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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

αS multimers in normal brain tissues and neural cells.(a) A fresh human cortical biopsy was crosslinked with either 1.75 mM DSP and reduced by βME (DSPr) or else with 1 mM DSG at increasing volume-to-protein ratio, followed by sequential extraction of PBS- and TX-100-soluble fractions. Each lane is one technical replicate from the biopsy. (b) Mouse brain, 1 mM DSG, PBS fraction; blots represent five independent experiments from different WT mice. (c) Mouse brain and human erythroleukemia (HEL) cells. DSG concentration gradients applied as indicated, and PBS fractions prepared; blots represent at least three independent experiments. (d) DIV13 rat neurons. 1.75 mM DSP/βME (DSPr, left panel), 0.5 mM DSG (middle) and 1 mM DSG (right) were applied, and PBS fractions prepared. Western blots for αS (Syn1 mAb), the monomeric proteins Parkin (Pkn), casein kinase 1α (CK1) and Ran, and the tetrameric proteins p53 and Drp1 (Drp); blots represent at least 3 independent experiments from different primary cultures. (e) Fluorescence microscopy of DIV13 rat neurons: endogenous αS in green (mAb 2F12) and transfected RFP (red), plus merged image; scale bar, 20 μm. (f) Fluorescence microscopy of virally transduced M17D cells: αS in red (mAb 2F12), GFP in green; scale bar, 5 μm. (g) Fluorescence microscopy of M17D cells transiently transfected with RFP plus YFP or αS-YFP or YFP αS complementation pairs, as indicated (empty vector control on far right). YFP in green, RFP in red, merge below; scale bar, 10 μm. (h) Quantification of multiple Venus-YFP complementation assays. A stable cell line M17D/VN-αS was transfected with DNA constructs expressing VC fused to αS (WT, A30P, E46K, G51D or A53T) or Ran (negative control). YFP complementation intensity relative to WT αS-VC transfection from N=8 independent experiments on 4 days using different DNA preparations;*P<0.05, **P<0.01, Student's t-test; relative to WT in this and subsequent figures, unless stated otherwise; error bars, s.d. Below are representative western blots for αS (Syn1 mAb), Ran, and the loading control β-actin.
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f1: αS multimers in normal brain tissues and neural cells.(a) A fresh human cortical biopsy was crosslinked with either 1.75 mM DSP and reduced by βME (DSPr) or else with 1 mM DSG at increasing volume-to-protein ratio, followed by sequential extraction of PBS- and TX-100-soluble fractions. Each lane is one technical replicate from the biopsy. (b) Mouse brain, 1 mM DSG, PBS fraction; blots represent five independent experiments from different WT mice. (c) Mouse brain and human erythroleukemia (HEL) cells. DSG concentration gradients applied as indicated, and PBS fractions prepared; blots represent at least three independent experiments. (d) DIV13 rat neurons. 1.75 mM DSP/βME (DSPr, left panel), 0.5 mM DSG (middle) and 1 mM DSG (right) were applied, and PBS fractions prepared. Western blots for αS (Syn1 mAb), the monomeric proteins Parkin (Pkn), casein kinase 1α (CK1) and Ran, and the tetrameric proteins p53 and Drp1 (Drp); blots represent at least 3 independent experiments from different primary cultures. (e) Fluorescence microscopy of DIV13 rat neurons: endogenous αS in green (mAb 2F12) and transfected RFP (red), plus merged image; scale bar, 20 μm. (f) Fluorescence microscopy of virally transduced M17D cells: αS in red (mAb 2F12), GFP in green; scale bar, 5 μm. (g) Fluorescence microscopy of M17D cells transiently transfected with RFP plus YFP or αS-YFP or YFP αS complementation pairs, as indicated (empty vector control on far right). YFP in green, RFP in red, merge below; scale bar, 10 μm. (h) Quantification of multiple Venus-YFP complementation assays. A stable cell line M17D/VN-αS was transfected with DNA constructs expressing VC fused to αS (WT, A30P, E46K, G51D or A53T) or Ran (negative control). YFP complementation intensity relative to WT αS-VC transfection from N=8 independent experiments on 4 days using different DNA preparations;*P<0.05, **P<0.01, Student's t-test; relative to WT in this and subsequent figures, unless stated otherwise; error bars, s.d. Below are representative western blots for αS (Syn1 mAb), Ran, and the loading control β-actin.

Mentions: We previously reported that the crosslinking pattern of endogenous αS in intact cells differed from the stochastic crosslinking of recombinant monomeric αS in vitro, in that the former strongly favoured the trapping of tetramers, with few or no intermediate dimers or trimers9. We now sought to crosslink αS in its endogenous state in normal brain tissue. We achieved this by mincing fresh brain with a mechanical tissue chopper, performing gentle centrifugal washes on the intact tissue pieces and then subjecting only these washed brain bits to crosslinking, thereby largely avoiding the tetramer-destabilizing effects of breaking cells open9 (see Methods and Supplementary Fig. 1). We applied this protocol to an optimal source of physiological αS: a fresh brain biopsy (elective surgery for focal epilepsy) from the cerebral cortex of a young patient free of any neurodegenerative process. After the mincing and centrifugal washes, we applied DSG (1 mM solution) at an increasing ratio of volume of crosslinking solution to brain protein, or else applied the reducible crosslinker DSP (2 mM solution) at a fixed ratio. The crosslinked tissue bits were then sonicated and separated (100,000 g spin) into PBS (‘cytosol') and Triton X-100 (TX-100; ‘membrane') fractions. Immunoblotting confirmed efficient crosslinking had occurred, as the cytosolic protein DJ-1 and the transmembrane protein VDAC were each trapped in their native dimeric states in a DSG dose-dependent manner (Fig. 1a, upper panels). As before, we fixed blots in paraformaldehyde17 to improve αS retention during blot washing16. Total αS levels were compared using the DSP-crosslinked-and-reduced sample, which we had shown previously was optimal for quantifying total cellular αS levels in crosslinking studies16. Enrichment of DJ-1 and absence of VDAC and synaptobrevin-2 established cytosol purity (Fig. 1a, upper panels). Immunoblotting confirmed our earlier findings in cultured neurons9: αS was detected in the biopsied human brain primarily as tetramers (αS60) and related multimers (αS80, αS100) by mAbs Syn1 and 15G7 (Fig. 1a). Antiserum C20 preferentially detected monomers, although its αS60:14 ratio was still 1:1 versus 2:1 using mAb Syn1 (Fig. 1a, lower panels). Including the αS80 and αS100 multimers with the αS60 tetramers showed the clear preponderance of αS multimers over monomers (3:1) in normal human brain. Similar to our findings in primary neurons9, the multimers were found overwhelmingly in the human brain cytosol (Fig. 1a, lower panels). In the membrane fraction, virtually only monomer was detected.


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)

αS multimers in normal brain tissues and neural cells.(a) A fresh human cortical biopsy was crosslinked with either 1.75 mM DSP and reduced by βME (DSPr) or else with 1 mM DSG at increasing volume-to-protein ratio, followed by sequential extraction of PBS- and TX-100-soluble fractions. Each lane is one technical replicate from the biopsy. (b) Mouse brain, 1 mM DSG, PBS fraction; blots represent five independent experiments from different WT mice. (c) Mouse brain and human erythroleukemia (HEL) cells. DSG concentration gradients applied as indicated, and PBS fractions prepared; blots represent at least three independent experiments. (d) DIV13 rat neurons. 1.75 mM DSP/βME (DSPr, left panel), 0.5 mM DSG (middle) and 1 mM DSG (right) were applied, and PBS fractions prepared. Western blots for αS (Syn1 mAb), the monomeric proteins Parkin (Pkn), casein kinase 1α (CK1) and Ran, and the tetrameric proteins p53 and Drp1 (Drp); blots represent at least 3 independent experiments from different primary cultures. (e) Fluorescence microscopy of DIV13 rat neurons: endogenous αS in green (mAb 2F12) and transfected RFP (red), plus merged image; scale bar, 20 μm. (f) Fluorescence microscopy of virally transduced M17D cells: αS in red (mAb 2F12), GFP in green; scale bar, 5 μm. (g) Fluorescence microscopy of M17D cells transiently transfected with RFP plus YFP or αS-YFP or YFP αS complementation pairs, as indicated (empty vector control on far right). YFP in green, RFP in red, merge below; scale bar, 10 μm. (h) Quantification of multiple Venus-YFP complementation assays. A stable cell line M17D/VN-αS was transfected with DNA constructs expressing VC fused to αS (WT, A30P, E46K, G51D or A53T) or Ran (negative control). YFP complementation intensity relative to WT αS-VC transfection from N=8 independent experiments on 4 days using different DNA preparations;*P<0.05, **P<0.01, Student's t-test; relative to WT in this and subsequent figures, unless stated otherwise; error bars, s.d. Below are representative western blots for αS (Syn1 mAb), Ran, and the loading control β-actin.
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f1: αS multimers in normal brain tissues and neural cells.(a) A fresh human cortical biopsy was crosslinked with either 1.75 mM DSP and reduced by βME (DSPr) or else with 1 mM DSG at increasing volume-to-protein ratio, followed by sequential extraction of PBS- and TX-100-soluble fractions. Each lane is one technical replicate from the biopsy. (b) Mouse brain, 1 mM DSG, PBS fraction; blots represent five independent experiments from different WT mice. (c) Mouse brain and human erythroleukemia (HEL) cells. DSG concentration gradients applied as indicated, and PBS fractions prepared; blots represent at least three independent experiments. (d) DIV13 rat neurons. 1.75 mM DSP/βME (DSPr, left panel), 0.5 mM DSG (middle) and 1 mM DSG (right) were applied, and PBS fractions prepared. Western blots for αS (Syn1 mAb), the monomeric proteins Parkin (Pkn), casein kinase 1α (CK1) and Ran, and the tetrameric proteins p53 and Drp1 (Drp); blots represent at least 3 independent experiments from different primary cultures. (e) Fluorescence microscopy of DIV13 rat neurons: endogenous αS in green (mAb 2F12) and transfected RFP (red), plus merged image; scale bar, 20 μm. (f) Fluorescence microscopy of virally transduced M17D cells: αS in red (mAb 2F12), GFP in green; scale bar, 5 μm. (g) Fluorescence microscopy of M17D cells transiently transfected with RFP plus YFP or αS-YFP or YFP αS complementation pairs, as indicated (empty vector control on far right). YFP in green, RFP in red, merge below; scale bar, 10 μm. (h) Quantification of multiple Venus-YFP complementation assays. A stable cell line M17D/VN-αS was transfected with DNA constructs expressing VC fused to αS (WT, A30P, E46K, G51D or A53T) or Ran (negative control). YFP complementation intensity relative to WT αS-VC transfection from N=8 independent experiments on 4 days using different DNA preparations;*P<0.05, **P<0.01, Student's t-test; relative to WT in this and subsequent figures, unless stated otherwise; error bars, s.d. Below are representative western blots for αS (Syn1 mAb), Ran, and the loading control β-actin.
Mentions: We previously reported that the crosslinking pattern of endogenous αS in intact cells differed from the stochastic crosslinking of recombinant monomeric αS in vitro, in that the former strongly favoured the trapping of tetramers, with few or no intermediate dimers or trimers9. We now sought to crosslink αS in its endogenous state in normal brain tissue. We achieved this by mincing fresh brain with a mechanical tissue chopper, performing gentle centrifugal washes on the intact tissue pieces and then subjecting only these washed brain bits to crosslinking, thereby largely avoiding the tetramer-destabilizing effects of breaking cells open9 (see Methods and Supplementary Fig. 1). We applied this protocol to an optimal source of physiological αS: a fresh brain biopsy (elective surgery for focal epilepsy) from the cerebral cortex of a young patient free of any neurodegenerative process. After the mincing and centrifugal washes, we applied DSG (1 mM solution) at an increasing ratio of volume of crosslinking solution to brain protein, or else applied the reducible crosslinker DSP (2 mM solution) at a fixed ratio. The crosslinked tissue bits were then sonicated and separated (100,000 g spin) into PBS (‘cytosol') and Triton X-100 (TX-100; ‘membrane') fractions. Immunoblotting confirmed efficient crosslinking had occurred, as the cytosolic protein DJ-1 and the transmembrane protein VDAC were each trapped in their native dimeric states in a DSG dose-dependent manner (Fig. 1a, upper panels). As before, we fixed blots in paraformaldehyde17 to improve αS retention during blot washing16. Total αS levels were compared using the DSP-crosslinked-and-reduced sample, which we had shown previously was optimal for quantifying total cellular αS levels in crosslinking studies16. Enrichment of DJ-1 and absence of VDAC and synaptobrevin-2 established cytosol purity (Fig. 1a, upper panels). Immunoblotting confirmed our earlier findings in cultured neurons9: αS was detected in the biopsied human brain primarily as tetramers (αS60) and related multimers (αS80, αS100) by mAbs Syn1 and 15G7 (Fig. 1a). Antiserum C20 preferentially detected monomers, although its αS60:14 ratio was still 1:1 versus 2:1 using mAb Syn1 (Fig. 1a, lower panels). Including the αS80 and αS100 multimers with the αS60 tetramers showed the clear preponderance of αS multimers over monomers (3:1) in normal human brain. Similar to our findings in primary neurons9, the multimers were found overwhelmingly in the human brain cytosol (Fig. 1a, lower panels). In the membrane fraction, virtually only monomer was detected.

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