The Inducing Effect Of Novel Compound FLZ On Heat Shock Proteins And Its Protective Effect On Dopaminergic System In Experimental Parkinson's Disease

Parkinson's disease (PD), an age-related neurodegenerative disorder, is the second most common progressive neurodegenerative brain disorder of humans, after Alzheimer's disease (AD). The neuropathological hallmarks are characterized by progressive and profound loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) with presence of eosinophillic, intracytoplamic, proteinaceous inclusions termed as Lewy bodies (LB) in surviving neurons, which lead to dysfunction of the dopaminergic neurons and relative domination of cholinergic function. All the above alterations finally result in development of dyskinesis. Clinical features of PD include resting tremor, rigidity, bradykinesia and postural instability.The etiology of PD is obscure, but it is believed that it may result from an environmental factor, a genetic causation or a combination of the two. The pathogenesis of PD is thought to include mitochondrial dysfunction, oxidative stress, protein misfolding and/or aggregation, and inflammation. Drugs currently used for PD therapy can only improve clinical symptoms but can not retard the progress of the disease. Therefore, new drugs which focus on ameliorating the symptoms of PD, on neuroprotection and neurorescue that can favourably delay the natural course of the disease are urgently needed.FLZ is a novel synthetic cyclic derivative of squamosamide. Our previous studies have demonstrated that FLZ has strong neuroprotective activity and improves the behavior deficits in experimental AD and PD models. In vitro assay, FLZ markedly reduced inflammation and apoptosis of neurons exposed to several kinds of neurotoxins. FLZ, therefore, is a promising drug candidate for treatment of PD. In this thesis, the neuroprotective effect and related mechanism of FLZ were further examined in SH-SY5Y cells, PD rat model and MN9D cells. This study includes three parts as below: Part I The inducing effect of FLZ on heat shock proteins and its protectective effect on SH-SY5Y cells intoxicated by MPP+ Heat shock proteins (HSPs) play an essential role in various neurodegenerative diseases. Manipulation of upregulation of HSPs in cells has been demonstrated to provide a therapeutic strategy to counteract the misfolding and aggregation of proteins that resulted in neurodegenerative disease. Our previous studies have shown that FLZ, a synthetic novel derivative of squamosamide from a Chinese herb, had potent neuroprotective effect against several experimental Parkinson's disease (PD) models. But the mechanism of its neuroprotective effect is still not clarified. The present study demonstrated that FLZ induced HSP27 and HSP70 protein and mRNA expression in a time- and dose-dependent manner in SH-SY5Y cells. Further studies showed that FLZ treatment stimulated the activation of heat shock factor 1 (HSF1) and its regulatory kinase Akt. Inactivation of Akt pathway by the PI3K inhibitor LY294002 blocked the expression of HSP27 and HSP70 induced by FLZ. Moreover, the inducing effects of FLZ on HSP27, HSP70 and HSF1 were all blocked by quercetin, an inhibitor of HSP biosynthesis. The cytoprotective effect of HSP27/HSP70 induced by FLZ against MPP+ was assessed in SH-SY5Y cells. The pretreatment of FLZ significantly induced the accumulations of HSP27/HSP70 and suppressed the apoptosis caused by MPP+ in SH-SY5Y cells. However, the protective effects of FLZ against MPP+ were significantly blocked by quercetin, which indicated that the cytoprotective action of FLZ against MPP+-induced apoptosis is at least partially mediated by its induction of HSP27/HSP70. These results provide new evidence for elucidating the mechanism of the neuroprotective effect of FLZ against PD.Part II FLZ protects against 6-hydroxydopamine-induced experimental Parkinson's disease model of rat and the underling mechanismsThe aim of this experiment was to study the neuroprotective effect of FLZ on the unilateral 6-hydroxydopamine (6-OHDA) lesion to the substantia nigra pars compacta (SNc) of rats. The successfully bult PD model was screened by apomorphine two weeks later after surgery. The rats were orally treated with FLZ 20 mg/kg,35 mg/kg and 50 mg/kg once a day for consecutive four weeks, and their dyskinesis was examined every two weeks. The neuropathology and biochemical alterations in the SNc and striatum of rats were detected by immunohistochemical and westernt-blot assay respectively. The DA concentration in the striatum of rats was detected by HPLC assay. The results showed that FLZ rescued dopaminergic neurons, restored striatal DA level, and thereby reversed behavioral asymmetry in the unilateral 6-OHDA lesioned rat model. In further studies, we found that FLZ reversed the down-regulation of phosphorylated TH and TH expression caused by 6-OHDA, while it had no effect on the activity of MAO-B which is the metabolic enzyme of DA, suggesting that FLZ increased the DA level mainly through increasing the expression of TH. We also found FLZ reversed the down-regulation of phosphorylation of Akt expression and up-regulation ofα-synuclein, caspase-3 and RTP801 expression caused by 6-OHDA, therefore FLZ facilitated the survival of neurons and thereby up-regulated the expression of TH. Moreover, FLZ facilitated the phosphorylation of TH through inhibiting the binding ofα-synuclein to TH, which negatively regulated the TH activity. These results demonstrate that FLZ is a promising drug candidate for the treatment of Parkinson's disease.PartⅢFLZ protects dopaminergic system in MN9D cells intoxicated by MPP+ and 6-hydroxydopamine and the underling mechanismsThe purpose of the present study was to further assess the protective effect of FLZ on dopaminergic system in MN9D cells intoxicated by MPP+ and 6-hydroxydopamine (6-OHDA) and to elucidate the underling mechanisms. The results showed that FLZ attenuated cell death and inhibited the decrease of DA level induced by MPP+ and 6-OHDA. In further studies, we found that FLZ reversed the down-regulation of phosphorylated TH expression caused by MPP+ and 6-OHDA, while it had no effect on the activity of MAO-B which is the metabolic enzyme of DA. We also found FLZ reversed the down-regulation of phosphorylation of Akt expression and up-regulation of RTP801 expression caused by MPP+ and 6-OHDA. Moreover, FLZ inhibited the overexpression ofα-synuclein induced by 6-OHDA, and promoted the phosphorylation of TH through inhibiting the binding ofα-synuclein to TH and facilitating the binding of 14-3-3ζto TH. The cytoprotective effect of Akt induced by FLZ against 6-OHDA was also assessed in MN9D cells. The protective effects of FLZ against 6-OHDA were significantly attenuated by the PI3K-Akt pathway inhibitor LY294002, which indicated that the cytoprotective action of FLZ against 6-OHDA is at least partially mediated by its activation of Akt. These in vitro results suggest that the therapeutic effect of FLZ on Parkinson's disease mainly through its protection on dopaminergic system.Conclusions:1. FLZ is a novel HSP27 and HSP70 inducer, and the neuroprotective effect of FLZ is mediated at least partly by its induction of HSP27 and HSP70. 2. FLZ inhibited the up-regulation of a-synuclein expression induced by 6-OHDA, and promoted the phosphorylation of TH through inhibiting the binding of a-synuclein to TH, facilitating the binding of 14-3-3ζto TH, increasing the synthesis of DA, and thereby protects against PD.3. FLZ protects agsinst PD mainly through activating Akt and inhibiting the expression of caspase-3 and RTP801. The cytoprotective action of FLZ is at least partially mediated by its activation of Akt.