iron, hypoxia and rls · uses iron as a means to bind oxygen and transport it to the tissues. •...

JamesR.Connor,Ph.D.andStephanieM.Patton,Ph.D.

Iron,HypoxiaandRLS

©2016RLSFoundation

• IntroductionofanewparadigmforBBBtransportandRegulation

BrainIronAcquisitioninRLS:

©2016RLSFoundation

Iron

Transferrin

Mitochondria

Tf Receptor

KeyBlood

Brain

Transferrin(Tf)transcytosistheworkingmodelforBBBirontransport:theconduitmodel

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?Ferritin

Endosome

Fe2+

TfR

DMT1pH

Fe3+

Fe2+

holoTf

Blood Brain

Astrocyte

Endothelial Cell

IronisreleasedinEC

apoTf

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©2016RLSFoundation

Transwell collagen membrane

BREC

Apical Chamber for additionOf substances for transport

CSFfromnormalorIDmonkeys

CanIronReleasefromtheBBBcellsberegulated?

©2016RLSFoundation

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59Fe(GammaCounts)

Baseline 1 2 3 4Time (hr)

IDA

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BBBBioassayforCSF

©2016RLSFoundation

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rin C

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CSFironstatussuggestsbrainisirondeficient:ironlevelsintheCSFnotdecreased:problemorcompensatoryresponse??

©2016RLSFoundation

Transwell insert

Microporous membrane

ApicalChamber

BasalChamberforplacementofCSF

BrainMicrovascularEndothelialCell

TransportofTransferrinandIronacrossamodeloftheBBBinRLS

©2016RLSFoundation

• CSFfromRLSpatientsdidnotaltertransportorreleaseinaninvitroBBBmodel– IstheprobleminRLSafailuretosignalregardingbrainironstatus?– Isthisrelatedtothesetpointconcept?

• Hemoglobin,notserumferritin,mayfunctionassystemicpredictorforbrainironuptake– Ishypoxicstatusmoreimportantthanironstatusforbrainironuptake?

Summary

©2016RLSFoundation

• Redbloodcells(erythrocytes)arethecelltypeinthebloodthatcarryoxygenfromthelungsthroughoutthebody.

• Approximately35%ofthetotalcontentoftheredbloodcellsismadeupofaproteincalledhemoglobin.

• Hemoglobinisametalloprotein,thatusesironasameanstobindoxygenandtransportittothetissues.

• Whenirondeficiencyoccurs,thesynthesisofheme,amajorbuildingblockofhemoglobin,isdecreasedandlessoxygencanbeboundandtransported.

Whatistherelationshipbetweenironandoxygen?

©2016RLSFoundation

• TofurtherexploretheimpactofcellularirondeficiencyinRLSneuromelanin cells,weproposedthehypothesisthatthelossofcellularironhomeostasisincellscouldelicitasurvivalresponseandactivationofsurvivalpathways.

• Onesuchsurvivalpathwayisactivationofhypoxiainduciblefactor1(HIF-1),whichregulatesexpressionofanumberofproteinsincludingthoseinvolvedinironhomeostasissuchasTfR,vascularendothelialgrowthfactor,anderythropoietin.

• Ironisaco-factorforHIFdegradation,thereforeirondeficiencywillleadtostabilizationofHIF-1αandactivationoftheHIFpathway.(seeHypoxiaResponsePathwaySchematic)

SoWhyExamineHypoxiaPathway?

©2016RLSFoundation(http://dx.doi.org/10.14348/molcells.2014.0150)

HypoxiaResponsePathway

©2016RLSFoundation

•DecreasedoxygenstatushasbeencorrelatedwithdecreasedIRP1levelsaswellasincreasedIRP2levels.(Hansonetal.,1999)

• EpidemiologicstudieshavedemonstratedahigherprevalenceofRLSinpatientsresidinginhigheraltitudes(Sevim etal.,2003andCastilloetal.,2006)

• SkeletalmusclemorphologyofpatientswithRLSdemonstratessignificantlylowerpredictedmaximaloxygenuptake(Larssonetal.,2007)

•IncreasedRLSprevalenceinpatientswithpulmonarydisease,includingchronicobstructivepulmonarydisease(LoCocoetal.,2009)andobstructivesleepapnea(Kapsimalis etal.,2009).

• Mostrecently,adirectclinicalmeasureofhypoxia,decreasedO2partialpressure,hasbeenreportedinthelegsofRLSpatients(Salminen etal.,2014)

PlausibilityoftheHypoxiaParadigm

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INDICATORSOFACTIVATIONOFTHEHYPOXICPATHWAYINTHESUBSTANTIANIGRA OFRLS

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ControlRLS

HIF-1αImmunostaininginSubstantiaNigra

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MICROVESSEL PERTURBATIONSINRLS

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RLS microvessel lysates contain significantly higher HIF-2α expression levels compared to control subjects (p <0.05).

Lines reflect the mean valuesData points are in triplicate

HIF-2αImmunoblotAnalysisofMicrovessel Lysates

©2016RLSFoundation

RLS microvessel lysates contain significantly higher vascular endothelial growth factor (VEGF) expression levels compared to control (p <0.00005).

Lines reflect the mean valuesData points are in triplicate

VEGFImmunoblotAnalysisofMicrovesselLysates

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PROTEOMICANALYSISOFTHECEREBROSPINALFLUIDOFPATIENTSWITHRESTLESSLEGSSYNDROME

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We sought to identify proteins that could prove valuable in understanding pathways affected in RLS/WED and identifying targets for treatment strategies.

ReasoningforCSFbiomarkeranalysispilotstudy

©2016RLSFoundation

ProteinsLevelsAlteredinRLSCSF

©2016RLSFoundation

• ThisstudyprovidedaCSFproteinprofileofRLSinwhichnumerousidentifiedproteins(VitaminDbindingprotein,ProstaglandinD2synthase,cystatinCandβ-hemoglobin)canbetiedtoactivationofthehypoxiaresponsepathway,thusfurthersupportingourconceptofhypoxiaactivationinRLS

• L-PGDSisupregulatedinthebrainsofsubjectswithhypoxic/ischemicencephalopathy(HIE).Also,inamousemodelofHIEthatL-PGDSandHIF-1αexpressionareco-localizedimplyingL-PGDSmayprotectneuronsfromhypoxicinsultbylocalsecretionofPGD2(Jordanetal.,2002).

• Astudyinwhichplasmaproteomesofhumanslivingatsealevelwerecomparedtothoselivingathighaltitude,VitaminDbindingproteinandβ-hemoglobinwerefoundtobeupregulatedinserumasaresultofhypoxia(Ahmadetal.,2013).

• CystatinChasbeendemonstratedtobeproducedinincreasedamountsbythechoroidplexusfollowinghypoxic/ischemicinsult(Palmetal.,1995).

Relationshipofbiomarkerproteinstohypoxia

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ALTERATIONSINHEARTRATE,MINUTEVENTILATIONANDFEMORALARTERYBLOODFLOWINRLS:APILOTSTUDY

©2016RLSFoundation

Alterations in Femoral Artery Blood Flow. This figure demonstrates the femoral artery blood flow in WED (blue) and control (orange) subjects. Error bars are standard error of the mean. n=18 (9 WED; 9 age- and gender-matched controls).

•Baseline femoral artery blood flow is increased 22.5% in RLS/WED subjects.

•RLS/WED subjects demonstrate only a 1.7% increase in blood flow following exposure to hypoxia.

•Control subjects demonstrate a 7.2% increase in blood flow following hypoxia.

FemoralArteryBloodFlowwithHypoxia

©2016RLSFoundation

Alterations in Heart Rate. This figure demonstrates the heart rate measurements in RLS/WED (blue) and control (orange) subjects. As shown in this figure, baseline heart rate is increased slightly, but not significantly in RLS/WED subjects as compared to their age-matched controls. Error bars are standard error of the mean. n=14 (7 WED; 7 age-matched controls).

HeartRateAlterationwithHypoxia

©2016RLSFoundation

Alterations in Minute Ventilation. This figure demonstrates the minute ventilation measurements in RLS/WED (blue) and control (orange) subjects. Error bars are standard error of the mean. n=14 (7 WED; 7 age-matched controls).

•Male RLS/WED subjects had an slightly elevated minute ventilation rate at baseline.

•Male RLS/WED subjects had a 65% increase in minute ventilation with hypoxic insult.

MinuteVentilationAlterationswithHypoxia

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•Activation of hypoxia pathway •Neuromelanin cells of the substantia nigra in RLS demonstrate:

• increased HIF

•Brain microvessels• increased HIF-2α • increased VEGF

DiscussionPoints– Hypoxiainthebrain

©2016RLSFoundation

•Elevated proteins in the CSF of patients with RLS also occur with hypoxia:

•Vitamin D binding protein, •Prostaglandin D2 synthase, •cystatin C •β-hemoglobin

•Decreased myelination and increased vascularization have been reported with constitutive HIF expression:

•Decreased CNPase, MBP, and PLP•Reduced volume by MRI •Elevated VEGF expression in brain microvessels

DiscussionPoints-hypoxiainthebrain

©2016RLSFoundation

• Increased erythropoetin in lymphocytes suggests activation of the hypoxia pathway.

• Physiologicalalterationswithnormoxia andhypoxiademonstratechangesconsistentwithHIFactivationintheperiphery:

• Increasedfemoralarterybloodflow• Increasedheartrate• Increasedminuteventilation

DiscussionPoints-hypoxiaintheperiphery

©2016RLSFoundation

Acknowledgements

Questions & Answers

JointheRLSFoundationatwww.rls.org

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