| Part one Dopamine D1 receptors regulate dendritic morphogenesis through Rac1 and RhoA in prefrontal cortex neuronsDrug addiction is a serious social problem anddefined as loss of control over compulsive drug taking despite horrendous adverse consequences.There exist many structural and functional changes in neural system during the development of drug addiction,one of which is the dendritic remodelingin some brain area.Many reports show that the dopamine signaling regulates the process of cocaine-induced dendrite remodeling.It was reported that the D1 and D3 dopamine receptors have opposite effect on neogenin and synaptotagmin gene expression,and those longterm changes in gene expression result in neuron restructuing and behavioral abnormalities.The Rho family of small GTPases,including Racl,RhoA,and Cdc42,are key regulators of the actin cytoskeleton rearrangement and play important roles in dendritic morphogenesis.These three proteins crosstalk with each other to regulate the rearrangement of cytoskeletal protein.It has been generally thought that RhoA and Rac1/Cdc42 have antagonistic effects on dendritic spine morphology and that Rac1/Cdc42 promotes the development of new spine,while RhoA inhibits their formation and maintenance.It has been reported that repeated dopamine treatment to neurons could mimic the biological effects of cocaine addiction in vivo,and this in vitro model system is useful for investigating cocaine-induced neuroadaptation.Similar with previous study,we set up a r PFCs model with repeated DA treatment,and found that repeated DA treatment could induce the structural remodeling of dendrites and spines in PFCs,which is very similar to the effect of cocaine in vivo.Meanwhile,we found that pretreatment of PFC neurons with D1 receptor inhibitor impaired the ability of DA to induce dendritic remodeling.Our data indicate that the D1 receptor is necessary and sufficient for mediating DA-induced dendritic remodeling.In addition,we found that regulation of dendritic remodeling by the DI receptor involves the modulation of Rac1 and RhoA activity and we provided evidence of crosstalk between Racl and RhoA in PFC neurons after DA treatment.These data provide fundamental insights into the signaling pathways controlling cocaine-induced structural plasticity,which has been implicated in the persistence of drug addiction.Results:1.Repeated dopamine treatment could mimic the cocaine addiction in vitro,make a neuron plasticity and a increase of synapse number.Compared with the saline grouP,the synapse number in repeated dopamine group is 53.44%increase,4.68±0.70 vs 3.05±0.28,P<0.001,the same as BDNF group.2.Using D1 dopamine receptor specific inhibitor SCH23390 to explore wtheter the D1 dopamine receptor is involved in the dopamine-induced dendrite remodeling.Models of chronic dopamine were injected SCH23390(10 ?mol/L)5 min before the infection of dopamine.And we fix the cells to have immunofluorescence after being stimulated by D1 dopamine receptor specific activator SKF81297(1 ?mol/L)for 15 min.In the prefrontal cortex area,the results showed that the dopamine-induced neuronal morphological changes will be significantly reversed by the application of D1 dopamine receptor inhibitor SCH23390.Synapse density is decreased by SCH23390.This indicates that D1 dopamine receptors played an important role in dopamine-induced dendrite remodeling.3.In order to explore whether Racl and RhoA affect the repeated dopamine induced dentritic remodeling,we conducted the lentiviral vector of the dominant negative effect varieties and the constitute activated varieties of Racl and RhoA.We fixed the cells to have immunofluorescence.The results showed that in the prefrontal cortex area,the infected RaclN17 significantly inhibit the increase of synapse number induced by repeated dopamine.While RaclL61 increase dopamine induced changes of synapse number,which suggests that Racl play a positive role in dopamine-induced dentritic remodeling.However,the infected RhoAN19 enhance the ability of dopamine to increase synapsedensity while RhoAL63 reduce this,which means RhoA play a negative role in dopamine-induced dendritic remodeling.Using Rac1 specific inhibitor NSC23766 and Rock specific inhibitor Y27632 to explore whether Rac1 and RhoA signaling moleculare is involved in the cocaine-induced dendrite remodeling.The prefrontal cortex cell were cultivated NSC23766(100 umol/L)and Y27632(100 ?mol/L)3 minutes before the stimulation by dopamine.We fixed the cells to have immunofluorescence.The results show that,the increase in synapse density of dopamine-induced were attenuated significantly when the cells were given NSC23776,which is the same with the case of virus-infected gorup.Butthere is not significant difference between Y27632 group and dopamine group in terms of statistics.4.The G?LISA analysis result show that,dopamine could induce an increase of Rac1 activity,while decrease the RhoA activity.And this response disappeared when D1 dopamine receptor specific inhibitor Sch23390 added,that means dopamine regulate Racl and RhoA via dopamine D1 receptor,and there have crosstalk between Rael and RhoA.Conclusion:The goal of the current study was to use cultures of primary PFC neurons to gain a better understanding of the molecular mechanisms underlying cocaine-induced dendritic rearrangement.We investigated the effects of repeated DA treatments on dendritic morphology changes in PFC neurons,and we identified Racl and RhoA as downstream effectors of D1 receptors during the regulation of dendritic morphogenesis.Importantly,we found that Dl receptor-regulated Racl and RhoA have distinct rolesin the regulation of dendritic morphogenesis after repeated DA treatments.Part two Activin B loaded multifunctional crosslinker based injectable hydrogel promoting neuroprotection in Parkinson's diseaseParkinson's disease PD)is a kind of progressive degeneration disease,mainly in the substantia nigra midbrain dopaminergic neuron,lack of the neurotransmitter is the main pathological biochemical basis of common movement disorder in the elderly.The main clinical symptoms are static tremor,muscle rigidity,bradykinesia,etc.Recent epidemiological survey showed that more than 65-year-old crowd incidence of Parkinson's disease in China is up to 1.7%(Tian et al,2011),the average survival from diagnosis to death is 15 years.The etiology and pathogenesis of Parkinson's disease is very complex and still not completely clear.All the treatment of Parkinson's disease is symptomatic treatment.Carrying out a research into the mechanism of Parkinson's disease,and further to find effective drug treatment,has profound social significance and great market prospect.The most popular clinical treatment of PD is the supplement of dopamine(DA)precursors synthesized levodopa(L-DOPA).However,a growing number of clinical cases found that after a long period of administration,the body's tolerance to L-DOPA gradually increased,the role of the drug gradually weakened,and showing a strong negative effect,weight gain and even played side effects of PD symptoms.In recent years,cytokines,in particular neurotrophic factor for the treatment of PD attract people's attention.Meanwhile,on the central nervous system,route of administration,means of administration,whether the medication can be absorped by neuron through the blood-brain barrier,as well as other issues of neurons is still not well resolved.The use of new biomaterials for intracerebral injection provides a new perspective,using good safety performance,good biological compatibility of biological material,not only can effectively wrapped cytokines into the brain,avoiding difficult caused by the blood-brain barrier,but with the biological material can degrade in the body,play a role in controlled releasing and stents.We used the MPP+-induced SY5Y cell model of Parkinson,s disease in vitro and intraperitoneal injection of MPTP mouse model of Parkinson's disease in vivo,to study the protective effect of activinB in Parkinson's disease,and stereotactically inject activin B-loaded hydrogels into the striatum of MPTP-induced mice.Histological and behavioral analysis were performed to investigate the neuroprotective of activinB-loaded hydrogel.Methods:1.Tthe hydrogel material was provided by Professor Malcolm Xing,University of Manitoba.We conducted a scanning electron microscope to observe the aperture,in vitro degradation experiments,water swelling experiments,and NMR analysis to detect the physical properties of the hydrogel.2.In vitro experiment:We use MPP+induced SY5Y cellbuild a cell model of Parkinson's disease.Experimental group given different concentrations of activinB,cell viability were detected by MTT test,hoechst nuclear staining was detected for cell activity in Parkinson's disease cellular models.3.In vivo experiment:We use 5 consecutive days by intraperitoneal injection of MPTP to build a mouse model of Parkinson,s disease,activinB were injected into mouse striatum by stereotactic injection,the 7,21,35 days after surgery,immunohistochemical staining of the striatum activinB to detect the remaining amount of activinB,and striatum TH+ dopaminergic neurons fiber density,to reflect the survival of striatal dopamine neurons.And select two representative behavioral detection methods,open field and rotarod test to reflect the regulatory function of the striatum.4.In vivo experiment:We use MPTP intraperitoneal injection induced Parkinson's disease mouse model,activinB loaded hydrogel were given by stereotactic injection into mouse striatum.7,21,35 days after surgery,immunohistochemical staining for activinB,striatum THH+dopaminergic neurons fiber density,and F4/80 positive cells to reflect the hydrogel biocompatibility.And open field test and rotarod test were carried out to reflect the regulatory function of the striatum.Results:1.The hydrogel we construct was a temperature sensitive hydrogels which were liquid state at 25 degrees and converted to a gel-like state when the temperature reaches 37 degrees.The pore size of hydrogel were 80-120um which was an excellent package carry cytokines into the body,in vitro effect of lysozyme may be about three weeks degradation,water can be 8-10 times the volume of the swollen,NMR scan analysis shows it has a stable structure.2.MPP+ can significantly induce apoptosis,cell activity decreased,nuclear fragmentation of nuclear apoptosis,thereby inducing Parkinson's disease model in vitro.ActivinB 10ng/ml,25ng/ml can significantly improve cell activity.3.It was found that,MPTP significantly reduced the density of TH+dopaminergic positive cells and induced a decreasedof dendritic spine density in striatum,reduce the density of TH+ dopaminergic neuron fibersin striatum,all have significant differences compared with the saline group.The behavioral analysis showed,compared with saline group,MPTP induced a decrease in total distance and average speed in open field test,and a decrease of rotarod time in rotarod test,all have statistically significant differences,suggesting that we successfully constructed Parkinson's diseasemice.model Stereotactic injection of activinB can protect neurons against MPTP damage,increased the density of TH+dopaminergic neuron fibersin striatum,and improvement the behavior performance.But this protective effect is limited to seven days after surgery,at 21 days after surgery,or even 35 days,the neuroprotective of activinB in Parkinson's disease mice disappeared,showing a symptoms of Parkinson's disease,and pathology changes.ICC for activinB showed that7days after surgey,activinB can be detect in the brain,but on 21 days and 35 days were not detected.4.Results in vivo:the hydrogel can significantly extend the lifetime of activinB in vivo.It can be detected 7,21,35 days after surgery in the brain,that means the hydrogel make a controlled release of activinB and maintain its active state.Anti-TH immunohistochemistry found that hydrogel+activinB enhance TH+ fiber density,there was a significant difference compared with MPTP group.Behavioral results showed that,compared with MPTP+saline group,the total distance,average speed and rotarod time have significant difference in MPTP+hydrogel+activinB group on days 7,21,35 after surgey.7 days after surgery,there were no significant differences between MPTP+activinB group and MPTP+hydrogel+activinB,but on days 21 and 35 after surgery,MPTP+hydrogel+activinB group showed a great advantage,have significant difference compared with MPTP+activinB group,not only in pathology but also in behavior test.5.Anti-F4/80 results show that around the hydrogel injection area can cause mild inflammation,the other surrounding tissue does not show any inflammatory reaction,that means the hydrogel have good tissue compatibility,and mild inflammatory cells can release a neuroprotective factor,thereby contributing to disease repair.Conclusion:activinB can protect dopaminergic neurons in MPTP-induced Parkinson'5 mouse model,and thus play a therapeutic role in Parkinson's disease.We build a safety and biodegradable biological hydrogel whichcan make sustained release of activinB in vivo,thrtrby extending the effective time of drug,achieve better teratment.Part three Magnetic nanoparticle induce neuron toxicity via p-JNK pathway in hippocampus and striatumNanoparticles as a novel bio-materials,have been used in many aspects of social life.Emergence of magnetic nanoparticles,because of its many unique features,small size,large contact area,high reactivity,and high load carrier herbs,attracting the attention of all walks of life.In the past many years,nanoparticles are widely used in machinery manufacturing industry,food processing industry,as well as cancer therapy,targeted drug delivery systems.In addition,because it can penetrate the blood brain barrier(blood-brain barrier,BBB),nanoparticles are widely used in the treatment of central nervous system diseases.So many applications will cause people'sconcerns of nanoparticles safety,whether it could cause body's microenvironment changes or organ tissue damage.During the environment we live,there have a large amoune of nanoparticles.It have been reported that inhaled nanoparticles can enter the human's brain and cause behavioral damage.Therefore,the study of the toxicity of nanoparticles,for better use of its advantages and avoid its shortcomings have important significance.Methods:1.Cell type:PC 12 cell lines,seeded in six-well platesat the density of 5×106 cells/ml,seeded in 96-well plates at the density of 5×104.2.Cells grouping:Saline control group,nanoparticle experimental group,the nanoparticle concentration in experimental group were 10-100ug/ml.3.Detection indicators:?CCK-8 cell activity assay:96-well plate cultured PC 12 cells were serum starved for 24h,incubated with different concentration of nanoparticle from 10 to 100ug/ml for 24h,cell viability was detected using CCK-8 kit;?cell viability analyzer for cell activity analysis:cells in serum starvation,followed by incubation with nanoparticle for 24h,digested,cell suspension was prepared,the machine reading the analysis of cell viability;?cell morphology:serum starved cells in six-well plates,cells in six-well plates incubated with 25,50,75,100 ug/ml nanoparticle for 24h,NGF were added to induced cell differentiation,five days later,cell morphological changes were observed for skeleton changes;?TEM:cells cultured insix-well plates were incubated with different concentration of nanoparticle(25,50,75,100ug/ml)for 24h.Then the cells were scraped,eentrifiugation,fixed,stained sections and scanning bytransmission electron microscopy;?western blotting:cells were cultured in six-well,and incubated with 25,50,75,100ug/ml nanoparticle for 24h.Cells were lysed by lysis buffer and cell extract proteins to detect the expression of TH.10min,30mm,60mm,120min after incubated with 100ug/ml nanoparticle,the p-JNK,t-JNK,p-P38,t-P38 protein expression were analysis.1.Animal models:C57 male mice,8-10 weeks of age,nanoparticle injected into mouse striatum and hippocampus using stereotactic technique.2.Animal groups:saline injection group,nanoparticle injection group.3.Detection indicators:CDHE:7 days and 14 days after surgery,mice were perfusion and the brain was removed,eonventional dehydration transparent embedded in paraffin,5-um sections for HE staining.? Anti-TH immunohistochemistry:7 days and 14 days after surgery,mice were perfusion and brain was removed,frozen sections were made to detect TH positive fiber density in striatum.?NEL staining:7 days and 14 days after surgery,mice were perfusion and the brain was removed,the apoptosis cells in striatum and hippocampus were detected by TUNEL assay kit.?Western blotting:after 10min,30min,60min,120minstereotactic injection of nanoparticle into striatum and hippocampus,tissue extracts were isolated,and the protein level of p-JNK,t-JNK,p-P38,t-P38 were detected.?Behavioral detection:7 days and 14 days after surgery,open field and rotarod test were carried out in striatum injected group,morris water maze test for hippocampus injected mice.Results:1.An in vitro experiment,CCK-8 test and cell viability analyzer results showed that,nanoparticle can induce apoptosis in a dose-dependent way.In 10-50ug/ml nanoparticle goups,cell activity are the same as saline group,have no significant difference(saline:98.91±0.62%;10ug/ml:99.12±0.32%;20ug/ml:98.68±0.70%;30ug/ml:99.11±0.32%;40ug/ml:98.72±0.51%;50ug/ml:98.95±0.59%,p>0.05).From the concentration of 60-200 ug/ml,leading an sianificance decrease of cell activity compared with the saline group(60 ug/ml:77.1±1.22%;70 ug/ml:74.1±2.50%;80 ug/ml:60.43±2.67%;90 ug/ml:53.25±3.43%;100 ug/ml:52.80±2.86%;200 ug/ml:27.01±1.82%F=941.695,p=0.000).Cell viability analyzer results also show,at the concentration of 25ug/ml and 50ug/ml nanoparticle,cell viability was not affected(saline:1.00±0.05;25ug/ml:0.99±0.01;50ug/ml:0.99±0.01),at the concentration of 75ug/ml and 100ug/ml nanoparticle(75ug/ml:0.74±0.03;100ug/ml:0.52±0.02),cell viability was affected,apoptosis increasedand the difference was statistically significant,p=0.000.After incubated with 25,50,75,100 ug/ml of nanoparticle,anti-TH western blotting analysis showed the high concentration of nanoparticle can lead to decrease of TH level,have significant difference compared with saline group and low concentration group,p=0.000.NGF can induce differentiation of PC 12 cells into neuron like cells,resulting in dendritic branching,given 100ug/ml of nanoparticle treated for 24h firsty,compared with NGF only group,the phenomenon of cell differentiation disappear,cells became round,reduces the ability to produce branched.This may be due to toxic effects on the cytoskeleton resulting nanoparticle induced.2.After incubated with saline,20ug/ml,50ug/ml,75ug/ml,100ug/ml of nanoparticle for 24h,cells were fixed and TEM experiments were take out.During 25 and 50 ug/ml,the cell present the normal absorption/endocytosis of the nanoparticle,the cell membrane integrity is not damaged,nucleus,mitochondria and other organelles are normal.But when 75,100 ug/ml,the cells no longer endocytosis nanoparticle as normal,cells appeared nuclear fragmentation,the membrane is no longer complete,mitochondrial swelling,broken,releasing large amounts of lysosomes,cells showed typical apoptotic state.Therefore,we speculate that the nanoparticle at low concentrations cannot affect the cell activity,but at high concentrations to stimulate cell apoptosis program started.3.Nanoparticle stereotactic injection into the two most important areas of the brain:the striatum and hippocampus,7 and 14 days after surgey,respectively,HE staining showed that occurs in the injection area and scattered around the magnetic nanoparticles,but compared with the saline group,no obvious morphological tissue damage,and no obvious tissue pressure or apoptosis.4.7 and 14 days after nanoparticle injected into the striatum,anti-TH immunohistochemistry found that nanoparticle can decrease TH-positive nerve fiber density of striatum(7d:53±6.61%vs 100±1.15%;14d:57±2.8%vs 100±1.79%).TUNEL apoptosis detection showed that,7 days after surgery,compared with the saline injection group,the number of apoptotic cells in the hippocampus 31.5±2.6,striatum 29.6±4.2,14 daysafter surgery,the number of apoptotic cells in the hippocampus 17.8±2.3,the number of apoptotic cells in the striatum area 19.3±2.1,P=0.000 compared with saline groups.5.In vitro experiments:after treatment of nanoparticle,protein extract at a timepoints of 10min,30min,60min,120min,western blotting analysis showed the expression of p-JNK were activated at 10min,and sustained in 120min,the expression of p-P38 had no changes.In vivo experiment,the hippocampus injection groupswere the same as the striatum injection group,1Omin after stereotactic injection of nanoparticle,expression of p-JNK were activatedand sustained in 120min,and p-P38 is not activated.6.After stereotactic injection into the brain striatum and hippocampus,respectively,on days 7 and days 14,behavioral test were carried out.Striatal injection group take the open field test and rotarod test,compared with the saline injection group,mice injected nanoparticle mice showed a decrease of total distance,average speed and rolling time.Hippocampus injection group take morris water maze behavioral testing,7 days after surgery,compared with the control group,the average swimming speed of the experimental group of mice was no significant difference(23.7±3.3 vs 22.2±2.1 cm/s,P=0.219),target quadrant statistically different from the percentage(24.0±9.2%vs 41.8±17.3%,t=3.075,p=0.006),the percentage of time the target quadrant also made a significant difference(26.2±9.2%vs 40.7? 19.4%,t=2.245,p=0.036).14 days after surgery,compared with the saline group,there were no significant difference between nanoparticle group and saline group(23.2±2.1 vs 23.1±3.2)in average swimming speed,but the percentage distance and timein the target quadrant made a significant difference(distance:21.3±7.3%vs 31.99±4.3%,t=4.159,p=0.000;time:26.6±6.9%vs 37.5±6.5%,t=3.792,P=0.001).Conclusions:Nanoparticle could induce cell apoptosis in a dose dependent way via activit p-JNK pathway,and make some changes on the behavior performance of mice. |
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