| Alzheimer's disease(AD)is one of neurodegenerative disease characterized by loss of memory and progressive cognitive impairment.According to the statistic data of WHO,the total number of dementia on the earth in 2010 was 35 millions.The new cases each year are about 7.7 millions,which mean there is a new dementia patient every 4 seconds and the number of dementia on the earth will be double every 20 years.As the most common type in dementia,Alzheimer's disease represents more than half of dementia on planet Earth.Following the increase with age,the incidentsof AD is augmenting.For example,there are 5%patients in 65-year old people,and the number would rise to one in three in 85-year people.As a progressive and irreversible disease,AD cause physiological and psychological problems to patients and bring serious burden both on psychology and economy.So AD is another killer to health of the old,except tumor,cardiovascular and cerebrovascular diseases.The main pathologic characteristics of AD are loss of neuron in cerebral cortex and hippocampus,the senile A ? plaque deposited outside of cell and high-phosphorylated tau fiber entanglement.Now many researches have revealed a fact that the oligomer of A ? is one of important pathologic factors to dysfunction of signal transduction in synapses and neuron death.However,the pathologic mechanism of A ? oligomer is still unclear.So it is necessary to explore the mechanism and affective factors,which are necessary foundation to find out effective therapy to AD.Part ONE ?-arrestin affect the neurotoxicity of A ?A ?,is a peptide fragment consist of 39-43 amino acid residues,which is cut by ?-secretase and ?-secretase abnormally from amyloid precursor protein(APP).The main form are A ? 1-40 and A ?1-42,but it has been demonstrated that it is the A ? 25-35 fragment which give neurotoxicity to A ?,any other A ? fragments which miss the whole A ? 25-35 fragment or contain A ? 35-25 fragment would loss the neurotoxicity.So it is necessary to find out the mechanism of A ? 25-35 neuropathy to reveal the development and progression of AD and explore methods to reduce it to find the effective therapy of AD.Arrestins,a kind of adaptors exist in cytoplasm and nuclear,are a small highly-conservative family consist of four proteins in mammals,visual arrestin,?-arrstin1,?-arrestin2 and rod arrestin.They are important proteins which have multifunction,such as inhibiting G protein signaling through blockade of the GPCR-G protein interaction in a process named "desensitization" and recruitment of GPCRs into intracellular compartments,which known as receptor internalization.Visual arrestin and rod arrestin are exclusively expressed in the retinal rods and cones.They are the key proteins mediated the desensitization of rhodopsin to regulate photosensibility of retina.Whereas the ?-arrestin 1 and ?-arrestin 2 are expressed invarious tissues,including brain.?-arrestins not only have been found to regulate GPCRs activities at desensitization,internalization,recycle and degradation,but also participate in cell development,migration and nuclear functions.There are some reports showing that ?-arrestin 1 or ?-arrestin2 affect production of A? from APP by ?-secretase and get a conclusion that ?-arrestins are pathologic to AD.However,Michela Guglielmotto reveal that A ? monomers or oligomers have different effects on apoptosis,only A ? oligomers can cause significant neuron death.So it is necessary to study how ?-arrestin 1 and 3-arrestin2 affect neurotoxicity of A ?.That is why we study ?-arrestins expression in AD patient's blood sample and different AD cell model and the role of ?-arrestins in neuron death induced by A ?.The conclusion is below.? A ? oligomer was neurotoxic,which induced endoplasmic reticulum stress and then kill neuron.1.Oligomer A ? significant decreased neuron viability.MTT showed that A ?markedly damage SH-SY5Y cells and PC 12 cells,and the toxicity was depended on concentration and time.2.Oligomer A? induced endoplasmic reticulum(ER)stress in neuron.After treatment of A ?,unfolded protein accumulated in the ER.Our confocal laser scanning microscope had observed 20 u M A ? induced ER swell and vacuolization after 8 hours treatment.Western blotting showed that the phosphorylation levels of eIF2 a was upregulated at 1h,and downregulated at 4h,when molecular chaperon protein GRP78 in ER began to increase,which were both time-dependence.The data gave us a conclusion that A ? induced unfolded protein accumulation in ER,leading to a time-dependence initiation of endoplasmic reticulum(ER)stress and stimulation of the unfolded protein response(UPR).A? 25-35 evoke an endoplasmic reticulum(ER)stress response.the phosphorylation status of the eukaryotic initiation factor(eIF2?)was initially upregulated after exposure to A? 25-35,and declined after 4 h,at which time cleaved PARP appeared in SH-SY5Y cells Increased expression of glucose-related protein 78(GRP78),was noticeable after 30 min of exposure to A?25-35,and was maintained at a high level during prolonged treatments in SH-SY5Y cells.3.Oligomer A? induced neuron apoptosis.Oligomer A ? can induced apoptosis in neuron dependent on dose and time.Western blotting showed that oligomer A ?induced a substrate PARP cut at A ? 8h treatment,which mean caspase3 is active at 8h.Therefore,apoptosis in neuron began.? The expression of ?-arrestins in AD patient's blood sample and different AD cell models was different with normal people blood sample and control of AD cell models.1.arrbl and arrb2 were lower expression in AD patient's blood sample.To study the expression of arrb1 and arrb2 in AD patients and normal people,we analyzed the difference on arrb1 and arrb2 transcription in two group blood samples by real-time PCR.The results showed that the mRNA levels of arrb1 and arrb2 was significantly reduced in AD group,which mean the transcription levels of arrb1/2 are inhibited in AD.2.?-arrestin1/2 levels were both lower in cells constantly express A ?.Western blotting showed ?-arrestinl/2 protein levels were downregulated in HEK293 cells constantly expressed A ? than in HEK293 cells without A? constant expression.What's more,the phosphorylation of ERK,a downstream protein of ?-arrestin,was also downregulated.From the data above,we can conclude that endogenous A ?affect the levels of ?-arrestin1 and ?-arrestin2.3.?-arrestinl and ?-arrestin2 were time-dependently expressed in cells treated by exogenous A ?.We add exogenous A? in SH-SY5Y cells and PC 12 cells as exogenous A ? cell model.Western blotting results showed that ?-arrestin2 was upregulated at 1h,downregulated at 4h and lasted until the cell death,and its downstream proteins phosphorylated ERK and phosphorylation AKT both follow the level of ?-arrestin2.?-arrestinl was slightly upregulated at 4h when ?-arrestin2 downregulated.The results showed that exogenous A ? affected ?-arrestin1 and ?-arrestin2 protein levels on time dependence.4.Protein levels of ?-arrestin1 and ?-arrestin2 were diverse in different cells.We take SH-SY5Y cells and PC12 cells as example,the western blotting results show that,compared with PC12 cells,?-arrestin1 protein level was lower and ?-arrestin2 level was higher in SH-SY5Y cells.The results tell us that ?-arrestinl was high expressed in PC 12 cells and ?-arrestin2 was high expressed in SH-SY5Y cells.? The role of ?-arrestinl in A ? induced neuron death was different with ?-arrestin2.1.In SH-SY5Y cells,arrb1-deletion by arrbl small silence RNA made cells more susceptible to A ?,and arrb2-deletion cells had a higher viability under A ?-treat than control cells.MTT experiments show that arrb1-deletion not only inhibited the multiplication capacity of cells,but also increase the neurotoxicity of A? to cells.However,the arrb2-deletion did not decrease proliferation of cell and keep a higher cell viability under A ?-treatment than control group.2.?-arrestinl inhibited proliferation-related regulators.Western blotting results showed that,in negative control group,A ? induced low level of ERK and AKT activation and PARP cleaved.However,after downregulation of ?-arrestinl,the phosphorylation levels of ERK and AKT lower than negative siRNA group of SH-SY5Y cell in control group,and A ? 25-38;intensified the effect.The result indicated the phosphorylation ERK and AKT regulated by ?-arrestin 1.PART TWO ?-arrestin1 affected the neurotoxicity of A?by participating the autophagy.Autophagy is a major intra-cytoplasmic protein degradation pathway.In autophagy,cytoplasmic contents are delivered,by double-membraned vesicles called autophagosomes,to the lysosome for degradation.The first morphologically characteristic structure in autophagy is the double-membraned,cup-shaped autophagosome precursor,called the phagophore,which engulfs substrates as its edges extend.After the phagophore edges close to form a vesicle,the completed autophagosomes traffic along microtubules to enable autophagosome-lysosome fusion,which leads to the degradation of the autophagic contents.Autophagy dysfunction may play a role in neurodegenerative diseases.Now,study revealed that ?-arrestin1/2 participated in the development of AD by affecting the production of A?,but the other mechanisms of ?-arrestinl/2 participate in AD unclear.We had known that ARRB1 mediates neuroprotection through coordination of beclinl-dependent autophagy in cerebral ischemia,which provide us an idea that whether ?-arrestin1/2 participates in the development of AD by autophagy.To explore the function of ?-arrestin1/2 in AD,we get the data below.?.? 25-35 induced autophagy activation.1.A? 25-35 evoke an endoplasmic reticulum(ER)stress response.the phosphorylation status of the eukaryotic initiation factor(eIF2a)was initially upregulated after exposure to A? 25-35,and declined after 4 h,at which time cleaved PARP appeared in SH-SY5Y cells Increased expression of glucose-related protein 78(GRP78),was noticeable after 30 min of exposure to AP25-35,and was maintained at a high level during prolonged treatments in SH-SY5Y cells.2.A? 25-35 induced activation of cytoprotective autophagy.Levels of LC3B-I and LC3B-II were significantly increased in SH-SY5Y cells after A ? 25-35 30 min treatment,and sharply dropped down at 1 h thereafter.Immunofluorescence staining further supported the observations that LC3B markedly increased after A? 25-35 stimulation for 1 h in SH-SY5Y cells and was eliminated after 8 h treatment.Next,we transfected SH-SY5Y cells with a pH-sensitive LC3-reporter plasmid containing tandem-tagged fluorescent proteins(mRFP-GFP-LC3)and take pictures by confocal microscopy.Within 1 h,increased autophagic flux occurred.After 4 h and 8 h treatments,disruption of lysosomes by AP25-35 led to impaired autophagosome-lysosome fusion and blocked autophagic flux.3.Activation of autophagy by AP25-35 served as a cytoprotective mechanism in SH-SY5Y cells.Knockdown of LC3B alone did not affect cell viability,whereas A?25-35-mediated cell death was facilitated when LC3B was depleted in SH-SY5Y cells.Similar results were observed in cells with reduced Beclin-1,another critical regulator of autophagy.The results indicate downregulation of autopahgy regulated proteins accelerated cell death induced by A ? 25-35,so activation of autophagy by AP25-35 served as a cytoprotective mechanism in SH-SY5Y cells.4.Activation of autophagy by A?25-35 served as a harmful mechanism in PC 12 cells.AP25-35-mediated cell death was alleviated when autophagy inhibitor 3-MA in PC 12 cells.The results indicated that inhibition of autophagy facilitated cell death of PC 12 cells,and activation of autophagy by A?25-35 served as a harmful mechanism in PC 12 cells.5.Autophagy levels were diverse in different cells.We take SH-SY5Y cells and PC 12 cells as example,the western blotting results show that,compared with PC 12 cells,LC3I and LC3II protein level was lower in SH-SY5Y cells.The results tell us that the basic autophagy level was lower in PC12 cells than in SH-SY5Y cells.??-arrestinl contributes to A? 25-35-induced autophagy in SH-SY5Y cells1.Expression of ?-arrestins was associated with a decreased conversion of LC3B-?/LC3B-? in AD cell model.?-arrestinl/2 might also be downregulated in HEK293-APPwt cells that constitutively overexpress APP and produce Ap.?-arrestin1 expression was slightly reduced in HEK293-APPwt cells that overexpressed A?,whereas the expression of ?-arrestin2 was lower than that of p-arrestinl in APPwt cells,and was associated with a decreased conversion of LC3B-?/LC3B-?.In exogenous AP25-35 AD cell model,A?25-35 was able to induce the expression of ?-arrestin 1 at 1 h,which then decreased in SH-SY5Y cells.Similar to ?-arrestin 1,the levels of ?-arrestin2 were upregulated at 1 h,and declined after prolonged treatment.Both ?-arrestin1/2 expression induced by A? 25-35 consistent with conversion of LC3B-?/LC3B-?,which indicated p-arrestin 1/2 consistent with autophagy induced by A ? 25-35.2.Autophagy was decreased by silencing ARRB1 in the presence of A?25-35.Our western blotting data show that depletion of ?-arrestin 1 resulted in a reduction of LC3B-?/LC3B-? and decreased levels of ATG7 and Beclin-1.LC3B fluorescence staining displayed a granular pattern in negative scramble siRNA controls after A?25-35 exposure for 1 h,whereas in siARRBl cells,the staining pattern was diffuse with reduced brightness.After treatment with A?25-35 for 8 h,the fluorescence intensity of LC3B was greatly decreased both in negative-control and siARRB1 cells,indicating that the arrbl knockdown impaired autophagy activation by AP25-35.3 Autophagy was no change by silencing ARRB2 in the presence of A?25-35.Our western blotting data show that depletion of ?-arrestin2 resulted in no change of LC3B-II/LC3B-I,and basic protein levels of ATG7 and Beclin-1,which mean?-arrestin2 had no role in A?25-35-activated autophagy.? A ? 25-35 damage the lysosome of cell,which cannot be relieved by ?-arrestins.1.A ? 25-35 inhibited the lysosome in cytoplasm.After staining by lysosomal tracker red,lysosome in cytoplasm show red puncta in confocal microscope pictures.The red punctas of lysosome in cells reduced after A ? 25-35 exposure,which related with the exposure time of A ? 25-35.The results show that A ? 25-35 reduced lysosome in cytoplasm.2.?-arrestins reduced A ? 25-35 toxicity not by affecting lysosome in cytoplasm.Lysosomal red punctas reduced a little after deletion of arrbl or arrb2,but deletion of arrbl/2 no change the lysosomal red punctas reduction after A ? 25-35 exposure,which mean ?-arrestins reduced A ? 25-35 toxicity not by affecting lysosome in cytoplasm.PART THREE Knockdown of ?-arrestin2 partially protects cells from A?25-35-induced apoptosis by facilitating a 7nAChR expression at the cell membrane.Acetylcholine receptor,one of the most important receptors in body,can be divided into M-type and N-type.Acetylcholine is its ligand.a 7nAChR,one of nicotinic acetylcholine receptors(nAChRs),a iron-channel receptor which widely distributed in the postsynaptic membrane of the neuron in the brain,was important for physiological neurotransmission.Loss of nicotinic acetylcholine receptors(nAChRs),seen in brains of patients with Alzheimer's disease(AD),and now one of the effective treatments,donepazel,improve the memory decline by active a 7nAChR in the postsynaptic membrane of the neuron.However,the desensitization of a 7nAChR put the treatment useless and facilitated neuron death in AD.So exploring the way to decrease desensitization of nAChR is needed.? ?7nAChR protect neuron.1.chrna7 was lower expression in AD patient's blood sample.To study the expression of chrna7 in AD patients and normal people,we analyzed the difference on chrna7 transcription in two group blood samples by real-time PCR.The results showed that the mRNA levels of chrna7 was significantly reduced in AD group,which mean the transcription levels of chrna7 are inhibited in AD.2.Nicotine,activating a7nAChR,offer protective effects from A? toxicity.The MTT data show that nicotine is a a7nAChR agonist that can activate a7nAChR and decrease A? toxicity.Knockdown of a7nAChR accelerated cell death,and cell viability as a result of nicotine treatment did not show recovery due to the deletion of the?7nAChR.A?25-35 induced cell death,and this cytotoxic effect was exacerbated by the depletion of a7nAChR.The data show that can nicotine activate a7nAChR and offer protective effects from A? toxicity.? ?-arrestin2,played a critical role in the accelerating neuron protection of nicotine from A? toxicity.1.Down-regulation of P-arrestinl have slight influence on the protection of nicotine.The cell viability assays revealed that silencing of arrbl exacerbated A?-induced toxicity,and nicotine pretreatment also did not improve the survival of cells exposed to A? when ?-arrestinl expression was low in cells.The data showed that compared with negative silence cells,nicotine change little in the cell membrane lacking?-arrestinl.2.Down-regulation of ?-arrestin2 have significant influence on the protection of nicotine.The cell viability assays revealed that silencing of arrb2 decreased A?-induced toxicity,and nicotine pretreatment also improve the survival of cells exposed to A?.When P-arrestin2 expression was low in cells,the data showed that compared with negative silence cells,nicotine significantly protect cells in the cell membrane lacking?-arrestin2.? ?-arrestin2 modulate a7nAChR expression on the cell membrane1.? treatment caused an increase in the expression of a7nAChR at the cell membrane.Flow cytometry detected that compared with negative cells,expression of a7nAChR increased in cells with A ? exposure.2.Down-regulation of ?-arrestinl have slight influence on the abundance of a7nAChR.Flow cytometry detected that compared with negative silence cells,expression of a7nAChR change a little in the cell membrane lacking ?-arrestinl.The cell viability assays revealed that silencing of arrbl exacerbated AP-induced toxicity,and nicotine pretreatment also did not improve the survival of cells exposed to A?when ?-arrestinl expression was low in cells.3.Down-regulation of ?-arrestin2 significantly enhanced the abundance of a7nAChR,which in turn leading to improved survival,and enhancement of nicotine-mediated protection against A?.PART FOUR Conclusions and InnovationI Conclusions1.arrbl and arrb2 were lower expression in AD patient's blood sample.2.?-arrestinl decreased the neurotoxicity of A ? and ?-arrestin2 had different role of A ? neurotoxicity in SH-SY5Y cells.3.?-arrestinl affected the neurotoxicity of A?by participating the autophagy in SH-SY5 Y cells4.?-arrestin2 modulate a7nAChR expression on the cell membrane in SH-SY5Y cells.? Innovation and defects1 It is the first report to reveal function of ?-arrestins in A ? induced cell death.2 It is the first time to report ?-arrestin1 affected neuron death by participating autophagy,but the mechanism need to explore further.3.It is the first time to report ?-arrestin2 affected neuron death by modulating a7nAChR expression on the cell membrane. |
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