Physiological Clearance Of Tau In The Periphery And Its Therapeutic Potential For Tauopathies

BackgroundTauopathies are progressive neurodegenerative disorders that include,but are not limited to,Alzheimer's disease?AD?,progressive supranuclear palsy?PSP?,corticobasal degeneration?CBD?,Pick's disease,certain forms of frontotemporal lobar degeneration?FTLD?,and chronic traumatic encephalopathy.Tau is an abundant,highly soluble intracellular protein that binds to microtubules and promotes their stability.However,in tauopathies,tau aggregates and accumulates in hyperphosphorylated neurofibrillary tangles?NFTs?that occur in neurons and dystrophic neurites.The amount of abnormally hyperphosphorylated tau,or tau pathology,is well correlated with neuronal loss,synaptic dysfunction,and cognitive decline.Unfortunately,effective therapies for tauopathies are lacking.It is suggested that intracellular pathogenic tau can be released into the intercellular space and then enter adjacent cells,where it seeds further tau aggregation and propagates the pathology.Blocking this transmission process by removal of pathogenic tau from the intercellular space represents a promising therapeutic approach for tauopathies.Recent studies suggest that brain-derived pathological tau can flow into the bloodstream.However,the subsequent consequences of tau efflux from the brain remain largely unknown.ObjectiveIn the present study,we aimed to investigate the physiological clearance of tau in the periphery and its potential and capacity of clearing pathological tau in the brain;whether enhancement of peripheral tau clearance could promote the efflux of central tau into blood and further reduce brain tau pathology.Materials and methods1.Physiological clearance of brain-derived tau in the periphery?1?Metabolic kinetics of tau in the brain:To explore the metabolic kinetics of tau in the brain and whether tau can efflux out of the brain into the blood,we injected ¹³¹I-labelled tau(¹³¹I-tau)into the cisterna magna of wild-type?WT?mice,and measured the dynamic radioactivity of ¹³¹I in the brain and blood at different timepoints.The half-life of tau in the brain was calculated.?2?Clearance kinetics of tau in the periphery:To explore the clearance kinetics of tau in the periphery,we injected ¹³¹I-tau into the tail vein of WT mice,and measured the dynamic radioactivity of ¹³¹I in the blood at different timepoints.The half-life of tau in the blood was calculated.?3?Localization of tau clearance in the periphery:To explore the location of tau clearance in the periphery,we injected ¹³¹I-tau into the cisterna magna or into the tail vein of WT mice,and measured the dynamic radioactivity of ¹³¹I in the brain and peripheral tissues and organs at different timepoints.?4?The physiological clearance of tau in the periphery in human:We enrolled a total of 20 patients who suffered from atrioventricular re-entrant tachycardia?AVRT?and underwent radiofrequency catheter ablation?RFCA?.Blood was collected from the inferior vena cava?IVC?proximal to the hepatic vein and right femoral artery?FA?,and plasma was separated.We measured plasma total tau?t-tau?concentration at the FA and IVC and compared tau levels in blood before and after flowing through viscera.2.The capacity of physiological tau clearance in the periphery on tau pathologies in the brain.?1?Consideration and establishment of parabiosis model:To explore the mechanism of peripheral clearance of tau,isochronic parabiosis was established between two age-and sex-matched WT mice.We injected ¹³¹I-tau into the cisterna magna of one parabiotic mouse?ipsilateral parabionts?and observed the distribution of ¹³¹I radioactivity in the brain and the periphery of both ipsilateral and contralateral parabiotic mice at different timepoints.Meanwhile we compared the change rate of ¹³¹I radioactivity in the brain and periphery between ipsilateral parabionts and single WT mice.?2?Parabiosis alleviates tau pathologies in the brain of pR5 mice:To investigate the capacity of physiological tau clearance in the periphery on tau pathologies in the brain,we established long-term isochronic parabiosis between pR5 mice and their sex-matched WT littermates.To examine the effects of parabiosis on tau pathologies in the brain,we measured and compared tau phosphorylation levels in different brain regions,differen t phosphorylation sites and different solubility fractions by immunohistochemical staining,western blot and ELISA assays in parabiotic pR5 and pR5 control mice.?3?Parabiosis alleviates neurodegeneration:To investigate the effects of parabiosis on neuroinflammation,we measured the levels of pro-inflammatory cytokines in brain homogenates and blood in parabiotic pR5 mice,WT and pR5 control mice without parabiosis by ELISA assays.To determine the effects of parabiosis on neurodegeneration,we examined the loss and apoptosis of neurons,neural regeneration,synapse injury by double immunofluorescence staining and western blot.To explore the effects of parabiosis on synaptic plasticity,we measured and compared the long-term potentiation?LTP?of the hippocampal CA1 region among parabiotic pR5 mice,WT and pR5 control mice without parabiosis by electrophysiology.3.Enhancing peripheral tau clearance facilitates tau efflux and clearance from the brain.?1?The potential of peritoneal dialysis on clearing pathological tau in the blood:To investigate whether peritoneal dialysis could clearance pathological tau in the blood,this study enrolled a total of 23 patients with chronic kidney disease?CKD?and underwent peritoneal dialysis for the first time.Blood were sampled before and immediately after4-hours-dialysis,and pT181-Tau levels was measured by ELISA.?2?The potential of peritoneal dialysis on clearing pathological tau in the brain:To investigate whether lowering blood tau levels can lead to a reduction in tau levels in the brain,we conducted a short-term peritoneal dialysis experiment and microdialysis operation with male,3-month-old pR5 mice,collected blood and microdialysis fluid samples at multiple timepoints,and examined the dynamic interaction between the blood tau and brain ISF tau levels by ELISA assay.ISF tau levels were reflected by the tau levels in microdialysis fluid.Results1.Physiological clearance of brain-derived tau in the periphery?1?Metabolic kinetics of tau in the brain:After cisterna magna injection,¹³¹I radioactivity in the brain declined sharply within 1 hour,and then declined gradually with time,suggesting that tau in the brain was cleared.The metabolic half-life of tau in the brain was about 3 hours.Additionally,high radioactivity levels were detected in whole blood 15minutes after injection,and declined with time,indicating that tau effluxed from the brain into the blood within minutes.It also indicates that peripheral system participates in the clearance of tau in the brain.?2?Clearance kinetics of tau in the periphery:After intravenous injection,¹³¹I radioactivity in the blood declined as time prolonged,indicating that tau was cleared in the periphery.Its metabolic half-life was 3.4 hours.However,the radioactivity of brain tissue was nealy undetectable after intravenous injection,suggesting that tau seldom transfers from the periphery into the brain,and tau in the periphery was mainly cleared by peripheral system,which further supports the physiological clearance of tau in the periphery.?3?Localization of tau clearance in the periphery:The results of ¹³¹I-tau injection through cisterna magna and tain vein consistently showed that the radioactivity in the periphery was primarily distributed in the blood and kidney,with a moderate presence in the liver,and declined over time in the blood and kidney,indicating that tau was cleared from these locations.Moreover,after cisterna magna injection,high radioactivity was detected in the periphery,and the radioactivity decreased with time;but after intravenous injection,the radioactivity of brain was nearly undetectable.These findings suggest that the peripheral system participates in the clearance of tau in the brain,whereas tau in the periphery is mainly cleared by peripheral system and rarely entered or cleared by the brain.?4?The physiological clearance of tau in the periphery in human:We found that total tau levels in blood from the inferior vena cava,which collects blood after it flows through the viscera,were lower than those in blood from the femoral artery artery,which collects blood before flowing through viscera,suggesting that tau protein in the blood was cleared when it went through the peripheral viscera under physiological conditions in humans.2.The capacity of physiological tau clearance in the periphery on tau pathologies in the brain.?1?Consideration and establishment of parabiosis model:At 15 minutes after cisterna magna injection,radioactivity was mainly detectable in the blood of contralateral mice.During the following 4 h,radioactivity levels in the blood,kidney,and liver increased remarkably over time.These results indicate that tau effluxed from the brain into the blood and was then transferred to contralateral mice via circulation,finally being cleared in the peripheral tissues and organs of contralateral mice.In ipsilateral parabionts,radioactivity in the brain,blood,and kidney declined faster than in singular mice,suggesting that parabiosis accelerated tau efflux from brain and its clearance in the periphery via adding another set of peripheral tissues and organs.These findings lay a theoretical foundation for the long-term parabiosis experiment.?2?Parabiosis alleviates tau pathologies in the brain of pR5 mice:Immunohistochemical assays showed that tau pathologies in the hippocampus,neocortex and amygdala were reduced in pa?3–6mo?pR5 mice.Western blot assays showed that the levels of tau phosphorylation at the Ser404?pS404?,Thr231?pT231?,Ser199?pS199?,and Thr181?pT181?epitopes were also reduced in pa?3–6mo?pR5 mice.ELISA assays showed that the levels of both pT231-tau and pT181-tau were reduced in the TBS fraction?TBS-soluble?as well as in the SDS fraction?TBS-insoluble?in parabiotic pR5 mice relative to controls.On the basis of ELISA analysis of pT231,we calculated that a singular peripheral system can remove approximately 19%of pathological tau from the brain in a pR5 mouse.?3?Parabiosis alleviates tau-related pathologies:The levels of TNF-??IFN-??IL-1?and IL-6 in brain homogenates of pR5 mice were significantly higher than those in WT mice.Both IFN-?and TNF-?were significantly reduced in the brains of pa?3–6 mo?pR5mice compared with control pR5 mice,suggesting that parabiosis rescues tau-mediated neuroinflammation.Compared with control pR5 mice,pa?3–6 mo?pR5 mice exhibited an increased proportion of staining for Map-2 in the hippocampal CA1 region and a decreased proportion of activated caspase-3 in the hippocampus CA3 region.Western blot assays showed that the immunoreactivities of synapse-related proteins,including SYP,SNAP-25,and VAMP-1,and proteins associated with neural regeneration,including NSE and DCX,were significantly elevated after parabiosis,indicating that parabiosis alleviated tau-mediated neurodegeneration in the brain.However,no significant difference in LTP was observed between parabiotic and control pR5 mice.This may be because the limited reduction of phosphorylated tau?<25%?was insufficient to improve LTP.3.Enhancing peripheral tau clearance facilitates tau efflux and clearance from the brain.?1?The potential of peritoneal dialysis on clearing pathological tau in the blood:ELISA assays showed that plasma pT181-Tau levels were significantly reduced after peritoneal dialysis,suggesting that peritoneal dialysis could effectively remove pathological tau from the blood,and peritoneal dialysis can be used as an approach of enhancing peripheral tau clearance.?2?The potential of peritoneal dialysis on clearing pathological tau in the brain:T-tau levels in the blood decreased immediately after peritoneal dialysis was started and reached the lowest levels 2–3 h later,followed by a slight and slow increase after peritoneal dialysis was stopped.Interestingly,the changes in tau levels in ISF paralleled those in the blood,indicating that the tau levels in ISF and blood were dynamically correlated.These findings suggest that lowering tau levels in the blood could induce a reduction in tau levels in the brain.ConclusionIn the present study,we for the first time reported the existence of physiological clearance of tau in the periphery.We showed that tau in the brain can efflux into the blood and is physiologically cleared in the periphery.The physiological clearance of tau in the periphery makes a significant contribution to pathological tau clearance from the brain.Approximately 19%of pathological tau burden in the brain were cleared in the perip hery.Enhancement of peripheral tau clearance could facilitate tau efflux and clearance from the brain,which provides new approaches for developing therapeutics for tauopathies.In conclusion,our study reveals the physiological contribution of the periphery to clearance of pathological brain tau and provides proof-of-concept evidence that therapies for tauopathies could focus on enhancing peripheral tau clearance.In addition,our findings highlight the importance of understanding the pathogenesis of neurodegenerative diseases and developing therapeutics for these diseases through a systemic approach.