Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3
Authors: Ted M Dawson
Michael Tianhao Ou
Joel Tyson
Ho Chul Kang
Senthilkumar S Karuppagounder
Jianmin Zhang
Sharon Lam
Hyejin Park
Han Seok Ko
Rafaella Araújo Gonçalves
Sung Ung Kang
Donghoon Kim
Chen Qi
James A Keiler
Xiling Yin
Rong Chen
Preston Ge
Xiaobo Mao
Tae-In Kam
Shu Zhang
Yu Liang
Seung Pil Yun
Yulan Xiong
Jinchong Xu
Dario A A Vignali
George Essien Umanah
Creg J Workman
Shaida A Andrabi
Nikhil Panicker
Saurav Brahmachari
Valina L Dawson
Joo-Ho Shin
Journal: Science (2016)
Discipline: Multidisciplinary
Keywords: Parkinson's disease; α-synuclein; LAG3; transmission; endocytosis
Affiliations: Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA 70130-2685, USA., Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA., Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Division of Pharmacology, Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 440-746, South Korea., Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Physiology, Ajou University School of Medicine, Suwon 443-721, South Korea., Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Xin Hua Hospital affiliated to Shanghai Jiaotong University School of Medicine, Shanghai 200092, China., Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Johns Hopkins Institute for NanoBio Technology, Johns Hopkins University, Baltimore, MD 21218, USA., Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA., Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA. Tumor Microenvironment Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232, USA., Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA 70130-2685, USA. Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.   ., Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA 70130-2685, USA.   ., Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Johns Hopkins Institute for NanoBio Technology, Johns Hopkins University, Baltimore, MD 21218, USA. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.   .

Abstract:  Emerging evidence indicates that the pathogenesis of Parkinson's disease (PD) may be due to cell-to-cell transmission of misfolded preformed fibrils (PFF) of α-synuclein (α-syn). The mechanism by which α-syn PFF spreads from neuron to neuron is not known. Here, we show that LAG3 (lymphocyte-activation gene 3) binds α-syn PFF with high affinity (dissociation constant = 77 nanomolar), whereas the α-syn monomer exhibited minimal binding. α-Syn-biotin PFF binding to LAG3 initiated α-syn PFF endocytosis, transmission, and toxicity. Lack of LAG3 substantially delayed α-syn PFF-induced loss of dopamine neurons, as well as biochemical and behavioral deficits in vivo. The identification of LAG3 as a receptor that binds α-syn PFF provides a target for developing therapeutics designed to slow the progression of PD and related α-synucleinopathies. 

Retracted: No

PubPeer comments:
 Interesting comments from Dr. Todd E. Golde (College of Medicine, University of Florida), http: www.alzforum.org news research-news immune-receptor-may-smuggle-synuclein-neurons-hasten-proteopathy 1. Posted: 03 Oct 2016 These data look quite interesting and the binding rather compelling. But I would caution that LAG3 is an immune checkpoint molecule whose expression in mice is thought to be largely restricted to T-cells and, in the brain, largely microglial cells. In humans, LAG3 expression data from RNAseq is less clear on individual cell types, but our own AMP-AD data suggest that again LAG3 in the AD and control brain is present at very low levels ( lt;1 CPM). So, I think the in vivo data may be reflective of a more complex mechanism than what is proposed here. Indeed, given the relative binding between LAG3 and APLP1 (~fivefold difference in affinity for u03b1-synuclein) and the fact that APLP1 is present at ~500-fold higher levels in the brain than LAG3, one would think further investigations into APLP1 are warranted. It is almost certain that both of these type 1 membrane proteins are shed into the media, and would act like decoy receptors. Again, this would seem to complicate the straightforward interpretation of this provocativeu00a0data. 2. Posted: 14 Oct 2016 Our consortium RNAseq data is available at synapse.org, along with other groupsu2019u00a0data. This data and RNAseq data from Ben Barres and colleagues at Stanford is consistent and pretty unequivocal. In humans, Lag3 RNA levels are ~500- to 1000-fold lower in the brain than APLP1. In the single study by Barres and colleagues, LAG3 RNA is almost undetectable. In mice itu2019s about 100-fold lower. In mice Lag3 is clearly a fairly selective microglial transcript (per the Barres study) and our data is consistent with that study. LAG3 is elevated in old APP mice (~threefold) and it falls into the microglial co-expression network. It has also previously been implicated as part of the microglialu00a0sensome. I think one has to be cautious in using ISH data from the Allen Brain Atlas, especially when there are data that simply don t jibe. APLP1, like APP, is very abundant in the brain. LAG3 is not. So the Allen data is almost certainlyu00a0flawed. , 
 Carefully read the paper. The authors used E15.5 (about peak time for microglia invasion into brain?) for preparing primary cortical neuronal culture, would the WT cortical neuron culture contained activated or differentiated mature microglia? Since differentiated mature microglia have high level LAG3, just a few microglia should be enough for the western blot. They should have shown us the immunocytochemistry of the cultures.u00a0 Would their enriched microglia from p1 mouse pups might have already dedifferentiated (they had kept for more than two weeks after isolation), or never ever had a chance to become mature, which led to their no detection of LAG3? And another thing is that non-specific LAG3 band which they claimed, shows in one blot but not the other. So confusing! , 
 Both mouse and human microglia data show LAG3 is one of the major microglia markers. How could the authors completely ignore it, and claim LAG3 was primarily expressed in neurons because LAG3 was not detectable in astrocytes or microglia . Is the LAG3 antibody specific? Zhang Y et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 2014 Sep 3;34(36):11929-47. doi: 10.1523 JNEUROSCI.1860-14.2014. Galatro TF et al., Transcriptomic analysis of purified human cortical microglia reveals age-associated changes. Nat Neurosci. 2017 Aug;20(8):1162-1171. doi: 10.1038 nn.4597. Epub 2017 Jul 3. ![file] , 
 Interesting comments by Dr. Ted Dawson, senior author of this paper, http: www.alzforum.org news research-news immune-receptor-may-smuggle-synuclein-neurons-hasten-proteopathy claming: the Allen Brain Atlas shows that LAG3 mRNA is localized primarily to neurons, including in the substantia nigra pars compacta. In addition, LAG3 has high mRNA levels in the brain consistent with the Workman et al., paper (see Allen Brain Atlas, http: mouse.brain-map.org experiment show?id=71920417). ![file] My understating is, simple in situ hybridization **can NOT** tell the cell type localization. The authors need to read the review by Kettenmann H, Kirchhoff F, Verkhratsky A. Microglia: new roles for the synaptic stripper. Neuron. 2013 Jan 9;77(1):10-8. doi: 10.1016 j.neuron.2012.12.023. The microglia in brain are also very abundant! (A) Throughout the central nervous system microglia (red) surveys neuronal networks (black) and astroglial (blue). (B) laser-scanning micrograph taken from an adult TgH(CX3CR1-EGFP) mouse brain in which microglia is labeled by expression of EGFP. , 
 #5  My understating is, simple in situ hybridization can NOT tell the cell type localization. I think that s maybe too general a statement. For some brain regions, some cell types have rather characteristic distributions and you can get can even get an idea of the sizes of the nuclei from the Allen Brain Atlas. In those cases one can be quite confident about some expression patterns. Consider pyramidal cells in the hippocampus, for instance. However, there are of course regions and cell types where the in situ would be equivocal at best. I don t have any knowledge about cellular distributions in the substantia nigra, to which you were perhaps referring specifically. , 
 ...the other issue of course is that in the Allen Brain Atlas n=1. , 
 #6 Not quite sure the nuclei size of the microglia, there are decent number of microglia in hippocampus, including dentate gyrus, please see ![file] (A) Throughout the central nervous system microglia (red) surveys neuronal networks (black) and astroglial (blue). (B) laser-scanning micrograph taken from an adult TgH(CX3CR1-EGFP) mouse brain in which microglia is labeled by expression of EGFP. Also, please check the Supplemental Video 1: 3D visualization of microglia in CX3CR1-EGFP mouse brain, it shows the individual microglia throughout the entire brain are evident. Chenchen Pan, Ruiyao Cai, Francesca Paola Quacquarelli, Alireza Ghasemigharagoz, Athanasios Lourbopoulos, Paweu00c5u0082 Matryba, Nikolaus Plesnila, Martin Dichgans, Farida Hellal Ali Ertu00c3u00bcrk. Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat Methods. 2016 Oct; , 
 The issue can be easily clarified by RNAseq. Just look at Ben Barres database http: www.brainrnaseq.org If you query LAG3 you see there is practically no RNA in neurons... , 
 Is there anyone following this paper u2018Pathological u03b1-synuclein transmission initiated by binding lymphocyte-activation gene 3u2019. pubmed: 27708076 doi: 10.1126 science.aah3374 . Just a few questions: 1) Are LAG3 primarily expressed in neurons in the central nervous system? From Maou2019s paper, it claims u2018LAG3 was primarily expressed in neurons because LAG3 was not detectable in astrocytes or microgliau2019. While LAG3, also known as CD223, is an immune checkpoint receptor. The mRNA expression is restricted to sparse cells in the adult brain cortex, and defined tracts at the base of the cerebellum and in the choroid plexus of dayu20047 postnatal brain (ref: Phenotypic analysis of the murine CD4-related glycoprotein, CD223 (LAG-3), Workman CJ et al., Eur J Immunol. 2002). u2018In situ hybridization analysis of adult brain has shown that LAG-3 mRNA is expressed in a restricted population of sparse cells throughout the cortex, but absent from the cerebellum. In contrast, very bright and dramatic hybridization was observed in dayu20047 postnatal brain, in defined tracts at the base of the cerebellum, in the developing white matter and in the choroid plexus. Although CD223 mRNA could not be found in human adult brain, this could be age related.u2019 Should not they do more solid experiment to confirm the expression in neurons instead of astrocytes or microglia? 2) Does LAG3 express on astrocytes, oligodendrocytes, and microglia? From u2018Immune Mediators of Central Nervous System Demyelination and Remyelinationu2019, a dissertation submitted to the faculty of the University of North Carolina at Chapel Hill, it shows LAG3 on a subpopulation of astrocytes, oligodendrocyte precursor cells. https: cdr.lib.unc.edu indexablecontent uuid:c751db89-2ae7-4225-88e0-c62eba36f255 3) LAG3 (CD223) is a potential cancer immunotherapeutic target, would the brain penetrable LAG3 antibodies affect the neuron function? There are some other evidence showing that LAG3 is not expressed on human neurons, for example, http: www.proteinatlas.org ENSG00000089692-LAG3 tissue Since finding of LAG3 as a key for pathological u03b1-synuclein transmission, which is a very big thing, it may really be necessary to confirm the LAG3 expression on neurons. , 
 Please rest assure that there is Lag3 protein in neurons. We are preparing a paper, which will be submitted soon. , 
 There following two papers are contradictory, so Confusing!!! 1) LAG3 is not expressed in human and murine neurons and does not modulate u03b1-synucleinopathies https: www.biorxiv.org content 10.1101 2021.04.25.441302v1 2) Aplp1 and the Aplp1-Lag3 Complex facilitates transmission of pathologic u03b1-synuclein https: www.biorxiv.org content 10.1101 2021.05.01.442157v1  The immunoreactivity of anti-GFP, which recognizes YFP co-localized with MAP2 immunostaining in the spinal cord, olfactory bulb, striatum, substantia nigra pars compacta, hippocampus, cortex, cerebellum and brainstem (fig. S3A). The lack of immunoreactivity of anti-GFP in WT mice indicates that the anti-GFP immunoreactivity is specific in Lag3L L-YFP mice (fig. S3B). In addition, anti-GFP colocalizes with IBA1 immunostaining, but not GFAP immunostaining (fig. S3B) ***Which one should we believe? Why the GFP antibody was used to stain YFP in the Dawson s paper? Artifacts?*** , 
 The same group and the same first author, they claimed in their 2016 Science paper, that u2018LAG3 was primarily expressed in neurons because LAG3 was not detectable in astrocytes or microgliau2019. (Science. 2016 Sep 30; 353(6307): aah3374), So many good labs had CLEARLY shown and approved that LAG3 is thought to be largely in microglia. If the LAG3 antibodies they used were specific, LAG3 expression in microglia should be detected in their 2016 Science paper, why they say  LAG3 was expressed in neurons but not in microglia? Now, Mao et al NEW preprint, they show the expression of LAG3 in microglia. (Aplp1 and the Aplp1-Lag3 Complex facilitates transmission of pathologic u03b1-synuclein, https: www.biorxiv.org content 10.1101 2021.05.01.442157v1 ) , 
 And interestingly, in this preprint (Aplp1 and the Aplp1-Lag3 Complex facilitates transmission of pathologic u03b1-synuclein, https: www.biorxiv.org content 10.1101 2021.05.01.442157v1 ), using LAG3-YFP transgenic mice to verify LAG3 protein expression in (mouse) neuron look good, but why the GFP antibody was used to stain YFP? Artifacts? The anti-GFP staining pattern was not convincing, if you look their nuclei counterstaining, it seems very like auto-fluorescence. Can this truly approve the neuronal expression of LAG3, why not do solid and convincing studies like another preprint  LAG3 is not expressed in human and murine neurons and does not modulate u03b1-synucleinopathies  The immunoreactivity of anti-GFP, which recognizes YFP co-localized with MAP2 immunostaining in the spinal cord, olfactory bulb, striatum, substantia nigra pars compacta, hippocampus, cortex, cerebellum and brainstem (fig. S3A). The lack of immunoreactivity of anti-GFP in WT mice indicates that the anti-GFP immunoreactivity is specific in Lag3L L-YFP mice (fig. S3B). In addition, anti-GFP colocalizes with IBA1 immunostaining, but not GFAP immunostaining (fig. S3B) , 
 A recent paper published on EMBO Mol Medicine calls into question the conclusions by Mao et al., 2016 https: www.embopress.org doi full 10.15252 emmm.202114745 , 
 Figure S9A: * Red boxes: Two panels appear to look very similar. Can the authors please check? ![file] , 
 #15 LAG3 is mainly expressed by microglia (Cell 174, 1015u20131030.e16 (2018). 10.1016 j.cell.2018.07.028; Science 2020;370:66u201369. DOI: 10.1126 science.abb8587). LAG3 expression can also be localized to oligodendrocytes and a subpopulation of astrocytes (Chen VS. Immune mediators of central nervous system demyelination and remyelination. University of North Carolina; 2008). The leading authors of this paper were just bravely claiming  **LAG3 was primarily expressed in neurons because LAG3 was not detectable in astrocytes or microglia** . The expression of LAG3 in neuronal cell types is not well supported at all by the other groups, for instance, Rimmerman, N., et al. Mol Psychiatry (2021). https: doi.org 10.1038 s41380-021-01338-0. Emmenegger et al found no evidence of LAG-3 in neuronal cell nor human brain samples using extensive multiple assays. **Even the binding between LAG-3 and PFFs was promiscuous and non-specific **(Emmenegger et al, https: doi.org 10.15252 emmm.202114745). , 
 #15 the Lag3 is not expressed in neurons. I stayed in Mao lab around 1 year , nobody could repeat the data. it s a big lie. Why this paper has not retracted yet, incredible. ,