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Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation.

Dettmer U, Newman AJ, Soldner F, Luth ES, Kim NC, von Saucken VE, Sanderson JB, Jaenisch R, Bartels T, Selkoe D - Nat Commun (2015)

Bottom Line: Neurons derived from an A53T patient have decreased tetramers.Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions.The other three fPD missense mutations likewise decrease tetramer:monomer ratios.

View Article: PubMed Central - PubMed

Affiliation: Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
β-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.

No MeSH data available.


Related in: MedlinePlus

A53T versus WT αS in hESC- and hiPSC-derived neurons.(a) Crosslinking analysis of non-induced (-Dox) and induced (+Dox) hESC-derived NPCs expressing WT or A53T αS: PBS-soluble fraction. DSP/βME-treated (DSPr) and DSG-treated samples were blotted for DJ-1 and αS (Syn1). (b) Quantification of the cytosolic αS60:14 ratios (N=3 different cultures, each analysed in parallel in duplicates, total n=6); ratios relative to WT αS. (c) Quantification of cytosolic αS60 and αS14 for both genotypes (normalized to WT αS60); NS, not significant. (d) Crosslinking analysis of neurons differentiated from human A53T iPSCs versus their genetically corrected isogenic WT line (WTcorr). PBS and TX-100 fractions of untreated (−), DSP/βME-treated and DSG-treated cells were probed for DJ-1 and αS (2F12). (e) DSG-crosslinked samples: cytosols blotted for αS (2F12, C20, Syn1) and DJ-1; * non-specific band detected only by Syn1 (ref. 9). (f) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by 2F12, C20 and Syn1 (N=4 cultures grown independently and analysed on different days in biological duplicates or triplicates; total n=10). (g) Densitometry of cytosolic αS60 (relative to WT) for both genotypes. (h) Densitometry of cytosolic αS14 (versus WT) for both genotypes. (i) Crosslinking analysis of neurons differentiated from WT hESCs or from a genetically engineered isogenic E46K line (E46Keng). TX-100 total protein lysates of DSG-treated cells were probed with casein kinase 1α (cytosolic monomer), DJ-1 (cytosolic dimer), VDAC (membrane-associated dimer) and αS (mAbs 2F12 and Syn1). The five panels on the left are from one experiment (N#1), the three panels on the right show the Syn1 western blots from three independent experiments (N#2-4, each quality-controlled by DJ-1 and VDAC western blots). *non-specific band detected only by Syn1 (ref. 9). (j) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by Syn1 mAb (N=4 cultures grown independently). (k) Densitometry of cytosolic αS60 (versus WT) for both genotypes. (l) Densitometry of cytosolic αS14 (versus WT) for both genotypes. *P<0.05, **P<0.01; Student's t-test (see Methods) for all quantifications shown; error bars, s.d.
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f5: A53T versus WT αS in hESC- and hiPSC-derived neurons.(a) Crosslinking analysis of non-induced (-Dox) and induced (+Dox) hESC-derived NPCs expressing WT or A53T αS: PBS-soluble fraction. DSP/βME-treated (DSPr) and DSG-treated samples were blotted for DJ-1 and αS (Syn1). (b) Quantification of the cytosolic αS60:14 ratios (N=3 different cultures, each analysed in parallel in duplicates, total n=6); ratios relative to WT αS. (c) Quantification of cytosolic αS60 and αS14 for both genotypes (normalized to WT αS60); NS, not significant. (d) Crosslinking analysis of neurons differentiated from human A53T iPSCs versus their genetically corrected isogenic WT line (WTcorr). PBS and TX-100 fractions of untreated (−), DSP/βME-treated and DSG-treated cells were probed for DJ-1 and αS (2F12). (e) DSG-crosslinked samples: cytosols blotted for αS (2F12, C20, Syn1) and DJ-1; * non-specific band detected only by Syn1 (ref. 9). (f) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by 2F12, C20 and Syn1 (N=4 cultures grown independently and analysed on different days in biological duplicates or triplicates; total n=10). (g) Densitometry of cytosolic αS60 (relative to WT) for both genotypes. (h) Densitometry of cytosolic αS14 (versus WT) for both genotypes. (i) Crosslinking analysis of neurons differentiated from WT hESCs or from a genetically engineered isogenic E46K line (E46Keng). TX-100 total protein lysates of DSG-treated cells were probed with casein kinase 1α (cytosolic monomer), DJ-1 (cytosolic dimer), VDAC (membrane-associated dimer) and αS (mAbs 2F12 and Syn1). The five panels on the left are from one experiment (N#1), the three panels on the right show the Syn1 western blots from three independent experiments (N#2-4, each quality-controlled by DJ-1 and VDAC western blots). *non-specific band detected only by Syn1 (ref. 9). (j) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by Syn1 mAb (N=4 cultures grown independently). (k) Densitometry of cytosolic αS60 (versus WT) for both genotypes. (l) Densitometry of cytosolic αS14 (versus WT) for both genotypes. *P<0.05, **P<0.01; Student's t-test (see Methods) for all quantifications shown; error bars, s.d.

Mentions: Our method of crosslinking fresh, minced brain tissue (Fig. 1; Supplementary Fig. 1.; Methods) presented the opportunity to analyse the effects on αS tetramers of a PD-causing αS mutation expressed in mouse brain. We examined the brains (Fig. 4a) of young αS-/- mice expressing relatively low levels of human WT (hWT) or A53T (hA53T) αS27. Comparing total αS levels among samples using both untreated and DSP+β-mercaptoethanol (βME)-treated samples confirmed that DSP+βME facilitates the quantitative detection of total αS immunoreactivity on blots (Fig. 4b)16. Consistent with the brain expression data reported for these mouse lines27, PBS extracts (‘cytosol') of the hA53T brain showed somewhat lower total αS than the hWT brain (Fig. 4b). The αS60:14 ratio was significantly reduced (P<0.05, Student's t-test, n=9) in hA53T brain cytosols (Fig. 4c), due to a greater decrease in αS60 tetramer than αS14 monomer in hA53T versus hWT (Fig. 4d). To show that this ratio difference was not an artifact of the somewhat lower total αS levels in the hA53T brain, we included longer (L) exposures for hA53T that were adjusted to match the αS14 intensity of the hWT sample (Fig. 4b: ‘expo. L'). Densitometry on both exposures led to closely similar results of >20% reduction, namely, an αS60:14 ratio for hA53T versus hWT of 69.4±20.2% (‘S') or 75.3±20.1% (‘L'), (P<0.05, n=9, Student's t-test), with no significant difference in this result between the two exposures (Fig. 4c). This lower αS60:14 ratio in the hA53T mouse brain cytosol was consistently observed with Syn1 and confirmed with antibodies 15G7 and C20 (Fig. 4e). As expected, the hA53T αS60:14 ratio was also reduced when we analysed total lysates of whole mouse brain (that is, homogenizing the crosslinked tissue directly in TX-100) (Fig. 5f). In the membrane (TX-100) fractions, we found no significant reduction of αS14 monomer levels in hA53T versus hWT; as in the fresh human brain (Fig. 1a), tetramers/multimers were very low in abundance in this fraction and were almost undetectable for hA53T (Fig. 4g,h).


Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation.

Dettmer U, Newman AJ, Soldner F, Luth ES, Kim NC, von Saucken VE, Sanderson JB, Jaenisch R, Bartels T, Selkoe D - Nat Commun (2015)

A53T versus WT αS in hESC- and hiPSC-derived neurons.(a) Crosslinking analysis of non-induced (-Dox) and induced (+Dox) hESC-derived NPCs expressing WT or A53T αS: PBS-soluble fraction. DSP/βME-treated (DSPr) and DSG-treated samples were blotted for DJ-1 and αS (Syn1). (b) Quantification of the cytosolic αS60:14 ratios (N=3 different cultures, each analysed in parallel in duplicates, total n=6); ratios relative to WT αS. (c) Quantification of cytosolic αS60 and αS14 for both genotypes (normalized to WT αS60); NS, not significant. (d) Crosslinking analysis of neurons differentiated from human A53T iPSCs versus their genetically corrected isogenic WT line (WTcorr). PBS and TX-100 fractions of untreated (−), DSP/βME-treated and DSG-treated cells were probed for DJ-1 and αS (2F12). (e) DSG-crosslinked samples: cytosols blotted for αS (2F12, C20, Syn1) and DJ-1; * non-specific band detected only by Syn1 (ref. 9). (f) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by 2F12, C20 and Syn1 (N=4 cultures grown independently and analysed on different days in biological duplicates or triplicates; total n=10). (g) Densitometry of cytosolic αS60 (relative to WT) for both genotypes. (h) Densitometry of cytosolic αS14 (versus WT) for both genotypes. (i) Crosslinking analysis of neurons differentiated from WT hESCs or from a genetically engineered isogenic E46K line (E46Keng). TX-100 total protein lysates of DSG-treated cells were probed with casein kinase 1α (cytosolic monomer), DJ-1 (cytosolic dimer), VDAC (membrane-associated dimer) and αS (mAbs 2F12 and Syn1). The five panels on the left are from one experiment (N#1), the three panels on the right show the Syn1 western blots from three independent experiments (N#2-4, each quality-controlled by DJ-1 and VDAC western blots). *non-specific band detected only by Syn1 (ref. 9). (j) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by Syn1 mAb (N=4 cultures grown independently). (k) Densitometry of cytosolic αS60 (versus WT) for both genotypes. (l) Densitometry of cytosolic αS14 (versus WT) for both genotypes. *P<0.05, **P<0.01; Student's t-test (see Methods) for all quantifications shown; error bars, s.d.
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f5: A53T versus WT αS in hESC- and hiPSC-derived neurons.(a) Crosslinking analysis of non-induced (-Dox) and induced (+Dox) hESC-derived NPCs expressing WT or A53T αS: PBS-soluble fraction. DSP/βME-treated (DSPr) and DSG-treated samples were blotted for DJ-1 and αS (Syn1). (b) Quantification of the cytosolic αS60:14 ratios (N=3 different cultures, each analysed in parallel in duplicates, total n=6); ratios relative to WT αS. (c) Quantification of cytosolic αS60 and αS14 for both genotypes (normalized to WT αS60); NS, not significant. (d) Crosslinking analysis of neurons differentiated from human A53T iPSCs versus their genetically corrected isogenic WT line (WTcorr). PBS and TX-100 fractions of untreated (−), DSP/βME-treated and DSG-treated cells were probed for DJ-1 and αS (2F12). (e) DSG-crosslinked samples: cytosols blotted for αS (2F12, C20, Syn1) and DJ-1; * non-specific band detected only by Syn1 (ref. 9). (f) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by 2F12, C20 and Syn1 (N=4 cultures grown independently and analysed on different days in biological duplicates or triplicates; total n=10). (g) Densitometry of cytosolic αS60 (relative to WT) for both genotypes. (h) Densitometry of cytosolic αS14 (versus WT) for both genotypes. (i) Crosslinking analysis of neurons differentiated from WT hESCs or from a genetically engineered isogenic E46K line (E46Keng). TX-100 total protein lysates of DSG-treated cells were probed with casein kinase 1α (cytosolic monomer), DJ-1 (cytosolic dimer), VDAC (membrane-associated dimer) and αS (mAbs 2F12 and Syn1). The five panels on the left are from one experiment (N#1), the three panels on the right show the Syn1 western blots from three independent experiments (N#2-4, each quality-controlled by DJ-1 and VDAC western blots). *non-specific band detected only by Syn1 (ref. 9). (j) Densitometry of the cytosolic αS60:14 ratios (relative to WT) as detected by Syn1 mAb (N=4 cultures grown independently). (k) Densitometry of cytosolic αS60 (versus WT) for both genotypes. (l) Densitometry of cytosolic αS14 (versus WT) for both genotypes. *P<0.05, **P<0.01; Student's t-test (see Methods) for all quantifications shown; error bars, s.d.
Mentions: Our method of crosslinking fresh, minced brain tissue (Fig. 1; Supplementary Fig. 1.; Methods) presented the opportunity to analyse the effects on αS tetramers of a PD-causing αS mutation expressed in mouse brain. We examined the brains (Fig. 4a) of young αS-/- mice expressing relatively low levels of human WT (hWT) or A53T (hA53T) αS27. Comparing total αS levels among samples using both untreated and DSP+β-mercaptoethanol (βME)-treated samples confirmed that DSP+βME facilitates the quantitative detection of total αS immunoreactivity on blots (Fig. 4b)16. Consistent with the brain expression data reported for these mouse lines27, PBS extracts (‘cytosol') of the hA53T brain showed somewhat lower total αS than the hWT brain (Fig. 4b). The αS60:14 ratio was significantly reduced (P<0.05, Student's t-test, n=9) in hA53T brain cytosols (Fig. 4c), due to a greater decrease in αS60 tetramer than αS14 monomer in hA53T versus hWT (Fig. 4d). To show that this ratio difference was not an artifact of the somewhat lower total αS levels in the hA53T brain, we included longer (L) exposures for hA53T that were adjusted to match the αS14 intensity of the hWT sample (Fig. 4b: ‘expo. L'). Densitometry on both exposures led to closely similar results of >20% reduction, namely, an αS60:14 ratio for hA53T versus hWT of 69.4±20.2% (‘S') or 75.3±20.1% (‘L'), (P<0.05, n=9, Student's t-test), with no significant difference in this result between the two exposures (Fig. 4c). This lower αS60:14 ratio in the hA53T mouse brain cytosol was consistently observed with Syn1 and confirmed with antibodies 15G7 and C20 (Fig. 4e). As expected, the hA53T αS60:14 ratio was also reduced when we analysed total lysates of whole mouse brain (that is, homogenizing the crosslinked tissue directly in TX-100) (Fig. 5f). In the membrane (TX-100) fractions, we found no significant reduction of αS14 monomer levels in hA53T versus hWT; as in the fresh human brain (Fig. 1a), tetramers/multimers were very low in abundance in this fraction and were almost undetectable for hA53T (Fig. 4g,h).

Bottom Line: Neurons derived from an A53T patient have decreased tetramers.Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions.The other three fPD missense mutations likewise decrease tetramer:monomer ratios.

View Article: PubMed Central - PubMed

Affiliation: Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
β-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.

No MeSH data available.


Related in: MedlinePlus