Endoplasmic reticulum stress, an important factor in the development of Parkinson's disease.

Similar to other types of neuronal degeneration, Parkinson's disease (PD) is characterized by the aggregation of a pathological protein, α-synuclein. The endoplasmic reticulum (ER) is the principal site of protein synthesis, quality control and degradation. Genetic mutants, environmental insults and other factors disturb ER balance and induce the accumulation of misfolded/unfolded proteins, which initiate ER stress and disturb normal cell function. ER stress perturbs Ca2+ homeostasis and initiates the activation of autophagy and inflammasomes, which have been identified as risk factors for the development of PD. However, the mechanisms by which ER stress contributes to the processed of PD pathogenesis and development remain unclear. This review summarizes current knowledge of ER stress and highlights the principal role of ER stress in PD pathogenesis which may help reveal novel sight to illustrate the pathomechanism of PD.

A new coumarin derivative, IMM-H004, attenuates okadaic acid-induced spatial memory impairment in rats.

AIM: A novel coumarin derivative 7-hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin (IMM-H004) has shown anti-apoptotic, anti-inflammatory and neuroprotective activities. In this study we investigated the effects of IMM-H004 on spatial memory in rats treated with okadaic acid (OKA), which was used to imitate Alzheimer's disease (AD)-like symptoms. METHODS: SD rats were administered IMM-H004 (8 mg·kg(-1)·d(-1), ig) or donepezil (positive control, 1 mg·kg(-1)·d(-1), ig) for 25 d. On d 8 and 9, OKA (200 ng) was microinjected into the right ventricle. Morris water maze test was used to evaluate the spatial memory impairments. Tau and ß-amyloid (Aß) pathology in the hippocampus was detected using Western blot and immunohistochemistry. TUNEL staining was used to detect cell apoptosis. RESULTS: OKA-treated rats showed significant impairments of spatial memory in Morris water maze test, which were largely reversed by administration of IMM-H004 or donepezil. Furthermore, OKA-treated rats exhibited significantly increased phosphorylation of tau, deposits of Aß protein and cell apoptosis in the hippocampus, which were also reversed by administration of IMM-H004 or donepezil. CONCLUSION: Administration of IMM-H004 or donepezil protects rats against OKA-induced spatial memory impairments via attenuating tau or Aß pathology. Thus, IMM-H004 may be developed as a therapeutic agent for the treatment of AD.

Environment-contact administration of rotenone: A new rodent model of Parkinson's disease.

Epidemiological studies suggest an association between pesticides and the incidence of Parkinson's disease (PD). Individuals are likely to be exposed to numerous natural or synthetic environmental agents by ingestion, inhalation, or skin contact. Here, we describe a novel environment-contact administration of rotenone model, in which male C57BL/6 mice (15 per group per time-point) were placed in one bedding-free, rotenone-applied cage for 2h every day over a period of 2-6 weeks, mimicking the common ways a person may be exposed to pesticides. Our results showed that rotenone exposure had no detrimental effect on body weights of mice during 6 weeks, nor did it cause systemic toxicity (HPLC analysis of rotenone in blood and brain, as well as complex I activity measurements in brain and muscle), but it caused significant impairments in motor function (open field test, pole test, and rotarod test) from 4 weeks that were responsive to apomorphine. Accordingly, rotenone caused significant dopamine depletion from the striatum (HPLC analysis), nigrostriatal degeneration (quantitative tyrosine hydroxylase immunohistochemistry and western blot), and accumulation of α-synuclein in the substantia nigra and striatum (α-synuclein immunohistochemistry) in a time-dependent manner. In addition, rotenone-exposed mice also developed deficits in gastrointestinal and olfactory function (fecal pellet output and buried food pellet test) prior to the motor dysfunction. Furthermore, we observed that α-synuclein accumulated in the anterior olfactory nucleus and the enteric nervous system at 2 weeks. In summary, this novel rotenone model was able to reproduce many key aspects of PD progression. Therefore, it provides new insight into how environmental factors could trigger PD and provides a useful tool for studying PD pathogenesis and testing neuroprotective strategies.

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.

IMM-H004, a novel coumarin derivative compound, attenuates the production of inflammatory mediatory mediators in lipopolysaccharide-activated BV2 microglia.

Therapeutic strategies designed to inhibit the activation of microglia may lead to significant advancement in the treatment of most neurodegenerative diseases. 7-hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin (IMM-H004) is a novel compound and has been reported exerting potent neuroprotective effects which may be related to anti-inflammation. In the present study, the anti-inflammatory effects of IMM-H004 were investigated in lipopolysaccharide (LPS)-treated BV2 microglia. Our observations indicated that treatment with IMM-H004 significantly inhibited BV2 microglia activation, protected PC12 cells and primary neurons against indirect toxicity mediated by exposure to conditioned medium (CM) from LPS-treated BV2 cells. Additionally, IMM-H004 significantly suppressed the release of TNF-α, IL-1ß and NO, and suppressed the expression of pro-inflammatory mediators and cytokines such as iNOS, COX-2, and IL-6 in LPS-stimulated BV2 microglia. The nuclear translocation of NF-κB and the phosphorylation level of JNK and p38 MAPK pathways were also inhibited by IMM-H004 in LPS-treated BV2 microglia. Moreover, IMM-H004 also was a strong selective OH scavenger whose effect was similar with vitamin C. Overall, our findings suggested that IMM-H004 might be a promising therapeutic agent for alleviating the progress of neurodegenerative diseases associated with microglia activation.

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

α-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).

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 Parkinson's 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.

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 Parkinson's 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 Parkinson's 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.