Cristina Malagelada

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.

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 Parkinsons 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.

Cell death pathways in Parkinson's disease: proximal triggers, distal effectors, and final steps.

Parkinson’s disease (PD) is a common neurodegenerative disorder. Neuronal cell death in PD is still poorly understood, despite a wealth of potential pathogenic mechanisms and pathways. Defects in several cellular systems have been implicated as early triggers that start cells down the road toward neuronal death. These include abnormal protein accumulation, particularly of alpha-synuclein; altered protein degradation via multiple pathways; mitochondrial dysfunction; oxidative stress; neuroinflammation; and dysregulated kinase signaling. As dysfunction in these systems mounts, pathways that are more explicitly involved in cell death become recruited. These include JNK signaling, p53 activation, cell cycle re-activation, and signaling through bcl-2 family proteins. Eventually, neurons become overwhelmed and degenerate; however, even the mechanism of final cell death in PD is still unsettled. In this review, we will discuss cell death triggers and effectors that are relevant to PD, highlighting important unresolved issues and implications for the development of neuroprotective therapies.

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 Parkinsons 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.

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 Parkinsons 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.

Puma and p53 Play Required Roles in Death Evoked in a Cellular Model of Parkinson Disease

Abstract6-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.

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

Parkinsons 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.

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.

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.

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.