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.
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
- 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
- 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.
- 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.