Lymphocyte‐Activation Gene 3 Facilitates Pathological Tau Neuron‐to‐Neuron Transmission

Abstract The spread of prion‐like protein aggregates is a common driver of pathogenesis in various neurodegenerative diseases, including Alzheimer's disease (AD) and related Tauopathies. Tau pathologies exhibit a clear progressive spreading pattern that correlates with disease severity. Clinical observation combined with complementary experimental studies has shown that Tau preformed fibrils (PFF) are prion‐like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several cell surface receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remain poorly understood. Here, it is shown that the lymphocyte‐activation gene 3 (Lag3) is a cell surface receptor that binds to PFF but not the monomer of Tau. Deletion of Lag3 or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron‐to‐neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lacking Lag3 selectively in neurons. These results identify neuronal Lag3 as a receptor of pathologic Tau in the brain，and for AD and related Tauopathies, a therapeutic target.


Figure S2 :
Figure S2: Binding of biotin-labeled Tau PFF to primary cortical neurons. a. Representative images of Tau PFF-biotin binding to wild-type (WT) mouse primary cortical neurons, Scale bar, 100μm.b.Quantification of binding of biotin, Tau-biotin monomer and heparin induced Tau-biotin (hep-Tau) PFF and Tau-biotin PFF to mouse primary cortical neurons (Tau-Biotin PFF KD = 276 nM), Data are the means ± SEM, n = 3 independent experiments.

Figure S3 :
Figure S3: Biotin-labeled Tau PFF can bind to Lag3 expressing SH-SY5Y cell surface receptor. a. Tau-biotin PFF binds to Lag3 overexpressing SH-SY5Y cell surface in a saturable manner, as a function of Tau biotin total concentration (monomer equivalent for PFF preparations).Scale bar, 100 μm.b.Immunoblot showing overexpression (OE) of Lag3 into SH-SY5Y cells in the cell surface binding assays.c.Immunoblot showing Lag3 expression level in WT and Lag3 knockout (Lag3 -/-) mouse primary cortical neurons.d.Representative images of Tau PFF-biotin binding to WT and Lag3 -/-mouse primary cortical neurons.Scale bar, 100 μm.e. Tau PFF binds to human recombinant LAG3 as assessed by ELISA.KD = 3.1 nM, n = 3.

Figure S4 :
Figure S4: Tau-biotin oligomer binding to Lag3 in SH-SY5Y cells. a. Flowchart for isolation of oligomeric Tau.b.Representative TEM images of Tau oligomers obtained by sucrose gradient.Globular Tau-biotin oligomers were obtained in 30% glucose gradient.Scale bar, 100 nm.c.Quantification of binding of Tau-biotin oligomer to empty and Lag3 plasmid transfected SH-SY5Y cells.Data are the means ± SEM, n = 3 independent experiments.d.Representative images of Tau-biotin oligomer binding to empty and Lag3 expressing plasmid transfected SH-SY5Y cell surface.Scale bar, 100 μm.

Figure S6 :
Figure S6: Heparin inhibits Tau PFF binding to Lag3 in primary cortical neurons. a. Representative images of Tau-biotin PFF binding in presence of increasing concentration of heparin (0-200 g/ml) to cell surface of primary cortical neurons.Scale bar, 100 μm.b.Quantification of binding of Tau-biotin PFF in presence of heparin, data are the means ± SEM, n = 3 independent experiments.c. representative images of Tau-biotin PFF binding in presence of increasing concentration of heparin (0-200 g/ml) to Lag3-expressing SH-SY5Y cell surface.Scale bar, 100 μm.d.Quantification of binding of Tau-biotin PFF in presence of heparin to SH-SY5Y cell surface.Data are the means ± SEM, n = 3 independent experiments.

Figure S7 :
Figure S7: Tau PFF amplified from tauopathy binding to Lag3. a. ThT fluorescence intensity at 485 nm for PBS, recombinant Tau PFF (Rec Tau) and 10% PSD and PiD patient seeded recombinant Tau PFF (n = 3).Error bars represent SEM.b.Representative images of Tau-biotin PFF to Lag3-expressing SH-SY5Y cell surface at 500 nM concentration.Data are the means ± SEM, n = 3 independent experiments, Scale bar, 100 μm.c.Quantification of binding of different Tau-biotin PFF strains to Lag3 expressing SHSY-5Y cells.d.Scatchard analysis.Data are the means ± SEM, n = 3 independent experiments.

Figure S8 :
Figure S8: Aβ-biotin PFF binding to Lag3 in SH-SY5Y cells. a. Representative images of Aβ-biotin PFF binding to empty and Lag3-expressing plasmid transfected SH-SY5Y cell surface b.Quantification of binding of Aβ fibrils to SH-SY5Y cell surface.Data are the means ± SEM, n = 3 independent experiments, Scale bar, 100 μm.

Figure S10 :
Figure S10: Tau PFF induced increase in intracellular Ca 2+ level is mediated by Lag3. a.Time series imaging of calcium-dependent fluorescence of WT and Lag3 -/-neurons.Neurons were treated with 1 µM Fluo-2 acetoxymethyl (AM) ester for 30 min followed by PBS or 500 nM Tau PFF.Live images were acquired for the indicated time period.Scale bar, 50 µM.b.Ca 2+ levels from WT and Lag3 -/-neurons without Tau PFF treatment.c.Fluo-2 acetoxymethyl (AM) associated fluorometric measurement showing changes in intracellular Ca 2+ signal in WT and Lag3 -/-neurons upon Tau PFF treatment.Ft fluorescence intensity of the indicator at t minutes, F0 is Fluorescence intensity at 0 min.Error bars represent SEM.

Figure S12 :
Figure S12: In vivo transmission and behavioral assessment of Tau PFF injected Lag3 L/L-N- /-mice. a. Representative distribution of Tau pathology (red dots) on coronal sections stained for AT8 distribution of Tau PFF-injected mice.b.Total stay duration in the empty cup and stranger cup in three chamber social interaction test.Student's t-test.*P < 0.01, c-e.Lag3 L/L-N-/-mice did not exhibit significant behavioral deficit in the Y-maze test.Specifically, number of entries in the new arm (c), percent time spent in the new arm (d), percentage of spontaneous alteration of behavior (e), Error bars represent SEM, n = 8 mice.