Studies On The Modulation Mechanism Of Iron On Parkinson's Disease In A Drosophila Model

Diet nutrition and food safety have become top issue.Increasing attention is being placed on healthy life and it's necessary to design more efficient and healthy food.Although essential trace elements are indispensable for living organisms,they cannot be generated or synthesized by the body and must be supplied through the diet.An imbalanced diet,and dyshomeostasis will lead to trace elements deficiency or excess and these changes may cause disease.Iron,one of the abundant trace element in the body,plays a key role in metabolism as a component of many enzymes,hormones,vitamin and nucleic acid.Researches have reported aberrant iron metabolism is associated with many diseases.Parkinson's disease(PD)is a neurodegenerative disease.The molecular etiology and pathology of this disease remain unclear,and there is still no effective treatment for halting this disease.It was reported that PD patients have abnormal iron levels.However,whether iron dyshomeostasis represents the initiation cause or merely the consequence is still unknown.We tend to explore iron modulation mechanism on PD.The findings may provide a basic theoretical support to improve PD progress and the feasibility of developing iron-related functional foods.Drosophila melanogaster,a classical model organism,has been extensively used in food nutrition and health recently because it offers an abundance of advantages,such as easy to feed,fast offspring turnover,rich phenotypes,considerable gene homology with humans and convenient genetic modulation.Our work has created Phosphatase and tensin homolog-induced putative kinase protein 1(PINK1)RNAi induced familial PD.The genetic,biochemical,physiological,molecular,behavioral techniques were used and genetic method was used to change d ZIP13 and Transferrin1(Tsf1)level.Behavioral test,physiological and biochemical methods were used to detect their effects on familial PD phenotypes.Biochemical and molecular biology methods were used to detect mitochondrial function.These results found that iron levels may affect mitochondrial respiratory chain enzyme activity,ATP generation and PD process.Besides,we have also created the rotenone induced sporadic PD model.Genetic method was used to change Tsf1 level.We study the effects of iron on PD phenotypes and oxidative damage.Further study was performed to explore the mechanism on molecular level.The main research contents and results are as follows:(1)To sdudy the effects of iron on familial PD,UAS/GAL4 system was performed to creat familial PD by knocking down PINK1 on muscle.d ZIP13 overexpression(d ZIP13 OE)and Transferrin1 RNA inteferance(Tsf1 RNAi)on muscle may rescue PINK1 RNAi induced PD progression,including muscle degeneration,abnormal wing posture and defective jumping.(2)PINK1 has many physiological functions.PINK1 works with Parkin in a common pathway involved in regulating mitophagy.To explore whether d ZIP13 OE or Tsf1 RNAi rescue PINK1 deficiency by regulating mitophagy,behavioral test was used to detect the effects of d ZIP13 OE or Tsf1 RNAi on Parkin deficiency induced phenotypes.However,d ZIP13 OE or Tsf1 RNAi can't rescue Parkin deficiency induced PD progression,including muscle degeneration,abnormal wing posture and defective jumping.These results suggested that d ZIP13 OE or Tsf1 RNAi may modulate PINK1 deficiency independent of mitophagy.Besides,we observed mitochondrial morphology and the colocalized signals with Autophagy-related gene 8?(Atg 8?)by using biochemistry and molecular biology techniques.These results indicated that d ZIP13 OE or Tsf1 RNAi can't rescue the decreased mitophagy levels and provided direct evidence that the modulation on PINK1 deficiency may independent of mitophagy.(3)Biochemical and physiological methods were used and we have found PINK1 RNAi reduced mitochondrial respiration ability and mitochondrial complexes activities.We measured Oxygen Consumption Rate(OCR)and mitochondrial complexes ?-? activities.The results found d ZIP13 OE or Tsf1 RNAi may ameliorate these changes,alleviate mitochondrial damage and affect mitochondrial function.(4)Our previous work has showed d ZIP13 and Tsf1 have iron metabolism function in the intestine.To further explore whether the rescue effects of d ZIP13 OE or Tsf1 RNAi may rely on iron levels,the iron levels were measured using Ferrozine assay and ICP-MS.The results of iron levles coupled with mitochondrial complexes activities presented the inference that d ZIP13 OE or Tsf1 RNAi may increase mitochondrial iron levels and lead to elevated mitochondrial complexes activities.In addition,we chose to reduce mitochondrial iron levels by knocking down Mitoferrin(Mfrn),an iron transporter transfer iron to mitochondria,and found Mfrn RNAi reduced the rescue effects of d ZIP13 OE or Tsf1 RNAi.These results demonstrated that d ZIP13 OE or Tsf1 RNAi rescued PINK1 deficiency by increasing iron level,improving mitochondrial respiratory chain function and ATP production.(5)To study the effects of iron on Sporadic PD.We created Sporadic PD by rotenone and used genetic methods to knockdown Tsf1 level.These results showed the reduced climbing capability,impaired jumping activity,shortened life span and dopaminergic neuronal loss were alleviated by Tsf1 RNAi.Further studies showed that rotenone decreased mitochondrial complex ? activity and increased reactive oxygen species(ROS)levels,while Tsf1 RNAi significantly restored mitochondrial function and attenuated oxidative stress injury.Moreover,we measured iron levels by using prussian blue staining and Ferrozine assay and observed the elevated iron-related phenotypes(i.e.,behavioral changes,oxidative stress levels and ATP levels).The results showed Tsf1 modulation on rotenone induced sporadic PD may directly rely on iron.The findings further illuminated that iron transporters modulatin may rescue sporadic PD.In summary,the present study used Drosophila melanogaster as a model organism and changed the iron transporters levels by using genetic method.We have found a new method to alleviate PD(i.e.,familial PD and sporadic PD)and explored the molecular mechanisms.Our work have shown d ZIP13 OE or Tsf1 RNAi may increase mitochondrial iron levels,mitochondrial respiration and complexes activity in muscle.That is to say,the elevated mitochondrial function may rescue PD progress.Besides,Tsf1 RNAi may decrease iron and ROS levels in brain and these effects may contribute to alleviate rotenone induced neurotoxicity.The study has shown that microelement modulation in vivo may ameliorate PD.We wish it act as guidance for diets and theoretical support for functional food development.