Steve Callaghan

Loss of the Parkinson’s Disease-linked gene DJ-1 perturbs mitochondrial dynamics

Growing evidence highlights a role for mitochondrial dysfunction and oxidative stress as underlying contributors to Parkinsons disease (PD) pathogenesis. DJ-1 (PARK7) is a recently identified recessive familial PD gene. Its loss leads to increased susceptibility of neurons to oxidative stress and death. However, its mechanism of action is not fully understood. Presently, we report that DJ-1 deficiency in cell lines, cultured neurons, mouse brain and lymphoblast cells derived from DJ-1 patients display aberrant mitochondrial morphology. We also show that these DJ-1-dependent mitochondrial defects contribute to oxidative stress-induced sensitivity to cell death since reversal of this fragmented mitochondrial phenotype abrogates neuronal cell death. Reactive oxygen species (ROS) appear to play a critical role in the observed defects, as ROS scavengers rescue the phenotype and mitochondria isolated from DJ-1 deficient animals produce more ROS compared with control. Importantly, the aberrant mitochondrial phenotype can be rescued by the expression of Pink1 and Parkin, two PD-linked genes involved in regulating mitochondrial dynamics and quality control. Finally, we show that DJ-1 deficiency leads to altered autophagy in murine and human cells. Our findings define a mechanism by which the DJ-1-dependent mitochondrial defects contribute to the increased sensitivity to oxidative stress-induced cell death that has been previously reported.

Cytoplasmic Pink1 activity protects neurons from dopaminergic neurotoxin MPTP

PTEN-induced putative kinase 1 (Pink1) is a recently identified gene linked to a recessive form of familial Parkinsons disease (PD). The kinase contains a mitochondrial localization sequence and is reported to reside, at least in part, in mitochondria. However, neither the manner by which the loss of Pink1 contributes to dopamine neuron loss nor its impact on mitochondrial function and relevance to death is clear. Here, we report that depletion of Pink1 by RNAi increased neuronal toxicity induced by MPP+. Moreover, wild-type Pink1, but not the G309D mutant linked to familial PD or an engineered kinase-dead mutant K219M, protects neurons against MPTP both in vitro and in vivo. Intriguingly, a mutant that contains a deletion of the putative mitochondrial-targeting motif was targeted to the cytoplasm but still provided protection against 1-methyl-4-phenylpyridine (MPP+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity. In addition, we also show that endogenous Pink1 is localized to cytosolic as well as mitochondrial fractions. Thus, our findings indicate that Pink1 plays a functional role in the survival of neurons and that cytoplasmic targets, in addition to its other actions in the mitochondria, may be important for this protective effect.

Role of Cdk5-Mediated Phosphorylation of Prx2 in MPTP Toxicity and Parkinson's Disease

We reported previously that calpain-mediated Cdk5 activation is critical for mitochondrial toxin-induced dopaminergic death. Here, we report a target that mediates this loss. Prx2, an antioxidant enzyme, binds Cdk5/p35. Prx2 is phosphorylated at T89 in neurons treated with MPP+ and/or MPTP in animals in a calpain/Cdk5/p35-dependent manner. This phosphorylation reduces Prx2 peroxidase activity. Consistent with this, p35-/- neurons show reduced oxidative stress upon MPP+ treatment. Expression of Prx2 and Prx2T89A, but not the phosphorylation mimic Prx2T89E, protects cultured and adult neurons following mitochondrial insult. Finally, downregulation of Prx2 increases oxidative stress and sensitivity to MPP+. We propose a mechanistic model by which mitochondrial toxin leads to calpain-mediated Cdk5 activation, reduced Prx2 activity, and decreased capacity to eliminate ROS. Importantly, increased Prx2 phosphorylation also occurs in nigral neurons from postmortem tissue from Parkinsons disease patients when compared to control, suggesting the relevance of this pathway in the human condition.

Calpain-Regulated p35/cdk5 Plays a Central Role in Dopaminergic Neuron Death through Modulation of the Transcription Factor Myocyte Enhancer Factor 2

The mechanisms underlying dopamine neuron loss in Parkinsons disease (PD) are not clearly defined. Here, we delineate a pathway by which dopaminergic loss induced by 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP) is controlled in vivo. We reported previously that calpains play a central required role in dopamine loss after MPTP treatment. Here, we provide evidence that the downstream effector pathway of calpains is through cyclin-dependent kinase 5 (cdk5)-mediated modulation of the transcription factor myocyte enhancer factor 2 (MEF2). We show that MPTP-induced conversion of the cdk5 activator p35 to a pathogenic p25 form is dependent on calpain activity in vivo. In addition, p35 deficiency attenuates MPTP-induced dopamine neuron loss and behavioral outcome. Moreover, MEF2 is phosphorylated on Ser444, an inactivating site, after MPTP treatment. This phosphorylation is dependent on both calpain and p35 activity, consistent with the model that calpain-mediated activation of cdk5 results in phosphorylation of MEF2 in vivo. Finally, we provide evidence that MEF2 is critical for dopaminergic loss because “cdk5 phosphorylation site mutant” of MEF2D provides neuroprotection in an MPTP mouse model of PD. Together, these data indicate that calpain-p35-p25/cdk5-mediated inactivation of MEF2 plays a critical role in dopaminergic loss in vivo.

DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway

Loss-of-function DJ-1 (PARK7) mutations have been linked with a familial form of early onset Parkinson disease. Numerous studies have supported the role of DJ-1 in neuronal survival and function. Our initial studies using DJ-1-deficient neurons indicated that DJ-1 specifically protects the neurons against the damage induced by oxidative injury in multiple neuronal types and degenerative experimental paradigms, both in vitro and in vivo. However, the manner by which oxidative stress-induced death is ameliorated by DJ-1 is not completely clear. We now present data that show the involvement of DJ-1 in modulation of AKT, a major neuronal prosurvival pathway induced upon oxidative stress. We provide evidence that DJ-1 promotes AKT phosphorylation in response to oxidative stress induced by H2O2 in vitro and in vivo following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Moreover, we show that DJ-1 is necessary for normal AKT-mediated protective effects, which can be bypassed by expression of a constitutively active form of AKT. Taken together, these data suggest that DJ-1 is crucial for full activation of AKT upon oxidative injury, which serves as one explanation for the protective effects of DJ-1.

The Parkinson's disease gene DJ-1 is also a key regulator of stroke-induced damage

Recent evidence has indicated that common mechanisms play roles among multiple neurological diseases. However, the specifics of these pathways are not completely understood. Stroke is caused by the interruption of blood flow to the brain, and cumulative evidence supports the critical role of oxidative stress in the ensuing neuronal death process. DJ-1 (PARK7) has been identified as the gene linked to early-onset familial Parkinsons disease. Currently, our work also shows that DJ-1 is central to death in both in Vitro and in Vivo models of stroke. Loss of DJ-1 increases the sensitivity to excitotoxicity and ischemia, whereas expression of DJ-1 can reverse this sensitivity and indeed provide further protection. Importantly, DJ-1 expression decreases markers of oxidative stress after stroke insult in Vivo, suggesting that DJ-1 protects through alleviation of oxidative stress. Consistent with this finding, we demonstrate the essential role of the oxidation-sensitive cysteine-106 residue in the neuroprotective activity of DJ-1 after stroke. Our work provides an important example of how a gene seemingly specific for one disease, in this case Parkinsons disease, also appears to be central in other neuropathological conditions such as stroke. It also highlights the important commonalities among differing neuropathologies.

Nuclear Factor-κB Modulates the p53 Response in Neurons Exposed to DNA Damage

Previous studies have shown that DNA damage-evoked death of primary cortical neurons occurs in a p53 and cyclin-dependent kinase-dependent (CDK) manner. The manner by which these signals modulate death is unclear. Nuclear factor-κB (NF-κB) is a group of transcription factors that potentially interact with these pathways. Presently, we show that NF-κB is activated shortly after induction of DNA damage in a manner independent of the classic IκB kinase (IKK) activation pathway, CDKs, ATM, and p53. Acute inhibition of NF-κB via expression of a stable IκB mutant, downregulation of the p65 NF-κB subunit by RNA interference (RNAi), or pharmacological NF-κB inhibitors significantly protected against DNA damage-induced neuronal death. NF-κB inhibition also reduced p53 transcripts and p53 activity as measured by the p53-inducible messages, Puma and Noxa, implicating the p53 tumor suppressor in the mechanism of NF-κB-mediated neuronal death. Importantly, p53 expression still induces death in the presence of NF-κB inhibition, indicating that p53 acts downstream of NF-κB. Interestingly, neurons cultured from p65 or p50 NF-κB-deficient mice were not resistant to death and did not show diminished p53 activity, suggesting compensatory processes attributable to germline deficiencies, which allow p53 activation still to occur. In contrast to acute NF-κB inhibition, prolonged NF-κB inhibition caused neuronal death in the absence of DNA damage. These results uniquely define a signaling paradigm by which NF-κB serves both an acute p53-dependent pro-apoptotic function in the presence of DNA damage and an anti-apoptotic function in untreated normal neurons.

ROS-dependent regulation of Parkin and DJ-1 localization during oxidative stress in neurons

Mutations in several genes, including Parkin, PTEN-induced kinase 1 (Pink1) and DJ-1, are associated with rare inherited forms of Parkinsons disease (PD). Despite recent attention on the function of these genes, the interplay between DJ-1, Pink1 and Parkin in PD pathogenesis remains unclear. In particular, whether these genes regulate mitochondrial control pathways in neurons is highly controversial. Here we report that Pink1-dependent Parkin translocation does occur in mouse cortical neurons in response to a variety of mitochondrial damaging agents. This translocation only occurs in the absence of antioxidants in the neuronal culturing medium, implicating a key role of reactive oxygen species (ROS) in this response. Consistent with these observations, ROS blockers also prevent Parkin recruitment in mouse embryonic fibroblasts. Loss of DJ-1, a gene linked to ROS management, results in increased stress-induced Parkin recruitment and increased mitophagy. Expression of wild-type DJ-1, but not a cysteine-106 mutant associated with defective ROS response, rescues this accelerated Parkin recruitment. Interestingly, DJ-1 levels increase at mitochondria following oxidative damage in both fibroblasts and neurons, and this process also depends on Parkin and possibly Pink1. These results not only highlight the presence of a Parkin/Pink1-mediated pathway of mitochondrial quality control (MQC) in neurons, they also delineate a complex reciprocal relationship between DJ-1 and the Pink1/Parkin pathway of MQC.

Essential role of cytoplasmic cdk5 and Prx2 in multiple ischemic injury models, in vivo

Recent evidence suggests that abnormal activation of cyclin-dependent kinase 5 (cdk5) is a critical prodeath signal in stroke. However, the mechanism(s) by which cdk5 promotes death is unclear. Complicating the role of cdk5 are the observations that cdk5 can exist in multiple cellular regions and possess both prosurvival and prodeath characteristics. In particular, the critical role of cytoplasmic or nuclear cdk5 in neuronal jury, in vivo, is unclear. Therefore, we determined where cdk5 was activated in models of ischemia and how manipulation of cdk5 in differing compartments may affect neuronal death. Here, we show a critical function for cytoplasmic cdk5 in both focal and global models of stroke, in vivo. Cdk5 is activated in the cytoplasm and expression of DNcdk5 localized to the cytoplasm is protective. Importantly, we also demonstrate the antioxidant enzyme Prx2 (peroxiredoxin 2) as a critical cytoplasmic target of cdk5. In contrast, the role of cdk5 in the nucleus is context-dependent. Following focal ischemia, nuclear cdk5 is activated and functionally relevant while there is no evidence for such activation following global ischemia. Importantly, myocyte enhancer factor 2D (MEF2D), a previously described nuclear target of cdk5 in vitro, is also phosphorylated by cdk5 following focal ischemia. In addition, MEF2D expression in this paradigm ameliorates death. Together, our results address the critical issue of cdk5 activity compartmentalization, as well as define critical substrates for both cytoplasmic and nuclear cdk5 activity in adult models of stroke.

Inactivation of Pink1 Gene in Vivo Sensitizes Dopamine-producing Neurons to 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and Can Be Rescued by Autosomal Recessive Parkinson Disease Genes, Parkin or DJ-1

Background: Mutations in Pink1 are associated with Parkinson disease. Results: Mouse Pink1 deficiency results in hypersensitivity to MPTP-induced dopaminergic neuronal loss, which can be rescued with expression of human Parkin or DJ-1. Conclusion: Pink1 gene can regulate response to exogenous stress. Significance: These results indicate how endogenous Pink1 plays an important role in management of exogenous stress in mouse brain. Mutations in the mitochondrial PTEN-induced kinase 1 (Pink1) gene have been linked to Parkinson disease (PD). Recent reports including our own indicated that ectopic Pink1 expression is protective against toxic insult in vitro, suggesting a potential role for endogenous Pink1 in mediating survival. However, the role of endogenous Pink1 in survival, particularly in vivo, is unclear. To address this critical question, we examined whether down-regulation of Pink1 affects dopaminergic neuron loss following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the adult mouse. Two model systems were utilized: virally delivered shRNA-mediated knockdown of Pink1 and germ line-deficient mice. In both instances, loss of Pink1 generated significant sensitivity to damage induced by systemic MPTP treatment. This sensitivity was associated with greater loss of dopaminergic neurons in the Substantia Nigra pars compacta and terminal dopamine fiber density in the striatum region. Importantly, we also show that viral mediated expression of two other recessive PD-linked familial genes, DJ-1 and Parkin, can protect dopaminergic neurons even in the absence of Pink1. This evidence not only provides strong evidence for the role of endogenous Pink1 in neuronal survival, but also supports a role of DJ-1 and Parkin acting parallel or downstream of endogenous Pink1 to mediate survival in a mammalian in vivo context.