Neuronal LAG3 facilitates pathogenic α-synuclein neuron-to-neuron propagation

Abstract

Lymphocyte activation gene 3 (LAG3) is a key receptor involved in the propagation of pathological proteins in Parkinson's disease (PD). This study investigates the role of neuronal LAG3 in mediating the binding, uptake, and propagation of α-synuclein (αSyn) preformed fibrils (PFFs). Using neuronal LAG3 conditional knockout mice and human induced pluripotent stem cells-derived dopaminergic (DA) neurons, we demonstrate that LAG3 expression is critical for pathogenic αSyn propagation. Our results show that the absence of neuronal LAG3 significantly reduces αSyn pathology, alleviates motor dysfunction, and inhibits neurodegeneration in vivo. Electrophysiological recordings revealed that αSyn PFFs induce pronounced neuronal hyperactivity in wild-type (WT) neurons, increasing firing rates in cell-attached and whole-cell configurations, and reducing miniature excitatory postsynaptic currents. In contrast, neurons lacking LAG3 resisted these electrophysiological effects. Moreover, treatment with an anti-human LAG3 antibody in human DA neurons inhibited αSyn PFFs binding and uptake, preventing pathology propagation. These findings confirm the essential function of neuronal LAG3 in mediating αSyn propagation and associated disruptions, identifying LAG3 as a potential therapeutic target for PD and related α-synucleinopathies.

Keywords: LAG3; PFFs; electrophysiology; neuron; α-synuclein.

Fig. 1. Neuronal LAG3 deletion attenuated αSyn PFFs-induced pathology propagation.

(A) Experimental design for αSyn PFFs stereotaxic injection and pathology assessment. Immunofluorescence (IF) and immunohistochemistry (IHC) were performed to track pS129-positive αSyn pathology. PFFs were injected into the striatum of LAG3^L/L-YFP (control) and LAG3^L/L-N−/− (neuronal LAG3 conditional knockout) mice, with comprehensive pathological and behavioral analyses conducted 6 months post-injection. (B) Spatial distribution of pS129-positive αSyn pathology following PFFs injection, visualized as red dots across brain sections. (C) Quantitative analysis of pS129 immunoreactivity intensity in multiple brain regions, including cerebral cortex (D), striatum (E), amygdala (F), substantia nigra (G), and entorhinal cortex (H). Statistical analysis performed using unpaired two-tailed Student's t-tests. ns, not statistically significant. Data presented as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001. n = 5 biologically independent mice per group. Scale bar, 100 μm.

Fig. 2. Attenuation of αSyn PFFs-induced behavioral deficits and neurodegeneration in neuronal LAG3 conditional knockout (LAG3^L/L-N−/−) mice.

Behavioral assessments at six months post αSyn PFFs stereotaxic intrastriatal injection: (A) Pole test, with a maximum descent time of 60 s to assess motor coordination and bradykinesia. (B) Forelimb grip strength test, (C) Four-limb grip strength test, (D) Rotarod test to evaluate motor performance and balance, and (E, F) Representative nest-building images following 16-hour nestlet introduction across experimental groups. Error bars represent mean ± SEM. Statistical significance determined by ANOVA followed by Tukey's multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. n = 10 biologically independent mice per group. (G) Representative photomicrographs of coronal mesencephalon sections showing tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN) region. Unbiased stereological quantification of (H) TH-positive and (I) Nissl-positive neurons in the SNpc region. Data presented as mean ± SEM; **p < 0.01, ***p < 0.001, ns, not significant, n = 6 biologically independent mice. Scale bar, 500 μm.

Fig. 3. αSyn PFFs increased firing frequencies in SNc DA neurons from WT mice, but not in LAG3^−/− mice.

(A-B) Schematic representation of midbrain slice incubation protocol. (A) Left, DA neuron location in SNc. Right: As the white arrow points, neurobiotin-filled cell (red) expressing tyrosine hydroxylase (TH, green) from SNc. Scale bar: 50 μm. (B) Experimental timeline for phosphate-buffered saline (PBS) or αSyn PFFs incubation in SNc slices and subsequent electrophysiological recordings. (C) Firing frequency of SNc DA neurons in PBS and αSyn PFFs-treated slices from WT and LAG3^−/− mice using cell-attached recording. WT mice-PBS, n = 8; LAG3^−/−-PBS mice, n = 3; WT mice-αSyn PFFs, n = 11; LAG3^−/− mice-αSyn PFFs, n = 6. (D) Whole-cell recording of SNc DA neuron firing frequency in PBS and αSyn PFFs-treated slices from WT and LAG3^−/− mice. Note the significant increase in firing frequency in the αSyn PFFs-treated WT group compared to PBS, whereas LAG3^−/− mice do not exhibit this increase. WT mice-PBS, n = 9; LAG3^−/−-PBS mice, n = 3; WT mice-αSyn PFFs, n = 13; LAG3^−/− mice-αSyn PFFs, n = 4. (E) Miniature EPSC frequency in SNc DA neurons from PBS and αSyn PFFs-treated WT and LAG3^−/− mice using whole-cell recording. WT mice-PBS, n = 9; LAG3^−/−-PBS mice, n = 6; WT mice-αSyn PFFs, n = 10; LAG3^−/− mice-αSyn PFFs, n = 6. (F) Representative traces of spontaneous firing activity and neuronal response to hyperpolarizing 200 pA current injection (2 s duration) following PBS and αSyn PFFs in vitro incubation. Scale bars: 20 mV and 200 ms. (G) αSyn PFFs induces increased SNc DA neuron excitability in WT mice, demonstrated by reduced current accommodation during incremental current injections (50, 100, 150, 200, 250, 300 pA). LAG3^−/− SNc DA neurons treated with PBS or αSyn PFFs maintain current accommodation similar to WT control conditions. WT mice-PBS, n = 15; LAG3^−/−-PBS mice, n = 4; WT mice-αSyn PFFs, n = 11; LAG3^−/− mice-αSyn PFFs, n = 4. Values expressed as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001, ns, not significant. Statistical significance determined by ANOVA with Tukey's post-hoc correction.

Fig. 4. Anti-human LAG3 (17B4) antibody blocks human αSyn PFFs binding, endocytosis, and pathology in induced pluripotent stem cell (iPSC)-derived DA neurons.

(A) αSyn-biotin PFFs binding to iPSC-derived DA neurons with mouse immunoglobulin G (mIgG) or 17B4 antibody treatment. Binding signal (red) quantified for individual cells. n = 9 cells for each group. Scale bar, 5 μm. (B) Quantification from n = 3 independent experiments. Statistical significance determined by Student's t-test. (C) mIgG and 17B4 antibodies blocking human LAG3 inhibit αSyn-biotin PFFs binding on iPSC-derived DA neurons. Colocalization of internalized αSyn-biotin PFFs with Rab7 assessed by confocal microscopy. Scale bar, 5 μm. (D) Quantification of colocalization from n = 3 independent experiments. (E) pS129 signal reduced by 17B4 antibodies in iPSC-derived DA neurons 2 weeks after αSyn PFFs treatment. Scale bar, 10 μm. (F) Quantification of pS129 levels from n = 3 independent experiments. Statistical significance determined by ANOVA with Tukey's post-hoc correction. Data presented as means ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.