SPAK inhibitor ZT-1a attenuates reactive astrogliosis and oligodendrocyte degeneration in a mouse model of vascular dementia.

BACKGROUND: Astrogliosis and white matter lesions (WML) are key characteristics of vascular contributions to cognitive impairment and dementia (VCID). However, the molecular mechanisms underlying VCID remain poorly understood. Stimulation of Na-K-Cl cotransport 1 (NKCC1) and its upstream kinases WNK (with no lysine) and SPAK (the STE20/SPS1-related proline/alanine-rich kinase) play a role in astrocytic intracellular Na+ overload, hypertrophy, and swelling. Therefore, in this study, we assessed the effect of SPAK inhibitor ZT-1a on pathogenesis and cognitive function in a mouse model of VCID induced by bilateral carotid artery stenosis (BCAS). METHODS: Following sham or BCAS surgery, mice were randomly assigned to receive either vehicle (DMSO) or SPAK inhibitor ZT-1a treatment regimen (days 14-35 post-surgery). Mice were then evaluated for cognitive functions by Morris water maze, WML by ex vivo MRI-DTI analysis, and astrogliosis/demyelination by immunofluorescence and immunoblotting. RESULTS: Compared to sham control mice, BCAS-Veh mice exhibited chronic cerebral hypoperfusion and memory impairments, accompanied by significant MRI DTI-detected WML and oligodendrocyte (OL) death. Increased activation of WNK-SPAK-NKCC1-signaling proteins was detected in white matter tissues and in C3d+ GFAP+ cytotoxic astrocytes but not in S100A10+ GFAP+ homeostatic astrocytes in BCAS-Veh mice. In contrast, ZT-1a-treated BCAS mice displayed reduced expression and phosphorylation of NKCC1, decreased astrogliosis, OL death, and WML, along with improved memory functions. CONCLUSION: BCAS-induced upregulation of WNK-SPAK-NKCC1 signaling contributes to white matter-reactive astrogliosis, OL death, and memory impairment. Pharmacological inhibition of the SPAK activity has therapeutic potential for alleviating pathogenesis and memory impairment in VCID.

A Novel Needle Mouse Model of Vascular Cognitive Impairment and Dementia.

Bilateral common carotid artery (CCA) stenosis (BCAS) is a useful model to mimic vascular cognitive impairment and dementia (VCID). However, current BCAS models have the disadvantages of high cost and incompatibility with magnetic resonance imaging (MRI) scanning because of metal implantation. We have established a new low-cost VCID model that better mimics human VCID and is compatible with live-animal MRI. The right and the left CCAs were temporarily ligated to 32- and 34-gauge needles with three ligations, respectively. After needle removal, CCA blood flow, cerebral blood flow, white matter injury (WMI) and cognitive function were measured. In male mice, needle removal led to ∼49.8% and ∼28.2% blood flow recovery in the right and left CCA, respectively. This model caused persistent and long-term cerebral hypoperfusion in both hemispheres (more severe in the left hemisphere), and WMI and cognitive dysfunction in ∼90% of mice, which is more reliable compared with other models. Importantly, these pathologic changes and cognitive impairments lasted for up to 24 weeks after surgery. The survival rate over 24 weeks was 81.6%. Female mice showed similar cognitive dysfunction, but a higher survival rate (91.6%) and relatively milder white matter injury. A novel, low-cost VCID model compatible with live-animal MRI with long-term outcomes was established.SIGNIFICANCE STATEMENT Bilateral common carotid artery (CCA) stenosis (BCAS) is an animal model mimicking carotid artery stenosis to study vascular cognitive impairment and dementia (VCID). However, current BCAS models have the disadvantages of high cost and incompatibility with magnetic resonance imaging (MRI) scanning due to metal implantation. We established a new asymmetric BCAS model by ligating the CCA to various needle gauges followed by an immediate needle removal. Needle removal led to moderate stenosis in the right CCA and severe stenosis in the left CCA. This needle model replicates the hallmarks of VCID well in ∼90% of mice, which is more reliable compared with other models, has ultra-low cost, and is compatible with MRI scanning in live animals. It will provide a new valuable tool and offer new insights for VCID research.

Intracerebroventricular Delivery of Recombinant NAMPT Deters Inflammation and Protects Against Cerebral Ischemia.

Our previous study indicated that nicotinamide phosphoribosyltransferase (NAMPT) is released from cells and might be an important extracellular neuroprotective factor in brain ischemia. Here, we tested whether NAMPT protects against ischemic brain injury when administered directly into the intracerebroventricular (ICV) compartment of the cranium. Recombinant NAMPT protein (2 µg) was delivered ICV in mice subjected to 45-min middle cerebral artery occlusion (MCAO), and the effects on infarct volume, sensorimotor function, microglia/macrophage polarization, neutrophil infiltration, and BBB integrity were analyzed. The results indicate that ICV administration of NAMPT significantly reduced infarct volume, retained its beneficial properties even when ICV administration was delayed by 6 h after MCAO, and improved neurological outcomes. NAMPT treatment inhibited pro-inflammatory microglia/macrophages, promoted microglia/macrophage polarization toward the anti-inflammatory phenotype, and reduced the infiltration of neutrophils into the perilesional area after brain ischemia. In vitro studies indicated that multiple pro-inflammatory microglial markers/cytokines were downregulated while multiple anti-inflammatory microglial markers/cytokines were induced in primary microglial cultures treated with NAMPT protein. NAMPT treatment also fortified the blood-brain barrier (BBB), as shown by reduced extravascular leakage of the small-molecule tracer Alexa Fluor 555 Cadaverine and larger-sized endogenous IgGs into brain parenchyma. Thus, NAMPT may protect against ischemic brain injury partly through a novel anti-inflammatory mechanism, which in turn maintains BBB integrity and reduces the infiltration of peripheral inflammatory cells. Taken together, these results provide validation of recombinant NAMPT delivery into the extracellular space as a potential neuroprotective strategy for stroke.

Promoting Neurovascular Recovery in Aged Mice after Ischemic Stroke - Prophylactic Effect of Omega-3 Polyunsaturated Fatty Acids.

The aged population is among the highest at risk for ischemic stroke, yet most stroke patients of advanced ages (>80 years) are excluded from access to thrombolytic treatment by tissue plasminogen activator, the only FDA approved pharmacological therapy for stroke victims. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) robustly alleviate ischemic brain injury in young adult rodents, but have not yet been studied in aged animals. This study investigated whether chronic dietary supplementation of n-3 PUFAs protects aging brain against cerebral ischemia and improves long-term neurological outcomes. Aged (18-month-old) mice were administered n-3 PUFA-enriched fish oil in daily chow for 3 months before and up to 8 weeks after 45 minutes of transient middle cerebral artery occlusion (tMCAO). Sensorimotor outcomes were assessed by cylinder test and corner test up to 35 days and brain repair dynamics evaluated immunohistologically up to 56 days after tMCAO. Mice receiving dietary supplementation of n-3 PUFAs for 3 months showed significant increases in brain ratio of n-3/n-6 PUFA contents, and markedly reduced long-term sensorimotor deficits and chronic ischemic brain tissue loss after tMCAO. Mechanistically, n-3 PUFAs robustly promoted post-ischemic angiogenesis and neurogenesis, and enhanced white matter integrity after tMCAO. The Pearson linear regression analysis revealed that the enhancement of neurogenesis and white matter integrity both correlated positively with improved sensorimotor activities after tMCAO. This study demonstrates that prophylactic dietary supplementation of n-3 PUFAs effectively improves long-term stroke outcomes in aged mice, perhaps by promoting post-stroke brain repair processes such as angiogenesis, neurogenesis, and white matter restoration.

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

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.

Effect of an inductive hydrogel composed of urinary bladder matrix upon functional recovery following traumatic brain injury.

Traumatic brain injury (TBI) is a major public health problem with no effective clinical treatment. Use of bioactive scaffold materials has been shown to be a promising strategy for tissue regeneration and repair in a number of injury models. Of these scaffold materials, urinary bladder matrix (UBM) derived from porcine bladder tissue, has demonstrated desirable properties for supporting and promoting the growth of neural cells in vitro, suggesting its potential as a scaffold for brain tissue repair in the treatment of TBI. Herein we evaluate the biocompatibility of UBM within brain tissue and the effects of UBM delivery upon functional outcome following TBI. A hydrogel form of UBM was injected into healthy rat brains for 1, 3, and 21 days to examine the tissue response to UBM. Multiple measures of tissue injury, including reactive astrocytosis, microglial activation, and neuron degeneration showed that UBM had no deleterious effects on normal brain. Following TBI, the brains were evaluated histologically and behaviorally between sham-operated controls and UBM- and vehicle-treated groups. Application of UBM reduced lesion volume and attenuated trauma-induced myelin disruption. Importantly, UBM treatment resulted in significant neurobehavioral recovery following TBI as demonstrated by improvements in vestibulomotor function; however, no differences in cognitive recovery were observed between the UBM- and vehicle-treated groups. The present study demonstrated that UBM is not only biocompatible within the brain tissue, but also can exert protective effects upon injured brain.

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.

GST P1, a novel downstream regulator of LRRK2, G2019S-induced neuronal cell death.

The enhanced neurotoxicity of the Parkinson's disease-associated LRRK2 mutant, G2019S, than its wild-type counter-part has recently been reported. Overexpression of LRRK2 (G2019S) in cultured neural cells results in caspase-3-dependent apoptosis via a yet undefined signaling pathway. Elucidation of the mechanism underlying LRRK2 (G2019S) neurotoxicity may offer new insights into the pathogenesis of Parkinson's disease. In this study, we identified glutathione s-transferase P1 (GSTP1) as a selective target whose expression is negatively regulated at the transcriptional levels via promoter hyper-methylation by LRRK2 (G2019S). Overexpression of LRRK2 (G2019S) in the human neuronal cell line SH-SY5Y markedly suppressed the expression of GSTP1 prior to any manifestation of cell death. Moreover, shRNA-mediated knockdown of endogenous GSTP1 expression exacerbated LRRK2 (G2019S) neurotoxicity, whereas overexpression of GSTP1 protected against LRRK2 (G2019S)-induced caspase-3 activation and neuronal apoptosis. In conclusion, the results suggest a previously undefined signaling mechanism underlying the neurotoxic effect of LRRK2 (G2019S), in which LRRK2 (G2019S) triggers oxidative stress in cells and, in turn, results in caspase-dependent apoptosis at least in part by suppressing the expression of GSTP1.

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 µg) 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 µg) 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.

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.

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 Parkinson's 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.

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.

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.

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.

Erythropoietin protects against 6-hydroxydopamine-induced dopaminergic cell death.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopaminergic neurons. In the present study, erythropoietin, a trophic factor that has both hematopoietic and neural protective characteristics, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. Using both the dopaminergic cell line, MN9D, and primary dopamine neurons, we show that erythropoietin (1-3 U/mL) is neuroprotective against the dopaminergic neurotoxin, 6-hydroxydopamine. Protection was mediated by the erythropoietin receptor, as neutralizing anti-erythropoietin receptor antibody abrogated the protection. Activation of Akt/protein kinase B (PKB), via the phosphoinositide 3-kinase pathway, is a critical mechanism in erythropoietin-induced protection, while activation of extracellular signal-regulated kinase (ERK)1/2 contributes only moderately. Indeed, transfection of constitutively active Akt/PKB into dopaminergic cells was sufficient to protect against cell death. Furthermore, erythropoietin diminished markers of apoptosis in MN9D cells, including caspase 9 and caspase 3 activation and internucleosomal DNA fragmentation, suggesting that erythropoietin interferes with the apoptosis-execution process. When erythropoietin was administered to mice unilaterally lesioned with 6-hydroxydopamine, it prevented the loss of nigral dopaminergic neurons and maintained striatal catecholamine levels for at least 8 weeks. Erythropoietin-treated mice also had significantly reduced behavioral asymmetries. These studies suggest that erythropoietin can be an effective neuroprotective agent for dopaminergic neurons, and may be useful in reversing behavioral deficits associated with Parkinson's disease.