DOI: 10.5061/dryad.5hqbzkhcw
title: Data from: Aplp1 interacts with Lag3 to facilitates transmission of pathologic α-synuclein

description: Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through
 binding to the lymphocyte-activation gene 3 (Lag3). Here we report that
 amyloid β precursor-like protein 1 (Aplp1) interacts with Lag3 that
 facilitates the binding, internalization, transmission, and toxicity of
 pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of
 dopaminergic neurons and the accompanying behavioral deficits induced by
 α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of
 α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the
 neurodegeneration induced by α-syn PFF in vivo. The identification of
 Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology advances
 our understanding of the molecular mechanism of cell-to-cell transmission
 of pathologic α-syn and provides additional targets for therapeutic
 strategies aimed at preventing neurodegeneration in Parkinson’s disease
 and related α-synucleinopathies.
description: # Aplp1 interacts with Lag3 to facilitates transmission of pathologic
 α-synuclein
 [https://doi.org/10.5061/dryad.5hqbzkhcw](https://doi.org/10.5061/dryad.5hqbzkhcw) **Brief summary of the dataset contents:** In this study, we investigated the role of amyloid precursor-like protein 1 (Aplp1) and lymphocyte activation gene 3 (Lag3) in the binding, internalization, and progression of pathological alpha-synuclein (α-syn) preformed fibrils (PFFs). Through a combination of in vitro cell culture models, structural biology techniques, and in vivo PD mouse models, we studied their roles in mediating the transmission and pathogenesis of pathological α-synuclein. We performed α-syn PFF binding assays in various cell lines transfected with Aplp1, Lag3, or their deletion mutants. Internalization assays were performed using fluorescently labeled α-syn PFFs in primary cortical neuron cultures from wild-type, Aplp1 knockout, Lag3 knockout, and Aplp1/Lag3 double knockout mice. Using mouse primary neuron cellular and animal models of Parkinson's disease (PD), we analyzed the interaction between Aplp1 and Lag3 and their contributions to α-synuclein-induced deficits. Our findings shows that Aplp1 interacts with Lag3, facilitating the binding, internalization, transmission, and toxicity of pathological α-synuclein. Additionally, nuclear magnetic resonance (NMR) spectroscopy was employed to demonstrate the interaction between Aplp1 and Lag3. In vivo studies demonstrate that an anti-Lag3 antibody disrupts the Aplp1-Lag3 interaction, preventing neurodegeneration induced by α-syn PFF injection. Knockout of both Aplp1 and Lag3 significantly reduces α-syn PFF binding, internalization, pathology, cell-to-cell transmission, both in vitro and in vivo. Furthermore, deletion of both Aplp1 and Lag3 rescues loss of dopaminergic (DA) neurons and associated behavioral deficits caused by α-syn PFF. ## Description of the data and file structure Files are labeled by figure and include imaging files, data sets in excel and graphs in GraphPad. Abbreviations include "WT" for wild type, "Quant" for quantification data, "Img" for image, "PFF" for pre-formed fibrils. All files included in this submission are raw data files generated from experiments performed for this manuscript.  **Figure 1** α-Syn PFF binds to Aplp1 Figure 1a: Schematic diagram of Aplp1 deletions mutants Figure 1b: Quantification of binding signals of deletion mutants of Aplp1 with α-syn-biotin PFF Figure 1c-d: Schematic diagram of Aplp1 chimeras Figure 1e: Schematic diagram of Lag3 and deletions mutants Figure 1f: Quantification of binding signals of deletion mutants of Lag3 with α-syn-biotin PFF  **Figure 2** Aplp1 mediates the endocytosis of α-syn PFF and subsequent pathology Figure 2a: Quantification of the intensity of the endocytosis of α-syn-pHrodo PFF in WT and Aplp1–/– neurons Figure 2b-c: Immunoblot and quantification analysis of α-syn-biotin PFF in the endolysosome fraction Figure 2d: Quantification of the intensity of the colocalization of α-syn-biotin PFF with Rab7 in WT and Aplp1–/– neurons Figure 2e: Quantification of co-localization of α-syn-biotin PFF and Rab7 in WT neurons with transient expression of full-length Aplp1 and deletion mutants Figure 2f: Quantification of co-localization signal of α-syn-biotin PFF in WT neurons with transient expression of Aplp1, chimeric Aplp1(E1)-Aplp2 and Aplp1(E1)-App Figure 2g: Immunostaining of anti-pS129 in WT and Aplp1–/– primary cortical neurons by Aplp1 and control lentivirus transduction, with the administration of α-syn PFF and PBS Figure 2h: Quantification of immunostaining (g) Figure 2i-k: Immunoblots in WT and Aplp1–/– neuron lysates of insoluble α-syn, pS129, soluble α-syn, and β-actin  **Figure 3** Aplp1 and Lag3 bind to each other Figure 3a: Lag3 pulls down Aplp1 by anti-Lag3 410C9 immunoprecipitation in WT mouse brain lysates, but not in Lag3–/– lysates Figure 3b: Aplp1 pulls down Lag3 by anti-Aplp1 CT11 immunoprecipitation in WT mouse brain lysates, but not in Aplp1–/– lysates Figure 3c-d: Mapping of the Lag3-binding domains in Aplp1 Figure 3e: Mapping of the Aplp1-binding domains in Lag3. Figure 3f-h: Identification of the interface of A1E1 (E1 domain of APLP1) binding to L3D2 (D2 domain of LAG3) Figure 3i: Validation of the NMR results Figure 3j: The scheme for the interaction among Aplp1, Lag3 and α-syn PFF  **Figure 4** The role of Aplp1-Lag3 interaction in mediating the binding, endocytosis of α-syn PFF, and subsequent pathology Figure 4a: Aplp1 and Lag3 account for greater than 40% of the binding of α-syn-biotin PFF to cortical neurons Figure 4b: Re-expression of Aplp1 and Lag3 together in Aplp1–/–/Lag3–/– cortical neurons led to significant enhancement binding that is greater than the sum of α-syn-biotin PFF binding to Aplp1 and Lag3 alone Figure 4c-d: Immunoblots of endosomal fractions isolated from WT, Aplp1–/–, Lag3–/–, and Aplp1–/–/Lag3–/– cortical neurons Figure 4e: APLP1 co-expression by transfection increased the uptake of α-syn-biotin in Lag3-transfected SH-SY5Y cells Figure 4f-g: Anti-Lag3 410C9 significantly disrupts the co-IP of FLAG-Aplp1 and Myc-Lag3 in HEK293FT cells Figure 4h-i: Anti-Lag3 410C9 (330 nM) reduced the internalization of α-syn-biotin PFF (1 μM) significantly more in WT cultures than in Lag3–/– cultures Figure 4j-l: Both the levels of the endosomal Aplp1 and Lag3 were significantly decreased by 410C9 in WT cortical neuron cultures  **Figure 5** The role of Aplp1-Lag3 interaction in α-syn pathology propagation, transmission, and neurotoxicity in vitro Figure 5a-b: Deletion of the Aplp1-Lag3 together significantly decreased (70%) pS129 immunostaining induced by α-syn PFF, compared to Aplp1–/–, Lag3–/–, or WT neurons Figure 5c-d: Immunostaining of anti-pS129 in Aplp1­­–/–/Lag3–/­– neurons, treated with α-syn PFF, transduced with Aplp1, Lag3, or Aplp1+Lag3 Figure 5e: Schematic of microfluidic neuron device with three chambers Figure 5f-i: Immunostaining images and quantification of pS129 signals in the transmission Figure 5j-k: PI/Hoechst staining for cell death in WT, Aplp1­­–/–, Lag3–/­–, and Aplp1­­–/–/Lag3–/­– cortical neuron cultures, treated with α-syn PFF  **Figure 6** Roles of Aplp1 and the Aplp1-Lag3 interaction in mediating α-syn PFF-induced neurodegeneration in vivo Figure 6a: Representative TH (tyrosine hydroxylase) immunohistochemistry and Nissl staining images of dopamine (DA) neurons in the SNpc of α-syn PFF-injected hemisphere in the WT, Aplp1–/–, and Aplp1–/–/Lag3–/– mice Figure 6b: Stereological counting of the number of TH- and Nissl-positive neurons in the substantia nigra via unbiased stereological analysis after 6 months of α-syn PFF injection in the WT, Aplp1–/–, and Aplp1–/–/Lag3–/– mice Figure 6c: DA concentrations in the striatum of α-syn PFF-injected mice and PBS-injected controls measured at 180 days by means of HPLC Figure 6d-e: Representative pS129 positive inclusions in the substantia nigra of WT, Aplp1–/–, and Aplp1–/–/Lag3–/– mice Figure 6f: Distribution of LB/LN-like pathology in the brain sections of α-syn PFF-injected WT, Aplp1–/–, and Aplp1–/–/Lag3–/– mice Figure 6g-h: Representative images of LB/LN-like pathology (the black box in panel H) and the quantification of pS129 intensity (green) from each coronal section (1–4) stained with pS129 Figure 6i-j: Assessments of the behavioral deficits measured by the pole test  **Figure 7** The role of anti-Lag3 in blocking α-syn PFF-induced neurodegeneration in vivo Figure 7a: Representative images of TH and Nissl staining of SNpc DA neurons of WT mice treated with mIgG or 410C9 at 6-months after intrastriatal injection of α-syn PFF injection Figure 7b: Stereology counts of data in a Figure 7c: DA concentrations in the striatum of α-syn PFF or PBS injected mice treated with 410C9 or mIgG Figure 7d-g: Grip strength test and pole test were performed and showed behavioral deficits were ameliorated by 410C9  Data Summary Table for Supplementary Figures  **Extended Data Fig. 1** Aplp1 binds to α-syn-biotin PFF but not monomer Extended Data Figure 1a: α-Syn-biotin PFF binding to SH-SY5Y cells expressing Aplp1 as a function of α-syn concentration with Scatchard analysis Extended Data Figure 1b: β-Amyloid PFF binds to Aplp1 in non-specific manner, but β-amyloid monomer does not exhibit appreciable binding to Aplp1 Extended Data Figure 1c-e: The membrane localization of Aplp1 and Lag3 mutants Extended Data Figure 1f: Live images of the endocytosis of α-syn-pHrodo PFF Extended Data Figure 1g: The co-localization of internalized α-syn-biotin PFF (red), Rab7 (green) and FLAG-Aplp1 (grey scale) in soma of WT and Aplp1–/– neuronal culture was assessed by means of confocal microscopy  **Extended Data Figure 2** α-Syn PFF binds to Aplp1 in cell surface binding assay Extended Data Figure 2a: Representative images of binding signals of deletion mutants of Aplp1 with α-syn-biotin PFF Extended Data Figure 2b: Representative images of binding of Aplp1(E1)-Aplp2 and Aplp1(E1)-App chimeras with α-syn-biotin PFF Extended Data Figure 2c: Representative images of binding of deletion mutants of Lag3 with α-syn-biotin PFF  **Extended Data Figure 3** Lag3 is expressed in neurons in WT mice Extended Data Figure 3a: Breeding strategy of the double knockout of Aplp1 and Lag3 (Aplp1­­–/–/Lag3–/­–) mice Extended Data Figure 3b: Genotyping confirmation of WT, Aplp1­­–/–, Lag3–/­– and Aplp1­­–/–/Lag3–/­– mice Extended Data Figure 3c-f: Co-Immunostaining of anti-Lag3 and anti-NeuN in four brain regions of WT and Aplp1­­–/–/Lag3–/­– mice  **Extended Data Figure 4** Lag3 is detected in the neurons by RNAscope Extended Data Figure 4a: Co-localization of Lag3 (red) inside neurons labelled by MAP2 (white) and microglia labelled by TMEM119 (green) in VMB region in WT and Lag3-/- mice Extended Data Figure 4b-c: Quantification of Lag3 positive cells in neurons and in microglia Extended Data Figure 4d: Co-localization of Lag3 (red) inside neurons labelled by MAP2 (white) and microglia labelled by TMEM119 (green) in cortex in WT and Lag3-/- mice Extended Data Figure 4e-f: Quantification of Lag3 positive cells in neurons and in microglia  **Extended Data Figure 5** Lag3 is expressed in neurons in Lag3L/L-YFP mice Extended Data Figure 5a-b: Cellular localization of Lag3 in a Lag3 Loxp reporter line with a YFP (yellow fluorescence protein) signal knocked into the Lag3 locus (Lag3L/L-YFP) Extended Data Figure 5c: A1E1 interacts with L3D3 Extended Data Figure 5d: Validation of the enrichment of the endolysosomes in the P3 fraction Extended Data Figure 5e-f: Validation of WT primary cortical neurons for Lag3 expression and Aplp1 expression with Tuj1, Oligo2, GFAP, IBA1 immunoblotting. Lag3-/- and Aplp1-/- primary cortical neurons are the negative controls, and WT brain lysate is the positive control Extended Data Figure 5g: Quantification shows no significant difference of Lag3 expression between WT and Aplp1-/- brain lysate with immunoblots. Extended Data Figure 5h: No significant difference of Aplp1 expression between WT and Lag3-/- brain lysate with immunoblots Extended Data Figure 5i: The co-staining of WGA (plasma membrane maker), Lag3, and Aplp1 in non-permeabilized WT and Aplp1-/-/Lag3-/- primary cortical neurons.  **Extended Data Figure** **6** α-syn PFF binding to Lag3 and Aplp1 Extended Data Figure 6a: Binding signal of α-syn-biotin PFF to WT, Aplp1­­–/–, Lag3–/­–, and Aplp1­­–/–/Lag3–/­– cortical neurons Extended Data Figure 6b: Binding images of α-syn-biotin PFF (63 nM) to Aplp1­­–/–/Lag3–/­– neurons transduced with Aplp1, Lag3, or Aplp1+Lag3 by lentivirus Extended Data Figure 6c: The uptake signal of α-syn-biotin PFF (31 nM and 1 μM) in Aplp1 nor Aplp1(mut9) transfected cells Extended Data Figure 6d: Epitope of anti-Lag3 410C9 for mouse Lag3 Extended Data Figure 6e: α-Syn PFF treatment of the cellular extract of FLAG-Aplp1 and Myc-Lag3 transfected HEK293FT cells significantly increases the co-IP of FLAG-Aplp1 and Myc-Lag3 Extended Data Figure 6f: α-Syn PFF increases the co-IP of Myc-Lag3 and FLAG-Aplp1 Extended Data Figure 6g: Anti-Lag3 (410C9) cannot significantly inhibit the uptake intensity of α-syn-biotin PFF in APLP1-expressing cells Extended Data Figure 6h: APLP1(mut9) can significantly increase the uptake intensity of α-syn-biotin PFF in Aplp1-/- primary cortical neurons Extended Data Figure 6i: The binding intensity of α-syn-biotin PFF (31 nM) to APLP1, Lag3, and APLP1-Lag3, and the inhibitory efficacy of 410C9 Extended Data Figure 6j: The uptake intensity of α-syn-biotin PFF (31 nM) by APLP1, Lag3, and APLP1-Lag3 , and the inhibitory efficacy of 410C9 Extended Data Figure 6k-l: APLP1 and Lag3(ΔD1) can increase binding and uptake of α-syn-biotin PFF (500 nM)  **Extended Data Figure 7** α-Syn PFF binding to primary neurons overexpressing Aplp1 and Lag3 Extended Data Figure 7a: Representative images of α-Syn PFF binding to WT primary cortical neurons and neurons overexpressing Aplp1, Lag3 or Aplp1 and Lag3 Extended Data Figure 7b: Quantification of α-syn-biotin PFF binding signals to WT, Aplp1, Lag3 overexpressing primary cortical neurons  **Extended Data Figure 8** Deletion of Aplp1 and the Aplp1-Lag3 prevent neurodegeneration induced by α-syn PFF Extended Data Figure 8a: Representative TH and Nissl staining images in the SNpc of α-syn PFF-injected hemisphere in the WT, App–/–, and Aplp1­­–/– Extended Data Figure 8b: Stereological counting of the number of Nissl- and TH-positive neurons in the substantia nigra via unbiased stereological analysis  Extended Data Figure 8c: Representative TH immunohistochemistry images in the striatum of α-syn PFF injected brain of WT, Aplp1­­–/–, and Aplp1­­–/–/Lag3–/­– mice Extended Data Figure 8d: Quantifications of TH-immunopositive fiber densities in the striatum Extended Data Figure 8e-g: Striatal metabolites levels in WT, Aplp1­­–/–, and Aplp1­­–/–/Lag3–/­– mice  **Extended Data Figure 9** Anti-Lag3 410C9 blocks neurodegeneration induced by α-syn PFF in vivo Extended Data Figure 9a: Body weight assessment of WT mice for 6-months after stereotaxic injection of α-syn PFF or PBS, and i.p.(intraperitoneal) treated with 410C9 or mIgG Extended Data Figure 9b: Ratio of 410C9 detected in CSF vs plasma was roughly 0.5% at 3 days Extended Data Figure 9c-d: 410C9 turnover in WT mice Extended Data Figure 9e-f: Co-localization of α-syn-biotin PFF with Rab7 is inhibited by 410C9 in vivo Extended Data Figure 9g-j: The representative images of pS129 immunostaining from α-syn PFF-injected WT mice with treatment of 410C9 or mIgG Extended Data Figure 9k-m: Immunoblot analysis of the striatum from α-syn PFF or PBS in WT mice Extended Data Figure 9n-p: Striatal metabolites levels of DOPAC, HVA, and 3M measured by HPLC-ECD