Neuroprotective Effects Of D-Ser~2-OXM In The MPTP Mouse Model Of Parkinson’s Disease

Backgroud:Parkinson’s disease（PD） is the second most common neurodegenerative disorder that is characterized by the progressive loss of dopaminergic neurons in the substantia nigra, axons and dopaminergic synaptic degradation in the striatum and aggregation of Lewy bodies in neurons. The etiology and pathogenesis of PD is generally thought to be related to mitochondrial oxidative stress, immune response, toxicity of excitatory amino acids, endoplasmic reticulum stress, cell apoptosis and autophagy. Recent studies have shown that Diabetes and PD share the common pathological physiological way. Risk of Parkinson’s disease onset in patients with diabetes increased significantly. It puts forward a new strategy based on the two similar disease and central role of GLP-1: Using drugs for the treatment of T2 DM to intervene in PD. Our early studies have shown that liraglutide, Lixisenatide and（Val8） GLP-1-gluPAL could significantly improve the abnormal behavior of PD mice and inhibit DAergic neurons apoptosis. D-Ser₂-OXM is a protease resistant oxyntomodulin analogue that activates the GLP-1 and the glucagon receptor, which has been developed to regulate the glucose metabolism and treat obesity. Furthermore, this dual-agonist strategy may show better effects than GLP-1 analogues. Here we demonstrate for the first time that such analogue can prevent or reverse the MPTP-induced loss of dopaminergic neurons. D-Ser₂-OXM may show neuroprotective properties in the MPTP mouse model of Parkinson’s disease.Part 1 Neuroprotective effects of D-Ser₂-OXM in subacute MPTP mouse model of Parkinson’s disease Objective:1. To investigate whether D-Ser₂-OXM could relieve behavioral disorders of PD mice and attenuate MPTP-induced DAergic neurons damage.2. To explore whether D-Ser₂-OXM could prevented or reversed the MPTP- induced activation of nerve inflammation, the reduction of the synaptic marker synapstophysin and anti-apoptotic molecule Bcl-2, the increase of pro-apoptotic protein Bax.3. To determine whether the neuroprotection of D-Ser₂-OXM against MPTP is mediated by the activation of the PI3K/Akt pathway. Methods:1. Animals were all randomly divided into 4 groups: control: NS（saline, i.p.） + NS（i.p.）; Oxy: NS（ip.） + Oxy（ip.）; MPTP: MPTP（ip.） + NS（ip.）; MPTP+Oxy: MPTP（ip.） + Oxy（ip.）. MPTP was in saline intraperitoneally injected daily（20 mg/kg i.p.） for 7 consecutive days, Saline or Oxy dissolved in normal saline were administered 30 min after each MPTP administration.2. Behavioral Test: The mice were observed whether develop tremor, vertical tail, gait instability and body stiffness and other symptoms. Motor function was evaluated by multiple paradigms, including open-field, rotarod, swimming test and traction test. After all MPTP administration, the rotarod was performed daily following treatments with each experimental schedule to measure behavioral change.3. The level of TH, GFAP, IBA-1, Synaptophysin were detected by Immunohistochemistry.4. Detect the number of apoptotic cells in the substantia nigra by TUNEL staining.5. Western Blotting detect the expression of TH, α-synuclein, Bax, Bcl-2, TNF-α, Synaptophysin, Akt, p-Aktser473. Results:1. The subacute PD animal model was successfully replicated by intraperitoneal injection of MPTP. We observed that D-Ser₂-OXM could improve the behavioral impairment symptoms caused by MPTP. The movement coordination and flexibility of mice were detected by spontaneous locomotion, Rotarod, swim activity, traction test. MPTP reduced significantly the exploratory behaviour as shown in numbers of rearings and the spontaneous locomotion, time stayed on the rolling bar, swim score and grip score of mice compared to controls. Treatment with Oxy reversed the impairment.2. Immunohistochemistry staining demonstrated that there were significant reductions in the level of SYN in SNpc and the number of TH-positive neurons in SNpc and striatum for the MPTP group compared with the control group. D-Ser₂-OXM treatment restored the level of SYN and the numbers of TH-positive neurons in MPTP-treated mice. GFAP, IBA-1 staining results showed that the numbers of GFAP, IBA-1 staining cells increased after MPTP-damaged. While the number of positive cells showed a tendency to recover after D-Ser₂-OXM treatment.3. TUNEL staining results showed that apoptotic cells displayed in the substantia nigra in each group. The number of TUNEL staining cells in the MPTP model group significantly increased compared with the control group. While the number of apoptotic cells showed a tendency to recover gradually after D-Ser₂-OXM treatment.4. The expression of TH、SYN and phosphorylation of the growth-factor kinase Akt were much reduced in the MPTP group compared to the control group. The expression of α-synuclein wasn’t increased after MPTP treatment. The expression of the pro-inflammatory cytokine TNF-α and the ratio of Bax/Bcl-2 were much enhanced in mice treated with MPTP. Oxy treatment partially normalised TNF-α levels, down-regulated ratio of Bax/Bcl-2, prevented the reduction in synapse numbers and restored levels of phosphorylated Akt. Conclusion:1. D-Ser₂-OXM could improve the behavioral disorders, reduce the DAergic neurons damage, increase the number of DAergic neurons survival in the substantia nigra and striatum.2. D-Ser₂-OXM could inhibit nerve inflammation, increase synapse numbers and regulate the expression of apoptotic protein, to protect the DAergic neurons in PD mice.3. Neuroprotection by D-Ser₂-OXM against MPTP is partially mediated by the activation of the PI3K/Akt pathway. Part 2 Neuroprotective effects of D-Ser₂-OXM in chronic MPTP mouse model of Parkinson’s disease Objective:To investigate whether D-Ser₂-OXM could reverse behavioral disorders and pathological changes in chronic MPTP mouse model of Parkinson’s disease. Methods:1. Animals were all randomly divided into 4 groups: control: NS（saline, i.p.） + NS（i.p.）; Oxy: NS（ip.） + Oxy（ip.）; MPTP: MPTP（ip.） + NS（ip.）; MPTP+Oxy: MPTP（ip.） + Oxy（ip.）. Mice were treated with 20mg/kg MPTP in saline subcutaneously, and 1h later were injected 250mg/kg probenecid in DMSO intraperitoneally every 3.5d over a period of 5 weeks. Saline or Oxy dissolved in normal saline were administered 30 min before each MPTP administration.2. Rotarod and Open field test were tested on 3th day after the last injection of MPTP.3. Immunohistochemistry was carried out to determine the number of TH. The expression level of TH, α-synuclein were detected by Western Blotting. Results:1. The chronic PD animal model was successfully replicated by intraperitoneal injection of MPTP. We observed that D-Ser₂-OXM could improve the behavioral impairment symptoms caused by MPTP. The movement coordination and flexibility of mice were detected by spontaneous locomotion and Rotarod test. MPTP reduced significantly the exploratory behaviour as shown in numbers of rearings and the spontaneous locomotion and time stayed on the rolling bar of mice compared to the controls. Treatment with D-Ser₂-OXM reversed the impairment.2. Immunohistochemistry staining demonstrated that there were significant reductions in the number of TH-positive neurons in SNpc and striatum for the MPTP group compared with the control group. D-Ser₂-OXM treatment restored the numbers of TH-positive neurons in MPTP-treated mice. The level of TH was much reduced by MPTP. D-Ser₂-OXM treatment partially prevented the reduction. The expression of α-synuclein was much enhanced in the MPTP group. Treatment with D-Ser₂-OXM completely blocked this. Conclusion:D-Ser₂-OXM could improve the behavioral disorders, reduce the DAergic neurons damage and decrease α-synuclein accumulation in PD mice.