| Iron is essential for various fundamental biological processes in the brain.During aging,iron deposits in specific brain regions associated with motor and cognitive impairment.Accumulating evidences suggest a role for iron dysmetabolism in the progress of multiple neurodegenerative diseases.Iron-associated physiological or pathological changes are traditionally viewed as being mediated by oxidative stress or iron responsive element,while the involvement of other regulatory mechanisms are rarely reported.In the present study,iron overload neuronal model was established on primary neurons treated with ferric iron.Exposure of primary neurons to excess iron caused reactive oxygen species accumulation,decreased ATP production and damaged neurites.Protein extracts from control and iron-treated primary neurons were subjected to label-free quantitative mass spectrometry analysis so as to evaluate the overall regulatory effects and molecular mechanisms of iron overload-related neuronal damage.Totally 336 differentially expressed proteins were identified between control and iron-treated group,with 82 of them classified as nervous system development and function-related proteins and 112 of them classified as neurological disease-associated proteins.Two of the top ten most remarkably regulated proteins,calsyntenin 1(Clstn1)and amyloid precursor protein(APP),were selected for further analysis because of their close association with neurodegenerative disorders.First,the influence of iron overload on Clstn1 was investigated in cultured primary cortical neurons.Clstn1 participates in nervous system development as well as learning and memory formation.It has been reported that the protein level of full length Clstn1 or its cleavage fragments were altered in a few neurodegenerative diseases.As far as known,there is no report about the relationship between iron and Clstn1.Proteomic analysis showed that iron treatment dramatically up-regulated the protein level of Clstn1,the change amplitude of which was ranked as the second biggest one among all differentially expressed proteins,with 20.502 change ratio as compared with control group.This result was further confirmed by western blot analysis.Mechanism study revealed that iron overload did not influence the transcription,glycosylation or cleavage of Clstn1 in primary cortical neurons.Alternatively,iron treatment altered the distribution of sAlcα,the N-terminal cleavage product of Clstn1,exhibited as reduced level of extracellular secreted sAlcα and increased level of cellular associated sAlcα.Further study demonstrated that the majority of the increased sAlcα in the cell lysates of iron-treated neurons was located in the plasma membrane.The binding property of sAlcα to the cellular surface of primary neurons was further confirmed by employing recombinant sAlcα.What was more,exogenous addition of sAlcα could ameliorate the neurite damage induced by iron overload.The current study provides the first demonstration of the association between iron and Clstn1 in primary neurons,and clarifies that iron overload caused membrane retention of sAlcα.In addition,our study indicates that sAlcα in secreted form may be beneficial for neuronal function while iron overload-induced reduced secretion of sAlcα may contribute to the iron overload associated neuronal damage.Further studies will be needed for drawing such conclusion.Similarly,the effect of iron overload on APP was studied in primary cortical neurons.APP is best known for its relevance to Alzheimer’s disease.Iron overload was reported to increase the expression of APP.Consistent with previous reports,proteomics analysis in the current study showed that iron treatment significantly up-regulated the protein level of APP.The following western blot results indicated that the increased level of APP might be largely due to the altered processing of APP.We then investigated the regulatory effects and molecular mechanisms of iron overload on APP processing in the primary cortical neurons.Iron treatment facilitated the non-amyloidogenic processing of APP manifested as the increase of CTFα However,the distribution of sAPPα in the primary cortical neurons was altered following iron treatment,with sAPPα detained in the cellular compartment instead of secreted into extracellular milieu.Meanwhile,the levels of APP amyloidogenic products including CTFβ in the cellular compartment,sAPPβ and Aβ in the extracellular media were all reduced by iron treatment.Further study found that the β-secretase activity in iron-treated neurons was significantly reduced as compared to that of control group,with no influence on the protein level of β-site amyloid precursor protein cleaving enzyme 1(BACE1).Mechanism study revealed that iron could directly inhibit BACE1 activity,as well as indirectly inhibit β-cleavage by promoting the interaction between sAPPα and BACE1.The current study further clarifies the regulatory effects of iron on APP non-amyloidogenic and amyloidogenic processing,and for the first time demonstrates that iron overload alters neuronal sAPPα distribution and directly inhibits BACE1 activity.As sAPPα and Aβ at physiological concentration were reported to possess neurotrophic effects,the dysregulation of sAPPα and Aβ caused by iron overload might play harmful role on neuronal function.Taken together,the current study explored the regulatory effects of iron overload on Clstn1 and APP in primary neurons on the basis of proteomic analysis.This study has discovered the relationship between iron and Clstn1,and further elucidated the regulatory effect of iron on APP processing,especially on the cellular distribution of their cleavage products,which sheds more light on the underlying mechanisms of iron overload associated neurological damage and provides important clues for the development of potential therapeutic strategy. |
