Synaptic hyperexcitability of cytomegalic pyramidal neurons contributes to epileptogenesis in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a developmental disorder associated with epilepsy, autism, and cognitive impairment. Despite inactivating mutations in the TSC1 or TSC2 genes and hyperactive mechanistic target of rapamycin (mTOR) signaling, the mechanisms underlying TSC-associated neurological symptoms remain incompletely understood. Here we generate a Tsc1 conditional knockout (CKO) mouse model in which Tsc1 inactivation in late embryonic radial glia causes social and cognitive impairment and spontaneous seizures. Tsc1 depletion occurs in a subset of layer 2/3 cortical pyramidal neurons, leading to development of cytomegalic pyramidal neurons (CPNs) that mimic dysplastic neurons in human TSC, featuring abnormal dendritic and axonal overgrowth, enhanced glutamatergic synaptic transmission, and increased susceptibility to seizure-like activities. We provide evidence that enhanced synaptic excitation in CPNs contributes to cortical hyperexcitability and epileptogenesis. In contrast, astrocytic regulation of synapse formation and synaptic transmission remains unchanged after late embryonic radial glial Tsc1 inactivation, and astrogliosis evolves secondary to seizures.

Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations.

Heterozygous mutations in GBA, the gene encoding the lysosomal enzyme glucosylceramidase beta/ß-glucocerebrosidase, comprise the most common genetic risk factor for Parkinson disease (PD), but the mechanisms underlying this association remain unclear. Here, we show that in GbaL444P/WT knockin mice, the L444P heterozygous Gba mutation triggers mitochondrial dysfunction by inhibiting autophagy and mitochondrial priming, two steps critical for the selective removal of dysfunctional mitochondria by autophagy, a process known as mitophagy. In SHSY-5Y neuroblastoma cells, the overexpression of L444P GBA impeded mitochondrial priming and autophagy induction when endogenous lysosomal GBA activity remained intact. By contrast, genetic depletion of GBA inhibited lysosomal clearance of autophagic cargo. The link between heterozygous GBA mutations and impaired mitophagy was corroborated in postmortem brain tissue from PD patients carrying heterozygous GBA mutations, where we found increased mitochondrial content, mitochondria oxidative stress and impaired autophagy. Our findings thus suggest a mechanistic basis for mitochondrial dysfunction associated with GBA heterozygous mutations. Abbreviations: AMBRA1: autophagy/beclin 1 regulator 1; BECN1: beclin 1, autophagy related; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chloroyphenylhydrazone; CYCS: cytochrome c, somatic; DNM1L/DRP1: dynamin 1-like; ER: endoplasmic reticulum; GBA: glucosylceramidase beta; GBA-PD: Parkinson disease with heterozygous GBA mutations; GD: Gaucher disease; GFP: green fluorescent protein; LC3B: microtubule-associated protein 1 light chain 3 beta; LC3B-II: lipidated form of microtubule-associated protein 1 light chain 3 beta; MitoGreen: MitoTracker Green; MitoRed: MitoTracker Red; MMP: mitochondrial membrane potential; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH transcription factor; NBR1: NBR1, autophagy cargo receptor; Non-GBA-PD: Parkinson disease without GBA mutations; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; ROS: reactive oxygen species; SNCA: synuclein alpha; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1/Porin: voltage dependent anion channel 1; WT: wild type.

Peptidyl arginine deiminase-4 activation exacerbates kidney ischemia-reperfusion injury.

Peptidyl arginine deiminase (PAD)4 is a nuclear enzyme that catalyzes the posttranslational conversion of arginine residues to citrulline. Posttranslational protein citrullination has been implicated in several inflammatory autoimmune diseases, including rheumatoid arthritis, colitis, and multiple sclerosis. Here, we tested the hypothesis that PAD4 contributes to ischemic acute kidney injury (AKI) by exacerbating the inflammatory response after renal ischemia-reperfusion (I/R). Renal I/R injury in mice increased PAD4 activity as well as PAD4 expression in the mouse kidney. After 30 min of renal I/R, vehicle-treated mice developed severe AKI with large increases in plasma creatinine. In contrast, mice pretreated with PAD4 inhibitors (2-chloroamidine or streptonigrin) had significantly reduced renal I/R injury. Further supporting a critical role for PAD4 in generating ischemic AKI, mice pretreated with recombinant human PAD4 (rPAD4) protein and subjected to mild (20 min) renal I/R developed exacerbated ischemic AKI. Consistent with the hypothesis that PAD4 regulates renal tubular inflammation after I/R, mice treated with a PAD4 inhibitor had significantly reduced renal neutrophil chemotactic cytokine (macrophage inflammatory protein-2 and keratinocyte-derived cytokine) expression and had decreased neutrophil infiltration. Furthermore, mice treated with rPAD4 had significantly increased renal tubular macrophage inflammatory protein-2 and keratinocyte-derived cytokine expression as well as increased neutrophil infiltration and necrosis. Finally, cultured mouse kidney proximal tubules treated with rPAD4 had significantly increased proinflammatory chemokine expression compared with vehicle-treated cells. Taken together, our results suggest that PAD4 plays a critical role in renal I/R injury by increasing renal tubular inflammatory responses and neutrophil infiltration after renal I/R.

The volatile anesthetic isoflurane increases endothelial adenosine generation via microparticle ecto-5'-nucleotidase (CD73) release.

Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5'-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation.

Renalase prevents AKI independent of amine oxidase activity.

AKI is characterized by increased catecholamine levels and hypertension. Renalase, a secretory flavoprotein that oxidizes catecholamines, attenuates ischemic injury and the associated increase in catecholamine levels in mice. However, whether the amine oxidase activity of renalase is involved in preventing ischemic injury is debated. In this study, recombinant renalase protected human proximal tubular (HK-2) cells against cisplatin- and hydrogen peroxide-induced necrosis. Similarly, genetic depletion of renalase in mice (renalase knockout) exacerbated kidney injury in animals subjected to cisplatin-induced AKI. Interestingly, compared with the intact renalase protein, a 20-amino acid peptide (RP-220), which is conserved in all known renalase isoforms, but lacks detectable oxidase activity, was equally effective at protecting HK-2 cells against toxic injury and preventing ischemic injury in wild-type mice. Furthermore, in vitro treatment with RP-220 or recombinant renalase rapidly activated Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinases and downregulated c-Jun N-terminal kinase. In summary, renalase promotes cell survival and protects against renal injury in mice through the activation of intracellular signaling cascades, independent of its ability to metabolize catecholamines, and we have identified the region of renalase required for these effects. Renalase and related peptides show potential as therapeutic agents for the prevention and treatment of AKI.

Selective deletion of the endothelial sphingosine-1-phosphate 1 receptor exacerbates kidney ischemia-reperfusion injury.

The role for the endothelial sphingosine-1-phosphate 1 receptor (S1P1R) in acute kidney injury (AKI) remains unclear as germline endothelial S1P1R deletion is embryonically lethal. Here, we generated conditional endothelial S1P1R deficiency by crossing mice with floxed S1P1R with mice expressing a tamoxifen-inducible form of Cre recombinase under the transcriptional control of the platelet-derived growth factor-ß (Pdgfß) gene. Mice with tamoxifen-induced deletion of endothelial S1P1R had increased renal tubular necrosis, inflammation, and impaired vascular permeability, as well as exacerbated renal tubular apoptosis after ischemic AKI compared with tamoxifen-treated wild-type mice. Moreover, endothelial S1P1R deletion resulted in increased hepatic injury after ischemic AKI. As a potential mechanism for exacerbated renal injury, conditional endothelial S1P1R-null mice had markedly reduced endothelial HSP27 expression compared with wild-type mice. Cultured glomerular endothelial cells treated with a specific S1P1R antagonist (W146) for 3 days also showed reduced HSP27 expression compared with vehicle-treated cells. Finally, mice treated with W146 for 3 days also showed reduced endothelial HSP27 expression as well as exacerbated renal and hepatic injury after ischemic AKI. Thus, our studies demonstrate a protective role for endothelial S1P1R against ischemic AKI most likely by regulating endothelial barrier integrity and endothelial HSP27 expression.

Critical role of interleukin-11 in isoflurane-mediated protection against ischemic acute kidney injury in mice.

BACKGROUND: Isoflurane releases renal tubular transforming growth factor-ß1 (TGF-ß1) and protects against ischemic acute kidney injury. Recent studies suggest that TGF-ß1 can induce a cytoprotective cytokine interleukin (IL)-11. In this study, the authors tested the hypothesis that isoflurane protects against ischemic acute kidney injury by direct induction of renal tubular IL-11 synthesis. METHODS: Human kidney proximal tubule cells were treated with 1.25-2.5% isoflurane or carrier gas (room air + 5% carbon dioxide) for 0-16 h. The authors also anesthetized C57BL/6 mice with 1.2% isoflurane or with equianesthetic dose of pentobarbital for 4 h. In addition, the authors subjected IL-11 receptor (IL-11R) wild-type, IL-11R-deficient, or IL-11 neutralized mice to 30-min renal ischemia followed by reperfusion under 4 h of anesthesia with pentobarbital or isoflurane (1.2%). RESULTS: Isoflurane increased IL-11 synthesis in human (approximately 300-500% increase, N = 6) and mouse (23 ± 4 [mean ± SD] fold over carrier gas group, N = 4) proximal tubule cells that were attenuated by a TGF-ß1-neutralizing antibody. Mice anesthetized with isoflurane showed significantly increased kidney IL-11 messenger RNA (13.8 ± 2 fold over carrier gas group, N = 4) and protein (31 ± 9 vs. 18 ± 2 pg/mg protein or approximately 80% increase, N = 4) expression compared with pentobarbital-anesthetized mice, and this increase was also attenuated by a TGF-ß1-neutralizing antibody. Furthermore, isoflurane-mediated renal protection in IL-11R wild-type mice was absent in IL-11R-deficient mice or in IL-11R wild-type mice treated with IL-11-neutralizing antibody (N = 4-6). CONCLUSION: In this study, the authors suggest that isoflurane induces renal tubular IL-11 via TGF-ß1 signaling to protect against ischemic acute kidney injury.

IL-11 is required for A1 adenosine receptor-mediated protection against ischemic AKI.

A1 adenosine receptor activation ameliorates ischemic AKI through the induction of renal proximal tubular sphingosine kinase-1. However, systemic adverse effects may limit A1 adenosine receptor-based therapy for ischemic AKI, indicating a need to identify alternative therapeutic targets within this pathway. Here, we evaluated the function of renal proximal tubular IL-11, a clinically approved hematopoietic cytokine, in A1 adenosine receptor-mediated induction of sphingosine kinase-1 and renal protection. Treatment of human proximal tubule epithelial (HK-2) cells with a selective A1 adenosine receptor agonist, chloro-N(6)-cyclopentyladenosine (CCPA), induced the expression of IL-11 mRNA and protein in an extracellular signal-regulated kinase-dependent manner, and administration of CCPA in mice induced renal synthesis of IL-11. Pretreatment with CCPA protected against renal ischemia-reperfusion injury in wild-type mice, but not in IL-11 receptor-deficient mice. Administration of an IL-11-neutralizing antibody abolished the renal protection provided by CCPA. Similarly, CCPA did not induce renal IL-11 expression or protect against renal ischemia-reperfusion injury in mice lacking the renal proximal tubular A1 adenosine receptor. Finally, treatment with CCPA induced sphingosine kinase-1 in HK-2 cells and wild-type mice, but not in IL-11 receptor-deficient or renal proximal tubule A1 adenosine receptor-deficient mice. Taken together, these results suggest that induction of renal proximal tubule IL-11 is a critical intermediary in A1 adenosine receptor-mediated renal protection that warrants investigation as a novel therapeutic target for the treatment of ischemic AKI.

Reynosin protects against neuronal toxicity in dopamine-induced SH-SY5Y cells and 6-hydroxydopamine-lesioned rats as models of Parkinson's disease: Reciprocal up-regulation of E6-AP and down-regulation of α-synuclein.

Aggregation of α-synuclein (ASYN) is considered a major determinant of neuronal loss in Parkinson's disease (PD). E6-associated protein (E6-AP), an E3 ubiquitin protein ligase, has been known to promote the degradation of α-synuclein. The aim of this study was to assess the effects of the sesquiterpene lactone reynosin on dopamine (DA)-induced neuronal toxicity and regulation of E6-associated protein and α-synuclein proteins in both in vitro and in vivo models of Parkinson's disease. Usi"ng flow cytometry and western blot analysis, we determined that reynosin significantly protected both against cell death from dopamine-induced toxicity in human neuroblastoma SH-SY5Y cells and against the loss of tyrosine hydroxylase (TH)-positive cells in 6-hydroxydopamine (6-OHDA)-lesioned rats (a rodent Parkinson's disease model system). In addition, reynosin made up-regulation of E6-associated protein expression and down-regulation of the over-expression of α-synuclein protein in both dopamine-treated SH-SY5Y cells and 6-hydroxydopamine-lesioned rats. These results suggest that the protective effect of reynosin against dopamine-induced neuronal cell death may be due to the reciprocal up-regulation of E6-associated protein and down-regulation of α-synuclein protein expression.

The volatile anesthetic isoflurane induces ecto-5'-nucleotidase (CD73) to protect against renal ischemia and reperfusion injury.

The volatile anesthetic isoflurane protects against renal ischemia and reperfusion injury by releasing renal tubular TGF-ß1. As adenosine is a powerful cytoprotective molecule, we tested whether TGF-ß1 generated by isoflurane induces renal tubular ecto-5'-nucleotidase (CD73) and adenosine to protect against renal ischemia and reperfusion injury. Isoflurane induced new CD73 synthesis and increased adenosine generation in cultured kidney proximal tubule cells and in mouse kidney. Moreover, a TGF-ß1-neutralizing antibody prevented isoflurane-mediated induction of CD73 activity. Mice anesthetized with isoflurane after renal ischemia and reperfusion had significantly reduced plasma creatinine and decreased renal tubular necrosis, neutrophil infiltration, and apoptosis compared with pentobarbital-anesthetized mice. Isoflurane failed to protect against renal ischemia and reperfusion injury in CD73-deficient mice, in mice pretreated with a selective CD73 inhibitor, or in mice treated with an adenosine receptor antagonist. The TGF-ß1-neutralizing antibody or the CD73 inhibitor attenuated isoflurane-mediated protection against HK-2 cell apoptosis. Thus, isoflurane causes TGF-ß1-dependent induction of renal tubular CD73 and adenosine generation to protect against renal ischemia and reperfusion injury. Modulation of this pathway may have important therapeutic implications to reduce morbidity and mortality arising from ischemic acute kidney injury.

Critical role of interleukin-17A in murine intestinal ischemia-reperfusion injury.

Intestinal ischemia-reperfusion (I/R) injury causes severe illness frequently complicated by remote multiorgan dysfunction and sepsis. Recent studies implicated interleukin-17A (IL-17A) in regulating inflammation, autoimmunity, and I/R injury. Here, we determined whether IL-17A is critical for generation of intestinal I/R injury and subsequent liver and kidney injury. Mice subjected to 30 min of superior mesenteric artery ischemia not only developed severe small intestinal injury (necrosis, apoptosis, and neutrophil infiltration) but also developed significant renal and hepatic injury. We detected large increases in IL-17A in the small intestine, liver, and plasma. IL-17A is critical for generating these injuries, since genetic deletion of IL-17A- or IL-17A-neutralizing antibody treatment markedly protected against intestinal I/R injury and subsequent liver and kidney dysfunction. Intestinal I/R caused greater increases in portal plasma and small intestine IL-17A, suggesting an intestinal source for IL-17A generation. We also observed that intestinal I/R caused rapid small intestinal Paneth cell degranulation and induced murine α-defensin cryptdin-1 expression. Furthermore, genetic or pharmacological depletion of Paneth cells significantly attenuated the intestinal I/R injury as well as hepatic and renal dysfunction. Finally, Paneth cell depletion significantly decreased small intestinal, hepatic, and plasma IL-17A levels after intestinal I/R. Taken together, we propose that Paneth cell-derived IL-17A may play a critical role in intestinal I/R injury as well as extraintestinal organ dysfunction.

Paneth cell-mediated multiorgan dysfunction after acute kidney injury.

Acute kidney injury (AKI) is frequently complicated by extrarenal multiorgan injury, including intestinal and hepatic dysfunction. In this study, we hypothesized that a discrete intestinal source of proinflammatory mediators drives multiorgan injury in response to AKI. After induction of AKI in mice by renal ischemia-reperfusion or bilateral nephrectomy, small intestinal Paneth cells increased the synthesis and release of IL-17A in conjunction with severe intestinal apoptosis and inflammation. We also detected significantly increased IL-17A in portal and systemic circulation after AKI. Intestinal macrophages appear to transport released Paneth cell granule constituents induced by AKI, away from the base of the crypts into the liver. Genetic or pharmacologic depletion of Paneth cells decreased small intestinal IL-17A secretion and plasma IL-17A levels significantly and attenuated intestinal, hepatic, and renal injury after AKI. Similarly, portal delivery of IL-17A in macrophage-depleted mice decreased markedly. In addition, intestinal, hepatic, and renal injury following AKI was attenuated without affecting intestinal IL-17A generation. In conclusion, AKI induces IL-17A synthesis and secretion by Paneth cells to initiate intestinal and hepatic injury by hepatic and systemic delivery of IL-17A by macrophages. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from AKI.

Interleukin-11 protects against renal ischemia and reperfusion injury.

Renal ischemia reperfusion (IR) injury causes renal tubular necrosis, apoptosis, and inflammation leading to acute and chronic kidney dysfunction. IL-11 is a multifunctional hematopoietic cytokine clinically approved to treat chemotherapy-induced thrombocytopenia. Recent studies suggest that IL-11 also has potent antiapoptotic and antinecrotic properties. In this study, we tested the hypothesis that exogenous IL-11 protects against renal IR injury and determined the mechanisms involved in renal protection. Pretreatment with human recombinant IL-11 (HR IL-11) or with long-acting site-specific polyethylene glycol (PEG)-conjugated human IL-11 analog (PEGylated IL-11) produced partial but significant protection against renal IR injury in mice. In addition, HR IL-11 or PEGylated IL-11 given 30-60 min after IR also provided renal protection in mice. Significant reductions in renal tubular necrosis and neutrophil infiltration as well as tubular apoptosis were observed in mice treated with HR IL-11 or PEGylated IL-11. Furthermore, HR IL-11 or PEGylated IL-11 decreased both necrosis and apoptosis in human proximal tubule (HK-2) cells in culture. Mechanistically, IL-11 increased nuclear translocation of hypoxia-inducible factor-1α (HIF-1α) and induced sphingosine kinase-1 (SK1) expression and activity in HK-2 cells. Moreover, selective HIF-1α inhibitors blocked IL-11-mediated induction of SK1 in HK-2 cells. Finally, HR IL-11 or PEGylated IL-11 failed to protect against renal IR injury in SK1-deficient mice. Together, our data show powerful renal protective effects of exogenous IL-11 against IR injury by reducing necrosis, inflammation, and apoptosis through induction of SK1 via HIF-1α.

A1 adenosine receptor allosteric enhancer PD-81723 protects against renal ischemia-reperfusion injury.

Activation of A(1) adenosine receptors (ARs) protects against renal ischemia-reperfusion (I/R) injury by reducing necrosis, apoptosis, and inflammation. However, extrarenal side effects (bradycardia, hypotension, and sedation) may limit A(1)AR agonist therapy for ischemic acute kidney injury. Here, we hypothesized that an allosteric enhancer for A(1)AR (PD-81723) protects against renal I/R injury without the undesirable side effects of systemic A(1)AR activation by potentiating the cytoprotective effects of renal adenosine generated locally by ischemia. Pretreatment with PD-81723 produced dose-dependent protection against renal I/R injury in A(1)AR wild-type mice but not in A(1)AR-deficient mice. Significant reductions in renal tubular necrosis, neutrophil infiltration, and inflammation as well as tubular apoptosis were observed in A(1)AR wild-type mice treated with PD-81723. Furthermore, PD-81723 decreased apoptotic cell death in human proximal tubule (HK-2) cells in culture, which was attenuated by a specific A(1)AR antagonist (8-cyclopentyl-1,3-dipropylxanthine). Mechanistically, PD-81723 induced sphingosine kinase (SK)1 mRNA and protein expression in HK-2 cells and in the mouse kidney. Supporting a critical role of SK1 in A(1)AR allosteric enhancer-mediated renal protection against renal I/R injury, PD-81723 failed to protect SK1-deficient mice against renal I/R injury. Finally, proximal tubule sphingosine-1-phosphate type 1 receptors (S1P(1)Rs) are critical for PD-81723-induced renal protection, as mice selectively deficient in renal proximal tubule S1P(1)Rs (S1P(1)R(flox/flox) PEPCK(Cre/-) mice) were not protected against renal I/R injury with PD-81723 treatment. Taken together, our experiments demonstrate potent renal protection with PD-81723 against I/R injury by reducing necrosis, inflammation, and apoptosis through the induction of renal tubular SK1 and activation of proximal tubule S1P(1)Rs. Our findings imply that selectively enhancing A(1)AR activation by locally produced renal adenosine may be a clinically useful therapeutic option to attenuate ischemic acute kidney injury without systemic side effects.

Regulatory effects of costunolide on dopamine metabolism-associated genes inhibit dopamine-induced apoptosis in human dopaminergic SH-SY5Y cells.

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic (DAergic) neurons in the substantia nigra and the subsequent depletion of dopamine (DA). This study assessed the protective effects of costunolide on DA-induced apoptosis in human DAergic SH-SY5Y cells, and its regulation of DA metabolism-associated gene and protein expression. Annexin V and propidium iodide (PI) staining using flow cytometric analysis (FACS) revealed that costunolide significantly protected human DAergic SH-SY5Y cells against DA-induced apoptosis. In addition, co-treatment of costunolide with DA in SH-SY5Y cells regulated DA metabolism-associated gene expression, as we observed an increase in both mRNA and protein levels of nuclear receptor related-1 (Nurr1), DA transporter (DAT), and vesicular monoamine transporter type 2 (VMAT2). In contrast, α-synuclein (ASYN) protein levels were decreased. Our findings suggest that costunolide has anti-apoptotic activity, presumably due to its regulatory effects on DA metabolism-associated genes. Therefore, costunolide could be considered as a candidate therapy for the treatment of Parkinson's disease.

Moracenin D from Mori Cortex radicis protects SH-SY5Y cells against dopamine-induced cell death by regulating nurr1 and α-synuclein expression.

In our efforts to find neuroprotective materials of plant origin, several compounds were isolated from Mori Cortex Radicis. The protective effect against dopamine-induced cell death was examined, and the subsequent effects on the levels of expression of Parkinson's disease-associated nurr1 and α-synuclein were evaluated in a dopamine-induced system. Five compounds were isolated and moracenin D protected cell death against dopamine-induction in human neuroblastoma SH-SY5Y cells. The effects of moracenin D on the levels of mRNA and protein expression of nurr1 and α-synuclein were subsequently examined using reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Treatment with moracenin D resulted in an up-regulation of nurr1 mRNA levels and a down-regulation of α-synuclein mRNA levels. Additionally, the α-synuclein protein expression was decreased in accordance with an increase in nurr1 protein expression. These results demonstrate that the protective effects of moracenin D were presumably due to the correlative effects on the up-regulation of nurr1 and down-regulation of α-synuclein expressions against dopamine induction. Therefore, moracenin D can be considered as a candidate for therapy for Parkinson's disease.

Neuroprotective effects of 3α-acetoxyeudesma-1,4(15),11(13)-trien-12,6α-olide against dopamine-induced apoptosis in the human neuroblastoma SH-SY5Y cell line.

Dopamine (DA), as a neurotoxin, can elicit severe Parkinson's disease-like syndrome by elevating intracellular reactive oxygen species (ROS) levels and apoptotic activity. We examined the inhibitory effects of 3α-acetoxyeudesma-1,4(15),11(13)-trien-12,6α-olide (AETO), purified from the leaves of Laurus nobilis L., on DA-induced apoptosis and α-synuclein (α-syn) formation in dopaminergic SH-SY5Y cells. AETO decreased the active form of caspase-3 and the levels of p53, which were accompanied by increased levels of Bcl-2 in a dose-dependent manner. Flow cytometric and Western blot analysis showed that AETO significantly inhibited DA-induced apoptosis along with suppression of intracellular tyrosinase activity, ROS generation, quinoprotein, and α-syn formation (P < 0.01). These results indicate that AETO inhibited DA-induced apoptosis, which is closely related to the suppression of intracellular tyrosinase activity and the formation of α-syn, ROS, and quinoprotein in SH-SY5Y cells.

Spirafolide from bay leaf (Laurus nobilis) prevents dopamine-induced apoptosis by decreasing reactive oxygen species production in human neuroblastoma SH-SY5Y cells.

Reactive oxygen species (ROS) are important mediators in many neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. This study tested the neuroprotective effects of spirafolide, a compound purified from the leaves of Laurus nobilis L. (Lauraceae), against dopamine (DA)-induced apoptosis in human neuroblastoma SH-SY5Y cells. Following a 24-h exposure of cells to DA (final conc., 0.6 mM), we observed a marked increase in apoptosis, increased generation of ROS and decreased cell viability. Pretreatment of the cells for 24 h with spirafolide (0.4, 2, and 10 µM) before exposure to DA notably increased cell survival (p < 0.01) and lowered intracellular ROS levels (p < 0.01). These results indicate that spirafolide has neuroprotective effects against DA toxicity. These effects may contribute to the treatment of neurodegenerative diseases.