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Rescue from tau-induced neuronal dysfunction produces insoluble tau oligomers.

Cowan CM, Quraishe S, Hands S, Sealey M, Mahajan S, Allan DW, Mudher A - Sci Rep (2015)

Bottom Line: Moreover, contrary to common belief, these tau oligomers were neither highly phosphorylated, and nor did they contain beta-pleated sheet structure.Whether these are inert or actively protective remains to be established.Nevertheless, this has wide implications for emerging therapeutic strategies such as those that target dissolution of tau oligomers as they may be ineffective or even counterproductive unless they act on the relevant toxic oligomeric tau species.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK.

ABSTRACT
Aggregation of highly phosphorylated tau is a hallmark of Alzheimer's disease and other tauopathies. Nevertheless, animal models demonstrate that tau-mediated dysfunction/toxicity may not require large tau aggregates but instead may be caused by soluble hyper-phosphorylated tau or by small tau oligomers. Challenging this widely held view, we use multiple techniques to show that insoluble tau oligomers form in conditions where tau-mediated dysfunction is rescued in vivo. This shows that tau oligomers are not necessarily always toxic. Furthermore, their formation correlates with increased tau levels, caused intriguingly, by either pharmacological or genetic inhibition of tau kinase glycogen-synthase-kinase-3beta (GSK-3β). Moreover, contrary to common belief, these tau oligomers were neither highly phosphorylated, and nor did they contain beta-pleated sheet structure. This may explain their lack of toxicity. Our study makes the novel observation that tau also forms non-toxic insoluble oligomers in vivo in addition to toxic oligomers, which have been reported by others. Whether these are inert or actively protective remains to be established. Nevertheless, this has wide implications for emerging therapeutic strategies such as those that target dissolution of tau oligomers as they may be ineffective or even counterproductive unless they act on the relevant toxic oligomeric tau species.

No MeSH data available.


Related in: MedlinePlus

Tau within GTO-like structures is largely unphosphorylated Western blots ofthe soluble fraction (S1) and the insoluble fraction (S3) from hTau{elavC155-Gal4/Y; UAS-hTau0N3R/ + }, hTau-Li and hTau– AR-A01448 treated fly brain lysates probed with an antibody thatdetects total tau (a and a”) and those that detect variousphospho-tau epitopes (b-g and b”-g”).Though there is a significant amount of tau in the insoluble fraction of drugtreated brain lysates (a”), it is largelyunphosphorylated at many sites(b”–g”). Signal at thesesites in the soluble fractions (b–g andb’–g’) provides apositive control for the antibodies, and shows that treatment withGSK-3β inhibitors LiCl and AR-A01448 reduced phosphorylation atmany of these sites: quantified in graphs(b’–g’). Graphs showthe average of 6 independent experiments; error bars are SEM;* p < 0.05. Blots were probedwith (a) dako polyclonal anti-tau (total tau), (b) anti-tau-1(unphosphorylated at 192–204), (c) anti-PHF-1 (pS396,pS404), (d) anti-AT8 (pS202, pT205), (e) anti-AT180 (pT231,pS235), (f) anti-MC1 (it is curious that this supposedly conformationspecific anti-body picks up its epitope after SDS-PAGE denaturation; it islikely that the epitope recognized here is largely denatured but nonethelessis disease predictive since others, like us have also shown similar WBimmunoreactivity in another Drosophila model of tauopathy10), (g) anti-pS262.
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f4: Tau within GTO-like structures is largely unphosphorylated Western blots ofthe soluble fraction (S1) and the insoluble fraction (S3) from hTau{elavC155-Gal4/Y; UAS-hTau0N3R/ + }, hTau-Li and hTau– AR-A01448 treated fly brain lysates probed with an antibody thatdetects total tau (a and a”) and those that detect variousphospho-tau epitopes (b-g and b”-g”).Though there is a significant amount of tau in the insoluble fraction of drugtreated brain lysates (a”), it is largelyunphosphorylated at many sites(b”–g”). Signal at thesesites in the soluble fractions (b–g andb’–g’) provides apositive control for the antibodies, and shows that treatment withGSK-3β inhibitors LiCl and AR-A01448 reduced phosphorylation atmany of these sites: quantified in graphs(b’–g’). Graphs showthe average of 6 independent experiments; error bars are SEM;* p < 0.05. Blots were probedwith (a) dako polyclonal anti-tau (total tau), (b) anti-tau-1(unphosphorylated at 192–204), (c) anti-PHF-1 (pS396,pS404), (d) anti-AT8 (pS202, pT205), (e) anti-AT180 (pT231,pS235), (f) anti-MC1 (it is curious that this supposedly conformationspecific anti-body picks up its epitope after SDS-PAGE denaturation; it islikely that the epitope recognized here is largely denatured but nonethelessis disease predictive since others, like us have also shown similar WBimmunoreactivity in another Drosophila model of tauopathy10), (g) anti-pS262.

Mentions: It is often presumed that tau aggregates including oligomers are composed ofphosphorylated tau. However we show here that decreased phosphorylation leads toformation of insoluble tau oligomers. Therefore we determined whether theseinsoluble oligomers are in fact phosphorylated by assessing the phosphorylationstatus of hTau in soluble and insoluble fractions from fly heads. We found thattau in the soluble fraction was highly phosphorylated in hTau0N3Rflies, at many of the sites hyper-phosphorylated in AD (htau lanes in Fig. 4b–g). As expected, GSK-3βinhibition significantly reduced tau phosphorylation at several of these sitesexcept at the ser262 site (htau-Li and htau-AR lanes in Fig.4b–g and Fig.4b’–g’ where reduction inphosphorylation in drug-treated hTau0N3R flies is quantified as apercentage of phosphorylation in untreated hTau0N3R flies).However, unlike the soluble tau fraction, the hTau in the insoluble (GTO)fraction was largely un-phosphorylated in all animals in which GTOs wereproduced (Fig.4b”–f”). Though there issignificantly less tau protein in the S3 fractions, the data in Fig. 4a” implies that the lack of phospho-tauimmunoreactivity in this fraction, was not due to undetectable total tau levels.This is because, in line with the biochemical analyses presented in Fig. 2, a non-phosphorylation dependent anti-tau antibodydetected significant amounts of tau in this S3 fraction (Fig.4a”). Additionally, equating the total tau levels of theS1, S2 and S3 fractions (by decreasing the amount of tau protein loaded in S1and S2 by several fold), still gave a positive signal with phospho-tauantibodies in these fractions without any signal in S3 (Supplementary Fig. 9), further implying thatthe lack of signal in S3 was not due to inadequate tau protein in S3. Thus, thetau proteins contained within the GTOs formed following GSK-3βinhibition are unlikely to be phosphorylated. These findings imply that tauoligomers formed in vivo don’t always need to comprise ofphosphorylated tau molecules; they can also be made up from non-phosphorylatedtau molecules.


Rescue from tau-induced neuronal dysfunction produces insoluble tau oligomers.

Cowan CM, Quraishe S, Hands S, Sealey M, Mahajan S, Allan DW, Mudher A - Sci Rep (2015)

Tau within GTO-like structures is largely unphosphorylated Western blots ofthe soluble fraction (S1) and the insoluble fraction (S3) from hTau{elavC155-Gal4/Y; UAS-hTau0N3R/ + }, hTau-Li and hTau– AR-A01448 treated fly brain lysates probed with an antibody thatdetects total tau (a and a”) and those that detect variousphospho-tau epitopes (b-g and b”-g”).Though there is a significant amount of tau in the insoluble fraction of drugtreated brain lysates (a”), it is largelyunphosphorylated at many sites(b”–g”). Signal at thesesites in the soluble fractions (b–g andb’–g’) provides apositive control for the antibodies, and shows that treatment withGSK-3β inhibitors LiCl and AR-A01448 reduced phosphorylation atmany of these sites: quantified in graphs(b’–g’). Graphs showthe average of 6 independent experiments; error bars are SEM;* p < 0.05. Blots were probedwith (a) dako polyclonal anti-tau (total tau), (b) anti-tau-1(unphosphorylated at 192–204), (c) anti-PHF-1 (pS396,pS404), (d) anti-AT8 (pS202, pT205), (e) anti-AT180 (pT231,pS235), (f) anti-MC1 (it is curious that this supposedly conformationspecific anti-body picks up its epitope after SDS-PAGE denaturation; it islikely that the epitope recognized here is largely denatured but nonethelessis disease predictive since others, like us have also shown similar WBimmunoreactivity in another Drosophila model of tauopathy10), (g) anti-pS262.
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Related In: Results  -  Collection

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f4: Tau within GTO-like structures is largely unphosphorylated Western blots ofthe soluble fraction (S1) and the insoluble fraction (S3) from hTau{elavC155-Gal4/Y; UAS-hTau0N3R/ + }, hTau-Li and hTau– AR-A01448 treated fly brain lysates probed with an antibody thatdetects total tau (a and a”) and those that detect variousphospho-tau epitopes (b-g and b”-g”).Though there is a significant amount of tau in the insoluble fraction of drugtreated brain lysates (a”), it is largelyunphosphorylated at many sites(b”–g”). Signal at thesesites in the soluble fractions (b–g andb’–g’) provides apositive control for the antibodies, and shows that treatment withGSK-3β inhibitors LiCl and AR-A01448 reduced phosphorylation atmany of these sites: quantified in graphs(b’–g’). Graphs showthe average of 6 independent experiments; error bars are SEM;* p < 0.05. Blots were probedwith (a) dako polyclonal anti-tau (total tau), (b) anti-tau-1(unphosphorylated at 192–204), (c) anti-PHF-1 (pS396,pS404), (d) anti-AT8 (pS202, pT205), (e) anti-AT180 (pT231,pS235), (f) anti-MC1 (it is curious that this supposedly conformationspecific anti-body picks up its epitope after SDS-PAGE denaturation; it islikely that the epitope recognized here is largely denatured but nonethelessis disease predictive since others, like us have also shown similar WBimmunoreactivity in another Drosophila model of tauopathy10), (g) anti-pS262.
Mentions: It is often presumed that tau aggregates including oligomers are composed ofphosphorylated tau. However we show here that decreased phosphorylation leads toformation of insoluble tau oligomers. Therefore we determined whether theseinsoluble oligomers are in fact phosphorylated by assessing the phosphorylationstatus of hTau in soluble and insoluble fractions from fly heads. We found thattau in the soluble fraction was highly phosphorylated in hTau0N3Rflies, at many of the sites hyper-phosphorylated in AD (htau lanes in Fig. 4b–g). As expected, GSK-3βinhibition significantly reduced tau phosphorylation at several of these sitesexcept at the ser262 site (htau-Li and htau-AR lanes in Fig.4b–g and Fig.4b’–g’ where reduction inphosphorylation in drug-treated hTau0N3R flies is quantified as apercentage of phosphorylation in untreated hTau0N3R flies).However, unlike the soluble tau fraction, the hTau in the insoluble (GTO)fraction was largely un-phosphorylated in all animals in which GTOs wereproduced (Fig.4b”–f”). Though there issignificantly less tau protein in the S3 fractions, the data in Fig. 4a” implies that the lack of phospho-tauimmunoreactivity in this fraction, was not due to undetectable total tau levels.This is because, in line with the biochemical analyses presented in Fig. 2, a non-phosphorylation dependent anti-tau antibodydetected significant amounts of tau in this S3 fraction (Fig.4a”). Additionally, equating the total tau levels of theS1, S2 and S3 fractions (by decreasing the amount of tau protein loaded in S1and S2 by several fold), still gave a positive signal with phospho-tauantibodies in these fractions without any signal in S3 (Supplementary Fig. 9), further implying thatthe lack of signal in S3 was not due to inadequate tau protein in S3. Thus, thetau proteins contained within the GTOs formed following GSK-3βinhibition are unlikely to be phosphorylated. These findings imply that tauoligomers formed in vivo don’t always need to comprise ofphosphorylated tau molecules; they can also be made up from non-phosphorylatedtau molecules.

Bottom Line: Moreover, contrary to common belief, these tau oligomers were neither highly phosphorylated, and nor did they contain beta-pleated sheet structure.Whether these are inert or actively protective remains to be established.Nevertheless, this has wide implications for emerging therapeutic strategies such as those that target dissolution of tau oligomers as they may be ineffective or even counterproductive unless they act on the relevant toxic oligomeric tau species.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK.

ABSTRACT
Aggregation of highly phosphorylated tau is a hallmark of Alzheimer's disease and other tauopathies. Nevertheless, animal models demonstrate that tau-mediated dysfunction/toxicity may not require large tau aggregates but instead may be caused by soluble hyper-phosphorylated tau or by small tau oligomers. Challenging this widely held view, we use multiple techniques to show that insoluble tau oligomers form in conditions where tau-mediated dysfunction is rescued in vivo. This shows that tau oligomers are not necessarily always toxic. Furthermore, their formation correlates with increased tau levels, caused intriguingly, by either pharmacological or genetic inhibition of tau kinase glycogen-synthase-kinase-3beta (GSK-3β). Moreover, contrary to common belief, these tau oligomers were neither highly phosphorylated, and nor did they contain beta-pleated sheet structure. This may explain their lack of toxicity. Our study makes the novel observation that tau also forms non-toxic insoluble oligomers in vivo in addition to toxic oligomers, which have been reported by others. Whether these are inert or actively protective remains to be established. Nevertheless, this has wide implications for emerging therapeutic strategies such as those that target dissolution of tau oligomers as they may be ineffective or even counterproductive unless they act on the relevant toxic oligomeric tau species.

No MeSH data available.


Related in: MedlinePlus