Upregulation of clusterin/apolipoprotein J in lactacystin-treated SH-SY5Y cells

Clusterin (apolipoprotein J) is a highly conserved, multifunctional, vertebrate glycoprotein. Several isoforms of clusterin have been described including the predominant secreted isoform (sCLU) and several nuclear isoforms (nCLU) associated with cell death. sCLU has been shown to bind a variety of partly unfolded, stressed proteins including those associated with Lewy bodies (LBs) in patients with Parkinson's disease (PD). The development of familial and sporadic PD has been associated with the ubiquitin-proteasome system (UPS) dysfunction and aberrant protein degradation. This suggests that failure of the UPS to degrade abnormal proteins may underlie nigral degeneration and LB formation in PD. The effects of toxin-mediated proteasomal impairment on changes in gene expression and cell viability were studied in differentiated SH-SY5Y cells. Clusterin expression was increased in cells exposed for 24 hr to the proteasomal inhibitor lactacystin (10 microM) as determined by gene microarray analysis. RT-PCR showed that sCLU, not nCLU, was the major clusterin isoform expressed in both control and lactacystin-treated cells. Western blot analysis identified statistically significant increases in sCLU in total cell lysates after 24 hr of lactacystin exposure and showed that sCLU fractionates with the endoplasmic reticulum. Time-course studies demonstrated that maximal decreases in proteasome activity (4 hr) preceded maximal increases in clusterin expression (24 hr). Together these data suggest that proteasome impairment results in the upregulation of sCLU in SH-SY5Y cells, supporting the hypothesis that the association of clusterin with LBs in PD may be related to UPS failure.     

Knockdown of uncoupling protein-5 in neuronal SH-SY5Y cells: Effects on MPP+-induced mitochondrial membrane depolarization, ATP deficiency, and oxidative cytotoxicity

Uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis by dissipating proton gradient across mitochondrial inner membrane. The physiological role of neuronal specific UCP5 is unknown. We explored the effects of reduced UCP5 expression on mitochondrial membrane potential (MMP), oxidative stress, ATP levels, and cell viability, under normal and MPP+-induced cytotoxic conditions, in human catecholaminergic SH-SY5Y cells. UCP5 expression was reduced by 56% by siRNA, compared to scrambled-siRNA controls. UCP5 knockdown induced apoptosis but did not affect basal levels of ATP, oxidative stress and MMP in the cells under normal conditions. However, UCP5 knockdown increased MPP+-induced cytotoxicity by 15% and oxidative stress levels by 40%, and partially restored MPP+-induced mitochondrial depolarization by 57%. UCP2 and UCP4 expression were unaffected by UCP5 knockdown. Exacerbation of cytotoxicity, oxidative stress and modification of MMP with reduced UCP5 expression in the face of MPP+ toxicity suggest that UCP5 might be physiologically important in the pathology of oxidative stress-induced neurodegeneration.     

Overexpression of amyloid precursor protein induces susceptibility to oxidative stress in human neuroblastoma SH-SY5Y cells

Summary. In Alzheimer’s disease amyloid β peptide (Aβ) produced from amyloid precursor protein (APP) is considered to induce cell death. To clarify the molecular mechanism underlying Aβ neurotoxicity, we established the cell line overexpressing wild or mutant (His684Arg) APP in human SH-SY5Y cells. This paper presents that overexpression of wild-APP in the cells (SH/w-APP) increased the levels of APP and Aβ_1–40 but not Aβ_1–42, and reduced Bcl-2 level and proteasome activity with increased susceptibility to oxidative stress. The intracellular levels of reactive oxygen species in SH/w-APP increased significantly by H₂O₂ treatment. The level of Bcl-2 protein, but not mRNA, was markedly decreased in SH/w-APP cells, which was inversely correlated with APP expression among subcloned SH/w-APP cells. These results indicate that increased expression of wild type APP renders neuronal cells more vulnerable to oxidative stress leading to cell death.     

Characterization of catechol-thioether-induced apoptosis in human SH-SY5Y neuroblastoma cells

Recent work has highlighted the involvement of a dopamine derivative, 5-S-cysteinyl-dopamine (CysDA), in neurodegeneration and apoptotic cell death. In this paper we study in further detail the apoptotic process activated by this catechol-thioether derivative of dopamine in SH-SY5Y neuroblastoma cells. CysDA activates a cascade of events by an initial perturbation of Calcium homeostasis in the cell. Cell treatment with the catechol-thioether induces an immediate rise in intracellular Ca(2+) concentration, as demonstrated by a shift in the indo-1 dye emission spectrum, and a sustained high calcium concentration at long times of incubation. Fluorescence microscopy data show that the treatment of cells induces mitochondrial transmembrane potential depolarization, a clear evidence of the onset of apoptotic process. Programmed cell death activation is also demonstrated by cytochrome c release from the mitochondria, by an increased activity of both caspase-8 and -9 and by the poly(ADP-ribose)polymerase (PARP-1) cleavage, yielding the typical 86 kDa fragment due to caspase-3 activity. Overall, our data support the hypothesis that CysDA may induce apoptotic death in neuronal cells, via an initial perturbation of calcium homeostasis in the cytosol.     

MPP+ induced apoptotic cell death in SH-SY5Y neuroblastoma cells: An electron microscope study

PD is a common, late-onset neurodegenerative disorder that results in part from the gradual loss of dopaminergic neurons in the substantia nigra pars compacta. The neurotoxin MPTP can induce PD-like clinical symptomatology and neuropathological destruction and, thus, has been used as a PD model. The human neuroblastoma cell line SH-SY5Y possesses many of the qualities of human neurons and, as such, has served as a model for them. Apoptosis is the mode of cell death induced in SH-SY5Y cells by MPTP, and this was confirmed with nick end labeling and bisbenzimide staining. Transmission electron microscopic analysis of the ultrastructural changes occurring in neurotoxin exposed SH-SY5Ys revealed many morphological characteristics consistent with apoptosis. These changes included plasmalemmal blebbing, altered cytosolic density, nuclear condensation and fragmentation, pronounced vacuole formation, ribosomal dispersion, and the disappearance of the golgi complex, microtubules, and smooth endoplasmic reticulum. Limited amounts of rough endoplasmic reticulum and mitochondria exhibited normal morphology throughout the apoptotic changes but then were disrupted during secondary necrotic changes. The in vitro induction of apoptosis by a parkinsonism neurotoxin might be reflective of the mechanisms of in vivo nigral degeneration occurring during PD. J. Neurosci. Res. 48:226–237, 1997. © 1997 Wiley-Liss, Inc.     

Minocycline protects SH-SY5Y cells from 6-hydroxydopamine by inhibiting both caspase-dependent and -independent programmed cell death

Minocycline, a tetracyclic antibiotic, exerts both antiinflammation by acting on microglia and a direct protection on neurons by inhibiting the apoptotic machinery at various levels. However, we are not aware of any study investigating the effects of minocycline on caspase-independent programmed cell death (PCD) pathways. This study investigated these alternative pathways in SH-SY5Y cells, a human dopaminergic cell line, challenged with 6-hydroxydopamine (6-OHDA). Minocycline exhibited neuroprotection and inhibition of the toxin-induced caspase-3-like activity, DNA fragmentation, and chromatin condensation, hallmarks of apoptosis. Moreover, we revealed that 6-OHDA also activated caspase-independent PCDs (such as paraptosis), which required de novo protein synthesis. Additionally, by separately monitoring caspase-dependent and caspase-independent pathways, we showed that inhibition of apoptosis only partially explained the protective effect of minocycline. Moreover, we observed that minocycline reduced the protein content of cells but, unexpectedly, increased the protein synthesis. These findings suggest that minocycline may actually increase protein degradation, so it may also accelerate the clearance of aberrant proteins. In conclusion, we report for the first time evidence indicating that minocycline may inhibit PCD pathways that are additional to conventional apoptosis.     

Pathological effects of glyoxalase I inhibition in SH-SY5Y neuroblastoma cells

In Alzheimer's disease (AD), in aging, and under conditions of oxidative stress, the levels of reactive carbonyl compounds continuously increase. Accumulating carbonyl levels might be caused by an impaired enzymatic detoxification system. The major dicarbonyl detoxifying system is the glyoxalase system, which removes methylglyoxal in order to minimize cellular impairment. Although a reduced activity of glyoxalase I was evident in aging brains, it is not known how raising the intracellular methylglyoxal level influences neuronal function and the phosphorylation pattern of tau protein, which is known to be abnormally hyperphosphorylated in AD. To simulate a reduced glyoxalase I activity, we applied an inhibitor of glyoxalase I, p-bromobenzylglutathione cyclopentyl diester (pBrBzGSCp(2)), to SH-SY5Y neuroblastoma cells to induce chronically elevated methylglyoxal concentrations. We have shown that 10 microM pBrBzGSCp(2) leads to a fourfold elevation of the methylglyoxal level after 24 hr. In addition, glyoxalase I inhibition leads to reduced cell viability, strongly retracted neuritis, increase in [Ca(2+)](i), and activation of caspase-3. However, pBrBzGSCp(2) did not lead to tau "hyper"-phosphorylation despite activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase but rather activated protein phosphatases 2 and induced tau dephosphorylation at the Ser(202)/Thr(205) and Ser(396)/Ser(404) epitopes. Preincubation with the carbonyl scavenger aminoguanidine prevented tau dephosphorylation, indicating the specific effect of methylglyoxal. Also, pretreatment with the inhibitor okadaic acid prevented tau dephosphorylation, indicating that methylglyoxal activates PP-2A. In summary, our data suggest that a reduced glyoxalase I activity mimics some changes associated with neurodegeneration, such as neurite retraction and apoptotic cell death.     

Synergistic effects of lipopolysaccharide and rotenone on dopamine neuronal damage in rats

Introduction

The etiology of Parkinson's disease (PD) is still unknown. Until now, oxidative stress and neuroinflammation play a crucial role in the pathogenesis of PD. However, the specific synergistic role of oxidative stress and neuroinflammation in the occurrence and development of PD remains unclear.

Methods

The changes in motor behavior, dopamine (DA) neurons quantification and their mitochondrial respiratory chain, glial cells activation and secreted cytokines, Nrf2 signaling pathway, and redox balance in the brain of rats were evaluated.

Results

Lipopolysaccharide (LPS)-induced neuroinflammation and rotenone (ROT)-induced oxidative stress synergistically aggravated motor dysfunction, DA neuron damage, activation of glial cells, and release of related mediators, activation of Nrf2 signaling and destruction of oxidative balance. In addition, further studies indicated that after ROT-induced oxidative stress caused direct damage to DA neurons, LPS-induced inflammatory effects had stronger promoting neurotoxic effects on the above aspects.

Conclusions

Neuroinflammation and oxidative stress synergistically aggravated DA neuronal loss. Furtherly, oxidative stress followed by neuroinflammation caused more DA neuronal loss than neuroinflammation followed by oxidative stress.     

Iron accelerates the conversion of dopamine-oxidized intermediates into melanin and provides protection in SH-SY5Y cells

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons in the substantia nigra (SN), and it has been suggested that dopamine is one of the main endogenous toxins in the genesis of PD. We demonstrated that thiol antioxidants (the reduced form of glutathione, N-acetyl-L-cysteine, and L-cysteine), which conjugate with one dopamine oxidation intermediate, o-quinone, provided almost complete protection from dopamine-mediated toxicity in SH-SY5Y, a human neuroblastoma cell line. In contrast, catalase partially provided protection against cell death caused by dopamine. These data suggest that the generation of dopamine oxidation intermediates, rather than hydrogen peroxide, plays a pivotal role in dopamine-induced toxicity. Iron accumulated in the SN of patients with PD can cause dopaminergic neuronal degeneration by enhancing oxidative stress. However, we found that iron reduced the total amounts of dopamine oxidation intermediates and enhanced the formation of melanin, a final product of dopamine oxidation. Also, addition of iron inhibited dopamine-induced cytotoxicity. These results suggest that iron can provide protection when it accelerates the conversion of dopamine oxidation intermediates.     

D-beta-hydroxybutyrate protects dopaminergic SH-SY5Y cells in a rotenone model of Parkinson's disease

It has been postulated that the pathogenesis of Parkinson's disease (PD) is associated with mitochondrial dysfunction. Rotenone, an inhibitor of mitochondrial complex I, provides models of PD both in vivo and in vitro. We investigated the neuroprotective effect of D-beta-hydroxybutyrate (bHB), a ketone body, against rotenone toxicity by using SH-SY5Y dopaminergic neuroblastoma cells. SH-SY5Y cells, differentiated by all-trans-retinoic acid, were exposed to rotenone at concentrations ranging from 0 to 1,000 nM. We evaluated cellular oxidation reduction by the alamarBlue assay, viability by lactate dehydrogenase (LDH) assay, and survival/death ratio by live/dead assays. Exposure to rotenone for 48 hr oxidized cells and decreased their viability and survival rate in a concentration-dependent manner. Pretreatment of cells with 8 mM bHB provided significant protection to SH-SY5Y cells. Whereas rotenone caused the loss of mitochondrial membrane potential, released cytochrome c into the cytosol, and reduced cytochrome c content in mitochondria, addition of bHB blocked this toxic effect. bHB also attenuated the rotenone-induced activation of caspase-9 and caspase-3. Administration of 0-10 mM 3-nitropropionic acid, a complex II inhibitor, also decreased the reducing power of SH-SY5Y cells measured by alamarBlue assay. Pretreatment with 8 mM bHB attenuated the decrease of alamarBlue fluorescence. These data demonstrated that bHB had a neuroprotective effect that supported the mitochondrial respiration system by reversing the inhibition of complex I or II. Ketone bodies, the alternative energy source in the mammalian brain, appear to have therapeutic potential in PD.     

Neurotoxicity assessment of triazole fungicides on mitochondrial oxidative respiration and lipids in differentiated human SH-SY5Y neuroblastoma cells

Indiscriminate overuse or occupational exposure to agricultural chemicals can lead to neurotoxicity. Many pesticides act to impair mitochondrial function which can lead to exacerbation of neurodegeneration. Triazole fungicides are applied to grain, fruit, and vegetable crops to combat mold and fungi and their use is increasing worldwide. Here, we assessed the in vitro toxicity of two widely used triazole fungicides, propiconazole and tebuconazole, to mitochondria using differentiated SH-SY5Y neuroblastoma cells as an in vitro cell model used in Parkinson’s disease research. Cell viability (based on ATP levels), mitochondrial membrane potential, oxidative respiration, and reactive oxygen species (ROS) were measured following fungicide treatments. Cell viability was decreased with 100 μM propiconazole after 24 and 48 h, while tebuconazole required higher doses to affect viability (−200 μM at 24 h). Mitochondrial membrane potential (MMP) was reduced with 50 μM propiconazole after 24 h while 200 μM tebuconazole reduced MMP. Oxidative respiration of SH-SY5Y cells was then measured using a XFe24 Flux analyzer and 100 μM propiconazole reduced basal respiration, oligomycin-induced ATP production, and FCCP-induced maximum respiration by −40−50%, while tebuconazole did not affect mitochondrial bioenergetics at the concentrations tested. Acute exposure to 100 μM propiconazole over 4 h did not immediately affect oxidative respiration in SH-SY5Y cells. ROS were not induced by propiconazole and tebuconazole up to 100 and 300 μM respectively. Based on these results, we focused our lipidomics investigations on SH-SY5Y exposed only to propiconazole, as lipid dysregulation is associated with mitochondrial dysfunction. Both 50 and 100 μM propiconazole altered the abundance of some ceramides, specifically reducing glucosylceramide non-hydroxyfatty acid-sphingosine (HexCer-NS) and increasing N-stearoyl-phytosphingosine (CerNP). Moreover, a recently discovered bioactive lipid called fatty acid ester of hydroxy fatty acid (FAHFA) was increased 5-fold, hypothesized to be a neuroprotective mechanism that has been demonstrated in other studies of human diseases. Additional lipids reduced in abundance included oxidized phosphatidylcholine (OxPC) and oxidized phosphatidylethanolamine (OxPE). There were no changes in cellular triacylglycerols nor total lipids with exposure to propiconazole. Taken together, this study provides insight into the toxicity of triazole fungicides in neuronal cells, which has implications for neurodegenerative diseases that involve the mitochondria such as Parkinson’s disease.     

Specific up-regulation of GADD153/CHOP in 1-methyl-4-phenyl-pyridinium-treated SH-SY5Y cells

Growth arrest DNA damage-inducible 153 (GADD153) expression was increased in 1-methyl-4-phenyl-pyridinium (MPP(+))-treated human SH-SY5Y neuroblastoma cells as determined by gene microarray analysis. GADD153 expression increased after 24 hr of MPP(+) (1 mM) exposure and preceded activation of caspase 3. Comparison of GADD153 expression among cultures treated with other toxins whose primary mode of action is either via mitochondrial impairment (rotenone) or via oxidative stress (6-hydroxydopamine or hydrogen peroxide) showed that GADD153 was uniquely up-regulated by MPP(+). Together these data suggest that a cellular mechanism distinct from mitochondrial impairment or oxidative stress contributes significantly to the up-regulation of GADD153 by MPP(+) and that GADD153 may function as an inducer of apoptosis following MPP(+) exposure. Published 2002 Wiley-Liss, Inc.     

Selective nitration of mitochondrial complex I by peroxynitrite: involvement in mitochondria dysfunction and cell death of dopaminergic SH-SY5Y cells

Summary. 3-Nitrotyrosine (3-NT) is a specific marker of protein nitration by peroxynitrite (ONOO⁻) produced from nitric oxide and superoxide. Increase in 3-NT containing protein (3-NT protein) was reported in brains from patients with some neurodegenerative disorders and aging. In this paper, intracellular localization of 3-NT protein was examined in dopaminergic SH-SY5Y cells using the selective antibody against protein-bound 3-NT. 3-NT protein was detected in plasma membrane/nucleus and mitochondria fractions, and interestingly in polypeptide composition of mitochondrial complex I. ONOO⁻-generating SIN-1 induced apoptotic cell death with concomitant increase in 3-NT protein and reduction in mitochondrial ATP synthesis. In addition, an inhibitor of proteasomes, carbobenzoxy-L-isoleucyl-γ-t-butyl-L-glutamyl-L-alanyl-L-leucinal, enhanced the effects of ONOO⁻. These results suggest that ONOO⁻ may induce mitochondrial dysfunction and cell death in neurons through nitration of mitochondrial complex I subunits. Received March 12, 2001; accepted September 26, 2001     

Mortalin inhibition in experimental Parkinson's disease

Among heat shock proteins, mortalin has been linked to the pathogenesis of Parkinson's disease. In the present work a rat model of Parkinson's disease was used to analyze the expression of striatal proteins and, more specifically, mortalin expression. The possible involvement of mortalin in Parkinson's disease pathogenesis was further investigated by utilizing an electrophysiological approach and pharmacological inhibition of mortalin in both the physiological and the parkinsonian states. Proteomic analysis was used to investigate changes in striatal protein expression in the 6‐hydroxydopamine rat model of Parkinson's disease. The electrophysiological effects of MKT‐077, a rhodamine‐123 analogue acting as an inhibitor of mortalin, were measured by field potential recordings from corticostriatal brain slices obtained from control, sham‐operated, and 6‐hydroxydopamine–denervated animals. Slices in the presence of rotenone, an inhibitor of mitochondrial complex I, were also analyzed. Proteomic analysis revealed downregulation of mortalin in the striata of 6‐hydroxydopamine–treated rats in comparison with sham‐operated animals. MKT‐077 reduced corticostriatal field potential amplitude in physiological conditions, inducing membrane depolarization and inward current in striatal medium spiny neurons. In addition, we observed that concentrations of MKT‐077 not inducing any electrophysiological effect in physiological conditions caused significant changes in striatal slices from parkinsonian animals as well as in slices treated with a submaximal concentration of rotenone. These findings suggest a critical link between mortalin function and mitochondrial activity in both physiological and pathological conditions mimicking Parkinson's disease. © 2011 Movement Disorder Society     

REM sleep deprivation generates cognitive and neurochemical disruptions in the intranigral rotenone model of Parkinson's disease

The recently described intranigral rotenone model of Parkinson's disease (PD) in rodents provides an interesting model for studying mechanisms of toxin‐induced dopaminergic neuronal injury. The relevance of this model remains unexplored with regard to sleep disorders that occur in PD. On this basis, the construction of a PD model depicting several behavioral and neurochemical alterations related to sleep would be helpful in understanding the association between PD and sleep regulation. We performed bilateral intranigral injections of rotenone (12 μg) on day 0 and the open‐field test initially on day 20 after rotenone. Acquisition phase of the object‐recognition test, executed also during day 20, was followed by an exact period of 24 hr of rapid eye movement (REM) sleep deprivation (REMSD; day 21). In the subsequent day (22), the rats were re‐exposed to the open‐field test and to the object‐recognition test (choice phase). After the last session of behavioral tests, the rat brains were immediately dissected, and their striata were collected for neurochemical purposes. We observed that a brief exposure to REMSD was able to impair drastically the object‐recognition test, similarly to a nigrostriatal lesion promoted by intranigral rotenone. However, the combination of REMSD and rotenone surprisingly did not inflict memory impairment, concomitant with a moderate compensatory mechanism mediated by striatal dopamine release. In addition, we demonstrated the existence of changes in serotonin and noradrenaline neurotransmissions within the striatum mostly as a function of REMSD and REMSD plus rotenone, respectively. © 2013 Wiley Periodicals, Inc.     

Chronic rotenone treatment induces behavioral effects but no pathological signs of parkinsonism in mice

It has been hypothesized that exposures to neurotoxic pesticides together with aging and genetic factors increase the risk for developing Parkinson's disease (PD) which is characterized by a progressive degeneration of the nigrostriatal dopaminergic pathway. Chronic treatment with the pesticide rotenone has been reported to induce parkinsonism in rats. Although transgenic mice (but not transgenic rats) are available to investigate the importance of environmental factors in genetically predisposed animals, the effects of chronic rotenone exposure have so far not been examined in intact mice. Therefore, we investigated the effects of chronic exposure to rotenone (2.5 or 4.0-5.0 mg/kg s.c. for 30-45 days) in mice aged 2.5, 5, or 12 months. During the treatment period, the effects on vitality and motor behavior were investigated. Furthermore, the toxicity of rotenone on dopaminergic nigrostriatal neurons and peripheral tissues was examined. In comparison with control mice, rotenone-treated mice had a decreased spontaneous motor activity, but the density of nigral dopaminergic neurons failed to show any significant changes, except for a tendency to decrease in old mice treated with 4 mg/kg. At the tested doses, rotenone caused a moderate hepatic fatty degeneration. The data indicate that rotenone is not able to cause the neuropathological characteristics of PD in mice under these testing paradigms, which were similar to those of the rotenone rat model. Further studies will have to clarify whether genetic mouse models of PD might be more sensitive to the neurotoxic effects of rotenone.     

Proteasome inhibitor model of Parkinson's disease in mice is confounded by neurotoxicity of the ethanol vehicle.

Defects in the ubiquitin-proteasome system have been implicated in Parkinson's Disease (PD). Recently, a rat model of PD was developed using a synthetic proteasome inhibitor (PSI), (Z-lle-Glu(OtBu)-Ala-Leu-al). We attempted to transfer this model to mouse studies, where genetics can be more readily investigated due to the availability of genetically modified mice. We treated C57BL/6 (B6) mice with six intraperitoneal injections of 6 mg/kg PSI in 50 mul of 70% ethanol over a 2-week-period. We found significant decreases in nigrostriatal dopamine in PSI-treated mice compared with saline-treated mice. However, we observed similar decreases in the ethanol-treated vehicle control group. Administration of ethanol alone led to significant long-term alterations in dopamine levels. Ethanol significantly eclipses the effects of PSI in the dopamine system, and therefore is a confounding vehicle for this model.