Dianbo Qu

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.

The role of Cdk5-mediated apurinic/apyrimidinic endonuclease 1 phosphorylation in neuronal death.

Accumulating evidence suggests that deregulated cyclin-dependent kinase 5 (Cdk5) plays a critical part in neuronal death. However, the pathogenic targets of Cdk5 are not fully defined. Here we demonstrate that the Cdk5 activator p35 interacts directly with apurinic/apyrimidinic endonuclease 1 (Ape1), a protein crucial for base excision repair (BER) following DNA damage. Cdk5 complexes phosphorylate Ape1 at Thr 232 and thereby reduces its apurinic/apyrimidinic (AP) endonuclease activity. Ape1 phosphorylation is dependent on Cdk5 in in vitro and in vivo. The reduced endonuclease activity of phosphorylated Ape1 results in accumulation of DNA damage and contributes to neuronal death. Overexpression of Ape1WT and Ape1T232A, but not the phosphorylation mimic Ape1T232E, protects neurons against MPP+/MPTP. Loss of Ape1 sensitizes neurons to death. Importantly, increased phosphorylated Ape1 was also observed in post-mortem brain tissue from patients with Parkinsons and Alzheimers diseases, suggesting a potential link between Ape1 phosphorylation and the pathogenesis of neurodegenerative diseases.

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.

The Chk1/Cdc25A pathway as activators of the cell cycle in neuronal death induced by camptothecin

Cell cycle regulators appear to play a paradoxical role in neuronal death. We have shown previously that cyclin-dependent kinases (CDKs), along with their downstream effectors, Rb (retinoblastoma) and E2F/DP1 (E2 promoter binding factor/deleted in polyposis 1), regulate neuronal death evoked by the DNA damaging agent camptothecin. However, the mechanism by which CDKs are activated in this model is unclear. The cell division cycle 25A (Cdc25A) phosphatase is a critical regulator of cell cycle CDKs in proliferating cells. In cortical neurons, we presently show that expression of Cdc25A promotes death even in the absence of DNA damage. Importantly, Cdc25A activity is rapidly increased during DNA damage treatment. Inhibition of Cdc25A blocks death and reduces cyclin D1-associated kinase activity and Rb phosphorylation. This indicates that endogenous Cdc25A activity is important for regulation of cell cycle-mediated neuronal death. We also examined how Cdc25A activity is regulated after DNA damage. Cultured embryonic cortical neurons have a significant basal activity of checkpoint kinase 1 (Chk1), a kinase that regulates cell cycle arrest. During camptothecin treatment of neurons, this activity is rapidly downregulated with a concomitant increase in Cdc25A activity. Importantly, expression of wild-type Chk1, but not kinase-dead Chk1, inhibits the camptothecin-induced increase in Cdc25A activity. In addition, Chk1 expression also promotes survival in the presence of the DNA-damaging agent. Together, our data suggest that a Chk1/Cdc25A activity participates in activation of a cell cycle pathway-mediated death signal in neurons. These data also define how a proliferative signal may be abnormally activated in a postmitotic environment.

Progressive dopaminergic cell loss with unilateral-to-bilateral progression in a genetic model of Parkinson disease.

DJ-1 mutations cause autosomal recessive early-onset Parkinson disease (PD). We report a model of PD pathology: the DJ1-C57 mouse. A subset of DJ-1–nullizygous mice, when fully backcrossed to a C57BL/6J background, display dramatic early-onset unilateral loss of dopaminergic (DA) neurons in their substantia nigra pars compacta, progressing to bilateral degeneration of the nigrostriatal axis with aging. In addition, these mice exhibit age-dependent bilateral degeneration at the locus ceruleus nucleus and display mild motor behavior deficits at aged time points. These findings effectively recapitulate the early stages of PD. Therefore, the DJ1-C57 mouse provides a tool to study the preclinical aspects of neurodegeneration. Importantly, by exome sequencing, we identify candidate modifying genes that segregate with the phenotype, providing potentially critical clues into how certain genes may influence the penetrance of DJ-1–related degeneration in mice.

The nuclear localization of SET mediated by impα3/impβ attenuates its cytosolic toxicity in neurons

SET is a multi‐functional protein in proliferating cells. Some of the proposed functions of SET suggest an important nuclear role. However, the nuclear import pathway of SET is also unknown and the function of SET in neurons is unclear. Presently, using cortical neurons, we report that the nuclear import of SET is mediated by an impα/impβ‐dependent pathway. Nuclear localization signal, 168KRSSQTQNKASRKR181, in SET interacts with impα3, which recruits impβ to form a ternary complex, resulting in efficient transportation of SET into nucleus. By in vitro nuclear import assay based on digitonin‐permeabilized neurons, we further demonstrated that the nuclear import of SET relies on Ran GTPase. We provide evidence that this nuclear localization of SET is important in neuronal survival. Under basal conditions, SET is predominately nuclear. However, upon death induced by genotoxic stress, endogenous SET decreases in the nucleus and increases in the cytoplasm. Consistent with a toxic role of SET in the cytoplasm, targeted expression of SET to the cytoplasm exacerbates death compared to wild type SET expression which is protective following DNA damage. Taken together, our results indicate that SET is imported into the nucleus through its association with impα3/impβ, and that localization of SET is important in regulation of neuronal death.

DJ-1 Interacts with and Regulates Paraoxonase-2, an Enzyme Critical for Neuronal Survival in Response to Oxidative Stress

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinsons disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2.

BAG2 Gene-mediated Regulation of PINK1 Protein Is Critical for Mitochondrial Translocation of PARKIN and Neuronal Survival

Emerging evidence has demonstrated a growing genetic component in Parkinson disease (PD). For instance, loss-of-function mutations in PINK1 or PARKIN can cause autosomal recessive PD. Recently, PINK1 and PARKIN have been implicated in the same signaling pathway to regulate mitochondrial clearance through recruitment of PARKIN by stabilization of PINK1 on the outer membrane of depolarized mitochondria. The precise mechanisms that govern this process remain enigmatic. In this study, we identify Bcl2-associated athanogene 2 (BAG2) as a factor that promotes mitophagy. BAG2 inhibits PINK1 degradation by blocking the ubiquitination pathway. Stabilization of PINK1 by BAG2 triggers PARKIN-mediated mitophagy and protects neurons against 1-methyl-4-phenylpyridinium-induced oxidative stress in an in vitro cell model of PD. Collectively, our findings support the notion that BAG2 is an upstream regulator of the PINK1/PARKIN signaling pathway.

Pim‐1 kinase as activator of the cell cycle pathway in neuronal death induced by DNA damage

DNA damage is a critical component of neuronal death underlying neurodegenerative diseases and injury. Neuronal death evoked by DNA damage is characterized by inappropriate activation of multiple cell cycle components. However, the mechanism regulating this activation is not fully understood. We demonstrated previously that the cell division cycle (Cdc) 25A phosphatase mediates the activation of cyclin‐dependent kinases and neuronal death evoked by the DNA damaging agent camptothecin. We also showed that Cdc25A activation is blocked by constitutive checkpoint kinase 1 activity under basal conditions in neurons. Presently, we report that an additional factor is central to regulation of Cdc25A phosphatase in neuronal death. In a gene array screen, we first identified Pim‐1 as a potential factor up‐regulated following DNA damage. We confirmed the up‐regulation of Pim‐1 transcript, protein and kinase activity following DNA damage. This induction of Pim‐1 is regulated by the nuclear factor kappa beta (NF‐κB) pathway as Pim‐1 expression and activity are significantly blocked by siRNA‐mediated knockdown of NF‐κB or NF‐κB pharmacological inhibitors. Importantly, Pim‐1 activity is critical for neuronal death in this paradigm and its deficiency blocks camptothecin‐mediated neuronal death. It does so by activating Cdc25A with consequent activation of cyclin D1‐associated kinases. Taken together, our results demonstrate that Pim‐1 kinase plays a central role in DNA damage‐evoked neuronal death by regulating aberrant neuronal cell cycle activation.

Regulation of the VHL/HIF-1 Pathway by DJ-1

DJ-1 (PARK7) is a gene linked to autosomal recessive Parkinson disease (PD). We showed previously that DJ-1 loss sensitizes neurons in models of PD and stroke. However, the biochemical mechanisms underlying this protective role are not completely clear. Here, we identify Von Hippel Lindau (VHL) protein as a critical DJ-1-interacting protein. We provide evidence that DJ-1 negatively regulates VHL ubiquitination activity of the α-subunit of hypoxia-inducible factor-1 (HIF-1α) by inhibiting HIF–VHL interaction. Consistent with this observation, DJ-1 deficiency leads to lowered HIF-1α levels in models of both hypoxia and oxidative stress, two stresses known to stabilize HIF-1α. We also demonstrate that HIF-1α accumulation rescues DJ-1-deficient neurons against 1-methyl-4-phenylpyridinium-induced toxicity. Interestingly, lymphoblast cells extracted from DJ-1-related PD patients show impaired HIF-1α stabilization when compared with normal individuals, indicating that the DJ-1–VHL link may also be relevant to a human context. Together, our findings delineate a model by which DJ-1 mediates neuronal survival by regulation of the VHL–HIF-1α pathway.