanin-containing neurons of the substantia nigra but not in cerebellar neurons. Evaluation of the potential role of RTP801 induction in our cellular model revealed that RTP801 overexpression is sufficient to promote death but does not further elevate death caused by 6-OHDA. Furthermore, RTP801 induction is requisite for death in our cellular PD models and in 6-OHDA-treated cultured sympathetic neurons in that its knockdown by short hairpin RNAs (shRNAs) is protective. The mechanism by which 6-OHDA and RTP801 induce neuron death appears to involve repression of mammalian target of rapamycin (mTOR) kinase activity, and such death is inhibited by shRNAs targeting TSC2 (tuberous sclerosis complex), a protein with which RTP801 interacts to block mTOR activation. Our findings thus suggest that the elevation of RTP801 we detect in PD substantia nigral neurons may mediate their degeneration and death and that RTP801 and its signaling cascade may be novel potential therapeutic targets for the disease.

Parkinson's disease (PD) is a common neurodegenerative disorder, for which there are no effective disease-modifying therapies. The transcription factor ATF4 (activating transcription factor 4) is induced by multiple PD-relevant stressors, such as endoplasmic reticulum stress and oxidative damage. ATF4 may exert either protective or deleterious effects on cell survival, depending on the paradigm. However, the role of ATF4 in the pathogenesis of PD has not been explored. We find that ATF4 levels are increased in neuromelanin-positive neurons in the substantia nigra of a subset of PD patients relative to controls. ATF4 levels are also upregulated in neuronal PC12 cells treated with the dopaminergic neuronal toxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+). To explore the role of ATF4 in cell survival in PD-relevant contexts, we either silenced or overexpressed ATF4 in cellular models of PD. In neuronal PC12 cells, silencing of ATF4 enhanced cell death in response to either 6-OHDA or MPP+. Conversely, overexpression of ATF4 reduced cell death caused by dopaminergic neuronal toxins. ATF4 was also protective against 6-OHDA-induced death of cultured mouse ventral midbrain dopaminergic neurons. We further show thatparkin, a gene associated with autosomal recessive PD, plays a critical role in ATF4-mediated protection. After treatment with 6-OHDA or MPP+, parkin protein levels fall, despite an increase in mRNA levels. ATF4 silencing exacerbates the toxin-induced reduction of parkin, whereas ATF4 overexpression partially preserves parkin levels. Finally, parkin silencing blocked the protective capacity of ATF4. These results indicate that ATF4 plays a protective role in PD through the regulation of parkin.