Immune Receptor May Smuggle α-Synuclein into Neurons, Hasten Proteopathy
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Researchers have identified a receptor that acts as a welcome mat for toxic α-synuclein particles on the surface of neurons. In the September 30 issue of Science, researchers led by Han Seok Ko, Valina Dawson, and Ted Dawson of Johns Hopkins University School of Medicine in Baltimore describe how lymphocyte activation gene-3 (LAG3), a member of the immunoglobulin superfamily of receptors, binds α-synuclein fibrils and triggers their endocytosis into neurons. Blocking or knocking out LAG3 in neuronal cultures or in animals mitigated the transmission of α-synuclein between neurons, and dampened accumulation as well as toxic effects of the fibrils on motor function. While much about the immune receptor’s role in α-synuclein transmission remains to be ironed out, the authors proposed it could make a useful therapeutic target to treat Parkinson’s and other synucleinopathies.
“If transmission of α-synuclein plays a role in the pathogenesis of PD, then interfering with the transmission would be expected to be disease-modifying,” Dawson told Alzforum. “Hopefully one day we can test this by blocking LAG3 in a clinical trial.” Because anti-LAG3 antibodies are already being tested as cancer treatments, the tools may already exist, he added.
LAG3 Opens the Door. LAG3 escorts α-synuclein fibrils into neurons (left), facilitating spread of the toxic protein. Cells treated with LAG3 antibodies (middle) or lacking the receptor (right) poorly take up the fibrils. [Image courtesy of Mao et al., Science 2016.]
Scientists believe α-synuclein spreads from neuron to neuron in a prion-like fashion. Aggregates of the normally cytoplasmic protein somehow exit neurons, gain entry into neighboring cells, and then corrupt healthy forms of the protein to misfold into toxic miscreants (see Aug 2009 news and Oct 2011 news). While such spread has been reported in mouse models and even in humans following transplants of fetal grafts of dopaminergic neurons (see Apr 2012 news; Nov 2012 news; and Apr 2008 news), the mechanisms that facilitate it, and the importance of transmission in the disease process, remain blurry (see Apr 2016 webinar). A recent study described a chaperone-mediated pathway that exports α-synuclein from the cell (see Jun 2016 news; Jun 2016 news), but offered no explanation for how the protein then gains access to neighboring neurons—a puzzle that researchers are also trying to solve for other cytoplasmic proteins, including tau, and for Aβ (see Apr 2016 webinar).
First author Xiaobo Mao and colleagues screened for receptors that ease the entry of α-synuclein fibrils into cells. Onto a library of cells overexpressing different transmembrane receptors, the researchers sprinkled preformed fibrils (PFFs) of α-synuclein conjugated with biotin. After probing the cells with biotin’s binding partner, streptavidin, three transmembrane proteins popped out: LAG3, neurexin 1b, and Aβ precursor-like protein 1 (APLP1). Of those three, LAG3 was the most selective for PFFs over α-synuclein monomers, so the researchers continued to investigate this receptor. They acknowledged that while the other two receptors were less specific for fibrils, they may also contribute to their entry into cells.
Dawson told Alzforum he was initially surprised by the discovery of LAG3 in the screen. “This is known for its role as an immune receptor on T cells,” Dawson said. “We wondered why it bound α-synuclein fibrils.” Indeed, LAG3 reportedly dampens T cell responses, which is why efforts are underway to block the receptor to unleash the full anti-cancer potential of T cells (see Nguyen et al., 2015; and clinical trials.gov). Despite its fame as an immune receptor, the researchers observed LAG3 expression in cortical neurons, but not in cultured microglia or astrocytes. The researchers found that α-synuclein fibrils bound to cortical neurons from wild-type mice, but less so to those from LAG3 knockouts.
Using a series of deletion mutants, the researchers ultimately homed in on residues 52 to 109, in the D1 domain of LAG3, as essential for fibril binding. As one of four immunoglobulin domains in LAG3, D1 also binds to MHC II molecules. In an accompanying perspective, Mathias Jucker of the University of Tübingen and Mathias Heikenwälder of the German Cancer Research Center in Heidelberg commented that these domains are rich in β-sheets, and tend to latch onto other proteins with similar attributes. The preponderance of β-sheets in α-synuclein fibrils could thus make them ideal LAG3 targets, they wrote.
What happens to α-synuclein after being ensnared by LAG3? To determine whether it then gained entry into the cell, the researchers attached the dye pHrodo red—which only fluoresces once inside acidic, endosomal compartments—to α-synuclein fibrils and added them to cortical neurons. The fibrils turned up in endosomes in wild-type cells, but less in LAG3 knockout neurons. Overexpressing LAG3 in either wild-type or knockout cells dramatically increased the fibrils’ entry into endosomes, and also boosted α-synuclein’s co-localization with Rab5, an endosomal marker.
The researchers next investigated whether LAG3-mediated entry of α-synuclein fibrils would exacerbate neuropathology. In wild-type cultures of cortical neurons treated with fibrils, the researchers observed a build-up of phosphorylated, insoluble α-synuclein, as well as malfunctions in calcium signaling, decrease in synaptic proteins, and cell death. All of these pathological hallmarks occurred to a much lesser extent in LAG3 knockout cells. Anti-LAG3 antibodies that blocked the receptor’s interaction with α-synuclein also suppressed these responses.
Is LAG3 required for transmission of α-synuclein fibrils between neurons? To find out, the researchers used a culture system with three sequential chambers, positioned in such a way that neurons in each could form connections with those in neighboring chambers, but added fibrils could not diffuse from one chamber to another. The researchers found that when α-synuclein fibrils were added to the first chamber, they spread to the second and ultimately the third chamber if all neurons expressed LAG3. However, if the middle chamber was either empty or occupied by cells lacking LAG3, α-synuclein largely failed to reach the third chamber. Adding anti-LAG3 antibodies to the middle chamber also prevented spread of the fibrils. These findings indicated that LAG3 played a role in passing α-synuclein fibrils between neurons.
Transmission Take-Down.
Injected α-synuclein fibrils (green) propagated into nearby dopaminergic neurons (red) in wild-type animals, but not LAG3 knockouts. [Image courtesy of Mao et al., Science 2016.]
Finally, the researchers wanted to determine whether LAG3 played a role in α-synuclein spread between neurons affected by Parkinson’s disease, and if that transmission was toxic. They injected α-synuclein fibrils directly into the dorsal striatum of wild-type or LAG3 knockout mice, then checked for spread of α-synuclein pathology into the neighboring substantia nigra pars compacta (SNpc) 30 and 180 days later. At both time points, mice lacking LAG3 had less than half as much α-synuclein in the substantia nigra as did wild-type mice. Dopamine neurons were also spared in LAG3 knockouts, while many died in the wild-type mice. Motor function defects accompanied the neuronal losses—wild-type mice displayed an odd clasping behavior when dangled by their tails, slid sloppily down a pole, and had poor grip strength. On the other hand, LAG3 knockout mice injected with fibrils performed similarly to mice injected with saline solution on all of these tests.
“This is a really good paper and helps address the top controversy in the field right now, namely whether α-synuclein spreads from neuron to neuron,” commented David Sulzer of Columbia University in New York. “This [LAG3] receptor provides a potential mechanism whereby endocytosis of α-synuclein is vastly enhanced.” Sulzer added that like any good study, the findings only spark more questions, including what happens to the fibrils after they are endocytosed, why only some types of neurons are susceptible to α-synuclein pathology, and whether the other receptors identified in the screen also play a role in α-synuclein transmission.
Todd Golde of the University of Florida in Gainesville viewed the data cautiously. He noted that while the researchers demonstrated a role for LAG3 in neurons in culture, more complex interactions, potentially involving other cell types, could be at play in the brain given the receptor’s role in immunity. He also pointed out that because much more APLP1 is expressed in the brain it may have a stronger effect on α-synuclein in vivo than LAG3 does. “It is almost certain that both of these type 1 membrane proteins are shed into the media, and would act like decoy receptors,” he added. “Again this would seem to complicate the straightforward interpretation of this provocative data.”
Despite all of the potential complications and myriad questions that arose from the data, Patrik Brundin of the Van Andel Research Institute in Grand Rapids, Michigan, was intrigued and impressed by the findings. “The study has identified a new potential therapeutic target for Parkinson’s disease and related synucleinopathies, and the future will tell if it is possible to develop strategies to slow disease progression in patients by interfering with LAG3,” he wrote to Alzforum.
Jucker and Heikenwälder shared Brundin’s enthusiasm: “Although many important issues remain to be resolved, the remarkable interaction of LAG3 and aggregated α-synuclein calls for additional research to determine the physiological function of LAG3 in the brain and to evaluate the potential of LAG3 as a therapeutic target for modifying the course of Lewy body dementia and Parkinson’s disease,” they wrote.—Jessica Shugart
References
News Citations
- Research Brief: α-synuclein Spoils the Neural Neighborhood
- Modeling Sporadic PD in a Dish?
- Synthetic Synuclein Corrupts Native Along Mouse Brain Networks
- Toxic Synuclein Corrupts Native in Wild-Type Mice
- Dopaminergic Transplants—Stable But Prone to Parkinson’s?
- Can’t Degrade That Pesky Misfolded Protein? Push It Off the MAPS
- Ushers of Propagation? More Evidence that Chaperones Evict Disease-Associated Proteins
Webinar Citations
Paper Citations
- Nguyen LT, Ohashi PS. Clinical blockade of PD1 and LAG3--potential mechanisms of action. Nat Rev Immunol. 2015 Jan;15(1):45-56. PubMed.
- Jucker M, Heikenwalder M. Immune receptor for pathogenic α-synuclein. Science. 2016 Sep 30;353(6307):1498-1499. PubMed.
External Citations
Further Reading
Primary Papers
- Mao X, Ou MT, Karuppagounder SS, Kam TI, Yin X, Xiong Y, Ge P, Umanah GE, Brahmachari S, Shin JH, Kang HC, Zhang J, Xu J, Chen R, Park H, Andrabi SA, Kang SU, Gonçalves RA, Liang Y, Zhang S, Qi C, Lam S, Keiler JA, Tyson J, Kim D, Panicker N, Yun SP, Workman CJ, Vignali DA, Dawson VL, Ko HS, Dawson TM. Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3. Science. 2016 Sep 30;353(6307) PubMed.
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Comments
Goizueta Institute @ Emory Brain Health
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 (<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 α-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 provocative data.
Associate Director of the Van Andel Research Institute, and Director of the Center for Neurodegenerative Science
The study by Mao and collaborators is extremely interesting. It addresses the issue of whether specific mechanisms govern neuronal uptake of α-synuclein fibrils from the extracellular space. The demonstrations that lymphocyte-activation gene 3 (LAG3) protein is a surface protein that binds α-synuclein fibrils, specifically in neurons, and that it is involved in the endocytosis of the fibrils are very exciting. The authors then used a variety of approaches to show that prion-like spread of α-synuclein aggregates is mitigated when LAG3 is depleted or blocked, and show this also applies when tested in vivo in animals. The study has identified a new potential therapeutic target for Parkinson's disease and related synucleinopathies, and the future will tell if it is possible to develop strategies to slow disease progression in patients by interfering with LAG3.
Johns Hopkins University School of Medicine
Todd Golde suggests that LAG3 expression is largely restricted to T-cells and, in the brain, microglia. It is true that LAG3 is expressed in both T-cells and the brain. Indeed, one of the first papers characterizing LAG3 showed that it is enriched not only in the thymus and spleen, but present at high levels in the brain as well (Workman et al., 2002). However, to suggest that it is largely in microglia is not supported by empirical evidence. For instance, 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). Moreover, the expression of mouse LAG3 is high in the hippocampus and cortex, with raw expression levels of 9.90 and 6.23, respectively. Unfortunately, the immunological reagents that are available for LAG3 are not of sufficient quality for immunolocalization in neuronal tissue, in that there is a high degree of staining in LAG3 knockout brains and cell cultures. As such, in lieu of immunohistochemistry, we showed, using western blots, that LAG3 is primarily expressed in neurons with undetectable levels in astrocytes and microglia (see Figure S5B of the paper). However, we cannot exclude the possibility that there may be very low or undetectable levels of LAG3 in astrocytes and microglia, or that LAG3 levels increase with activation of astrocytes or microglia. Future studies will be required to determine if LAG3 is expressed at detectable levels in activated astrocytes or microglia. For now, the best evidence indicates that LAG3 is predominantly expressed in neurons.
Golde also suggests that APLP1 levels in the brain are 500-fold greater than LAG3. It is very likely that APLP1 is expressed at higher levels than LAG3, but the fold expression over LAG3 is probably orders of magnitude less. According to Allen Brain Atlas, the raw expression levels of APLP1 in the hippocampus and cortex are 37.34 and 32.85 respectively, which are only modestly higher than LAG3. Although one certainly cannot directly compare mRNA levels of APLP1 and LAG3 to make definitive conclusions, the Allen Brain Atlas mRNA data, and our western blot findings in both mouse and human neurons, indicate that APLP1 is likely to be modestly more abundant than LAG3, but certainly not over 500-fold.
It is true, as Golde suggests, that things may be more complicated than they appear since both the extracellular domain of APLP1 and LAG3 are shed into the media. Moreover, as we report and suggest, LAG3 may not be the only way in which pathologic α-synuclein gets into cells. These and other questions await further experimentation.
We agree with Patrik Brundin that our study identified a new potential therapeutic target and we are looking forward to the day when we test whether interfering with LAG3 is a disease-modifying therapy for Parkinson’s disease.
References:
Workman CJ, Rice DS, Dugger KJ, Kurschner C, Vignali DA. Phenotypic analysis of the murine CD4-related glycoprotein, CD223 (LAG-3). Eur J Immunol. 2002 Aug;32(8):2255-63. PubMed.
Goizueta Institute @ Emory Brain Health
Our consortium RNAseq data is available at synapse.org, along with other groups’ data.
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 it’s 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 microglial sensome.
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 certainly flawed.
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