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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

Intact-cell crosslinking of αS expressed at varying levels.(a) DSG-crosslinking analysis of an αS DNA gradient (0-8 μg per 6-cm culture dish). Western blots for αS (Syn1) and endogenous DJ-1. (b) DSG samples: αS60 intensities plotted against αS14 (densitometry). Highest value in each series was set to 1; graph shows mean data for N=5 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (2 exps.), 2, 4, 8 μg DNA (1 exp.) and 4, 8 μg DNA (2 exps.); data points generated in the same experiment are indicated by identical symbols. (c) αS60:αS14 ratios for the same samples as in 2b. (d) DSP-crosslinking analysis of an αS DNA gradient (0; 1-8 μg); western blots (Syn1) for αS in non-reduced and βME-reduced samples (e) Quantification of DSP samples: αS60 versus αS14. Highest value in each series was set to 1; N=3 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (1 exp.), 2, 4, 8 μg (1 exp.) and 4, 8 μg (1 exp.). (f) αS60:14 ratios for the same samples as in (e). (g) ELISA analysis of αS DNA gradients (1, 2, 4, 8 μg) transfected into M17D cells compared to human cortical homogenate (PBS fraction). Below: western blots for 0 and 8 μg DNA transfection versus human brain homogenate (red). N=2 for gradients (different days, different DNA preps) and brain homogenates. (h) ELISA of αS WT and fPD mutant transfectants (8 μg per 6-cm dish), PBS fraction. Graph: concentrations versus αS WT set to 1 (N=10 independent transfections on 4 different days using at least 4 different DNA preps per αS variant). tf'ed, transfected. (i) ELISA of αS WT and fPD mutant transfectants after 1 mM DSG crosslinking and sequential extraction (PBS→PBS/1%Triton→2% LDS→88% formic acid=FA). Graph: concentrations relative to the PBS fractions of the respective αS variant set to 1.
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f2: Intact-cell crosslinking of αS expressed at varying levels.(a) DSG-crosslinking analysis of an αS DNA gradient (0-8 μg per 6-cm culture dish). Western blots for αS (Syn1) and endogenous DJ-1. (b) DSG samples: αS60 intensities plotted against αS14 (densitometry). Highest value in each series was set to 1; graph shows mean data for N=5 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (2 exps.), 2, 4, 8 μg DNA (1 exp.) and 4, 8 μg DNA (2 exps.); data points generated in the same experiment are indicated by identical symbols. (c) αS60:αS14 ratios for the same samples as in 2b. (d) DSP-crosslinking analysis of an αS DNA gradient (0; 1-8 μg); western blots (Syn1) for αS in non-reduced and βME-reduced samples (e) Quantification of DSP samples: αS60 versus αS14. Highest value in each series was set to 1; N=3 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (1 exp.), 2, 4, 8 μg (1 exp.) and 4, 8 μg (1 exp.). (f) αS60:14 ratios for the same samples as in (e). (g) ELISA analysis of αS DNA gradients (1, 2, 4, 8 μg) transfected into M17D cells compared to human cortical homogenate (PBS fraction). Below: western blots for 0 and 8 μg DNA transfection versus human brain homogenate (red). N=2 for gradients (different days, different DNA preps) and brain homogenates. (h) ELISA of αS WT and fPD mutant transfectants (8 μg per 6-cm dish), PBS fraction. Graph: concentrations versus αS WT set to 1 (N=10 independent transfections on 4 different days using at least 4 different DNA preps per αS variant). tf'ed, transfected. (i) ELISA of αS WT and fPD mutant transfectants after 1 mM DSG crosslinking and sequential extraction (PBS→PBS/1%Triton→2% LDS→88% formic acid=FA). Graph: concentrations relative to the PBS fractions of the respective αS variant set to 1.

Mentions: In light of these disadvantages of relying solely on YFP complementation for αS multimer quantification, we sought to rigorously standardize our intact-cell crosslinking protocol as a second independent method (Methods). We transfected M17D cells with increasing amounts of WT αS cDNA and applied a constant DSG amount at 40 hr post transfection. By confirming equal DJ-1 dimer:monomer ratios in the same cytosols as an internal standard for crosslinking efficiency in all experiments (Fig. 2a, left panel), we observed a constant αS60:14 ratio across the range of αS expression levels we tested (Fig. 2a, right panel). Plotting the relative intensities of αS60 and αS14 in multiple independent experiments revealed a highly linear relationship of the two (R2=0.97) (Fig. 2b), and the apparent αS60:14 ratio (using Syn1 for detection) was a constant 1.2 in this series of experiments (Fig. 2c). (For the three multimer species combined (αS60+80+100), their ratio to monomer was 2.8.) We next tested the reducible crosslinker DSP (Fig. 2d) and again found a highly linear correlation (R2=0.97) between αS60 and αS14 levels (Fig. 2e), here with a constant ratio of 0.9 (Fig. 2f). Analysis of αS80 and αS100 revealed a similarly linear ratio to monomer across this range of αS expression (Supplementary Fig. 4), but we focused our further analyses on the αS60 tetramer as our standard readout for four reasons: an independent biophysical method (analytical ultracentrifugation) originally revealed that the principal α-helical multimer purified from fresh human erythrocytes under non-denaturing conditions has a molecular weight of 58 kDa, exactly that of four monomers7; all of our many intact-cell crosslinking experiments to date identified αS60 as the most abundant and consistent cellular multimer7916; mass spectrometry confirmed here that αS60 contains only αS (Supplementary Fig. 5); and for quantification, αS60 is representative of all physiological αS multimers we detect (for example, Supplementary Fig. 4). The constant αS60:14 ratio across a wide range of cellular αS levels (Fig. 2c,f) was important for our subsequent analysis of fPD mutations: even if αS expression levels differ somewhat among mutants for biological or technical reasons, comparisons of the ratios should be meaningful.


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)

Intact-cell crosslinking of αS expressed at varying levels.(a) DSG-crosslinking analysis of an αS DNA gradient (0-8 μg per 6-cm culture dish). Western blots for αS (Syn1) and endogenous DJ-1. (b) DSG samples: αS60 intensities plotted against αS14 (densitometry). Highest value in each series was set to 1; graph shows mean data for N=5 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (2 exps.), 2, 4, 8 μg DNA (1 exp.) and 4, 8 μg DNA (2 exps.); data points generated in the same experiment are indicated by identical symbols. (c) αS60:αS14 ratios for the same samples as in 2b. (d) DSP-crosslinking analysis of an αS DNA gradient (0; 1-8 μg); western blots (Syn1) for αS in non-reduced and βME-reduced samples (e) Quantification of DSP samples: αS60 versus αS14. Highest value in each series was set to 1; N=3 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (1 exp.), 2, 4, 8 μg (1 exp.) and 4, 8 μg (1 exp.). (f) αS60:14 ratios for the same samples as in (e). (g) ELISA analysis of αS DNA gradients (1, 2, 4, 8 μg) transfected into M17D cells compared to human cortical homogenate (PBS fraction). Below: western blots for 0 and 8 μg DNA transfection versus human brain homogenate (red). N=2 for gradients (different days, different DNA preps) and brain homogenates. (h) ELISA of αS WT and fPD mutant transfectants (8 μg per 6-cm dish), PBS fraction. Graph: concentrations versus αS WT set to 1 (N=10 independent transfections on 4 different days using at least 4 different DNA preps per αS variant). tf'ed, transfected. (i) ELISA of αS WT and fPD mutant transfectants after 1 mM DSG crosslinking and sequential extraction (PBS→PBS/1%Triton→2% LDS→88% formic acid=FA). Graph: concentrations relative to the PBS fractions of the respective αS variant set to 1.
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Show All Figures
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f2: Intact-cell crosslinking of αS expressed at varying levels.(a) DSG-crosslinking analysis of an αS DNA gradient (0-8 μg per 6-cm culture dish). Western blots for αS (Syn1) and endogenous DJ-1. (b) DSG samples: αS60 intensities plotted against αS14 (densitometry). Highest value in each series was set to 1; graph shows mean data for N=5 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (2 exps.), 2, 4, 8 μg DNA (1 exp.) and 4, 8 μg DNA (2 exps.); data points generated in the same experiment are indicated by identical symbols. (c) αS60:αS14 ratios for the same samples as in 2b. (d) DSP-crosslinking analysis of an αS DNA gradient (0; 1-8 μg); western blots (Syn1) for αS in non-reduced and βME-reduced samples (e) Quantification of DSP samples: αS60 versus αS14. Highest value in each series was set to 1; N=3 independent experiments of 1, 2, 3, 4, 6, 8 μg DNA (1 exp.), 2, 4, 8 μg (1 exp.) and 4, 8 μg (1 exp.). (f) αS60:14 ratios for the same samples as in (e). (g) ELISA analysis of αS DNA gradients (1, 2, 4, 8 μg) transfected into M17D cells compared to human cortical homogenate (PBS fraction). Below: western blots for 0 and 8 μg DNA transfection versus human brain homogenate (red). N=2 for gradients (different days, different DNA preps) and brain homogenates. (h) ELISA of αS WT and fPD mutant transfectants (8 μg per 6-cm dish), PBS fraction. Graph: concentrations versus αS WT set to 1 (N=10 independent transfections on 4 different days using at least 4 different DNA preps per αS variant). tf'ed, transfected. (i) ELISA of αS WT and fPD mutant transfectants after 1 mM DSG crosslinking and sequential extraction (PBS→PBS/1%Triton→2% LDS→88% formic acid=FA). Graph: concentrations relative to the PBS fractions of the respective αS variant set to 1.
Mentions: In light of these disadvantages of relying solely on YFP complementation for αS multimer quantification, we sought to rigorously standardize our intact-cell crosslinking protocol as a second independent method (Methods). We transfected M17D cells with increasing amounts of WT αS cDNA and applied a constant DSG amount at 40 hr post transfection. By confirming equal DJ-1 dimer:monomer ratios in the same cytosols as an internal standard for crosslinking efficiency in all experiments (Fig. 2a, left panel), we observed a constant αS60:14 ratio across the range of αS expression levels we tested (Fig. 2a, right panel). Plotting the relative intensities of αS60 and αS14 in multiple independent experiments revealed a highly linear relationship of the two (R2=0.97) (Fig. 2b), and the apparent αS60:14 ratio (using Syn1 for detection) was a constant 1.2 in this series of experiments (Fig. 2c). (For the three multimer species combined (αS60+80+100), their ratio to monomer was 2.8.) We next tested the reducible crosslinker DSP (Fig. 2d) and again found a highly linear correlation (R2=0.97) between αS60 and αS14 levels (Fig. 2e), here with a constant ratio of 0.9 (Fig. 2f). Analysis of αS80 and αS100 revealed a similarly linear ratio to monomer across this range of αS expression (Supplementary Fig. 4), but we focused our further analyses on the αS60 tetramer as our standard readout for four reasons: an independent biophysical method (analytical ultracentrifugation) originally revealed that the principal α-helical multimer purified from fresh human erythrocytes under non-denaturing conditions has a molecular weight of 58 kDa, exactly that of four monomers7; all of our many intact-cell crosslinking experiments to date identified αS60 as the most abundant and consistent cellular multimer7916; mass spectrometry confirmed here that αS60 contains only αS (Supplementary Fig. 5); and for quantification, αS60 is representative of all physiological αS multimers we detect (for example, Supplementary Fig. 4). The constant αS60:14 ratio across a wide range of cellular αS levels (Fig. 2c,f) was important for our subsequent analysis of fPD mutations: even if αS expression levels differ somewhat among mutants for biological or technical reasons, comparisons of the ratios should be meaningful.

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