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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

Multimers/toxicity/inclusions with 1-3 E46K-like mutations.(a) Schematic of human αS sequence showing engineered mutations (color-coded). Highly conserved KTKEGV motifs underlined, less conserved motifs dotted. (b) DSG-treated M17D cells expressing the indicated αS variants lysed in PBS/1% TX-100; graph (αS60:14 ratios by western blot densitometry) and western blots (Syn1, anti-DJ-1) represent N=3 experiments on different days. For this and the following graphs, WT set to 1 unless stated otherwise. (c) Analogous to (b) but 1 or 3 E→R substitutions. (d) Analogous to (b) using lentiviral pools; long and short western blot exposures, blots cut once as indicated. (e) Venus-YFP complementation assay by automated fluorescence reading. M17D/VN-αS cells transfected with αS-VC WT or mutant or Ran-VC (negative control). YFP fluorescence relative to WT; N=6 independent experiments on 3 different days using different DNA preparations. Representative western blots for αS (pAb C20) plus loading control β-actin. (f) Venus-YFP complementation assay by fluorescence microscopy; αS-VC (always WT) and 3 indicated VN-αS mutants were co-expressed (or not: -) in M17D cells; representative bright-field or fluorescent images and corresponding western blots (Syn1; β-actin as a loading control; blots cut as indicated). N=8 independent transfections on 3 different days. (g) Cytotoxicity assays: trypan blue exclusion for live cell count (N=18) relative to WT αS, Toxilight assay for adenylate kinase (a.k.) release relative to Bax (N=12), and western blot for cleaved PARP (representative of 6 independent experiments), each transfected as indicated or mock (-); plus western blots for αS (2F12) and β-actin (total lysates PBS/1% TX-100). (h) Western blots for αS (Syn1) and VDAC in TX-100-soluble fractions of M17D cells transfected as indicated. (i) Fluorescence microscopy of live M17D cells co-expressing RFP plus indicated αS-YFP variants; scale bar, 10 μm. Percentages of cells with inclusions were counted in a blinded fashion (right: N=3; 100 cells each). (j) Fluorescence microscopy of rat neurons (DIV14) transfected with indicated untagged αS variants; immunofluorescence with human-specific mAb 15G7; scale bar, 20 μm. Percentages of cells showing inclusions, blinded counting (right: N=3; 100 cells each). *P<0.05, **P<0.01; one-sided ANOVA (see Methods) for all quantifications shown; error bars, s.d.
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f6: Multimers/toxicity/inclusions with 1-3 E46K-like mutations.(a) Schematic of human αS sequence showing engineered mutations (color-coded). Highly conserved KTKEGV motifs underlined, less conserved motifs dotted. (b) DSG-treated M17D cells expressing the indicated αS variants lysed in PBS/1% TX-100; graph (αS60:14 ratios by western blot densitometry) and western blots (Syn1, anti-DJ-1) represent N=3 experiments on different days. For this and the following graphs, WT set to 1 unless stated otherwise. (c) Analogous to (b) but 1 or 3 E→R substitutions. (d) Analogous to (b) using lentiviral pools; long and short western blot exposures, blots cut once as indicated. (e) Venus-YFP complementation assay by automated fluorescence reading. M17D/VN-αS cells transfected with αS-VC WT or mutant or Ran-VC (negative control). YFP fluorescence relative to WT; N=6 independent experiments on 3 different days using different DNA preparations. Representative western blots for αS (pAb C20) plus loading control β-actin. (f) Venus-YFP complementation assay by fluorescence microscopy; αS-VC (always WT) and 3 indicated VN-αS mutants were co-expressed (or not: -) in M17D cells; representative bright-field or fluorescent images and corresponding western blots (Syn1; β-actin as a loading control; blots cut as indicated). N=8 independent transfections on 3 different days. (g) Cytotoxicity assays: trypan blue exclusion for live cell count (N=18) relative to WT αS, Toxilight assay for adenylate kinase (a.k.) release relative to Bax (N=12), and western blot for cleaved PARP (representative of 6 independent experiments), each transfected as indicated or mock (-); plus western blots for αS (2F12) and β-actin (total lysates PBS/1% TX-100). (h) Western blots for αS (Syn1) and VDAC in TX-100-soluble fractions of M17D cells transfected as indicated. (i) Fluorescence microscopy of live M17D cells co-expressing RFP plus indicated αS-YFP variants; scale bar, 10 μm. Percentages of cells with inclusions were counted in a blinded fashion (right: N=3; 100 cells each). (j) Fluorescence microscopy of rat neurons (DIV14) transfected with indicated untagged αS variants; immunofluorescence with human-specific mAb 15G7; scale bar, 20 μm. Percentages of cells showing inclusions, blinded counting (right: N=3; 100 cells each). *P<0.05, **P<0.01; one-sided ANOVA (see Methods) for all quantifications shown; error bars, s.d.

Mentions: Whereas A53T occurs in a unique stretch within αS, E46K is located within a canonical KTKEGV motif, which occurs at least six times in αS with slight variation (Fig. 6a), resembles motifs in apolipoprotein A1, and may facilitate interactions with lipids31. These facts suggested an intriguing opportunity to validate our central hypothesis: we reasoned that the multimer-destabilizing effect of E46K (Figs 1h and 3) might be amplified by analogous mutations in other KTKEGV motifs. We analysed αS constructs with one, two or three E46K-like substitutions, targeting the highly conserved KTKEGV motifs #3 and #5 flanking the fPD E46K-harbouring motif #4 (Fig. 6a). Intact-cell crosslinking of transfected M17D cells revealed a striking ‘dose-dependent' destabilization of αS60 by each additional E→K substitution, with the triple-K mutant almost abolishing cellular tetramers (Fig. 6b; Supplementary Fig. 9 for all uncropped blots in this section). To exclude an artificial effect of these lysine substitutions on the lysine-directed DSG crosslinking reaction itself, we expressed analogous arginine mutations (that is, 1R=E46R, and 3R), which also change the negative charge to positive but do not offer additional sites for our lysine-directed DSG crosslinker. Like E→K, the E→R substitutions in these three adjacent αS repeat motifs caused similar sharp reductions of the αS60:14 ratio (Fig. 6c). Importantly, the αS14 monomer remained well expressed for all constructs. We confirmed these findings in independent pools of lentivirus-transduced E→K M17D cells (Fig. 6d), obtaining closely similar decreases in the αS60:14 ratio despite some variability in total αS expression. The stepwise decrease in tetramer level was associated with a stepwise increase in monomer level, strongly supporting our overall hypothesis.


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)

Multimers/toxicity/inclusions with 1-3 E46K-like mutations.(a) Schematic of human αS sequence showing engineered mutations (color-coded). Highly conserved KTKEGV motifs underlined, less conserved motifs dotted. (b) DSG-treated M17D cells expressing the indicated αS variants lysed in PBS/1% TX-100; graph (αS60:14 ratios by western blot densitometry) and western blots (Syn1, anti-DJ-1) represent N=3 experiments on different days. For this and the following graphs, WT set to 1 unless stated otherwise. (c) Analogous to (b) but 1 or 3 E→R substitutions. (d) Analogous to (b) using lentiviral pools; long and short western blot exposures, blots cut once as indicated. (e) Venus-YFP complementation assay by automated fluorescence reading. M17D/VN-αS cells transfected with αS-VC WT or mutant or Ran-VC (negative control). YFP fluorescence relative to WT; N=6 independent experiments on 3 different days using different DNA preparations. Representative western blots for αS (pAb C20) plus loading control β-actin. (f) Venus-YFP complementation assay by fluorescence microscopy; αS-VC (always WT) and 3 indicated VN-αS mutants were co-expressed (or not: -) in M17D cells; representative bright-field or fluorescent images and corresponding western blots (Syn1; β-actin as a loading control; blots cut as indicated). N=8 independent transfections on 3 different days. (g) Cytotoxicity assays: trypan blue exclusion for live cell count (N=18) relative to WT αS, Toxilight assay for adenylate kinase (a.k.) release relative to Bax (N=12), and western blot for cleaved PARP (representative of 6 independent experiments), each transfected as indicated or mock (-); plus western blots for αS (2F12) and β-actin (total lysates PBS/1% TX-100). (h) Western blots for αS (Syn1) and VDAC in TX-100-soluble fractions of M17D cells transfected as indicated. (i) Fluorescence microscopy of live M17D cells co-expressing RFP plus indicated αS-YFP variants; scale bar, 10 μm. Percentages of cells with inclusions were counted in a blinded fashion (right: N=3; 100 cells each). (j) Fluorescence microscopy of rat neurons (DIV14) transfected with indicated untagged αS variants; immunofluorescence with human-specific mAb 15G7; scale bar, 20 μm. Percentages of cells showing inclusions, blinded counting (right: N=3; 100 cells each). *P<0.05, **P<0.01; one-sided ANOVA (see Methods) for all quantifications shown; error bars, s.d.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4490410&req=5

f6: Multimers/toxicity/inclusions with 1-3 E46K-like mutations.(a) Schematic of human αS sequence showing engineered mutations (color-coded). Highly conserved KTKEGV motifs underlined, less conserved motifs dotted. (b) DSG-treated M17D cells expressing the indicated αS variants lysed in PBS/1% TX-100; graph (αS60:14 ratios by western blot densitometry) and western blots (Syn1, anti-DJ-1) represent N=3 experiments on different days. For this and the following graphs, WT set to 1 unless stated otherwise. (c) Analogous to (b) but 1 or 3 E→R substitutions. (d) Analogous to (b) using lentiviral pools; long and short western blot exposures, blots cut once as indicated. (e) Venus-YFP complementation assay by automated fluorescence reading. M17D/VN-αS cells transfected with αS-VC WT or mutant or Ran-VC (negative control). YFP fluorescence relative to WT; N=6 independent experiments on 3 different days using different DNA preparations. Representative western blots for αS (pAb C20) plus loading control β-actin. (f) Venus-YFP complementation assay by fluorescence microscopy; αS-VC (always WT) and 3 indicated VN-αS mutants were co-expressed (or not: -) in M17D cells; representative bright-field or fluorescent images and corresponding western blots (Syn1; β-actin as a loading control; blots cut as indicated). N=8 independent transfections on 3 different days. (g) Cytotoxicity assays: trypan blue exclusion for live cell count (N=18) relative to WT αS, Toxilight assay for adenylate kinase (a.k.) release relative to Bax (N=12), and western blot for cleaved PARP (representative of 6 independent experiments), each transfected as indicated or mock (-); plus western blots for αS (2F12) and β-actin (total lysates PBS/1% TX-100). (h) Western blots for αS (Syn1) and VDAC in TX-100-soluble fractions of M17D cells transfected as indicated. (i) Fluorescence microscopy of live M17D cells co-expressing RFP plus indicated αS-YFP variants; scale bar, 10 μm. Percentages of cells with inclusions were counted in a blinded fashion (right: N=3; 100 cells each). (j) Fluorescence microscopy of rat neurons (DIV14) transfected with indicated untagged αS variants; immunofluorescence with human-specific mAb 15G7; scale bar, 20 μm. Percentages of cells showing inclusions, blinded counting (right: N=3; 100 cells each). *P<0.05, **P<0.01; one-sided ANOVA (see Methods) for all quantifications shown; error bars, s.d.
Mentions: Whereas A53T occurs in a unique stretch within αS, E46K is located within a canonical KTKEGV motif, which occurs at least six times in αS with slight variation (Fig. 6a), resembles motifs in apolipoprotein A1, and may facilitate interactions with lipids31. These facts suggested an intriguing opportunity to validate our central hypothesis: we reasoned that the multimer-destabilizing effect of E46K (Figs 1h and 3) might be amplified by analogous mutations in other KTKEGV motifs. We analysed αS constructs with one, two or three E46K-like substitutions, targeting the highly conserved KTKEGV motifs #3 and #5 flanking the fPD E46K-harbouring motif #4 (Fig. 6a). Intact-cell crosslinking of transfected M17D cells revealed a striking ‘dose-dependent' destabilization of αS60 by each additional E→K substitution, with the triple-K mutant almost abolishing cellular tetramers (Fig. 6b; Supplementary Fig. 9 for all uncropped blots in this section). To exclude an artificial effect of these lysine substitutions on the lysine-directed DSG crosslinking reaction itself, we expressed analogous arginine mutations (that is, 1R=E46R, and 3R), which also change the negative charge to positive but do not offer additional sites for our lysine-directed DSG crosslinker. Like E→K, the E→R substitutions in these three adjacent αS repeat motifs caused similar sharp reductions of the αS60:14 ratio (Fig. 6c). Importantly, the αS14 monomer remained well expressed for all constructs. We confirmed these findings in independent pools of lentivirus-transduced E→K M17D cells (Fig. 6d), obtaining closely similar decreases in the αS60:14 ratio despite some variability in total αS expression. The stepwise decrease in tetramer level was associated with a stepwise increase in monomer level, strongly supporting our overall hypothesis.

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