Pathogenic Protein Spread Lets Think Again Webinar Transcript

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01 Oct 2016

Researchers take identified a receptor that acts as a welcome mat for toxic α-synuclein particles on the surface of neurons. In the September 30 outcome of Science, researchers led by Han Seok Ko, Valina Dawson, and Ted Dawson of Johns Hopkins University Schoolhouse of Medicine in Baltimore describe how lymphocyte activation factor-iii (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 every bit toxic effects of the fibrils on motor function. While much about the immune receptor'due south function in α-synuclein transmission remains to be ironed out, the authors proposed information technology could make a useful therapeutic target to treat Parkinson'due south and other synucleinopathies.

"If manual of α-synuclein plays a part in the pathogenesis of PD, then interfering with the manual would be expected to be disease-modifying," Dawson told Alzforum. "Hopefully one twenty-four hour period we can examination this past blocking LAG3 in a clinical trial." Because anti-LAG3 antibodies are already being tested every bit 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 (correct) poorly take upwardly the fibrils. [Prototype courtesy of Mao et al., Science 2016.]

Scientists believe α-synuclein spreads from neuron to neuron in a prion-similar fashion. Aggregates of the normally cytoplasmic protein somehow exit neurons, gain entry into neighboring cells, and then corrupt good for you 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 post-obit transplants of fetal grafts of dopaminergic neurons (see Apr 2012 news; Nov 2012 news; and Apr 2008 news), the mechanisms that facilitate information technology, and the importance of manual in the disease process, remain blurry (meet Apr 2016 webinar). A recent written report described a chaperone-mediated pathway that exports α-synuclein from the cell (meet Jun 2016 news; Jun 2016 news), merely offered no explanation for how the protein then gains admission to neighboring neurons—a puzzle that researchers are besides trying to solve for other cytoplasmic proteins, including tau, and for Aβ (encounter 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 i (APLP1). Of those three, LAG3 was the near selective for PFFs over α-synuclein monomers, then the researchers continued to investigate this receptor. They acknowledged that while the other ii 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 (run into Nguyen et al., 2015; and clinical trials.gov). Despite its fame as an immune receptor, the researchers observed LAG3 expression in cortical neurons, only 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, equally essential for fibril bounden. As ane of four immunoglobulin domains in LAG3, D1 also binds to MHC Two 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 later on existence ensnared by LAG3? To make up one's mind whether it then gained entry into the cell, the researchers attached the dye pHrodo blood-red—which only fluoresces in one case 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 additional α-synuclein'due south 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, too 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'due south interaction with α-synuclein as well suppressed these responses.

Is LAG3 required for transmission of α-synuclein fibrils betwixt neurons? To find out, the researchers used a culture organization with 3 sequential chambers, positioned in such a style that neurons in each could grade connections with those in neighboring chambers, but added fibrils could not lengthened from one chamber to another. The researchers found that when α-synuclein fibrils were added to the offset sleeping room, they spread to the second and ultimately the 3rd chamber if all neurons expressed LAG3. However, if the centre chamber was either empty or occupied by cells lacking LAG3, α-synuclein largely failed to reach the tertiary bedchamber. Calculation anti-LAG3 antibodies to the middle sleeping accommodation also prevented spread of the fibrils. These findings indicated that LAG3 played a role in passing α-synuclein fibrils between neurons.

Transmission Take-Downwards.

Injected α-synuclein fibrils (green) propagated into nearby dopaminergic neurons (red) in wild-type animals, but not LAG3 knockouts. *[Paradigm courtesy of Mao et al., Science 2016.]*

Finally, the researchers wanted to decide whether LAG3 played a part in α-synuclein spread between neurons affected past Parkinson's disease, and if that transmission was toxic. They injected α-synuclein fibrils directly into the dorsal striatum of wild-blazon 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 on. At both time points, mice lacking LAG3 had less than one-half as much α-synuclein in the substantia nigra as did wild-type mice. Dopamine neurons were as well spared in LAG3 knockouts, while many died in the wild-blazon mice. Motor part defects accompanied the neuronal losses—wild-type mice displayed an odd clasping beliefs 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 Academy 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 simply spark more than questions, including what happens to the fibrils after they are endocytosed, why just some types of neurons are susceptible to α-synuclein pathology, and whether the other receptors identified in the screen also play a part in α-synuclein transmission.

Todd Golde of the Academy of Florida in Gainesville viewed the information cautiously. He noted that while the researchers demonstrated a function for LAG3 in neurons in civilization, more complex interactions, potentially involving other prison cell types, could be at play in the brain given the receptor'southward function in amnesty. He also pointed out that considering much more APLP1 is expressed in the brain information technology may have a stronger effect on α-synuclein in vivo than LAG3 does. "It is about certain that both of these type 1 membrane proteins are shed into the media, and would act like decoy receptors," he added. "Over again this would seem to complicate the straightforward estimation 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 Yard Rapids, Michigan, was intrigued and impressed by the findings. "The study has identified a new potential therapeutic target for Parkinson's affliction and related synucleinopathies, and the hereafter will tell if it is possible to develop strategies to slow illness progression in patients by interfering with LAG3," he wrote to Alzforum.

Jucker and Heikenwälder shared Brundin's enthusiasm: "Although many important issues remain to exist 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 illness," they wrote.—Jessica Shugart

References

News Citations

Enquiry Brief: α-synuclein Spoils the Neural Neighborhood 10 Aug 2009
Modeling Sporadic PD in a Dish? ten October 2011
Constructed Synuclein Corrupts Native Along Mouse Encephalon Networks 26 Apr 2012
Toxic Synuclein Corrupts Native in Wild-Type Mice 16 Nov 2012
Dopaminergic Transplants—Stable Just Prone to Parkinson's? 12 Apr 2008
Can't Degrade That Pesky Misfolded Protein? Push It Off the MAPS 17 Jun 2016
Ushers of Propagation? More Evidence that Chaperones Evict Disease-Associated Proteins 24 Jun 2016