RTP801 mediates transneuronal toxicity in culture via extracellular vesicles.

Extracellular vesicles (EVs) play a crucial role in intercellular communication, participating in the paracrine trophic support or in the propagation of toxic molecules, including proteins. RTP801 is a stress-regulated protein, whose levels are elevated during neurodegeneration and induce neuron death. However, whether RTP801 toxicity is transferred trans-neuronally via EVs remains unknown. Hence, we overexpressed or silenced RTP801 protein in cultured cortical neurons, isolated their derived EVs (RTP801-EVs or shRTP801-EVs, respectively), and characterized EVs protein content by mass spectrometry (MS). RTP801-EVs toxicity was assessed by treating cultured neurons with these EVs and quantifying apoptotic neuron death and branching. We also tested shRTP801-EVs functionality in the pathologic in vitro model of 6-Hydroxydopamine (6-OHDA). Expression of RTP801 increased the number of EVs released by neurons. Moreover, RTP801 led to a distinct proteomic signature of neuron-derived EVs, containing more pro-apoptotic markers. Hence, we observed that RTP801-induced toxicity was transferred to neurons via EVs, activating apoptosis and impairing neuron morphology complexity. In contrast, shRTP801-EVs were able to increase the arborization in recipient neurons. The 6-OHDA neurotoxin elevated levels of RTP801 in EVs, and 6-OHDA-derived EVs lost the mTOR/Akt signalling activation via Akt and RPS6 downstream effectors. Interestingly, EVs derived from neurons where RTP801 was silenced prior to exposing them to 6-OHDA maintained Akt and RPS6 transactivation in recipient neurons. Taken together, these results suggest that RTP801-induced toxicity is transferred via EVs, and therefore, it could contribute to the progression of neurodegenerative diseases, in which RTP801 is involved.

CD300f immune receptor contributes to healthy aging by regulating inflammaging, metabolism, and cognitive decline.

Emerging evidence suggests that immune receptors may participate in many aging-related processes such as energy metabolism, inflammation, and cognitive decline. CD300f, a TREM2-like lipid-sensing immune receptor, is an exceptional receptor as it integrates activating and inhibitory cell-signaling pathways that modulate inflammation, efferocytosis, and microglial metabolic fitness. We hypothesize that CD300f can regulate systemic aging-related processes and ultimately healthy lifespan. We closely followed several cohorts of two strains of CD300f-/- and WT mice of both sexes for 30 months and observed an important reduction in lifespan and healthspan in knockout mice. This was associated with systemic inflammaging, increased cognitive decline, reduced brain glucose uptake observed by 18FDG PET scans, enrichment in microglial aging/neurodegeneration phenotypes, proteostasis alterations, senescence, increased frailty, and sex-dependent systemic metabolic changes. Moreover, the absence of CD300f altered macrophage immunometabolic phenotype. Taken together, we provide strong evidence suggesting that myeloid cell CD300f immune receptor contributes to healthy aging.

STAT3 and REDD1: an unconventional story of gene repression.

The non-canonical functions of the transcription factor STAT3 have been poorly studied in comparison to its canonical mechanisms of gene expression activation. Here, Köhler et al. put the spotlight on a novel unconventional repressing mechanism of STAT3 over the REDD1 gene, named DDIT4. These findings are crucial to expand the knowledge of the stress-induced short-lived REDD1 protein that inactivates mTOR and the consequences of this fine-tuned regulation in the context of pathological conditions such as cancer or neurodegenerative diseases.

Increased Phospho-AKT in Blood Cells from LRRK2 G2019S Mutation Carriers.

The purpose of this study was to investigate whether  differential phosphorylation states of blood markers can identify patients with LRRK2 Parkinson's disease (PD). We assessed phospho(P)-Ser-935-LRRK2 and P-Ser-473-AKT levels in peripheral blood cells from patients with G2019S LRRK2-associated PD (L2PD, n = 31), G2019S LRRK2 non-manifesting carriers (L2NMC, n = 26), idiopathic PD (iPD, n = 25), and controls (n = 40, total n = 122). We found no differences at P-Ser-935-LRRK2 between groups but detected a specific increase of P-Ser-473-AKT levels in all G2019S carriers, either L2PD or L2NMC, absent in iPD. Although insensitive to LRRK2 inhibition, our study identifies P-Ser-473-AKT as an endogenous candidate biomarker for peripheral inflammation in G2019S carriers using accessible blood cells. ANN NEUROL 2022;92:888-894.

Synaptic RTP801 contributes to motor-learning dysfunction in Huntington's disease.

RTP801/REDD1 is a stress-responsive protein that mediates mutant huntingtin (mhtt) toxicity in cellular models and is up regulated in Huntington's disease (HD) patients' putamen. Here, we investigated whether RTP801 is involved in motor impairment in HD by affecting striatal synaptic plasticity. To explore this hypothesis, ectopic mhtt was over expressed in cultured rat primary neurons. Moreover, the protein levels of RTP801 were assessed in homogenates and crude synaptic fractions from human postmortem HD brains and mouse models of HD. Finally, striatal RTP801 expression was knocked down with adeno-associated viral particles containing a shRNA in the R6/1 mouse model of HD and motor learning was then tested. Ectopic mhtt elevated RTP801 in synapses of cultured neurons. RTP801 was also up regulated in striatal synapses from HD patients and mouse models. Knocking down RTP801 in the R6/1 mouse striatum prevented motor-learning impairment. RTP801 silencing normalized the Ser473 Akt hyperphosphorylation by downregulating Rictor and it induced synaptic elevation of calcium permeable GluA1 subunit and TrkB receptor levels, suggesting an enhancement in synaptic plasticity. These results indicate that mhtt-induced RTP801 mediates motor dysfunction in a HD murine model, revealing a potential role in the human disease. These findings open a new therapeutic framework focused on the RTP801/Akt/mTOR axis.

Increased Levels of Rictor Prevent Mutant Huntingtin-Induced Neuronal Degeneration.

Rictor associates with mTOR to form the mTORC2 complex, which activity regulates neuronal function and survival. Neurodegenerative diseases are characterized by the presence of neuronal dysfunction and cell death in specific brain regions such as for example Huntington's disease (HD), which is characterized by the loss of striatal projection neurons leading to motor dysfunction. Although HD is caused by the expression of mutant huntingtin, cell death occurs gradually suggesting that neurons have the capability to activate compensatory mechanisms to deal with neuronal dysfunction and later cell death. Here, we analyzed whether mTORC2 activity could be altered by the presence of mutant huntingtin. We observed that Rictor levels are specifically increased in the striatum of HD mouse models and in the putamen of HD patients. Rictor-mTOR interaction and the phosphorylation levels of Akt, one of the targets of the mTORC2 complex, were increased in the striatum of the R6/1 mouse model of HD suggesting increased mTORC2 signaling. Interestingly, acute downregulation of Rictor in striatal cells in vitro reduced mTORC2 activity, as shown by reduced levels of phospho-Akt, and increased mutant huntingtin-induced cell death. Accordingly, overexpression of Rictor increased mTORC2 activity counteracting cell death. Furthermore, normalization of endogenous Rictor levels in the striatum of R6/1 mouse worsened motor symptoms suggesting an induction of neuronal dysfunction. In conclusion, our results suggest that increased Rictor striatal levels could counteract neuronal dysfunction induced by mutant huntingtin.

Chelerythrine promotes Ca2+-dependent calpain activation in neuronal cells in a PKC-independent manner.

BACKGROUND: Chelerythrine is widely used as a broad range protein kinase C (PKC) inhibitor, but there is controversy about its inhibitory effect. Moreover, it has been shown to exert PKC-independent effects on non-neuronal cells. METHODS: In this study we investigated possible off-target effects of chelerythrine on cultured cortical rodent neurons and a neuronal cell line. RESULTS: We found that 10µM chelerythrine, a commonly used concentration in neuronal cultures, reduces PKC and cAMP-dependent protein kinase substrates phosphorylation in mouse cultured cortical neurons, but not in rat primary cortical neurons or in a striatal cell line. Furthermore, we found that incubation with chelerythrine increases pERK1/2 levels in all models studied. Moreover, our results show that chelerythrine promotes calpain activation as assessed by the cleavage of spectrin, striatal-enriched protein tyrosine phosphatase and calcineurin A. Remarkably, chelerythrine induces a concentration-dependent increase in intracellular Ca2+ levels that mediates calpain activation. In addition, we found that chelerythrine induces ERK1/2- and calpain-independent caspase-3 activation that can be prevented by the Ca2+ chelator BAPTA-AM. CONCLUSIONS: This is the first report showing that chelerythrine promotes Ca2+-dependent calpain activation in neuronal cells, which has consequences for the interpretation of studies using this compound. GENERAL SIGNIFICANCE: Chelerythrine is still marketed as a specific PKC inhibitor and extensively used in signal transduction studies. We believe that the described off-target effects should preclude its use as a PKC inhibitor in future works.

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 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.

ATF4 protects against neuronal death in cellular Parkinson's disease models by maintaining levels of parkin.

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 that parkin, 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.

Akt as a victim, villain and potential hero in Parkinson's disease pathophysiology and treatment.

There are two major purposes of this essay. The first is to summarize existing evidence that irrespective of the initiating causes, neuron death and degeneration in Parkinson's disease (PD) are due to the common feature of failure of signaling by Akt, a kinase involved in neuron survival and maintenance of synaptic contacts. The second is to consider possible means by which such a failure of Akt signaling might be benignly prevented or reversed in neurons affected by PD, so as to treat PD symptoms, block disease progression, and potentially, promote recovery.

RTP801/REDD1 regulates the timing of cortical neurogenesis and neuron migration.

The generation, differentiation, and migration of newborn neurons are critical features of normal brain development that are subject to both extracellular and intracellular regulation. However, the means of such control are only partially understood. Here, we show that expression of RTP801/REDD1, an inhibitor of mTOR (mammalian target of rapamycin) activation, is regulated during neuronal differentiation and that RTP801 functions to influence the timing of both neurogenesis and neuron migration. RTP801 levels are high in embryonic cortical neuroprogenitors, diminished in newborn neurons, and low in mature neurons. Knockdown of RTP801 in vitro and in vivo accelerates cell cycle exit by neuroprogenitors and their differentiation into neurons. It also disrupts migration of rat newborn neurons to the cortical plate and results in the ectopic localization of mature neurons. On the other hand, RTP801 overexpression delays neuronal differentiation. These findings suggest that endogenous RTP801 plays an essential role in temporal control of cortical development and in cortical patterning.

Rapamycin protects against neuron death in in vitro and in vivo models of Parkinson's disease.

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.

Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways.

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 IIgamma (CaMKIIgamma) and JNK. Remarkably, CaMKIIgamma 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 CaMKIIgamma-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.

Activation of caspase-8 by tumour necrosis factor receptor 1 is necessary for caspase-3 activation and apoptosis in oxygen-glucose deprived cultured cortical cells.

TNF-alpha has been reported to be relevant in stroke-induced neuronal death. However the precise function of TNF-alpha in brain ischemia remains controversial since there are data supporting either a detrimental or a protective effect. Here we show that TNF-alpha is released after oxygen-glucose deprivation (OGD) of cortical cultures and is a major contributor to the apoptotic death observed without affecting the OGD-mediated necrotic cell death. In this paradigm, apoptosis depends on TNF-alpha-induced activation of caspase-8 and -3 without affecting the activation of caspase-9. By using knock-out mice for TNF-alpha receptor 1, we show that the activation of both caspase-3 and -8 by TNF-alpha is mediated by TNF-alpha receptor 1. The pro-apoptotic role of TNF-alpha in OGD is restricted to neurons and microglia, since astrocytes do not express either TNF-alpha or TNF-alpha receptor 1. Altogether, these results show that apoptosis of cortical neurons after OGD is mediated by TNF-alpha/TNF-alpha receptor 1.

RTP801 is induced in Parkinson's disease and mediates neuron death by inhibiting Akt phosphorylation/activation.

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.

17beta-estradiol does not protect cerebellar granule cells from excitotoxicity or apoptosis.

Mounting evidences have suggested that 17beta-estradiol (E2) could have a neuroprotective action in the CNS. In the present study, we wanted to study whether this estrogen was able to protect cerebellar granule cells (CGCs) from apoptosis or excitotoxicity. Our results suggest that E2 has no anti-apoptotic effect in CGCs cultures. The lack of phosphoinositide 3-kinase/Akt pathway activation in CGCs cultures could be on the basis of the failure of estradiol to protect CGCs from potassium-deprivation and ceramide-mediated apoptosis. Moreover, E2 does not protect CGCs from glutamate-mediated death despite activating the extracellular signal regulated kinase kinase/extracellular signal regulated kinase pathway, which suggests that extracellular signal regulated kinase kinase/extracellular signal regulated kinase pathway activation is not sufficient to sustain an estrogen-mediated neuroprotective effect in CGCs cultures. By contrast, we found that the estrogen had a significant neuroprotective effect against hydrogen peroxide-mediated neuronal death. This effect was due to the antioxidant properties of the chemical structure of estradiol, as the biological inactive isomer 17alpha-estradiol was also able to reduce hydrogen peroxide-mediated neuronal death.

RTP801 is elevated in Parkinson brain substantia nigral neurons and mediates death in cellular models of Parkinson's disease by a mechanism involving mammalian target of rapamycin inactivation.

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.

N-methyl-D-aspartate blocks activation of JNK and mitochondrial apoptotic pathway induced by potassium deprivation in cerebellar granule cells.

During the postnatal development of cerebellum, lack of excitatory innervation from the mossy fibers results in cerebellar granule cell (CGC) apoptosis during the migration of the cells toward the internal granule cell layer. Accordingly, CGCs die by apoptosis when cultured in physiological KCl concentrations (5 mm; K5), and they survive in the presence of depolarizing conditions such as high KCl concentration (25 mm; K25) or N-methyl-D-aspartate (NMDA). We have recently shown that NMDA is able to exert a long lasting neuroprotective effect when added to immature (2 days in vitro) CGC cultures by inhibition of caspase-3 activity. Here we show that NMDA- and K25-mediated neuroprotection is associated with an increase in the levels of Bcl-2, an inhibition of K5-mediated increase in Bax, and the inhibition of the release of apoptogenic factors from mitochondria such as Smac/DIABLO and cytochrome c. Moreover, we have shown that similar effects are observed when c-Jun N-terminal kinases (JNKs) are inhibited and that treatment of CGC cultures with NMDA blocks K5-mediated JNK activation. These results allow us to postulate that the inhibition of JNK-mediated release of apoptogenic factors from mitochondria is involved in the NMDA protection from K5-mediated apoptosis of CGCs.

Puma and p53 play required roles in death evoked in a cellular model of Parkinson disease.

6-Hydroxydopamine (6-OHDA) is widely used in vivo and in vitro to mimic the selective neuronal degeneration that characterizes Parkinson disease (PD). To uncover candidate genes that may mediate neuron death in PD, we previously used SAGE to identify transcripts that are rapidly induced by 6-OHDA in neuronally differentiated PC12 cells. Among induced pro-apoptotic genes was that encoding the BH3-only protein PUMA. Here, we confirm that 6-OHDA induces both PUMA mRNA and protein. 6-OHDA additionally induced Bim, another pro-apoptotic BH3-only protein. Using specific siRNAs, we demonstrate that PUMA, but not Bim, is required for death evoked by 6-OHDA. PUMA is a target of p53, a transcription factor activated by 6-OHDA. Involvement of p53 in 6-OHDA evoked death was confirmed by the protective actions of a DN p53 and pifithrin alpha, inhibitors of p53 signaling. Our findings thus indicate that p53 and PUMA play required roles in a cellular model of PD.