The Neuroprotective Effects And Mechanism Of Optimized α-Synuclein DNA Vaccine Immunize MPTP Inducing On Chronic Mouse Model Of Parkinson's Disease

BackgoundParkinson's disease (PD) is the second most common neurodegenerative disease, which affects over 65 years old among more than 1% of the population. It's can't be ignored which concerned about the health of the elder of more than 60 years making up about 10% of total population.The major pathological markers of PD is the abnormal accumulation ofα-synuclein (α-syn) in substantia nigra(SN) as the main component of Lewy bodies. Abnormalα-syn aggregation in PD involved in dopaminergic (DA) neuron degeneration process, and abnormal accumulation ofα-syn in familial and sporadic PD pathogenesis has played an important role. So far a fundamental treatment hasn't been found for the PD. In recent years, the DNA vaccine in the prevention and treatment of degenerative diseases show attractive prospects. Our research's pre-experiment successfully constructed a humanα-syn DNA vaccine which experimented on mammals, and obverted thatα-syn DNA vaccine can obtain a better effect on chronic MPTP mouse PD model. Therefore, further optimization of vaccine to improve performance, as well as the protection mechanisms of neurons will be a focus on research.In the natural state, a-syn conformation consists of a large number of disordered non-closed to the isomer mixture, is riches in hydrophobic region of natural extension protein: N-terminal (1~60), it contains four incomplete repeats, which are easy to form spiral amphoteric alone. NAC area (61~95), which is a sequence of a-syn in the strongest of hydrophobic section; Carboxy-terminal (96~140), which is rich in acidic amino acids such as proline, with large number of negative charge, and maintain a-syn to a random coil in the normal state. So do these three structures have different immune potency? Do they have an impact on the different immunization? It remains our exploration and study.At the early stage of our research, in spite of hα-syn DNA vaccine immunotherapy to chronic MPTP mouse PD model has achieved successfully. However,α-syn is the molecular weight of 14kD, containing 140 amino acid residues which composed of small molecule protein, because ofα-syn covers small molecular weight, as well as immunological activity of the weak, all those made it needs a larger quantity inducing a sufficient immune response. Ig Kappa chain signal peptide (Ig ksp) can increased the movement from the intracellular to the extracellular cell of hα-syn, and increased secretion capacity, it will contribute to the improvement of antibody titer to degrade and remove more abnormalα-syn protein. Therefore, add mouse Ig ksp gene sequence based on hα-syn, building a highly efficient secretion of hα-syn-based eukaryotic expression vector, and enhancing the effect of immunotherapy, increasing the safety of vaccines, those is the key research project.The study clearly shows that the abnormalα-syn aggregation can be degraded by ubiquitin-proteasome system, as well as lysosomal degradations.The block ofα-syn pathway of degradation may lead to an abnormalα-syn aggregation to result in the formation of Lewy bodies. The application of protease inhibitors which inhibit proteasome activity can produce dose-dependent selective death of DA neurons. Lysosome also plays a key role in the process ofα-syn degradation. The Lysosomal pathway is a major protective mechanism againstα-syn oligomer-mediated toxicity, and lysosomal degradation pathway could remove the accumulation ofα-syn. Masliah found that the recombinant hα-syn immunized the hα-syn transgenic mice produced the antibody againstα-syn that can be removed by means of lysosomal pathway, will decrease the aggregation ofα-syn in DA neuron cell body and synapse. Does the optimization of hα-syn DNA vaccine can play a neuroprotective effect by increasing the degradation of abnormal accumulation ofα-syn? The combination of antigen and antibody bound to catabolism through the lysosomal pathway and UPS? It also needs a further study. The Immune inflammatory response links closely to the pathogenesis of PD. So PD can be considered to be a dynamic process of imflammation change. The activation and proliferation of MI is the most important embodiments of central nervous system (CNS) response. The activation of MI in SN can be sustained by the abnormalα-syn, which would contribute to the degeneration of DA neurons and finally lead to the occurrence of PD. The activatation and proliferation of MI can induce inflammatory cytokines, such asα-tumor necrosis factor (TNF-α), reactive oxygen species, IL-1β, IL-6, etc. Conversely, the inflammatory cytokines can exacerbate the activation of MI, which involve in the process of brain inflammation. For this reason, the inflammation acts a main role in the pathophysiology of PD process. If the DNA vaccine palys a role in the protection of neurons, what is the impact imposed by the inflammatory characterisctis in PD pathology? We will get a better understanding of the mechanism of vaccine if we have the clarification of the process of the activated MI and the regulation of inflammatory response.Research purposesAccording to the folding feature of a-syn, we construct the three groups of the optimized high secreted human alpha-synuclein DNA vaccine and immunize the chronic MPTP mouse PD model, to observe the neurological effects of immune protection, and explore the role of mechanisms. The main contents including:①Amplified humanα-syn 1-140,84-140,34-140 gene with the Ig ksp gene is cloned into the plasmid pVAX1, get the three groups of the recombinant plasmid,then expression in mammalian cells and identification of its biological activity;②Observe the effectiveness of immunization,the specificity and different characteristics of immunization;③Immunize the mouse model of PD, observe the neuroprotective effect ,lysosomal function, the effects of inflammatory response and regulation after immunization in the process of PD;④Observe the effect of Proteasome inhibitors and lysosome inhibitors on DA neurons in normal mice by stereotactic brain injection into SN in order to blockα-syn degradation pathway, observe the different doses and time of lysosomal inhibitors for the characteristics of behavior,TH,Cath D on the DA neurons ;⑤Observe the lysosome and the proteasome on the role of the hα-syn DNA vaccine-induced protection of neuron-specific antibodies generated by immunization, the characteristic of degrading and removing the overexpression ofα-syn or the characteristic of apoptosis.Methods1. The construction and expression of recombinant plasmid. Amplify the gene ofα-syn 1-140,84-140,34-140 from the recombinant plasmid in human and synthesize the sequence Ig ksp,then the two fusion gene will be cloned into plasmid pVAX1 to construct the three groups of recombinant plasmid namely, A group is pVAX1-Igk-hαS1-140; B group is pVAX1-Igk-hαS84-140; C group is pVAX1-Igk-hαS34-140.Restriction endonuclease digestion analysis and DNA sequencing.Transfer the recombinant plasmid into E.coliDH5α, extract the plasmid and transfecte COS-7 cells to assay its biological activity using Western blot.2. The activity of recombinant plasmid induced humoral immune We use the recombinant plasmid A, B, C of optimized DNA vaccines immunize the healthy C57/BL mice in the musculus quadriceps, every time 100μg/100ul, every three weeks to strengthen the immunity, totally five times. Take those mice blood before the first of immunization and two weeks after immunization; separate the serum for antibody determination. Immune mouse blood serum and the expression of mouseα-syn brain tissue neutralization reaction are used in order to observe immune serum's specificity.3. Observe the neuroprotective effect optimizated DNA vaccine. C57BL mouse is injected MPTP twice every week, totally 10 times in order to the establishment of MPTP chronic mouse PD model; immunize three groups of vaccines. Observate the number of DA TH cell and the expression levels ofα-syn, the inflammatory reaction as well as the lysosome cathepsin D.4. Observation the characteristics of lysosome and proteasome inhibitors on DA neurons in health mice stereotactic injection into the right SN with different doses of lysosome inhibitor chloroquine phosphate(CQP), as well as the proteasome inhibitor MG-132.Observate the apomorphine(APO)-induced rotational behavior change; detecte the number of SN TH positive cells , the levels ofα-syn expression and cath D changes in different time periods after injection of lysosome inhibitors or proteasome inhibitors.5. Explore the vaccine's influence to the degradation and elimination of a-syn after the block of the lysosome and the proteasome pathway C57BL mouse is injected with MPTP, 25mg/kg?d,twice every week, totally 10 times. Immunize the the chronic MPTP PD mouse with the vaccine of pVAX1-Igk-hαS1-140.Three days before the third immunization, stereotactic micro-injection into the right SN with 100μmol/L lysosome inhibitors and proteasome inhibitor. Detecte the SN TH, cell morphological changes and the impact on apoptosisResults1. Construction and expression of recombinant plasmidsΑ-syn 1-140, 84-140, 34-140 genes are amplified successfully, and Ig ksp gene is synthesized and the fused gene of the two is cloned into the pVAX1. The size, direction and sequence were completely correct with endonuclease digestion analysis and DNA sequencing.The recombinant plasmids are constructed successfully, and expressed effectively in mammalian cells COS-7with better biological activity.2. The induction of humoral immunity by recombinant plasmids Three recombinant plasmids and empty plasmid pVAX1 are preparated largescally. A higher antibody titer was produced in animals, pVAX1-Igk-hαS1-140:(3.76±0.33)×103,pVAX1-Igk-hαS84-140:(3.87±0.12)×103,pVAX1-Igk-hαS34-140:(3.79±0.22)×103. There was no obvious differences between them (p>0.05), while compared to empty plasmid (p<0.05). The minimized serum can generate immunohistochemical reaction with mouse brain tissue over-expressedα-syn.3. Observe the protective effect of immunity by DNA vaccine Compare with empty plasmid, three vaccines elevate the ability of crawling pole, elevate TH-positive cells to 60-65%,diminute a-syn to 44-47%, and augmente Cath D 46-50%(p>0.05).Compared with PBS,the TNF-a decrease 27-55% (P<0.05). B,C vaccine show the higher safety.4. observe the characteristics of lysosome, proteasome inhibitors effect on DA neurons in healthy mice .(1) there have rotational behaviors 1 or 2 times occasionally below the 200μmol / L of lysosome inhibitor.More pronounced rotary behavior was shown in only 400μmol / L group. There is significant difference between proteasome inhibitor and 400μmol / L lysosome inhibitor group. Rotational behavior is shown in proteasome inhibitor group after the analepsia even needn't APO, while lysosome inhibition group needed it. (2)An obvious variablely reduction in the number of TH-positive neurons in the damaged lateral SN is indicated with TH immunohistochemistry. In the third week after stereotactic injection, the degree of the damaged TH is aggratated according to the dose from the lysosome 25μmol/L to 400μmol/L.The gradual return of the amount of dopamine is discovered after stereotactic injection 100μmol/L from one week to forth week. (3) Contrast with the normal brain tissue in the SN of the results of cathepsin D, the damaged side of the SN region of cath D-positive cells exists in varying decreased degrees significantly. The number of the positive expression of cathepsin D is significantly reduced with the accumulating dose in the Lysosomes from the third week of 25μmol/L to 400μmol/L dose of stereotactic group, contrast with the number of damaged TH, we found that cathepsin D expression with the TH-positive of damage is directly proportional.5 Explore the characteristics of a-syn on channel blockers after DNA vaccineNo significant difference is found between PBS group and others in the number of TH-expression neurons in the damaged lateral SN. To compare with normal lateral, the degree of damage is lighter in the lysosome inhibitor group, heavier in the proteasome inhibitor group, heaviest in the lysosomal and proteasome inhibitor group but still local expression of the number of TH-positive is observed in the injection. TH and Hoechst staining showe that part of a large number of positive nuclei in SN were irregular shrinkage, nuclear fragmentation, some cracking to multiple apoptotic bodies. Mice in each group in the SN can be seen in the nuclear morphology, and non-injection side of the nuclear morphology, PBS group were not significantly different. And non-injected side compared with less damage to the lysosome inhibitor, proteasome inhibitor slightly heavy damage, and lysosomal proteasome inhibitor increases the relative heavier harm.Conclusion1. The three eukaryotic expression plasmids pVAX1-Igk-hαS1-140, pVAX1-Igk-hαS84-140, pVAX1-Igk-hαS34-140 were successfully constructed and showed better biological activity.2. Optimized DNA vaccines produced higher antibody titers and the specificity ofα-syn.3. Optimized DNA vaccine were clear abnormal brain accumulation ofα-syn in the chronic mouse model of PD, and reduced the over-expression ofα-syn. PVAX1-Igk-hαS84-140 fusion plasmid showed better immune effect than the other two groups.4. Lysosome inhibitor can induce rotational behaviors, but less than proteasome. Lysosome inhibitor and proteasome inhibitor on DA neuronal damage depend on the dose and time. The positively correlated between the damage levels of DA neurons and the expression of cath D.5. Optimized DNA vaccines have a good neuroprotective effect on DA neurons, and apoptosis damaged by the lysosome inhibitors and proteasome inhibitor.