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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

Transgenic hA53T versus hWT αS expressed in αS-/- mouse brain.(a) Whole brains from both genotypes immediately before mincing. (b) Minced brain bits from both mouse genotypes were subjected to crosslinking, and PBS-soluble (‘cytosolic') fractions blotted (Syn1). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples (in technical triplicate) are shown at identical short (S) exposures of hWT and hA53T samples (left panel) or a longer (L) exposure of the hA53T samples (right panel: exposure matched to αS14 intensity of hWT αS in the left panel). (c) Densitometry of the cytosolic αS60:14 ratios (relative to hWT, set to 1) based on both S and L exposures (N=3 mice of each genotype analysed on different days in triplicates of separate brain-bit samples, total n=9); NS, not significant. (d) Densitometry of cytosolic αS60 and αS14 bands in both genotypes based on identical exposures (N=3, n=9); values relative to hWT αS60. (e) DSG crosslinked mouse brain samples: cytosols blotted for αS (Syn1, 15G7, C20) and DJ-1; Ponceau-staining of the blot membrane is on left. DJ-1 served as control for equal crosslinking efficiency and equal loading. (f) Minced brain bits from both genotypes: TX-100 total homogenates (cytosolic and membrane proteins). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples in triplicates (Syn1 mAb, right panel). Left panel, Ponceau-staining of the membrane. (g) DSG crosslinking of PBS-insoluble TX-100-soluble (TX) fractions of brain from both genotypes in triplicates. (h) Densitometry of αS60 and αS14 in the TX fractions (N=2 mice of each genotype analysed on different days in triplicates of separate brain bits, total n=6); values relative to those of hWT αS14. *P<0.05, **P<0.01; Student's t-test (see Methods) for all quantifications shown; error bars, s.d.
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f4: Transgenic hA53T versus hWT αS expressed in αS-/- mouse brain.(a) Whole brains from both genotypes immediately before mincing. (b) Minced brain bits from both mouse genotypes were subjected to crosslinking, and PBS-soluble (‘cytosolic') fractions blotted (Syn1). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples (in technical triplicate) are shown at identical short (S) exposures of hWT and hA53T samples (left panel) or a longer (L) exposure of the hA53T samples (right panel: exposure matched to αS14 intensity of hWT αS in the left panel). (c) Densitometry of the cytosolic αS60:14 ratios (relative to hWT, set to 1) based on both S and L exposures (N=3 mice of each genotype analysed on different days in triplicates of separate brain-bit samples, total n=9); NS, not significant. (d) Densitometry of cytosolic αS60 and αS14 bands in both genotypes based on identical exposures (N=3, n=9); values relative to hWT αS60. (e) DSG crosslinked mouse brain samples: cytosols blotted for αS (Syn1, 15G7, C20) and DJ-1; Ponceau-staining of the blot membrane is on left. DJ-1 served as control for equal crosslinking efficiency and equal loading. (f) Minced brain bits from both genotypes: TX-100 total homogenates (cytosolic and membrane proteins). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples in triplicates (Syn1 mAb, right panel). Left panel, Ponceau-staining of the membrane. (g) DSG crosslinking of PBS-insoluble TX-100-soluble (TX) fractions of brain from both genotypes in triplicates. (h) Densitometry of αS60 and αS14 in the TX fractions (N=2 mice of each genotype analysed on different days in triplicates of separate brain bits, total n=6); values relative to those of hWT αS14. *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)

Transgenic hA53T versus hWT αS expressed in αS-/- mouse brain.(a) Whole brains from both genotypes immediately before mincing. (b) Minced brain bits from both mouse genotypes were subjected to crosslinking, and PBS-soluble (‘cytosolic') fractions blotted (Syn1). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples (in technical triplicate) are shown at identical short (S) exposures of hWT and hA53T samples (left panel) or a longer (L) exposure of the hA53T samples (right panel: exposure matched to αS14 intensity of hWT αS in the left panel). (c) Densitometry of the cytosolic αS60:14 ratios (relative to hWT, set to 1) based on both S and L exposures (N=3 mice of each genotype analysed on different days in triplicates of separate brain-bit samples, total n=9); NS, not significant. (d) Densitometry of cytosolic αS60 and αS14 bands in both genotypes based on identical exposures (N=3, n=9); values relative to hWT αS60. (e) DSG crosslinked mouse brain samples: cytosols blotted for αS (Syn1, 15G7, C20) and DJ-1; Ponceau-staining of the blot membrane is on left. DJ-1 served as control for equal crosslinking efficiency and equal loading. (f) Minced brain bits from both genotypes: TX-100 total homogenates (cytosolic and membrane proteins). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples in triplicates (Syn1 mAb, right panel). Left panel, Ponceau-staining of the membrane. (g) DSG crosslinking of PBS-insoluble TX-100-soluble (TX) fractions of brain from both genotypes in triplicates. (h) Densitometry of αS60 and αS14 in the TX fractions (N=2 mice of each genotype analysed on different days in triplicates of separate brain bits, total n=6); values relative to those of hWT αS14. *P<0.05, **P<0.01; Student's t-test (see Methods) for all quantifications shown; error bars, s.d.
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f4: Transgenic hA53T versus hWT αS expressed in αS-/- mouse brain.(a) Whole brains from both genotypes immediately before mincing. (b) Minced brain bits from both mouse genotypes were subjected to crosslinking, and PBS-soluble (‘cytosolic') fractions blotted (Syn1). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples (in technical triplicate) are shown at identical short (S) exposures of hWT and hA53T samples (left panel) or a longer (L) exposure of the hA53T samples (right panel: exposure matched to αS14 intensity of hWT αS in the left panel). (c) Densitometry of the cytosolic αS60:14 ratios (relative to hWT, set to 1) based on both S and L exposures (N=3 mice of each genotype analysed on different days in triplicates of separate brain-bit samples, total n=9); NS, not significant. (d) Densitometry of cytosolic αS60 and αS14 bands in both genotypes based on identical exposures (N=3, n=9); values relative to hWT αS60. (e) DSG crosslinked mouse brain samples: cytosols blotted for αS (Syn1, 15G7, C20) and DJ-1; Ponceau-staining of the blot membrane is on left. DJ-1 served as control for equal crosslinking efficiency and equal loading. (f) Minced brain bits from both genotypes: TX-100 total homogenates (cytosolic and membrane proteins). Untreated (-), DSP/βME-treated (DSPr) and DSG-treated samples in triplicates (Syn1 mAb, right panel). Left panel, Ponceau-staining of the membrane. (g) DSG crosslinking of PBS-insoluble TX-100-soluble (TX) fractions of brain from both genotypes in triplicates. (h) Densitometry of αS60 and αS14 in the TX fractions (N=2 mice of each genotype analysed on different days in triplicates of separate brain bits, total n=6); values relative to those of hWT αS14. *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