Research Article

Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3

See allHide authors and affiliations

Science  30 Sep 2016:
Vol. 353, Issue 6307, aah3374
DOI: 10.1126/science.aah3374
  • LAG3 deletion or antibodies to LAG3 delay α-synuclein PFF transmission.

    Compared with wild-type neurons, binding and endocytosis of α-synuclein PFF is dramatically reduced with antibodies to LAG3 or when LAG3 is deleted, resulting in delayed pathologic α-synuclein transmission and toxicity. Illustration credit: I-Hsun Wu

  • Fig. 1 α-Syn PFF binds to LAG3.

    (A) Individual clones from a library consisting of 352 individual cDNAs encoding transmembrane proteins (GFC-transfection array panel, Origene) were transfected into SH-SY5Y cells, and the relative binding signals of human α-syn PFF to individual transmembrane proteins are shown. Positive candidates are LAG3 (NM_002286), NRNX1 (NM_138735), and APLP1 (NM_005166). (B) Mouse α-syn-biotin monomer and α-syn-biotin PFF–binding affinity to SH-SY5Y cells expressing the indicated proteins. LAG3* Kd assessment was performed without Triton X-100 (TX-100). All other experiments were performed with 0.1% TX-100. Transmembrane proteins similar to the candidates were also tested. Quantification of bound α-syn-biotin PFF to the candidates was performed with ImageJ. Kd values are means ± SEM and are based on monomer equivalent concentrations. Selectivity was calculated by dividing Kd (monomer) by Kd (PFF). Binding of α-syn-biotin monomer was detected at a concentration of 3000 nM, but binding was not saturable. (C) (Top) α-Syn-biotin monomer or α-syn-biotin PFF binding to LAG3-overexpressing SH-SY5Y cells as a function of total α-syn concentration in 0% TX-100 or 0.1% TX-100 conditions (monomer equivalent for PFF preparations). (Bottom) Scatchard analysis. Kd = 71 nM (0% TX-100) and 77 nM (0.1% TX-100); data are the means ± SEM, n = 3 independent experiments. (D) Binding of α-syn-biotin PFF to cultured cortical neurons (21 days in vitro) is reduced by LAG3 knockout (LAG3−/−), as assessed by means of alkaline phosphatase assay. α-Syn-biotin PFF WT-Kd = 374 nM, LAG3−/−-Kd = 449 nM, estimated Kd for neuronal LAG3 (dotted line, ΔLAG3 = wild type minus LAG3−/−) is 103 nM. Data are the means ± SEM, n = 3 independent experiments. *P < 0.05, Student’s t test. Power (1 – β error probability) = 1. (E) Specificity of LAG3 binding with α-syn-biotin PFF (fig. S4). Tau-biotin PFF (fig. S8), β-amyloid-biotin oligomer, and β-amyloid-biotin PFF (fig. S9) are negative controls.

  • Fig. 2 Endocytosis of α-syn PFF is dependent on LAG3.

    (A) Live image analysis of the endocytosis of α-syn-pHrodo PFF. α-Syn PFF was conjugated with a pH-dependent dye (pHrodo red), in which fluorescence increases as pH decreases from neutral to acidic environments. White arrowheads indicate nontransfected WT or LAG3−/− neurons, and white arrows indicate LAG3-transfected neurons. Scale bar, 10 μm. (B) Quantification of (A), cell number (5-46) from n = 3 independent experiments. (C) Internalized α-syn-biotin PFF colocalizes with Rab5. Colocalization of internalized α-syn-biotin PFF and Rab5 was assessed by means of confocal microscopy. Scale bar, 10 μm. (D) Quantification of (C), cell number (13-32) from n = 4 independent experiments. One-way analysis of variance (ANOVA) with Tukey’s correction. Data in (B) and (D) are as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Power (1 – β error probability) = 1.

  • Fig. 3 α-Syn PFF induced pathology is reduced by deletion of LAG3 in vitro.

    (A) WT and LAG3−/− primary cortical neurons at 7 days in vitro were treated with α-syn PFF or PBS. LAG3 was overexpressed via Lentivirus (LV) transduction in WT or LAG3−/− neurons at 4 days in vitro. Three days after transduction, cultures 7 days in vitro were treated with α-syn PFF or PBS. All the cultures were fixed 10 days after treatment in 4% PFA. Neurons were stained with rabbit mAb MJF-R13 (8-8) for P-α-syn. Scale bar, 40 μm. (B) Quantification of (A), n = 5 independent experiments, each performed in duplicate. Values are given as the means ± SEM. Statistical significance was determined by using one-way ANOVA followed with Tukey’s correction; ***P < 0.001. Power (1 – β error probability) = 1. (C) Immunoblots in WT and LAG3−/− neuron lysates of misfolded α-syn, P-α-syn, synapsin II, SNAP25, and LAG3. β-actin served as a loading control. WT and LAG3−/− neuron lysates were sequentially extracted in 1% TX-100 (TX-soluble) followed by 2% SDS (TX-insoluble) 14 days after α-syn PFF treatment. α-Syn PFF recruited endogenous α-syn into TX-insoluble and hyperphosphorylated aggregates, which was ameliorated by deletion of LAG3. α-Syn PFF caused a reduction in levels of SNAP25 and synapsin II compared with PBS 14 days after treatment. Deletion of LAG3 prevented PFF-induced synaptic protein loss. (D to G) Quantification of (C). Values are given as means ± SEM, n = 3 independent experiments. Statistical significance was determine by using one-way ANOVA followed by Tukey’s correction; *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 α-Syn PFF transmission is reduced by deletion of LAG3 in vitro.

    (A) Schematic representation of the three chambers in which neurons were cultured: chamber 1 (C1), chamber 2 (C2), and chamber 3 (C3) (top); or C1 and C3 (bottom). (B) α-Syn PFF was added to C1 of the microfluidic device. On day 14, P-α-syn was detected in C2 and C3 when neurons were present in all three chambers. Transmission to C3 is not detectable when neurons are not present in C2. Scale bar, 100 μm. (C) Quantification of immunofluorescent images in (B). Data are the means ± SEM, n = 3 independent experiments. One-way ANOVA followed by Sidak’s correction; ***P < 0.001 versus C1. Power (1 – β error probability) = 1. (D) Schematic of microfluidic neuron device with three chambers to separate neurons seeded in three chambers. (E) Transmission of pathologic P-α-syn from C1 to C2 to C3 14 days after addition of α-syn PFF in C1. The different combinations of neurons tested in C2, listed as C1-(C2)-C3, are WT-(WT)-WT, WT-(WT+LAG3)-WT, WT-(LAG3−/−)-WT, WT-(LAG3−/−+LAG3)-WT. Scale bar, 10 μm. (F) Quantification of (E). Values are given as means ± SEM, n = 3 independent experiments. Statistical significance was determine by using one-way ANOVA followed by Tukey’s correction; *P < 0.05, **P < 0.01, ***P < 0.001. Power (1-β error probability) = 1.

  • Fig. 5

    Antibodies to LAG3 block α-syn PFF binding, endocytosis, pathology, and transmission. (A) C9B7W and 410C9 (both 50 μg/mL), antibodies to LAG3, block the binding of α-syn-biotin PFF (500 nM) on SH-SY5Y cells expressing LAG3. Scale bar, 50 μm. (Right) Quantification of images in (A). Data are the means ± SEM, n =3 independent experiments, Student’s t test; ***P < 0.001. (B) C9B7W and 410C9 (both 50 μg/mL) reduced the endocytosis of α-syn-biotin PFF (1 μM) in primary cortical neurons 12 days in vitro. Rab7 was used to confirm the isolation of endosomes. (C) P-α-syn as detected with rabbit mAb MJF-R13 (8-8) was reduced by antibodies to LAG3 in primary cortical neurons. Scale bar, 50 μm. (Bottom) Quantification of images in (C). Data are the means ± SEM, n = 3 independent experiments, one-way ANOVA followed by Tukey’s correction; **P < 0.01. (D) 410C9 delays α-Syn PFF transmission in neurons. (Top left) A schematic representation of the three microchambers in which neurons were cultured in C1, C2, and C3. α-Syn PFF was added to C1 of the microfluidic device on day 7. Mouse IgG or 410C9 (both 50 μg/mL) was added to C2 on day 7. α-Syn PFF transmission was detected via P-α-syn immunostaining in C2 and C3 on day 21. Scale bar, 10 μm. (Top right) Quantification of images at bottom. Data are the means ± SEM, n = 3 independent experiments, one-way ANOVA followed by Tukey’s correction. ***P < 0.001; n.s., nonsignificant.

  • Fig. 6 α-Syn PFF–induced pathology is reduced by deletion of LAG3 in vivo.

    (A) Representative P-α-syn immunostaining and quantification in the substantia nigra par compacta (SNpc) of WT and LAG3−/− mice sacrificed at 30 and 180 days after intrastriatal α-syn PFF injection. Data are the means ± SEM, n = 5 to 9 mice per group, one-way ANOVA with Sidak’s correction. (B) Stereology counts from TH immunostaining and Nissl staining of SNpc DA neurons of WT and LAG3−/− mice at 180 days after intrastriatal α-syn PFF, α-syn monomer, or PBS injection. Data are the mean number of cells per region ± SEM, n = 5 to 9 mice per group, one-way ANOVA with Dunnett’s correction. (C) DA concentrations in the striatum of α-syn PFF–injected mice and PBS-treated controls measured at 180 days by means of HPLC. Data are the means ± SEM, n = 5 to 8 mice per group, one-way ANOVA with Tukey’s correction. (D and E) 180 days after α-syn PFF injection, the pole test and grip strength were performed in WT or LAG3−/− mice injected with PBS or α-syn PFF. Behavioral abnormalities in the pole test and grip strength induced by α-syn PFF injection were ameliorated in LAG3−/− mice. Data are the means ± SEM, n = 7 to 9 mice per group for behavioral studies. Statistical significance was determined by using one-way ANOVA with Tukey’s correction; *P < 0.05, **P < 0.01, ***P < 0.001; n.s., nonsignificant. Power (1 – β error probability) = 1.

Supplementary Materials

  • Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3

    Xiaobo Mao, Michael Tianhao Ou, Senthilkumar S. Karuppagounder, Tae-In Kam, Xiling Yin, Yulan Xiong, Preston Ge, George Essien Umanah, Saurav Brahmachari, Joo-Ho Shin, Ho Chul Kang, Jianmin Zhang, Jinchong Xu, Rong Chen, Hyejin Park, Shaida A. Andrabi, Sung Ung Kang, Rafaella Araújo Gonçalves, Yu Liang, Shu Zhang, Chen Qi, Sharon Lam, James A. Keiler, Joel Tyson, Donghoon Kim, Nikhil Panicker, Seung Pil Yun, Creg J. Workman, Dario A. A. Vignali, Valina L. Dawson, Han Seok Ko, Ted M. Dawson

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Figs. S1 to S24
    • Tables S1 to S2
    • References

Navigate This Article