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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 fPD-linked αS missense mutations.(a) DSG crosslinking analysis of M17D cells transiently transfected with αS WT or the indicated mutations. Western blots for endogenous DJ-1 and transfected αS in duplicate (Syn1); each lane is one transfection. (b) Analagous to Fig. 2a, but using the reducible crosslinker DSP: upper panel, non-reduced: bottom panel, βME-reduced (Syn1). (c) DSG and DSP crosslinking, plus meta-analysis of both: intensity of αS60 alone (upper panel) or αS60+80+100 (lower panel) is graphed relative to WT αS (set to 1) (DSG: N=8 experiments each done in biological duplicates (n=2) on different days, total n=16; DSP: N=4, n=9). For better visibility in this and the following graphs, only one-direction error bars are shown. (d) DSG and DSP crosslinking plus meta-analysis: levels of αS60 alone (upper panel) or αS60+80+100 together (lower) relative to WT αS; (e) level of αS14 alone relative to WT αS. (f) Representative western blots of DSG crosslinking of 3x (H50Q-G51D-A53T) and 4x (3x+E46K) compound fPD mutants relative to G51D alone. (g) Quantification of αS60:14 ratio for 3x and 4x compound fPD mutants relative to G51D alone (N=4, n=8). *P<0.05, **P<0.01; one-sided ANOVA (see Methods) for all quantifications shown; error bars, s.d.
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f3: Intact-cell crosslinking of fPD-linked αS missense mutations.(a) DSG crosslinking analysis of M17D cells transiently transfected with αS WT or the indicated mutations. Western blots for endogenous DJ-1 and transfected αS in duplicate (Syn1); each lane is one transfection. (b) Analagous to Fig. 2a, but using the reducible crosslinker DSP: upper panel, non-reduced: bottom panel, βME-reduced (Syn1). (c) DSG and DSP crosslinking, plus meta-analysis of both: intensity of αS60 alone (upper panel) or αS60+80+100 (lower panel) is graphed relative to WT αS (set to 1) (DSG: N=8 experiments each done in biological duplicates (n=2) on different days, total n=16; DSP: N=4, n=9). For better visibility in this and the following graphs, only one-direction error bars are shown. (d) DSG and DSP crosslinking plus meta-analysis: levels of αS60 alone (upper panel) or αS60+80+100 together (lower) relative to WT αS; (e) level of αS14 alone relative to WT αS. (f) Representative western blots of DSG crosslinking of 3x (H50Q-G51D-A53T) and 4x (3x+E46K) compound fPD mutants relative to G51D alone. (g) Quantification of αS60:14 ratio for 3x and 4x compound fPD mutants relative to G51D alone (N=4, n=8). *P<0.05, **P<0.01; one-sided ANOVA (see Methods) for all quantifications shown; error bars, s.d.

Mentions: To determine whether the fPD-causing αS mutations A30P, E46K, G51D and A53T (tested by fluorescence complementation in Fig. 1h) and the recently discovered H50Q26 alter the tetramer:monomer ratio in intact cells, we initially transfected human M17D neural cells with WT αS or one of these five fPD mutants and performed DSG crosslinking and quantitative western blots of the cytosols (Fig. 3a, top panel). Trapping of the endogenous DJ-1 dimer confirmed equal crosslinking across samples (Fig. 3a, middle panel; Supplementary Fig. 7 for uncropped blots). We identified Syn1 as the optimal antibody to detect αS because it is widely used and commercially available (unlike 15G7 or our 2F12), it does not show a preference for monomeric or multimeric αS (C20 reacts more with monomers, and 211 and LB509 do not detect multimers9), and it gave the clearest blots of the αS transfectants. Thus, statistical analyses of the experiments that follow used Syn1, but importantly, other αS antibodies confirmed all key findings in this section. In many iterative experiments, we detected similar or slightly reduced αS expression levels for the mutants versus WT, except for A30P and G51D, which showed variable but modestly higher αS levels (Fig. 3a,b; compare with Fig. 1h). These variations were well within the αS expression range found not to alter the αS60:14 ratio (see Fig. 2). Densitometry of multiple independent experiments after intact-cell crosslinking with DSG (exemplified by Fig. 3a) or DSP (exemplified by Fig. 3b) and a meta-analysis of the two agents revealed highly significant decreases (P<0.01, n=25, one-sided analysis of variance (ANOVA), percentages±s.d.; statistical details in Fig. 3 legend and Methods) in the αS60:14 ratio for A30P (77.5±15.5% of WT, that is, a 22.5% decrease), E46K (61.7±10.1% of WT, a 38.3% decrease), G51D (47.8±10.0% of WT, a 52.2% decrease) and A53T (66.5±10.4% of WT, a 33.5% decrease). H50Q caused a smaller (83.7±14.4% of WT, 16.3% decrease) but still significant (P<0.05) reduction (Fig. 3c, upper panel). The same significant effects were observed when the ratio of all multimeric species (αS60+80+100 combined) relative to αS14 was analysed (Fig. 3c, lower panel). We observed decreases versus WT in αS60 levels (Fig. 3d, upper panel) or αS60+80+100 levels (Fig. 3d, lower panel) for E46K, G51D and A53T, while the αS60 levels for A30P and H50Q were not significantly changed from WT. αS14 monomer levels increased significantly versus WT for A30P and G51D (Fig. 3e). Strikingly, engineered compound fPD mutants ‘3x' (H50Q+G51D+A53T) and ‘4x' (H50Q+G51D+A53T+E46K) further decreased the αS60:14 ratio compared with G51D alone, causing clear-cut elevations of free monomers (Fig. 3f,g). We then tested the overall finding—reduction of relative cellular multimer levels by fPD mutants—in a system that is closer to the steady-state situation in neurons, namely stable lentivirus transduction of the neural M17D cells with WT, E46K or G51D αS. The fPD mutants again reduced the cytosolic multimer:monomer ratio (due to both a decrease in multimers and increase in monomers versus WT), and sequential extractions again showed that non-cytosolic fractions contained only minor αS amounts (Supplementary Fig. 7).


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 fPD-linked αS missense mutations.(a) DSG crosslinking analysis of M17D cells transiently transfected with αS WT or the indicated mutations. Western blots for endogenous DJ-1 and transfected αS in duplicate (Syn1); each lane is one transfection. (b) Analagous to Fig. 2a, but using the reducible crosslinker DSP: upper panel, non-reduced: bottom panel, βME-reduced (Syn1). (c) DSG and DSP crosslinking, plus meta-analysis of both: intensity of αS60 alone (upper panel) or αS60+80+100 (lower panel) is graphed relative to WT αS (set to 1) (DSG: N=8 experiments each done in biological duplicates (n=2) on different days, total n=16; DSP: N=4, n=9). For better visibility in this and the following graphs, only one-direction error bars are shown. (d) DSG and DSP crosslinking plus meta-analysis: levels of αS60 alone (upper panel) or αS60+80+100 together (lower) relative to WT αS; (e) level of αS14 alone relative to WT αS. (f) Representative western blots of DSG crosslinking of 3x (H50Q-G51D-A53T) and 4x (3x+E46K) compound fPD mutants relative to G51D alone. (g) Quantification of αS60:14 ratio for 3x and 4x compound fPD mutants relative to G51D alone (N=4, n=8). *P<0.05, **P<0.01; one-sided ANOVA (see Methods) for all quantifications shown; error bars, s.d.
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f3: Intact-cell crosslinking of fPD-linked αS missense mutations.(a) DSG crosslinking analysis of M17D cells transiently transfected with αS WT or the indicated mutations. Western blots for endogenous DJ-1 and transfected αS in duplicate (Syn1); each lane is one transfection. (b) Analagous to Fig. 2a, but using the reducible crosslinker DSP: upper panel, non-reduced: bottom panel, βME-reduced (Syn1). (c) DSG and DSP crosslinking, plus meta-analysis of both: intensity of αS60 alone (upper panel) or αS60+80+100 (lower panel) is graphed relative to WT αS (set to 1) (DSG: N=8 experiments each done in biological duplicates (n=2) on different days, total n=16; DSP: N=4, n=9). For better visibility in this and the following graphs, only one-direction error bars are shown. (d) DSG and DSP crosslinking plus meta-analysis: levels of αS60 alone (upper panel) or αS60+80+100 together (lower) relative to WT αS; (e) level of αS14 alone relative to WT αS. (f) Representative western blots of DSG crosslinking of 3x (H50Q-G51D-A53T) and 4x (3x+E46K) compound fPD mutants relative to G51D alone. (g) Quantification of αS60:14 ratio for 3x and 4x compound fPD mutants relative to G51D alone (N=4, n=8). *P<0.05, **P<0.01; one-sided ANOVA (see Methods) for all quantifications shown; error bars, s.d.
Mentions: To determine whether the fPD-causing αS mutations A30P, E46K, G51D and A53T (tested by fluorescence complementation in Fig. 1h) and the recently discovered H50Q26 alter the tetramer:monomer ratio in intact cells, we initially transfected human M17D neural cells with WT αS or one of these five fPD mutants and performed DSG crosslinking and quantitative western blots of the cytosols (Fig. 3a, top panel). Trapping of the endogenous DJ-1 dimer confirmed equal crosslinking across samples (Fig. 3a, middle panel; Supplementary Fig. 7 for uncropped blots). We identified Syn1 as the optimal antibody to detect αS because it is widely used and commercially available (unlike 15G7 or our 2F12), it does not show a preference for monomeric or multimeric αS (C20 reacts more with monomers, and 211 and LB509 do not detect multimers9), and it gave the clearest blots of the αS transfectants. Thus, statistical analyses of the experiments that follow used Syn1, but importantly, other αS antibodies confirmed all key findings in this section. In many iterative experiments, we detected similar or slightly reduced αS expression levels for the mutants versus WT, except for A30P and G51D, which showed variable but modestly higher αS levels (Fig. 3a,b; compare with Fig. 1h). These variations were well within the αS expression range found not to alter the αS60:14 ratio (see Fig. 2). Densitometry of multiple independent experiments after intact-cell crosslinking with DSG (exemplified by Fig. 3a) or DSP (exemplified by Fig. 3b) and a meta-analysis of the two agents revealed highly significant decreases (P<0.01, n=25, one-sided analysis of variance (ANOVA), percentages±s.d.; statistical details in Fig. 3 legend and Methods) in the αS60:14 ratio for A30P (77.5±15.5% of WT, that is, a 22.5% decrease), E46K (61.7±10.1% of WT, a 38.3% decrease), G51D (47.8±10.0% of WT, a 52.2% decrease) and A53T (66.5±10.4% of WT, a 33.5% decrease). H50Q caused a smaller (83.7±14.4% of WT, 16.3% decrease) but still significant (P<0.05) reduction (Fig. 3c, upper panel). The same significant effects were observed when the ratio of all multimeric species (αS60+80+100 combined) relative to αS14 was analysed (Fig. 3c, lower panel). We observed decreases versus WT in αS60 levels (Fig. 3d, upper panel) or αS60+80+100 levels (Fig. 3d, lower panel) for E46K, G51D and A53T, while the αS60 levels for A30P and H50Q were not significantly changed from WT. αS14 monomer levels increased significantly versus WT for A30P and G51D (Fig. 3e). Strikingly, engineered compound fPD mutants ‘3x' (H50Q+G51D+A53T) and ‘4x' (H50Q+G51D+A53T+E46K) further decreased the αS60:14 ratio compared with G51D alone, causing clear-cut elevations of free monomers (Fig. 3f,g). We then tested the overall finding—reduction of relative cellular multimer levels by fPD mutants—in a system that is closer to the steady-state situation in neurons, namely stable lentivirus transduction of the neural M17D cells with WT, E46K or G51D αS. The fPD mutants again reduced the cytosolic multimer:monomer ratio (due to both a decrease in multimers and increase in monomers versus WT), and sequential extractions again showed that non-cytosolic fractions contained only minor αS amounts (Supplementary Fig. 7).

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