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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

The amount of insoluble tau detected from hTau0N3R-expressingDrosophila is increased dramatically after treatment withGSK-3β inhibitors.Western blots of aqueous-soluble fraction (S1), detergent-soluble fraction(S2) and insoluble fraction (S3) probed for anti-hTau. Samples in lanes1–3 are from heads of hTau0N3R flies (hTau– {elavC155-Gal4/Y ;UAS-hTau0N3R/ + }),hTau0N3R flies treated with 20 mM lithium(hTau-Li), hTau0N3R flies treated with20 μM AR-A01448 (hTau-AR), and flies co-expressinghTau0N3R with dominant negative shaggy(hTau;sggDN{elavC155-Gal4/Y; UAS-hTau0N3R/+;UAS-sggDN/ + }).The fourth lane in the third panel (labelled “3xTmouse”) is a 10-fold dilution of sample from triple-transgenicmouse, used as a positive control for insoluble tau. Bar charts ina’–d’ are quantifications of blots in a– d presented as a percentage of total tau{S3/(S1 + S2 + S3)} in eachgenotype. Treatment with Li or AR-AR01448 did not alter the amount of taudetected in either the S1 (a,a’) or S2 fractions(b,b’). However treatment with Li, AR-AR01448(c,c’) or co-expression of dominant negativeshaggy (hTau;sggDN)(d,d’) significantly increased the amount of taudetected in the insoluble S3 fraction. (error bars are standard error ofmean and n = 5 for each genotype/treatment;*p < 0.05 by Students t-test).
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f2: The amount of insoluble tau detected from hTau0N3R-expressingDrosophila is increased dramatically after treatment withGSK-3β inhibitors.Western blots of aqueous-soluble fraction (S1), detergent-soluble fraction(S2) and insoluble fraction (S3) probed for anti-hTau. Samples in lanes1–3 are from heads of hTau0N3R flies (hTau– {elavC155-Gal4/Y ;UAS-hTau0N3R/ + }),hTau0N3R flies treated with 20 mM lithium(hTau-Li), hTau0N3R flies treated with20 μM AR-A01448 (hTau-AR), and flies co-expressinghTau0N3R with dominant negative shaggy(hTau;sggDN{elavC155-Gal4/Y; UAS-hTau0N3R/+;UAS-sggDN/ + }).The fourth lane in the third panel (labelled “3xTmouse”) is a 10-fold dilution of sample from triple-transgenicmouse, used as a positive control for insoluble tau. Bar charts ina’–d’ are quantifications of blots in a– d presented as a percentage of total tau{S3/(S1 + S2 + S3)} in eachgenotype. Treatment with Li or AR-AR01448 did not alter the amount of taudetected in either the S1 (a,a’) or S2 fractions(b,b’). However treatment with Li, AR-AR01448(c,c’) or co-expression of dominant negativeshaggy (hTau;sggDN)(d,d’) significantly increased the amount of taudetected in the insoluble S3 fraction. (error bars are standard error ofmean and n = 5 for each genotype/treatment;*p < 0.05 by Students t-test).

Mentions: First, we hypothesized that if GTO-like structures were forming then we wouldidentify them using commonly used biochemical assays used to detect insolubletau oligomers. Indeed the presence of tau immuno-positive material within thestacking gel of brain lysates from drug treated hTau0N3R fliessuggested that insoluble tau species were present but were unable to enter theSDS-resolving gel (Supplementary Fig.6). To explore this further, we fractionated brain lysates intoaqueous-soluble (S1), detergent-soluble (S2) and detergent-insoluble (S3)fractions using established protocols for identifying insoluble tauoligomers2439 (Supplementary Fig. 7 shows that this protocol detects insoluble tau).In all hTau0N3R flies, whether drug treated or not, the majorityof hTau was in the S1 aqueous-soluble (Fig. 2a) or S2detergent-soluble fractions (Fig. 2b). The fraction of tau(as a percentage of total tau) found in either S1 or S2 was the same for allhTau0N3R flies, whether drug treated or not (Fig. 2a’). In contrast, the amount of tau found in theinsoluble S3 fraction changed significantly following drug treatment. Whereasonly a very small amount of tau was detected in the insoluble S3 fraction inuntreated flies (Fig. 2c htau lane and Fig.2c’ white bar), this increased dramatically, almost bythree-fold, after LiCl or AR-A01448 treatment (Fig. 2chtau-Li and htau-AR lanes and Fig 2c’ lightgray and dark gray bars). To confirm that the protocol used to generate the S3fraction was indeed enriching for insoluble aggregated tau, it was used tofractionate tau from transgenic mice where such tau is found in abundance. Asexpected, insoluble tau was detected with our protocol from tau transgenic brainhomogenate (Fig. 2c 3xT lane). To verify that the increasein insoluble tau was caused by GSK-3β inhibition and not any otherdrug action, we assessed tau solubility inhTau0N3R;SggDN flies. Co-expression ofSggDN led to a significant increase in insoluble tau, provingthat this effect was mediated by GSK-3β inhibition (Fig. 2d and d’). These results were corroborated using asecond anti-tau antibody, which confirmed that the material picked up in the S3fraction of drug treated hTau0N3R flies was indeed insoluble tau(Supplementary Fig. 6b). Asmentioned above, this effect is specific to GSK-3β becausedavunetide/NAP treatment neither produced GTO-like structures38nor increased insoluble tau levels (Supplementary Fig. 6c). This data shows that pharmacological orgenetic reduction of GSK-3β-mediated tau phosphorylation leads toincreased tau levels and formation of structures containing insoluble tau.


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)

The amount of insoluble tau detected from hTau0N3R-expressingDrosophila is increased dramatically after treatment withGSK-3β inhibitors.Western blots of aqueous-soluble fraction (S1), detergent-soluble fraction(S2) and insoluble fraction (S3) probed for anti-hTau. Samples in lanes1–3 are from heads of hTau0N3R flies (hTau– {elavC155-Gal4/Y ;UAS-hTau0N3R/ + }),hTau0N3R flies treated with 20 mM lithium(hTau-Li), hTau0N3R flies treated with20 μM AR-A01448 (hTau-AR), and flies co-expressinghTau0N3R with dominant negative shaggy(hTau;sggDN{elavC155-Gal4/Y; UAS-hTau0N3R/+;UAS-sggDN/ + }).The fourth lane in the third panel (labelled “3xTmouse”) is a 10-fold dilution of sample from triple-transgenicmouse, used as a positive control for insoluble tau. Bar charts ina’–d’ are quantifications of blots in a– d presented as a percentage of total tau{S3/(S1 + S2 + S3)} in eachgenotype. Treatment with Li or AR-AR01448 did not alter the amount of taudetected in either the S1 (a,a’) or S2 fractions(b,b’). However treatment with Li, AR-AR01448(c,c’) or co-expression of dominant negativeshaggy (hTau;sggDN)(d,d’) significantly increased the amount of taudetected in the insoluble S3 fraction. (error bars are standard error ofmean and n = 5 for each genotype/treatment;*p < 0.05 by Students t-test).
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f2: The amount of insoluble tau detected from hTau0N3R-expressingDrosophila is increased dramatically after treatment withGSK-3β inhibitors.Western blots of aqueous-soluble fraction (S1), detergent-soluble fraction(S2) and insoluble fraction (S3) probed for anti-hTau. Samples in lanes1–3 are from heads of hTau0N3R flies (hTau– {elavC155-Gal4/Y ;UAS-hTau0N3R/ + }),hTau0N3R flies treated with 20 mM lithium(hTau-Li), hTau0N3R flies treated with20 μM AR-A01448 (hTau-AR), and flies co-expressinghTau0N3R with dominant negative shaggy(hTau;sggDN{elavC155-Gal4/Y; UAS-hTau0N3R/+;UAS-sggDN/ + }).The fourth lane in the third panel (labelled “3xTmouse”) is a 10-fold dilution of sample from triple-transgenicmouse, used as a positive control for insoluble tau. Bar charts ina’–d’ are quantifications of blots in a– d presented as a percentage of total tau{S3/(S1 + S2 + S3)} in eachgenotype. Treatment with Li or AR-AR01448 did not alter the amount of taudetected in either the S1 (a,a’) or S2 fractions(b,b’). However treatment with Li, AR-AR01448(c,c’) or co-expression of dominant negativeshaggy (hTau;sggDN)(d,d’) significantly increased the amount of taudetected in the insoluble S3 fraction. (error bars are standard error ofmean and n = 5 for each genotype/treatment;*p < 0.05 by Students t-test).
Mentions: First, we hypothesized that if GTO-like structures were forming then we wouldidentify them using commonly used biochemical assays used to detect insolubletau oligomers. Indeed the presence of tau immuno-positive material within thestacking gel of brain lysates from drug treated hTau0N3R fliessuggested that insoluble tau species were present but were unable to enter theSDS-resolving gel (Supplementary Fig.6). To explore this further, we fractionated brain lysates intoaqueous-soluble (S1), detergent-soluble (S2) and detergent-insoluble (S3)fractions using established protocols for identifying insoluble tauoligomers2439 (Supplementary Fig. 7 shows that this protocol detects insoluble tau).In all hTau0N3R flies, whether drug treated or not, the majorityof hTau was in the S1 aqueous-soluble (Fig. 2a) or S2detergent-soluble fractions (Fig. 2b). The fraction of tau(as a percentage of total tau) found in either S1 or S2 was the same for allhTau0N3R flies, whether drug treated or not (Fig. 2a’). In contrast, the amount of tau found in theinsoluble S3 fraction changed significantly following drug treatment. Whereasonly a very small amount of tau was detected in the insoluble S3 fraction inuntreated flies (Fig. 2c htau lane and Fig.2c’ white bar), this increased dramatically, almost bythree-fold, after LiCl or AR-A01448 treatment (Fig. 2chtau-Li and htau-AR lanes and Fig 2c’ lightgray and dark gray bars). To confirm that the protocol used to generate the S3fraction was indeed enriching for insoluble aggregated tau, it was used tofractionate tau from transgenic mice where such tau is found in abundance. Asexpected, insoluble tau was detected with our protocol from tau transgenic brainhomogenate (Fig. 2c 3xT lane). To verify that the increasein insoluble tau was caused by GSK-3β inhibition and not any otherdrug action, we assessed tau solubility inhTau0N3R;SggDN flies. Co-expression ofSggDN led to a significant increase in insoluble tau, provingthat this effect was mediated by GSK-3β inhibition (Fig. 2d and d’). These results were corroborated using asecond anti-tau antibody, which confirmed that the material picked up in the S3fraction of drug treated hTau0N3R flies was indeed insoluble tau(Supplementary Fig. 6b). Asmentioned above, this effect is specific to GSK-3β becausedavunetide/NAP treatment neither produced GTO-like structures38nor increased insoluble tau levels (Supplementary Fig. 6c). This data shows that pharmacological orgenetic reduction of GSK-3β-mediated tau phosphorylation leads toincreased tau levels and formation of structures containing insoluble tau.

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