Bingjie Xie

A newly discovered neurotoxin ADTIQ associated with hyperglycemia and Parkinson's disease.

BACKGROUND Diabetes is associated with an increased risk of Parkinsons disease (PD). Number of studies have suggested that methylglyoxal (MGO) induced by diabetes is related to PD. However, very little is known about its molecular mechanism. On other hand, 1-acetyl-6, 7- dihydroxyl-1, 2, 3, 4- Tetrahydroisoquinoline(ADTIQ) is a dopamine (DA)-derived tetrahydroisoquinoline (TIQ), a novel endogenous neurotoxins, which was first discovered in frozen Parkinsons disease human brain tissue. While ADTIQ precursor methylglyoxal was also found in diabetic patients related to the glucose metabolism and diabetic patients. METHODS LC-MS/MS, 1H NMR and infrared spectroscopy identified the structure of ADTIQ. The Annexin V-FITC/PI, MTT and western blot analysis were used to measure the neurotoxicity of ADTIQ. The levels of ADTIQ and methylglyoxal were detected by LC-MS/MS. RESULTS Here we report the chemical synthesis of ADTIQ, demonstrate its biosynthesis in SH-SY5Y neuroblastoma cell line and investigate its role in the pathogenesis of PD. In addition, a significant increase in the level of ADTIQ was detected in the brains of transgenic mice expressing mutant forms (A53T or A30P) of α-synuclein. ADTIQ also reduced the cell viability and induced mitochondrial apoptosis in dopaminergic cells, suggesting that ADTIQ acts as an endogenous neurotoxin and potentially involved in the pathogenesis of PD. Methylglyoxal, a major byproduct of glucose metabolism and abnormalities in glucose metabolism could influence the levels of ADTIQ. Consistent with the hypothesis, increased levels of ADTIQ and methylglyoxal were detected in the striatum of diabetic rats and SH-SY5Y cells cultured in the presence of high glucose concentrations. CONCLUSIONS Increased levels of ADTIQ could be related with Hyperglycemia and death of dopaminergic neurons. GENERAL SIGNIFICANCE The increased levels of ADTIQ could be a reason of dopamine neuron dysfunction in diabetes. Therefore, ADTIQ may play a key role in increasing the risk for PD in patients with diabetes.

Alpha-synuclein overexpression induced mitochondrial damage by the generation of endogenous neurotoxins in PC12 cells

Alpha-synuclein is one of the important components of Lewy body which involved in neuropathology of Parkinsons disease (PD). The relationship between α-synuclein and cell death is still unclear. In the study, PC12 cell, stably over expressing α-synuclein model was used, and we investigated the level of intracellular oxidative stress, dopamine and endogenous neurotoxin. The results showed that the level of oxidative stress and intracytoplasmic dopamine (DA) was increased in cells over expressing α-synuclein compared with normal PC12 cells. Simultaneously, additional generation of endogenous neurotoxins 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) and 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolin (NM-salsolinol) was detected and this phenomenon was exacerbated after exposed to H₂O₂ for 24 h, but mitigated when treated with dopamine synthesis inhibitors. The presence of endogenous neurotoxins exacerbated α-synuclein induced mitochondrial damage. These results suggest that the endogenous neurotoxins may become a bridge between α-synuclein and cell death.

Glial U87 cells protect neuronal SH-SY5Y cells from indirect effect of radiation by reducing oxidative stress and apoptosis

Recent studies have demonstrated the role of indirect effect of radiation in neurodegeneration. However, the role of glial cells in neuroprotection against indirect effect of radiation is still not clear, although they are known to protect neurons under stress conditions in central nervous system. Our study showed that indirect effect of radiation increased the oxidative stress that further enhances the expression of key apoptotic proteins and leads to neuronal cell death. We also investigated the indirect effect of radiation on neuronal cells in the presence of glial cells in a transwell co-culture system, while our analysis was focused on neuronal cells. Irradiated cell-conditioned medium (ICCM) was used as source of indirect radiation and neuroprotective effect was analyzed by various endpoints. It was observed that ICCM-induced reactive oxidative species level was significantly reduced in SH-SY5Y cells co-cultured with glial U87 cells, which might help to maintain the integrity of mitochondrial membrane potential. Increased levels of antioxidant enzyme superoxide dismutase and antioxidant glutathione were observed in SH-SY5Y cells co-cultured with glial U87 cells. Moreover, it was also observed that co-culture with glial cells inhibits the expression of ICCM-induced apoptotic proteins, i.e. Bax, cytochrome c, and caspase-3 in SH-SY5Y cells. Hence, it can be speculated that in co-culture system glial cells may protect the neuronal SH-SY5Y cells by reducing the ICCM-induced oxidative stress and apoptotic death.

Methylglyoxal increases dopamine level and leads to oxidative stress in SH-SY5Y cells

More and more studies have suggested that methylglyoxal (MGO) induced by type-2 diabetes is related to Parkinsons disease (PD). However, little is known about the molecular mechanism. In this study, we explored the MGO toxicity in neuroblastoma SH-SY5Y cells. Neurotoxicity of MGO was measured by mitochondrial membrane potential, malondialdehyde, and methylthiazoletetrazolium assays. The levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) were detected by liquid chromatography-mass spectrometry/mass spectrometry. The expressions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) were detected by reverse transcriptase polymerase chain reaction and western blot analysis. The results showed that MGO induced an increase in TH and DAT expressions in SH-SY5Y neuroblastoma cells, while the levels of dopamine, DOPAC, and endogenous neurotoxin salsolinol also increased. Aminoguanidine (AG) is an inhibitor of MGO. It was found that AG could decrease the reactive oxygen species (ROS) level induced by MGO, but could not inhibit an increase of TH, DAT and dopamine. The increase of dopamine, DOPAC and salsolinol levels could lead to high ROS and mitochondrial damage. This study suggests that ROS caused by dopamine could contribute to the damage of dopaminergic neurons when MGO is increased during the course of diabetes.

Arterial vascular cell line expressing SSAO: a new tool to study the pathophysiology of vascular amine oxidases

Semicarbazide-sensitive amine oxidase (SSAO) widely exists in nature, mainly expressed at significant levels in vasculature. It plays a detrimental role in vascular diseases, particularly atherosclerosis, which occurs mainly in arteries. Herein we for the first time present SSAO expression in arterial lineage of vascular cell line, i.e., human umbilical arterial endothelial cell (HUAEC). Firstly, two commercially available gene transfection reagents were compared to determine high transfection efficiency and then the expression behavior of HUAEC:SSAO was characterized. Furthermore, our model was also been compared with commonly used human embryonic kidney (HEK) cell transfected with the same vector. For enzymatic assay, an in-house developed highly sensitive high performance liquid chromatography electron spray ionization mass spectrometry method was applied. Results indicated that the maximal transfection efficiency in HUAEC was detected by JetPEI™ and transfected protein was expressed at membrane and cytosol of different clones. No significant variations were observed in HUAEC between cell passages 1 and 7, although HEK cell displayed twofold higher SSAO expression level than HUAEC. The transfected SSAO was shown to be released into the cell-culture medium. Both cellular and released types of SSAO exhibited monomer and dimer structural forms. The cytotoxicity determination exhibited large number of viable cells after transfection with JetPEI™. Differential expression characterization of this new cell line demonstrates the correct behavior of SSAO in arterial endothelial cells and also provides a real physiological environment to elucidate the unclear role of this enzyme. In addition, our cellular model could partly solve the problems raised by the loss of enzyme expression found in cultured endothelial cells. This model could also be a useful tool for proteomic base study, screening of interacting protein and analysis of compounds that could modify its activity for therapeutic purposes.

Determination of Endogenous Neurotoxin 1-Acetyl-6,7-Dihydroxyl-1,2,3,4-Tetrahydroisoquinoline in Rat Substantia Nigra by High Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

The endogenous neurotoxin, 1-acetyl-6,7-dihydroxyl-1,2,3,4 –tetrahydro isoquinoline (ADTIQ), is acatecholamine isoquinoline, indicating its possible involvement in Parkinsons disease. Moreover, it has been assumed to be a bridge between diabetes and Parkinsons disease because metabolic synthesis resembles glucose metabolism. Therefore, the accurate determination of ADTIQ could play an important role in diagnosis of diabetes and Parkinsons disease. Herein, a sensitive and precise method was established to determine ADTIQ by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry in multiple reaction monitoring mode. The [M+H]+ precursor product ions for ADTIQ were 208.1/190.1 and linear range of calibration curve was from 1.0 to 250.0 nM. The limit of detection and limit of quantification of the developed method were 0.2 nM and 1 nM, respectively. The inter-day, intra-day precision, accuracy, and recoveries were acceptable for biological samples. Furthermore, the reported method was used to determine ADTIQ levels in the substantia nigra of rats. The results indicated that the developed method was highly sensitive and accurate for the determination of ADTIQ.