MANF Alleviates The Dopaminogenic Neuronal Lesions Induced By6-hydroxydopamine In Rats

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized byresting tremor, muscle rigidity, and bradykinesia. The movement disorder is mainly aconsequence of degeneration of dopaminergic neurons in substantial nigral parscompacta (SNpc) which results in the severe loss of dopamine in brain. MesencephalicAstrocyte-Derived Neurotrophic Factor (MANF) is one kind of highly conservedneurotrophic factor, which was originally named as Arginine-rich Mutated in EarlyStage Tumors or Arginine-rich Protein (ARMET or ARP). MANF can specifically protect dopamine neurons in vitro, and endoplasmic reticulum(ER) stress can upregulateits expression. Whether MANF has the therapeutic effects on the restoration of theneuronal function in PD is not clear. In this study, we have investigated the therapeuticeffects of recombinant human MANF on PD rats.OBJECTIVE:To observe whether MANF has the therapeutic effects on the restoration of6-OHDA-induced dopaminergic neuronal lesions in SNpc of rat midbrain.METHODS:To express and purify the recombinant human MANF, pET28a-MANF(+) wastransformed into the competent cells of DH5α, then the plasmid was extracted out andtransformed into the competent cells of BL21followed by enlarging cultivation. TheHis-tagged fusion protein was expressed under IPTG induction. Then it was extractedout and purified by affinity chromatography through the Ni-beads column. The purifiedprotein was then concentrated and its concentration was detected. The aliquots werekept at-80°C.The rat model of PD was prepared by injecting6-OHDA into the striatums of the ratswith a stereotaxic frame. The purified protein of MANF, with three doses of5μg,10μgand20μg, or PBS (as a negative control) was injected into the same site of striatums ofrats. Selegiline was intraperitoneally injected as a positive control.The behavioral performance of the rat was tested by counting the times ofapomorphine-induced rotational activity from lesion side to the intact side within30min. The number of tyrosine hydroxylase (TH) positive neurons and fibers insubstantial nigra (SN) and striatum (STR) in each group of rats were observed by immunohistochemistry. The number of cleaved Caspase-3positive cells in SN weretested in the group of PBS and MANF10μg by immunohistochemistry as well.RESULTS:1. The expression and purification of His-tagged MANF protein.His-tagged MANF was soluble in the lysis of E. col.; We had obtained high purificationand concentration of MANF fusion protein for this study.2. The preparation and screening of rat PD model.In this experiment,60rats were used for preparing PD model and8rats for normalcontrol. We found that43rats among60rats was successfully developed into PD modeland the rats’ rotational behavior induced by apomorphine was observed3weeks afterthe lesion. For screening the PD models, the rotation times (from lesion side to intactside) must be more than210. Then the PD rats were randomly divided into5groups:PBS control (negative control), SEL control (positive control), and MANF groups withthe doses of5g,10g, and20g. There were8-9rats in each group.3. Effects of MANF on the behavior of PD rats.The rotational behavior of the rats were detected4times after the6-OHDA injection.We counted the times of the rats’ rotation within30min at5min after theintraperitoneal injection of apomorphine. The results showed that, the times of therotation of all the PD rats were declined, which were significantly delined aftertreatment with MANF (10μg) and SEL (P<0.01), but not PBS. However, there was nostatistical significance between the groups of MANF and SEL (P>0.05). The other twogroups MANF (5μg and20μg) showed no difference, compared with the group of PBS(P>0.05).4. Effects of MANF on the the number of TH positive neurons in SN The TH positive neurons in the lesioned side of SN in the group of PBS wassignificantly less than that in the intact side. After the administration of MANF, thesurvival of TH positive neurons in the lesioned side of SN in each dose of MANF wasobviously restored. The group of MANF (10μg) showed the best effect (P<0.01)among the groups.5. Effects of MANF on the average integral optical density (AIOD) of the TH positivenerve fibre in striatumThe AIOD of the TH positive nerve fibre in the lesioned side of striatum was severlyreduced in the PD rats receiving PBS injection, compared with that in the intact side.After treatment with MANF and SEL, the amount of TH positive nerve fibre in eachtherapeutic group was significantly increased, and the effect of MANF with the dose of10μg were the most significant (P<0.01).6. Effects of MANF on the numbers of cleaved Caspase-3positive cells in SNThe number of cleaved Caspase-3positive cells in the lesion side of SN in the group ofPBS was more than the intact side obviously; In the group of MANF10μg, the numberof cleaved Caspase-3positive cells in the lesion side of SN was slightly more than theother side, but less than that of the lesion side in the group of PBS.CONCLUSIONS:MANF can alleviate the dopaminogenic neuronal lesion in PD rat induced by6-OHDA,which suggests that MANF may be considered as a potential agent for PD treatment.