| ALS,or amyotrophic lateral sclerosis,is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord.The progressive degeneration of the motor neurons in ALS eventually leads to their demise.When the motor neurons die,the ability of the brain to initiate and control muscle movement is lost.With voluntary muscle action progressively affected,people may lose the ability to speak,eat,move and breathe.There are two different types of ALS,sporadic and familial.Sporadic,which is the most common form of the disease,accounts for 90 to 95 percent of all cases.It may affect anyone,anywhere.Familial ALS(FALS)accounts for 5 to 10 percent of all cases.Familial ALS means the disease is inherited.In those families,there is a 50% chance each offspring will inherit the gene mutation and may develop the disease.In recent years,the non-cell-autonomous effect caused by the dysfunction of glial cells has attracted people's attention.The non-cell-autonomous effect of neurons refers to the neuronal lesion caused by the dysfunction of non-neuronal cells.Studies have shown that astrocytes play an important role in neurodegenerative diseases including ALS.Astrocytes are complex,highly differentiated cells that tile the entire central nervous system(CNS)in a contiguous fashion and make numerous essential contributions to normal function in the healthy CNS,including regulation of blood flow,provision of energy metabolites to neurons,participation in synaptic function and plasticity,and maintenance of the extracellular balance of ions,fluid balance and transmitters.In addition,astrocytes respond to all forms of CNS insults such as infection,trauma,ischemia and neurodegenerative disease by a process commonly referred to as dysfunctional astrocytes,which involves changes in their molecular expression and morphology,and in severe cases,scar formation.The occurrence of neuroinflammation is one of the steady-state responses of brain tissue in emergency,which is mainly caused by the activation of astrocytes and microglia.When neuroinflammation occurs,the expression level of pro-inflammatory factors in the central nervous system increases,such as cytokines,inflammatory chemokines,second messengers,etc.Studies have shown that inflammatory cytokines can induce neurodegeneration of neurons,and autophagy plays an important role in inflammatory response.Autophagy plays an important role in maintaining homeostasis in vivo.Autophagy may be helpful to inhibit the inflammatory response because it can disturb the stability of intracellular environment.On the one hand,autophagy can clear the factors that cause inflammatory response,such as damaged mitochondria;on the other hand,autophagy can protect cells by inhibiting inflammatory complexes.Inflammatory cytokines can also regulate autophagy by affecting the special receptors attached to the plasma membrane.The role of autophagy in inflammatory response has not been determined.Therefore,we hypothesized that autophagy damage may be the core of the pathogenesis of ALS,and the increased secretion of inflammatory factors in astrocytes caused by autophagy damage may be one of the important reasons for the non-cell-autonomous effect of astrocytes on motor neurons in ALS.Part One Reprogramming of patients' somatic cells to obtain ALS astrocytes in vitroObjective: To reprogram peripheral blood mononuclear cells(PBMC)of three ALS patients and three health donors into iPSC using Sendai virus carrying four transcription factors as vectors,and then detect cell quality,pluripotency and differentiation ability by PCR,karyotype analysis,STR detection,immunofluorescence and flow cytometry,in order to provide high quality cell source for further research.To induce ALS specific iPSC to differentiate into neural stem cells,and then to establish a stable method to obtain high-purity mature astrocytes.Methods:1.To obtain ALS specific iPSC by reprogramming PBMCPeripheral blood samples from three ALS patients and three healthy controls were used.The PBMC were extracted by Ficoll centrifugation.Using Sendai virus as vector,four reprogramming transcription factors were introduced into PBMC and reprogrammed into iPSC.2.IPSCs obtained by reprogramming have homology,pluripotency and multi differentiation ability.3.IPSC can differentiate into high purity neural stem cells after 7 days of neural inductionIn this study,iPSCs from three ALS patients and three healthy controls were used to obtain NSCs with high purity.NSCs of P3 generation were divided into control group and patient group.Immunofluorescence technique was used to detect neural stem cell markers,such as Nestin,SOX2 and PAX6.Fluorescence microscope was used to observe the imaging,calculate the fluorescence intensity,and analyze the protein expression levels between control group and patient group.4.Neural stem cells can be induced to differentiate into mature astrocytes with high purity within 8 weeks in vitroNSCs from control group and patient group were cultured in astrocyte induction medium for about 21 days,during which the NSCs were purified by differential centrifugation,differential adhesion and oscillation.The astrocytes were cultured in astrocyte medium for another 35 days.The astrocytes cultured for 8 weeks were divided into control group and patient group.Immunofluorescence technique was used to detect the mature astrocyte markers of GFAP and S100?.Fluorescence microscope was used to observe the imaging,calculate the fluorescence intensity,and analyze the protein expression difference between control group and patient group.Results:1.ALS specific iPSCs were obtained by reprogramming PBMCIn this study,we used peripheral blood from three patients with clinically confirmed ALS and three healthy donors,and extracted 2 ml of peripheral blood from the median cubital vein.PBMC were extracted by Ficoll centrifugation method.PBMCs cultured for 5 days were selected as reprogramming cells and transfected with Sendai virus carrying four reprogramming factors.The clones grew to the size that could be picked up about 20 days after transfection.2.The reprogrammed iPSCs have homology,pluripotency and multi differentiation ability1)PCR results showed that the reprogrammed iPSCs had no residual virus after passage,which ensured the reprogramming quality;2)karyotype analysis showed that the cell karyotype did not change;3)STR detection showed that the reprogrammed iPSCs had the same origin as the source cells PBMC;4)immunofluorescence staining and flow cytometry showed that the pluripotent cell markers of iPSCs were positive,and there was no statistical difference in fluorescence intensity between groups(P > 0.05).5)Immunofluorescence staining showed that the specific markers of iPSC differentiation were positive.3.IPSC can differentiate into high purity neural stem cells after one week of neural inductionIn this study,iPSCs from three ALS patients and three healthy controls were used to obtain NSCs with high purity.NSCs of P3 generation were divided into control group and patient group.Immunofluorescence technique was used to detect neural stem cell markers,such as Nestin,SOX2 and PAX6.Fluorescence microscope was used to observe the imaging,calculate the fluorescence intensity,and analyze the protein expression levels between control group and patient group.4.Neural stem cells can be induced to differentiate into mature astrocytes with high purity within 8 weeks in vitro.NSCs from control group and patient group were cultured in astrocyte induction medium for about 21 days,during which the NSCs were purified by differential centrifugation,differential adhesion and oscillation.The astrocytes were cultured in astrocyte medium for another 35 days.The astrocytes cultured for 8 weeks were divided into control group and patient group.Immunofluorescence technique was used to detect the mature astrocyte markers of GFAP and S100?.Fluorescence microscope was used to observe the imaging,calculate the fluorescence intensity,and analyze the protein expression difference between control group and patient group.Conclusions:1.ALS specific iPSCs were obtained by reprogramming PBMC.2.The reprogrammed iPSCs have homology,pluripotency and multi-differentiation ability.3.IPSC can differentiate into high purity neural stem cells after one week of neural induction in vitro.4.Neural stem cells can be induced to differentiate into mature astrocytes with high purity within 8 weeks in vitro.Part Two Effect of ALS astrocytes on motoneurons through secretion of inflammatory factorsObjective: The effect of ALS astrocytes on motoneurons and the secretion level of inflammatory factors in the astrocyte culture medium were detected by using astrocyte conditioned medium as the experimental path,and then the effect of ALS astrocytes on motoneurons was explored through the stimulation of exogenous inflammatory factors.Methods:1.IPSC can differentiate into high purity motoneurons in 4 weeks.In this study,iPSCs from a healthy subject was induced to differentiate into MNP after 12 days in the motor neuron induction medium,and the specific markers of MNP,Olig2 and Pax6,were detected by immunofluorescence;the motor neurons were obtained after 16 days of induction and differentiation,and the expression of Ch AT and TUJ were detected by immunofluorescence.2.The effect of ALS astrocyte conditioned medium on motoneuron cell viabilityThe experiment was divided into four groups: normal medium group,fresh medium group,control ACM group and patient ACM group.In normal medium group,the basic medium for motor neuron induction was placed in three empty containers,and the medium was collected after standing in the cell incubator for 48h;in fresh medium group,the basic medium for motor neuron induction was directly used;in control group,the basic medium for motor neuron induction was used;in control ACM group,the basic medium of motor neuron induction was placed in the mature astrocytes with 90%confluence from three healthy control sources,and the culture medium was collected 48 hours later;in patient ACM group,the basic medium of motor neuron induction was placed in the mature astrocytes with 90% confluence from three ALS patients,and the culture medium was collected 48 hours later.The effects of astrocyte conditioned medium and negative control on motoneuron viability were detected by MTT assay3.The changes of IL1?,IL6 and TNF? secretion in ALS astrocytesMature astrocytes with 90% confluence were selected and divided into healthy control group(control group)and ALS patient group(patient group).After 48 hours of motor neuron basic medium,the medium of each group was collected,and the contents of IL1?,IL6 and TNF? in the medium were detected by ELISA.4.Effects of inflammatory factors IL1?,IL6 and TNF? on motoneuron cell viabilityThe experiment was divided into four groups: control ACM group,patient ACM group,fresh medium group and cytokines medium group.For the preparation of control ACM group,patient ACM group and fresh medium group,refer to 2.6.1 of Part II.In the cytokines medium group,according to the results of the previous part,the dissolved IL1?,IL6 and TNF? storage solutions were added to the basic medium for motor neuron induction,and the final concentrations were 120 pg/ml,185 pg/ml and 180 pg/ml,respectively.The effects of inflammatory factors and astrocyte conditioned medium on motoneuron viability were detected by MTT assay.Results:1.IPSC can differentiate into high purity motoneurons in 28 days.In the experiment,IPSC from a healthy control was induced to differentiate into MNP after 12 days in the motor neuron induction medium,and the specific markers of MNP,Olig2 and Pax6,were detected by immunofluorescence technique;the motor neurons were obtained after 16 days of induction and differentiation,and the expression of Ch AT and TUJ were detected by immunofluorescence technique.2.Effect of ALS specific astrocyte conditioned medium on motoneuron cell viabilityMTT results showed that compared with control ACM group(0.647±0.070),patient ACM group(0.493±0.032)could significantly reduce the activity of motoneurons.There was no significant difference between normal medium group(0.673±0.047),fresh medium group(0.677±0.050)and control ACM group(0.647±0.070)(P > 0.05)3.The changes of IL1?,IL6 and TNF? secretion in ALS astrocytesELISA results of IL1? showed that compared with control group(18.96±8.49),the relative secretion level of patient group(39.94±9.10)increased,the difference was statistically significant(P < 0.05);ELISA results of IL6 showed that compared with control group(119.30±4.62),the relative secretion level of patient group(186.30±7.84)increased,the difference was statistically significant(P < 0.05);ELISA results of TNF? showed that compared with control group(105.60±8.80),the relative secretion level of patient group(179.90±27.45)increased,and the difference was statistically significant(P < 0.05).4.Effects of inflammatory factors IL1?,IL6 and TNF? on motoneuron cell viabilityMTT results showed that compared with control ACM group(0.630±0.044),patient ACM group(0.510±0.026)and cytokines medium group(0.473±0.047)could significantly reduce the activity of motoneurons(P< 0.05).There was no significant difference between fresh medium group(0.737±0.050)and control ACM group(0.630±0.044),between cytokines medium group(0.473±0.047)and patient ACM group(0.510±0.026)(P >0.05)Conclusions:1.IPSC can differentiate into high purity motoneurons after 28 days.2.ALS astrocyte conditioned medium can reduce the activity of motoneurons.3.The secretion of IL1?,IL6 and TNF? increased in ALS astrocytes.4.Inflammatory factors IL1?,IL6 and TNF? can reduce the cell activity of motoneurons.Part Three Regulation of inflammatory factors secretion by ALS astrocytes via m TOR autophagy pathwayObjective: To observe the changes of autophagy activity of ALS specific astrocytes by immunofluorescence,Western blot and transmission electron microscope.Immunofluorescence and Western blot were used to detect the changes of autophagy activity and the expression of m TOR pathway protein under the intervention of classical autophagy activator rapamycin and autophagy inhibitor 3-MA.Methods:1.Changes of autophagy function of ALS specific astrocytesThe mature astrocytes of healthy control group(control group)and ALS patients group(patient group)were selected.1)After the confluence reached70%-80%,the protein of cells was extracted for Western blot detection,and the expression levels of p62 and LC3 proteins in the two groups were determined with the expression of GAPDH as the internal reference;2)immunofluorescence staining was performed,and the images were collected by fluorescence microscope,and the number of p62 positive fluorescent puncta was automatically counted;3)After fixation,astrocyte ultrathin sections were made,and the number of autophagosomes was counted.2.ALS specific astrocytes affect autophagy through m TOR pathwayMature astrocytes with 70%-80% confluence were divided into healthy control group(control group),healthy control + rapamycin group(control +rapamycin group),healthy control + 3-methyladenine group(control + 3-MA group),ALS patients group(patient group),ALS patients + rapamycin group(patient + rapamycin group)and ALS patients + 3-methyladenine group(patient + rapamycin group)+3-MA group).1)Detection of protein expression levels of m TOR/p-m TOR,ULK1/p-ULK1,Beclin1/p-Beclin1 in ALS specific astrocytes: Western blot was used to detect the protein of control group and patient group,and BD imaging system was used to develop the images.The expression levels of m TOR/p-m TOR,ULK1/p-ULK1,Beclin1/p-Beclin1 were determined with GAPDH as internal reference;2)Effects of rapamycin and3-MA on the expression of m TOR/p-m TOR,ULK1/p-ULK1,Beclin1/p-Beclin1 in ALS specific astrocytes.Control + rapamycin group,control +3-MA group,patient + rapamycin group and patient + 3-MA group were pretreated with rapamycin and 3-MA respectively for 48 hours.The protein of each group was collected for Western blot detection and BD imaging system was used to develop the images.The expression of GAPDH was used as internal reference to determine m TOR/p-m TOR,ULK1/p-ULK1,Beclin1/p-Beclin1 protein expression level;3)Effects of rapamycin and 3-MA on the expression of p62 and LC3 proteins in ALS specific astrocytes: the protein was extracted and detected by Western blot,developed by BD imaging system,and the expression levels of p62 and LC3 protein in the two groups were determined by GAPDH expression as internal reference;immunofluorescence staining was performed,images were collected by fluorescence microscope,and p62 positive fluorescence points were automatically counted.3.The cell-autonomous regulation of ALS specific astrocytes on the secretion of inflammatory cytokines IL1?,IL6 and TNF? through autophagyMature astrocytes with 90% confluence were divided into healthy control group(control group),healthy control + rapamycin group(control +rapamycin group),healthy control + 3-methyladenine group(control+3-MA group),ALS patients group(patient group),ALS patients + rapamycin group(patient + rapamycin group)and ALS patients + 3-methyladenine group(patient + 3-MA group).Control + rapamycin group,control + 3-MA group,patient + rapamycin group and patient + 3-MA group were pretreated with rapamycin and 3-MA respectively for 48 hours.The medium of each group was collected and the contents of IL-1?,IL6 and TNF? in the medium were detected by ELISA.Results:1.Changes of autophagy function of ALS specific astrocytesCompared with control group(28.94±7.61),patient group(53.89±5.41)had more p62 positive signal points(P < 0.05).Western blot results showed that compared with control group(0.84±0.19),the relative level of p62 protein band optical density in patient group(1.45±0.12)was increased,and the difference was statistically significant(P < 0.05).Compared with control group(4.63±0.48),the relative level of LC3-?/LC3-?protein band optical density in patient group(0.33±0.02)was increased,and the difference was statistically significant(P < 0.05).The results of transmission electron microscopy showed that the number of autophagosomes in the patient group(1.22±0.28)was significantly higher than that in the control group(4.25±0.31)(P < 0.05).2.ALS specific astrocytes affect autophagy through m TOR pathwayWestern blot analysis of m TOR showed that,compared with control group(0.97±0.17),the relative protein expression levels of patient group(1.50±0.14)and control + 3-MA group(1.50±0.08)were significantly higher(P < 0.05),while control + rapamycin group(1.46±0.04)had no significant difference(P > 0.05);Compared with patient group(1.50±0.14),patient +rapamycin group(1.45±0.20)and patient + 3-MA group(1.50±2.28)(P >0.05).Western blot analysis of p-m TOR showed that,compared with control group(1.94±0.08),the relative expression levels of patient group(1.04±0.28)and control + 3-MA group(0.83±0.26)were decreased,the difference was statistically significant(P < 0.05),control + rapamycin group(1.86±0.07)had no statistical difference(P > 0.05);compared with patient group(1.04±0.28),the relative expression levels of patient + rapamycin group(2.07±0.55)were increased,the difference was statistically significant.There was no significant difference between patient group(1.04±0.28)and patient + 3-MA group(0.52±0.26)(P > 0.05).Western blot analysis of ULK1 showed that,compared with control group(1.80±0.15),the relative expression level of patient group(1.26±0.10)was significantly lower(P < 0.05),but control + rapamycin group(1.80±0.25)and control + 3-MA group(1.50±0.05)had no significant difference(P > 0.05);compared with patient group(1.26±0.10),the relative expression level of patient + rapamycin group(1.97±0.13)was significantly lower(P < 0.05).But patient + 3-MA group(1.27±0.25)had no significant difference(P > 0.05).Western blot results of p-ULK1 showed that there was no significant difference among control group(1.34±0.28),control + rapamycin group(1.58±0.16),control + 3-MA group(1.83±0.10),patient group(1.61±0.44),patient + rapamycin group(1.45±0.54)and patient + 3-MA group(1.22±0.18)(P > 0.05).Western blot results of Beclin1 showed that there was no significant difference among control group(1.11±0.30),control + rapamycin group(1.51±0.14),control + 3-MA group(1.44±0.10),patient group(1.07±0.28),patient + rapamycin group(1.43±0.12)and patient + 3-MA group(1.61±0.11)(P > 0.05).Western blot results of p-Beclin1 showed that,compared with the control group(1.66±0.40),the relative expression levels of patient group(0.65±0.22)and control + 3-MA group(0.62±0.11)were significantly lower(P < 0.05);the control + rapamycin group(1.16±0.11)had no significant difference(P >0.05);compared with the patient group(0.65±0.22),the relative expression levels of patient + rapamycin group(1.37±0.14)were significantly lower(P <0.05).But there was no significant difference between patient group(0.65±0.22)and patient + 3-MA group(0.62±0.11)(P > 0.05).Western blot results of p62 showed that,compared with control group(0.84±7.61),the relative expression level of p62 protein in patient group(1.45±0.12)and control + 3-MA group(1.44±0.18)was increased(P < 0.05),but control + rapamycin group(0.74±0.19)had no significant difference(P >0.05);compared with patient group(1.45±0.12),the relative expression level of p62 protein in patient + rapamycin group(0.85±0.30)was decreased(P <0.05).Patient + 3-MA group(1.81±0.14)had no difference(P < 0.05).Western blot results of LC3 showed that,compared with control group(4.63±0.48),patient group(0.33±0.02),control + rapamycin group(1.78±0.03)and control + 3-MA group(1.37±0.14)had significantly lower LC3-?/LC3-?expression;The relative expression level of LC3-?/LC3-?in patient group(0.33±0.02)was lower than patient + rapamycin group(1.59±0.09),while there was no difference between patient group(0.33±0.02)and patient + 3-MA group(0.60±0.14)(P < 0.05).Compared with control group(28.94±7.61),the positive signal puncta of p62 in patient group(53.89±5.41)and control + 3-MA group(51.22±8.26)increased significantly(P < 0.05),but there was no significant difference between control group(28.94±7.61)and control + rapamycin group(27.11±5.59)(P > 0.05);compared with patient group(53.89±5.41),the positive signal puncta of p62 in patient + rapamycin group(32.50±4.17)decreased significantly(P < 0.05).There was no difference between patient group(53.89±5.41)and patient + 3-MA group(54.11±9.00)(P < 0.05).3.The cell-autonomous regulation of ALS specific astrocytes on the secretion of inflammatory cytokines IL1?,IL6 and TNF? through autophagyELISA results of IL1? showed that compared with the control group(22.37±10.06),the relative secretion level of patient group(58.15±6.91)and control + 3-MA group(53.89±11.65)increased,the difference was statistically significant(P < 0.05),the control + rapamycin group(17.91±13.34)had no significant difference with control group(P > 0.05);compared with the patient group(58.15±6.91),the relative secretion level of the patient + rapamycin group(22.74±7.62)had no significant difference(P > 0.05).The patient +3-MA group(57.92±11.90)had no significant difference from the patient group(P > 0.05).ELISA results of IL6 showed that compared with the control group(119.30±4.62),the relative secretion level of patient group(186.30±7.84)and control + 3-MA group(188.30±16.27)increased,the difference was statistically significant(P < 0.05),the control + rapamycin group(75.22±39.15)had no significant difference(P > 0.05);compared with the patient group(186.30±7.84),the patient + rapamycin group(111.20±27.71)had no significant difference(P > 0.05),the difference was statistically significant(P < 0.05),the patient + 3-MA group(193.70±14.37)had no significant difference(P > 0.05).ELISA results of TNF? showed that compared with the control group(105.60±8.80),the relative secretion level of patient group(179.90±27.45)and control + 3-MA group(173.50±22.49)increased,the difference was statistically significant(P < 0.05),the control + rapamycin group(58.70±9.21)had no significant difference(P > 0.05);compared with the patient group(179.90±27.45),the patient + rapamycin group(111.60±9.21)had no significant difference(P > 0.05),the difference was statistically significant(P< 0.05),the patient + 3-MA group(178.40±21.36)had no significant difference(P > 0.05).Conclusions:1.The autophagy activity of ALS astrocytes decreased.2.ALS astrocytes regulate autophagy through m TOR pathway.3.Astrocytes regulate the secretion of IL1?,IL6 and TNF? through m TOR autophagy pathway. |
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