Zhongfang Weng

Leptin Protects against 6-Hydroxydopamine-induced Dopaminergic Cell Death via Mitogen-activated Protein Kinase Signaling

The death of midbrain dopaminergic neurons in sporadic Parkinson disease is of unknown etiology but may involve altered growth factor signaling. The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death. In vitro studies using the murine dopaminergic cell line MN9D showed that leptin attenuated 6-OHDA-induced apoptotic markers, including caspase-9 and caspase-3 activation, internucleosomal DNA fragmentation, and cytochrome c release. ERK1/2 phosphorylation (pERK1/2) was found to be critical for mediating leptin-induced neuroprotection, because inhibition of the MEK pathway blocked both the pERK1/2 response and the pro-survival effect of leptin in cultures. Knockdown of the downstream messengers JAK2 or GRB2 precluded leptin-induced pERK1/2 activation and neuroprotection. Leptin/pERK1/2 signaling involved phosphorylation and nuclear localization of CREB (pCREB), a well known survival factor for dopaminergic neurons. Leptin induced a marked MEK-dependent increase in pCREB that was essential for neuroprotection following 6-OHDA toxicity. Transfection of a dominant negative MEK protein abolished leptin-enhanced pCREB formation, whereas a dominant negative CREB or decoy oligonucleotide diminished both pCREB binding to its target DNA sequence and MN9D survival against 6-OHDA toxicity. Moreover, in the substantia nigra of mice, leptin treatment increased the levels of pERK1/2, pCREB, and the downstream gene product BDNF, which were reversed by the MEK inhibitor PD98059. Collectively, these data provide evidence that leptin prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of Parkinson disease.

Leptin neuroprotection in the CNS: mechanisms and therapeutic potentials

Leptin is well known as a hormone important in the central control of appetitive behaviors via receptor‐mediated actions in the hypothalamus, where leptin adjusts food intake to maintain homeostasis with the body’s energy stores. Recent evidence has shown that leptin and its receptors are widespread in the CNS and may provide neuronal survival signals. This review summarizes our current knowledge of how leptin functions in the brain and then focuses on the ability of leptin to mitigate neuronal damage in experimental models of human neurological disorders. Damage to the brain by acute events such as stroke, or long‐term loss of neurons associated with neurodegenerative diseases, including Parkinson’s and Alzheimer’s disease, may be amenable to treatment using leptin to limit death of susceptible cells. Leptin‐mediated pro‐survival signaling is now known to prevent the death of neurons in these models. The signaling cascades that leptin generates are shared by other neuroprotective molecules including insulin and erythropoietin, and are thus a component of the neurotrophic effects mediated by endogenous hormones. Coupled with evidence that leptin dysregulation in human disease also results in enhanced neuronal susceptibility to damage, development of leptin as a therapeutic methodology is an attractive and viable possibility.

Hsp27 Protects against Ischemic Brain Injury via Attenuation of a Novel Stress-Response Cascade Upstream of Mitochondrial Cell Death Signaling

Heat shock protein 27 (Hsp27), a recently discovered member of the heat shock protein family, is markedly induced in the brain after cerebral ischemia and other injury states. In non-neuronal systems, Hsp27 has potent cell death-suppressing functions. However, the mechanism of Hsp27-mediated neuroprotection has not yet been elucidated. Using transgenic and viral overexpression of Hsp27, we investigated the molecular mechanism by which Hsp27 exerts its neuroprotective effect. Overexpression of Hsp27 conferred long-lasting tissue preservation and neurobehavioral recovery, as measured by infarct volume, sensorimotor function, and cognitive tasks up to 3 weeks following focal cerebral ischemia. Examination of signaling pathways critical to neuronal death demonstrated that Hsp27 overexpression led to the suppression of the MKK4/JNK kinase cascade. While Hsp27 overexpression did not suppress activation of an upstream regulatory kinase of the MKK/JNK cascade, ASK1, Hsp27 effectively inhibited ASK1 activity via a physical association through its N-terminal domain and the kinase domain of ASK1. The N-terminal region of Hsp27 was required for neuroprotective function against in vitro ischemia. Moreover, knockdown of ASK1 or inhibition of the ASK1/MKK4 cascade effectively inhibited cell death following neuronal ischemia. This underscores the importance of this kinase cascade in the progression of ischemic neuronal death. Inhibition of PI3K had no effect on Hsp27-mediated neuroprotection, suggesting that Hsp27 does not promote cell survival via activation of PI3K/Akt. Based on these findings, we conclude that overexpression of Hsp27 confers long-lasting neuroprotection against ischemic brain injury via a previously unexplored association and inhibition of ASK1 kinase signaling.

Peroxiredoxin-2 protects against 6-hydroxydopamine-induced dopaminergic neurodegeneration via attenuation of the apoptosis signal-regulating kinase (ASK1) signaling cascade.

The peroxiredoxin (PRX) family of antioxidant enzymes helps maintain the intracellular reducing milieu and suppresses apoptosis in non-neuronal cells. However, whether PRX can inhibit neuronal apoptosis through specific signaling mechanisms remains poorly understood. Induction of PRX2, the most abundant neuronal PRX, occurs in Parkinsons disease (PD) patient brains, but its functional impact is unclear. In the present study, we used the dopaminergic (DA) toxin 6-hydroxydopamine (6-OHDA) to model PD and explore the protective effect and mechanisms of PRX on DA neurons. Of the 2-cysteine PRXs that were tested in MN9D DA neurons, endogenous PRX2 was most beneficial to cell survival. Lentivirus-mediated PRX2 overexpression conferred marked in vitro and in vivo neuroprotection against 6-OHDA toxicity in DA neurons, and preserved motor functions involving the dopamine system in mouse. In addition to its role as an antioxidant enzyme, PRX2 exhibited anti-apoptotic effects in DA neurons via suppression of apoptosis signal-regulating kinase (ASK1)-dependent activation of the c-Jun N-terminal kinase/c-Jun and p38 pro-death pathways, which are also activated in DA neurons of postmortem PD brains. PRX2 inhibited 6-OHDA-induced ASK1 activation by modulating the redox status of the endogenous ASK1 inhibitor thioredoxin (Trx). PRX2 overexpression maintained Trx in a reduced state by inhibiting the cysteine thiol-disulfide exchange, thereby preventing its dissociation from ASK1. This study describes a previously undefined mechanism by which redox-sensitive molecules signal via apoptotic pathways in response to PD-relevant toxic stress in DA neurons. Our results also suggest that PRX2 and ASK1 may be potential targets for neuroprotective intervention in PD.

Phosphorylation of HSP27 by Protein Kinase D Is Essential for Mediating Neuroprotection against Ischemic Neuronal Injury

Heat shock protein 27 (HSP27) (or HSPB1) exerts cytoprotection against many cellular insults, including cerebral ischemia. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical downstream target of HSP27 conferring the neuroprotective effects of HSP27 against neuronal ischemia. However, the function of HSP27 is highly influenced by posttranslational modification, with differential cellular effects based on phosphorylation at specific serine residues. The role of phosphorylation in neuronal ischemic neuroprotection is currently unknown. We have created transgenic mice and viral vectors containing HSP27 mutated at three critical serine residues (Ser15, Ser78, and Ser82) to either alanine (HSP27-A, nonphosphorylatable) or aspartate (HSP27-D, phosphomimetic) residues. Under both in vitro and in vivo neuronal ischemic settings, overexpression of wild-type HSP27 (HSP27) and HSP27-D, but not HSP27-A, was neuroprotective and inhibited downstream ASK1 signaling pathways. Consistently, overexpressed HSP27 was phosphorylated by endogenous mechanisms when neurons were under ischemic stress, and single-point mutations identified Ser15 and Ser82 as critical for neuroprotection. Using a panel of inhibitors and gene knockdown approaches, we identified the upstream kinase protein kinase D (PKD) as the primary kinase targeting HSP27 directly for phosphorylation. PKD and HSP27 coimmunoprecipitated, and inhibition or knockdown of PKD abrogated the neuroprotective effects of HSP27 as well as the interaction with and inhibition of ASK1 signaling. Together, these data demonstrate that HSP27 requires PKD-mediated phosphorylation for its suppression of ASK1 cell death signaling and neuroprotection against ischemic injury.

Pharmacological Induction of Heme Oxygenase-1 by a Triterpenoid Protects Neurons Against Ischemic Injury

Background and Purpose— Heme oxygenase-1 (HO-1) is an inducible Phase 2 enzyme that degrades toxic heme; its role in cerebral ischemia is not fully understood. We hypothesize that chemically induced HO-1 upregulation with the novel triterpenoid CDDO-Im (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline), a robust inducer of Phase 2 genes, protects neurons against ischemic injury. Methods— Using 3 different models of ischemia, including oxygen–glucose deprivation in neuronal cultures, global ischemia in rats, and focal ischemia in mice, we determined (1) whether CDDO-Im induces HO-1 expression and protects against ischemic injury; and (2) whether HO-1 inhibition disrupts the neuroprotective effect of CDDO-Im. Results— CDDO-Im treatment (50–300 nmol/L) resulted in 8-fold HO-1 upregulation in cultured neurons and protected against oxygen–glucose deprivation. The protection was abolished when the cultures were transfected with nuclear factor (erythroid-derived 2) like-2–shRNA or coincubated with tin protoporphyrin IX, a specific HO-1 inhibitor. In the rat model of global ischemia, intracerebroventricular infusion of CDDO-Im (0.5–1.5 &mgr;g) augmented HO-1 expression in hippocampal neurons and resulted in significant increases in CA1 neuronal survival after global ischemia. To further strengthen the clinical relevance of the CDDO-Im treatment, we tested its effects in the mouse model of temporary focal ischemia (60 minutes). Postischemic intraperitoneal injection of CDDO-Im (10–100 &mgr;g) enhanced HO-1 expression and significantly reduced neurological dysfunction and infarct volume. Intracerebroventricular infusion of tin protoporphyrin IX reduced the neuroprotective effect of CDDO-Im against global and focal ischemia. Conclusions— CDDO-Im confers neuroprotection against ischemic injury by upregulating HO-1, suggesting that enhance of HO-1 expression may be a legitimate strategy for therapeutic intervention of stroke.

Neuronal NAMPT is released after cerebral ischemia and protects against white matter injury

Nicotinamide phosphoribosyltransferase (NAMPT) has been implicated in neuroprotection against ischemic brain injury, but the mechanism underlying its protective effect remains largely unknown. To further examine the protective effect of NAMPT against ischemic stroke and its potential mechanism of action, we generated a novel neuron-specific NAMPT transgenic mouse line. Transgenic mice and wild-type littermates were subjected to transient occlusion of the middle cerebral artery (MCAO) for 60 minutes. Neuron-specific NAMPT overexpression significantly reduced infarct volume by 65% (P = 0.018) and improved long-term neurologic outcomes (P ≤ 0.05) compared with littermates. Interestingly, neuronal overexpression of NAMPT increased the area of myelinated fibers in the striatum and corpus callosum, indicating that NAMPT protects against white matter injury. The mechanism of protection appeared to be through extracellular release of NAMPT. First, NAMPT was secreted into the extracellular medium by primary cortical neurons exposed to ischemia-like oxygen–glucose deprivation (OGD) in vitro. Second, conditioned medium from NAMPT-overexpressing neurons exposed to OGD protected cultured oligodendrocytes from OGD. Third, the protective effects of conditioned medium were abolished by antibody-mediated NAMPT depletion, strongly suggesting that the protective effect is mediated by the extracellular NAMPT released into in the medium. These data suggest a novel neuroprotective role for secreted NAMPT in the protection of white matter after ischemic injury.

HSP27 Protects the Blood-Brain Barrier Against Ischemia-Induced Loss of Integrity

Loss of integrity of the blood-brain barrier (BBB) in stroke victims initiates a devastating cascade of events including extravasation of blood-borne molecules, water, and inflammatory cells deep into brain parenchyma. Thus, it is important to identify mechanisms by which BBB integrity can be maintained in the face of ischemic injury in experimental stroke. We previously demonstrated that the phylogenetically conserved small heat shock protein 27 (HSP27) protects against transient middle cerebral artery occlusion (tMCAO). Here we show that HSP27 transgenic overexpression also maintains the integrity of the BBB in mice subjected to tMCAO. Extravasation of endogenous IgG antibodies and exogenous FITC-albumin into the brain following tMCAO was reduced in transgenic mice, as was total brain water content. HSP27 overexpression abolished the appearance of TUNEL-positive profiles in microvessel walls. Transgenics also exhibited less loss of microvessel proteins following tMCAO. Notably, primary endothelial cell cultures were rescued from oxygen-glucose deprivation (OGD) by lentiviral HSP27 overexpression according to four viability assays, supporting a direct effect on this cell type. Finally, HSP27 overexpression reduced the appearance of neutrophils in the brain and inhibited the secretion of five cytokines. These findings reveal a novel role for HSP27 in attenuating ischemia/reperfusion injury - the maintenance of BBB integrity. Endogenous upregulation of HSP27 after ischemia in wild-type animals may exert similar protective functions and warrants further investigation. Exogenous enhancement of HSP27 by rational drug design may lead to future therapies against a host of injuries, including but not limited to a harmful breach in brain vasculature.

APE1/Ref-1 facilitates recovery of gray and white matter and neurological function after mild stroke injury

Significance AP endonuclease-1 (APE1)/redox effector factor-1 (Ref-1) is an essential DNA repair enzyme that has been difficult to study mechanistically because of embryonic lethality in conventional knockout animals. Thus, we generated a conditional APE1 knockout model to examine the protective role of endogenous APE1 in experimental stroke. Induced APE1 knockout in adulthood greatly exacerbated neuron and oligodendrocyte loss after mild ischemic stroke and prevented the intrinsic, long-term recovery of sensorimotor function and spatial learning and memory. APE1 knockout also aggravated ischemia-induced destruction of myelin and impairment of axon conduction in white matter. We conclude that APE1 dictates fundamental life and death decisions in both gray and white matter and plays an indispensable role in intrinsic recovery after mild ischemic injury. A major hallmark of oxidative DNA damage after stroke is the induction of apurinic/apyrimidinic (AP) sites and strand breaks. To mitigate cell loss after oxidative DNA damage, ischemic cells rapidly engage the base excision-repair proteins, such as the AP site-repairing enzyme AP endonuclease-1 (APE1), also named redox effector factor-1 (Ref-1). Although forced overexpression of APE1 is known to protect against oxidative stress-induced neurodegeneration, there is no concrete evidence demonstrating a role for endogenous APE1 in the long-term recovery of gray and white matter following ischemic injury. To address this gap, we generated, to our knowledge, the first APE1 conditional knockout (cKO) mouse line under control of tamoxifen-dependent Cre recombinase. Using a well-established model of transient focal cerebral ischemia (tFCI), we show that induced deletion of APE1 dramatically enlarged infarct volume and impaired the recovery of sensorimotor and cognitive deficits. APE1 cKO markedly increased postischemic neuronal and oligodendrocyte degeneration, demonstrating that endogenous APE1 preserves both gray and white matter after tFCI. Because white matter repair is instrumental in behavioral recovery after stroke, we also examined the impact of APE1 cKO on demyelination and axonal conduction and discovered that APE1 cKO aggravated myelin loss and impaired neuronal communication following tFCI. Furthermore, APE1 cKO increased AP sites and activated the prodeath signaling proteins, PUMA and PARP1, after tFCI in topographically distinct manners. Our findings provide evidence that endogenous APE1 protects against ischemic infarction in both gray and white matter and facilitates the functional recovery of the central nervous system after mild stroke injury.

Apurinic/apyrimidinic endonuclease 1 upregulation reduces oxidative DNA damage and protects hippocampal neurons from ischemic injury.

AIMS Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme that participates in base-excision repair of oxidative DNA damage and in the redox activation of transcription factors. We tested the hypothesis that APE1 upregulation protects neuronal structure and function against transient global cerebral ischemia (tGCI). RESULTS Upregulation of APE1 by low-dose proton irradiation (PI) or by transgene overexpression protected hippocampal CA1 neurons against tGCI-induced cell loss and reduced apurinic/apyrimidinic sites and DNA fragmentation. Conversely, APE1 knockdown attenuated the protection afforded by PI and ischemic preconditioning. APE1 overexpression inhibited the DNA damage response, as evidenced by lower phospho-histone H2A and p53-upregulated modulator of apoptosis levels. APE1 overexpression also partially rescued dendritic spines and attenuated the decrease in field excitatory postsynaptic potentials in hippocampal CA1. Presynaptic and postsynaptic markers were reduced after tGCI, and this effect was blunted in APE1 transgenics. The Morris water maze test revealed that APE1 protected against learning and memory deficits for at least 27 days post-injury. Animals expressing DNA repair-disabled mutant APE1 (D210A) exhibited more DNA damage than wild-type controls and were not protected against tGCI-induced cell loss. INNOVATION This is the first study that thoroughly characterizes structural and functional protection against ischemia after APE1 upregulation by measuring synaptic markers, electrophysiological function, and long-term neurological deficits in vivo. Furthermore, disabling the DNA repair activity of APE1 was found to abrogate its protective impact. CONCLUSION APE1 upregulation, either endogenously or through transgene overexpression, protects DNA, neuronal structures, synaptic function, and behavioral output from ischemic injury.