Deregulation Of Iron Metabolism In Astrocytes Are Involved In Neuron Has Iron Deposit In Parkinson's Disease

Parkinson's disease?PD?is a neurodegenerative disorder characterized in its late phase by the sustained loss of dopaminergic neurons in the substantia nigra pars compacta?SNpc?.Clinically PD is characterized by bradykinesia,rigidity,rest tremor and postural instability.The mechanisms underlying PD pathogenesis have not been revealed yet.Multiple factors might be involved,such as genetic mutation,environmental factors,aging,et al.In animal models and PD patients,selective deposition of iron in the substantia nigra?SN?has been observed,while no iron content increases in other brain regions.The most studies focused on iron deposition in dopaminer neurons of PD,in fact,a variety of gliocytes also plays an important role in cellular iron homeostasis.In addition to the sustained loss of dopaminergic neurons in the SNpc,the activation and proliferation of astrocytes also present in animal models and PD patients.They have a high iron tolerance.When iron loaded,neurons,microglia and oligodendrocytes have iron deposit,but astrocytes does not have iron deposit in the same situation.In our previous studies,we reported that in neurons,6-hydroxydopamine?6-OHDA?activate iron regulatory protein 1?IRP1?,increased the expression of divalent metal transporter-1?DMT1?and decreased the expression of ferroportin 1?FPN1?,led to increased iron influx and decreased iron efflux.In astrocytes,6-OHDA activated hypoxia-inducible factor-2??HIF-2??,increased both DMT1 and FPN1 expression,increased iron influx and efflux.Therefore,the mechanisms of iron metabolism regulations in neurons and astrocytes were different.Astrocytes may play as“iron pump”,but the mechanisms remain elusive.HIFs are involved in regulating iron homeostasis and are heterodimers consisting of two helix-loop-helix-containing subunits:HIF-?,a regulatory subunit,and HIF-1?,which is constitutively expressed.Therefore,studies on HIFs mainly focus on HIF-1?and HIF-2?.Binding of HIF-2?to the promoter region of the hypoxia response elements?HREs?of DMT1 and FPN1 increases their expression.Protein kinase C?PKC?modulates the HIF pathway in the human cervical cancer cell line HeLa and PKC?plays an important role in regulating HIFs.In astrocytes,whether PKC pathway is involved in 6-OHDA activation of HIF-2??Moreover,does the enhanced iron traffic in astrocytes become the source of iron deposition in dopaminergic neurons in SN of PD?In this study,using primary cultured rat astrocytes,PKC pathway in regulating HIF-2?was investigated.Transwell was used to establish a co-culture system of astrocytes and neurons,and the interaction and mechanism of iron metabolism in astrocytes and neurons were observed.The results were as follow:1.PKC pathway was involved in the actiavation of HIF-2?in 6-OHDA-treated astrocytes.?Expression of HIF-2?increased after primary cultured astrocytes were treated with 0.2?M PMA for 24 h,compared with the control group?P<0.05?.Pretreatment with PKC inhibitor Bisl decreased the expression of HIF-2?in PMA-treated cells,compared with the PMA group?P<0.05?.The expression of DMT1 increased in 0.2 ?M PMA-treated primary cultured astrocytes,compared with the control group?P<0.05?.Pretreatment with Bisl decreased the expression of DMT1 in PMA-treated cells,compared with the PMA group?P<0.05?.Expression of FPN1 increased in 0.2?M PMA-treated primary cultured astrocytes,compared with the control group?P<0.05?.Pretreatment with Bisl decreased the expression of FPN1 in PMA-treated cells,compared with the PMA group?P<0.05?.n=6.?Expression of HIF-2?increased after primary cultured astrocytes were treated with 10?M 6-OHDA for 24 h,compared with the control group?P<0.05?.Pretreatment with Bisl decreased the expression of HIF-2?in 6-OHDA-treated cells,compared with the 6-OHDA group?P<0.05?.Expression of DMT1 increased in 10?M 6-OHDA-treated primary cultured astrocytes,compared with the control group?P<0.05?.Pretreatment with Bisl decreased the expression of DMT1 in 6-OHDA-treated cells,compared with the 6-OHDA group?P<0.05?.Expression of FPN1 increased in 10?M 6-OHDA-treated primary cultured astrocytes,compared with the control group?P<0.05?.Pretreatment with Bisl decreased the expression of FPN1 in 6-OHDA-treated cells,compared with the 6-OHDA group?P<0.05?.n=6.2.ROS/RNS activating PKC?were involved in the activation of HIF-2?in 6-OHDA-treated astrocytes.?Expression of HIF-2?increased significantly after primary cultured astrocytes were treated with 10?M 6-OHDA for 24 h,compared with the control group?P<0.01?.Pretreatment with NAC decreased expression of HIF-2?in 6-OHDA-treated primary cultured astrocytes,compared with the 6-OHDA group?P<0.05?.Pretreatment with 1 mM L-NAME decreased expression of HIF-2?in 6-OHDA-treated primary cultured astrocytes,compared with the 6-OHDA group?P<0.05?.The expression of DMT1 increased in10?M 6-OHDA-treated primary cultured astrocytes,compared with the control group?P<0.05?.Pretreatment with NAC decreased expression of DMT1 in 6-OHDA-treated cells,compared with the 6-OHDA group?P<0.05?.Pretreatment with L-NAME decreased significantly expression of DMT1 in 6-OHDA-treated cells,compared with the 6-OHDA group?P<0.01?.Expression of FPN1 increased in 10?M 6-OHDA-treated primary cultured astrocytes,compared with the control group?P<0.05?.Pretreatment with NAC decreased expression of FPN1 in 6-OHDA-treated cells,cmpared with the 6-OHDA group?P<0.05?.Pretreatment with L-NAME decreased expression of FPN1 in 6-OHDA-treated cells,compared with the 6-OHDA group?P<0.05?.n=6.?PKC?phosphorylation increased after primary cultured astrocytes were treated with 10?M 6-OHDA for 24 h,compared with the control group?P<0.05?.Pretreatment with NAC decreased PKC?phosphorylation in 6-OHDA-treated primary cultured astrocytes,compared with the 6-OHDA group?P<0.05?.Pretreatment with 1 mM L-NAME decreased PKC?phosphorylation in 6-OHDA-treated primary cultured astrocytes,compared with the 6-OHDA group?P<0.05?.n=6.3.6-OHDA accelerated iron traffic in astrocytes,which was the source of iron deposited in neurons?In astrocytes-neuron co-cultured system,compared with the AS+FAC/N+6-OHDA group,when astrocytes were treated with 6-OHDA?the AS+6-OHDA+FAC/N+6-OHDA group?,Fe²⁺content increased in VM neurons?P<0.05?.Compared with the AS+6-OHDA/N+6-OHDA group,When astrocytes were pretreated with FAC?the AS+6-OHDA+FAC/N+6-OHDA group?,Fe²⁺content increased in VM neurons?P<0.05?.Compared with the AS+6-OHDA+FAC/N group,when VM neurons were treated with 6-OHDA?the AS+6-OHDA+FAC/N+6-OHDA group?,Fe²⁺content significantly increased in VM neurons?P<0.01?.Compared with the AS/N group,when astrocytes and neurons were treated with 6-OHDA,and astrocytes were pretreated with FAC?the AS+6-OHDA+FAC/N+6-OHDA group?,Fe²⁺content increased in VM neurons?P<0.05?.n=6.?In astrocytes-neurons co-cultured system,compared with the AS/N group,when VM neurons were treated with 6-OHDA?the AS/n+6-OHDA group?,the expression of IRP1 increased in VM neurons?P<0.05?.Compared with the AS/N group,when astrocytes were treated with 6-OHDA?the AS+6-OHDA/N group?,the expression of IRP1 increased in VM neurons?P<0.05?.Compared with the AS/N group,when astrocytes and VM neurons were both treated with 6-OHDA?the AS+6-OHDA/N+6-OHDA group?,the expression of IRP1 increased significantly in VM neuron?P<0.01?.n=6.?In astrocytes-neurons co-cultured system,compared with the AS/N group,when VM neurons were treated with 6-OHDA?the AS/N+6-OHDA group?,the expression of DMT1 increased significantly in VM neurons?P<0.01?.Compared with the AS/N group,when astrocytes were treated with 6-OHDA?the AS+6-OHDA/N group?,the expression of DMT1 increased significantly in VM neurons?P<0.01?.Compared with the AS/N group,when astrocytes and neurons were both treated with 6-OHDA?the AS+6-OHDA/N+6-OHDA group?,the expression of DMT1 increased in VM neurons?P<0.05?.In astrocytes-neurons co-cultured system,compared with the AS/N group,when VM neurons treated with 6-OHDA?the AS/N+6-OHDA group?,the expression of FPN1 decreased significantly in VM neurons?P<0.01?.Compared with the AS/N group,when astrocytes were treated with 6-OHDA?the AS+6-OHDA/N group?,the expression of FPN1 decreased in VM neurons?P<0.05?.Compared with the AS/N group,when astrocytes and neurons were both treated with 6-OHDA?the AS+6-OHDA/N+6-OHDA group?,the expression of FPN1 decreased significantly in VM neurons?P<0.01?.n=6.4.6-OHDA generated in DA neurons enhanced iron traffic in astrocytes.?In astrocytes and neurons co-cultured system,the Fe²⁺content was unchanged in AS/N+6-OHDA group,compared with AS/N group?P>0.05?.Fe²⁺content was unchanged in AS+6-OHDA/N group,compared with AS/N group?P>0.05?.Fe²⁺ content was unchanged in AS+6-OHDA/N+6-OHDA group,compared with AS/N group?P>0.05?.Fe²⁺content was unchanged in AS+FAC/N group,compared with AS/N group?P>0.05?.Fe²⁺content was unchanged in AS+FAC/N+6-OHDA group,compared with AS/N group?P>0.05?.Fe²⁺content was unchanged in AS+6-OHDA+FAC/N group,compared with AS/N group?P>0.05?.Fe²⁺content was unchanged in AS+6-OHDA+FAC/N+6-OHDA group,compared with AS/N group?P>0.05?.n=6.?In astrocytes-neurons co-cultured system,compared with the AS/N group,when VM neurons were treated with 6-OHDA?the AS/N+6-OHDA group?,the expression of HIF-2?increased in astrocytes?P<0.05?.Compared with the AS/N group,when astrocytes were treated with 6-OHDA?AS+6-OHDA/N group?,the expression of HIF-2?increased in astrocytes?P<0.05?.Compared with the AS/N group,when astrocytes and VM neurons were treated with 6-OHDA?the AS+6-OHDA/N+6-OHDA group?,the expression of HIF-2?increased in astrocytes?P<0.05?.n=6.?In astrocytes and neurons co-cultured system,compared with the AS/N group,when VM neurons were treated with 6-OHDA?the AS/N+6-OHDA group?,the expression of DMT1 increased in astrocytes?P<0.05?.Compared with the AS/N group,when astrocytes were treated with 6-OHDA?the AS+6-OHDA/N group?,the expression of DMT1 increased in astrocytes?P<0.05?.Compared with the AS/N group,when astrocytes and VM neurons were both treated with 6-OHDA?the AS+6-OHDA/N+6-OHDA group?,the expression of DMT1 increased in astrocytes?P<0.05?.B.In astrocytes and neurons co-cultured system,compared with the AS/N group,when VM neurons were treated with 6-OHDA?the AS/N+6-OHDA group?,the expression of FPN1 increased in astrocytes?P<0.05?.Compared with the AS/N group,when astrocytes were treated with 6-OHDA?the AS+6-OHDA/N group?,the expression of FPN1 increased significantly in astrocytes?P<0.01?.Compared with the AS/N group,when astrocytes and VM neurons were treated with 6-OHDA?the AS+6-OHDA/N+6-OHDA group?,the expression of FPN1 increased significantly in astrocytes?P<0.01?.n=6.In conclusion,our data indicate that PKC pathway is involved in 6-OHDA activation of HIF-2?in astrocytes,which may be associated with ROS and NO activation of PKC?.Astrocytes is“iron pump”,6-OHDA accelerates iron traffic in astrocytes,which is the source of iron deposited in neurons.6-OHDA generated in DA neurons enhances iron traffic in astrocytes,leading to iron deposit in DA neurons in the SN.