Kai-Li Ma

Overexpression of Human E46K Mutant α-Synuclein Impairs Macroautophagy via Inactivation of JNK1-Bcl-2 Pathway

Parkinson’s disease (PD) is pathologically characterized by selective loss of dopaminergic neurons in the midbrain and the existence of intracellular protein inclusions termed Lewy bodies, largely composed of α-synuclein. Genetic studies have revealed that rare point mutations in the gene encoding α-synuclein including A30P, A53T, and E46K are associated with familial forms of PD, indicating a pathological role for mutant α-synuclein in PD etiology. However, the mechanisms underlying the neuronal toxicity of mutant α-synuclein are still to be elucidated. Growing evidence has suggested a deleterious effect of mutant α-synuclein on the autophagy-lysosome pathway. In this study, we discovered that overexpression of human E46K mutant α-synuclein impaired macroautophagy in mammalian cells. Our data showed that overexpression of E46K mutant α-synuclein impaired autophagy at an early stage of autophagosome formation via the c-Jun N-terminal kinase 1 (JNK1)-Bcl-2 but not the mammalian target of rapamycin (mTOR) pathway. Overexpressed E46K mutant α-synuclein inhibited JNK1 activation, leading to a reduced Bcl-2 phosphorylation and increased association between Bcl-2 and Beclin1, further disrupting the formation of Beclin1/hVps34 complex, which is essential for autophagy initiation. Furthermore, overexpression of E46K mutant α-synuclein increased the vulnerability of differentiated PC12 cells to rotenone treatment, which would be partly due to its inhibitory effects on autophagy. Our findings may shed light on the potential roles of mutant α-synuclein in the pathogenesis of PD.

Targeted Overexpression of α-Synuclein by rAAV2/1 Vectors Induces Progressive Nigrostriatal Degeneration and Increases Vulnerability to MPTP in Mouse

Mutations, duplication and triplication of α-synuclein genes are linked to familial Parkinson’s disease (PD), and aggregation of α-synuclein (α-syn) in Lewy bodies (LB) is involved in the pathogenesis of the disease. The targeted overexpression of α-syn in the substantia nigra (SN) mediated by viral vectors may provide a better alternative to recapitulate the neurodegenerative features of PD. Therefore, we overexpressed human wild-type α-syn using rAAV2/1 vectors in the bilateral SN of mouse and examined the effects for up to 12 weeks. Delivery of rAAV-2/1-α-syn caused significant nigrostriatal degeneration including appearance of dystrophic striatal neurites, loss of nigral dopaminergic (DA) neurons and dissolving nigral neuron bodies in a time-dependent manner. In addition, the α-syn overexpressed mice also developed significant deficits in motor function at 12 weeks when the loss of DA neurons exceeded a threshold of 50%. To investigate the sensitivity to neurotoxins in mice overexpressing α-syn, we performed an MPTP treatment with the subacute regimen 8 weeks after rAAV injection. The impact of the combined genetic and environmental insults on DA neuronal loss, striatal dopamine depletion, dopamine turnover and motor dysfunction was markedly greater than that of either alone. Moreover, we observed increased phosphorylation (S129), accumulation and nuclear distribution of α-syn after the combined insults. In summary, these results reveal that the overexpressed α-syn induces progressive nigrostriatal degeneration and increases the susceptibility of DA neurons to MPTP. Therefore, the targeted overexpression of α-syn and the combination with environmental toxins may provide valuable models for understanding PD pathogenesis and developing related therapies.

The nuclear accumulation of alpha-synuclein is mediated by importin alpha and promotes neurotoxicity by accelerating the cell cycle

α-Synuclein (α-syn), a 14 kDa pre-synaptic protein, is widely involved in the Parkinsons disease (PD) pathogenesis. Recent studies have shown that the nuclear accumulation of α-syn might have a toxic effect. The main purpose of the present study was to explore which amino acid residues in α-syn are associated with its nuclear accumulation, the molecule(s) mediated the nuclear import of α-syn, and the role of α-syn accumulated in the nucleus. It has been noted that the nuclear import of α-syn may be mediated by importin α and that both the amino acid residues 1-60 and 103-140 of α-syn were indispensable for its nuclear import. After imported into the nucleus, the accumulated α-syn played a toxic role in both the PC12 cells and the C57 mice. Furthermore, α-syn-nuclear localization signal-injected mice showed behavioral symptoms associated with PD. Further studies performed in vitro showed that the toxicity of α-syn in the nucleus might be due to an interference of the cell cycle. Thus, it can be concluded that α-syn can accumulate in nucleus, which is mediated by importin α, and promote neurotoxicity by accelerating the cell cycle.

Expression of chemokine-like factor 1 after focal cerebral ischemia in the rat.

Cerebral ischemic injury is associated with the induction of a series of pro-inflammatory mediators such as cytokines and chemokines. The chemokine-like factor 1(CKLF1), as a novel human cytokine, displays chemotactic activities in a wide spectrum of leukocytes. The present study was conducted to determine if CKLF1 was produced in the brain of rats subjected to transient middle cerebral artery occlusion (TMCAO). Therefore, RT-PCR, Western blot and immunohistochemistry were utilized to characterize the expression of CKLF1 at different times after TMCAO. The result showed that almost no expression of CKLF1 was found in the sham-operated or contralateral cerebral cortex and hippocampus. CKLF1 expression significantly increased in the ischemic cerebral cortex and hippocampus, elevating at 12 h and peaking at 2 days after reperfusion. CKLF1 positive staining was mainly present in the cerebral cortex, hippocampus, thalamus, and hypothalamus. These results demonstrate that the expression of CKLF1 increases after focal cerebral ischemia in rat brain. Thus, CKLF1 may be a potential therapeutic target for cerebral ischemia.

DJ-1 regulating PI3K-Nrf2 signaling plays a significant role in bibenzyl compound 20C-mediated neuroprotection against rotenone-induced oxidative insult

Oxidative stress is thought to be involved in the development of Parkinsons disease (PD). We previously reported that 20C, a bibenzyl compound isolated from Gastrodia elata, possesses antioxidative properties, but its in-depth molecular mechanisms against rotenone-induced neurotoxicity remains unknown. Recent studies indicate that without intact DJ-1, nuclear factor erythroid 2-related factor (Nrf2) protein becomes unstable, and the activity of Nrf2-mediated downstream antioxidant enzymes are thereby suppressed. In this study, we showed that 20C clearly protected PC12 and SH-SY5Y cells against rotenone-induced oxidative injury. Furthermore, 20C markedly up-regulated the levels of DJ-1, which in turn activated phosphoinositide-3-kinase (PI3K)/Akt signaling and inhibited glycogen synthase kinase 3β (GSK3β) activation, eventually promoted the nuclear translocation of Nrf2 and induced the expression of hemeoxygenase-1 (HO-1). The antioxidant effects of 20C could be partially blocked by ShRNA-mediated knockdown of DJ-1 and inhibition of the PI3K/Akt pathways with Akt1/2 kinase inhibitor, respectively. Conclusively, our findings confirm that DJ-1 is necessary for 20C-mediated protection against rotenone-induced oxidative damage, at least in part, by activating PI3K/Akt signaling, and subsequently enhancing the nuclear accumulation of Nrf2. The findings from our investigation suggest that 20C should be developed as a novel candidate for alleviating the consequences of PD in the future.

α-Synuclein is prone to interaction with the GC-box-like sequence in vitro.

Abstractα-Synuclein (α-syn) is a presynaptic protein that is widely implicated in the pathophysiology of Parkinson’s disease, a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons and the formation of Lewy bodies. Evidence suggests that α-syn could be imported into the nucleus and subsequently disrupt normal neuronal function. The existence of α-syn in the nucleus provides the possibility of interaction with DNA leading to gene transcript regulation. Thus, CD spectra were used to determine the specific DNA sequence with which α-syn is most likely to interact. Our results indicated that α-syn was prone to preferentially interact with the GC-box-like sequence in vitro at a ratio of 2:1 or less (α-syn: the GC-box-like sequence).

Deletion in exon 5 of the SNCA gene and exposure to rotenone leads to oligomerization of α-synuclein and toxicity to PC12 cells.

α-Synuclein (α-syn) is a presynaptic protein that is widely implicated in the pathophysiology of Parkinsons disease (PD). Recently, four α-syn isoforms that are produced by alternative splicing have been described, they are α-syn140, α-syn126, α-syn112, and α-syn98. The stable cell lines which expressed the four α-syn isoforms respectively were obtained, and the aggregation formation of these α-syn isoforms and their associated toxicity to PC12 cell were investigated. The results of this study indicate that over-expression of α-syn isoforms alone in dopaminergic cells have no effect on the formation of α-syn oligomeric species and cell viabilities. When exposed to rotenone, these cell lines which over expressed exon 5-lacking form of α-syn isoforms showed the formation of oligomeric species and toxicity to PC12 cells.

Over-expression of α-synuclein 98 triggers intracellular oxidative stress and enhances susceptibility to rotenone

The α-synuclein protein is a major component of Lewy bodies found in the brains of patients with Parkinsons disease (PD). Recently, α-synuclein 98 (α-syn98), a small isoform of the wild type protein was isolated. The neurotoxicity of this protein was assessed by over-expressing α-syn98 in dopaminergic cells. Enhanced expression of α-syn98 was insufficient to adversely affect the survival of neurons or to promote aggregation of the protein. However, when exposed to rotenone, α-syn98 over-expressing dopaminergic cells demonstrated significantly increased cytotoxicity and aggregate formation. Furthermore, we found enhanced basal ROS production and MDA levels in α-syn98 over-expressing neurons. High basal oxidative stress induced by α-syn98, combined with oxidative stress caused by rotenone treatment, promoted aggregate formation and significantly decreased cell viability. These data indicate that α-syn98 can enhance the susceptibility of dopaminergic neurons to oxidative insults by raising steady-state levels of oxidative stress.

Nurr1: A vital participant in the TLR4-NF-κB signal pathway stimulated by α-synuclein in BV-2 cells

&NA; Parkinsons disease (PD) is a multi‐factorial neurodegenerative disease. Abnormal &agr;‐synuclein protein aggregate and sustained microglia activation contribute to the pathogenic processes of PD. However, the relationship between &agr;‐synuclein and microglia‐mediated neuroinflammation remains unclear. We purified &agr;‐synuclein after overexpression in Escherichia coli and then used it to stimulate BV‐2 cells or primary microglia cells from wild type or toll‐like receptor 4 (TLR4)‐defective mice. Enzyme linked immunosorbent assay (ELISA) and real‐time PCR results confirmed that &agr;‐synuclein could enhance the production of tumor necrosis factor &agr; (TNF‐&agr;) through TLR4 activation. Western blotting results confirmed the involvement of the TLR4/PI3K/AKT/GSK3&bgr; signal pathway in the inflammatory response. Nuclear factor kappa B (NF‐&kgr;B) could translocate to the nucleus, promoting the expression of TNF‐&agr; when stimulated by &agr;‐synuclein in BV‐2 cells. Nurr1 suppressed the production of TNF‐&agr; via interaction with NF‐&kgr;B/p65 and inhibiting its nuclear translocation. In addition, both NF‐&kgr;B and Nurr1 appeared to be regulated by the TLR4‐mediated signal pathway. Our work demonstrated that TLR4 recognized &agr;‐synuclein and activated downstream signaling mechanisms leading to the release of pro‐inflammatory mediators that are contra‐balanced by Nurr1 expression. In conclusion, Nurr1 is a novel participant in the neuroinflammation stimulated by &agr;‐synuclein, thus the regulation of Nurr1 may be a novel neuroprotective target for PD treatment. HIGHLIGHTS&agr;‐Synuclein enhanced the production of TNF‐&agr; through TLR4 activation.TLR4/PI3K/AKT/GSK3&bgr; signal pathway is involved in the inflammatory response.&agr;‐Synuclein induced nuclear translocation of NF‐&kgr;B/p65 in BV‐2 cells.Nurr1 may suppress the production of TNF‐&agr; via interaction with NF‐&kgr;B/p65.Both NF‐&kgr;B and Nurr1 are regulated by the TLR4‐mediated signal pathway.

Quantitative Proteomic Analysis of Hepatic Tissue of T2DM Rhesus Macaque

Type 2 diabetes mellitus (T2DM) is a metabolic disorder that severely affects human health, but the pathogenesis of the disease remains unknown. The high-fat/high-sucrose diets combined with streptozotocin- (STZ-) induced nonhuman primate animal model of diabetes are a valuable research source of T2DM. Here, we present a study of a STZ rhesus macaque model of T2DM that utilizes quantitative iTRAQ-based proteomic method. We compared the protein profiles in the liver of STZ-treated macaques as well as age-matched healthy controls. We identified 171 proteins differentially expressed in the STZ-treated groups, about 70 of which were documented as diabetes-related gene in previous studies. Pathway analyses indicated that the biological functions of differentially expressed proteins were related to glycolysis/gluconeogenesis, fatty acid metabolism, complements, and coagulation cascades. Expression change in tryptophan metabolism pathway was also found in this study which may be associations with diabetes. This study is the first to explore genome-wide protein expression in hepatic tissue of diabetes macaque model using HPLC-Q-TOF/MS technology. In addition to providing potential T2DM biomarkers, this quantitative proteomic study may also shed insights regarding the molecular pathogenesis of T2DM.