Synuclein Snatch: Antibodies Snag Protein En Route to Next Neuron

In another step forward for passive immunotherapy, researchers have shown that antibodies to α-synuclein can rescue neurons and improve symptoms in a mouse model of sporadic Parkinson’s disease. Writing in the June 11 Cell Reports online, senior author Virginia Lee and colleagues at the University of Pennsylvania’s Perelman  School of Medicine in Philadelphia suggest that their antibody grabs malformed synuclein as it traverses the synapse between neurons. Lee told Alzforum that immunotherapy should be high on the list of PD treatment options.

“I was not overwhelmed by the magnitude of the effect, but it is a start,” said Todd Golde of the University of Florida in Gainesville, who was not involved in the work. “It adds to the evidence that antibodies against synuclein can have some moderate efficacy.” Other research groups are also attempting passive immunotherapy to soak up pathogenic proteins in Parkinson’s and other neurodegenerative conditions (Bae et al., 2012; Masliah et al., 2011; Yanamandara et al., 2013).

First author Hien Tran, now at Isis Pharmaceuticals, Inc., in Carlsbad, California, tried the antibody treatment in a PD model developed by the Lee lab (see Nov 2012 news story). It uses injection of synthetic synuclein fibrils into the dorsal striatum of wild-type mice. Over time, synuclein inclusions appear in areas connected to the injection site, suggesting that the pathology travels from cell to cell, converting normal synuclein into the aggregate-prone form, as is thought to occur in human brains (Braak et al., 2003). This model mimics the sporadic disease that the majority of PD patients have. However, Golde and colleagues were recently unable to replicate it, a discrepancy some experts attribute to slight differences in how each lab prepared the synuclein starter fibrils (see Apr 2014 news story). 

Tran injected synuclein fibrils into the mice and waited a week for the pathology to get started. She then began weekly, intraperitoneal treatment with the antibody Syn303. Control mice received non-specific IgG. Tran tested grip strength and coordination by measuring how long the animals could hang from a wire. The anti-synuclein-treated animals consistently outperformed the controls at one, three, and six months after starting immunotherapy. When Tran sacrificed the animals at six months, she observed 30 percent fewer synuclein-positive aggregates in the substantia nigra pars compacta of the group, compared with controls, and 40 percent fewer inclusions in the amygdala. The anti-synuclein-treated mice also lost fewer dopaminergic neurons than the control IgG animals.

Golde said the results were relatively predictable, since the researchers added the disease-inducing fibrils and then added antibodies to stop them. He said it confirms that Lee’s injection model can be used to test therapeutics.

The Mechanism in Question
These results left Lee and Tran wondering which form of α-synuclein was subject to antibody attack: the original seeds or converted cellular synuclein. The antibodies might grab the original synthetic fibrils before they initially enter cells. Or, those synthetic fibrils could convert the native cellular synuclein to a pathological form, and the antibodies could capture these new seeds as they travel between neurons. Tran tried to distinguish these possibilities using cultured primary hippocampal neurons, where synthetic synuclein fibrils can seed aggregation (Volpicelli-Daley et al., 2011). 

First, Tran asked if the antibody blocked cellular uptake of implanted seeds. Adding anti-synuclein antibody before the fibrils dramatically reduced pathology, Lee said. Tran also used immunofluorescence to show that antibody treatment prevented fibril entry.

However, Lee noted that synuclein seeds are not waiting in the extracellular space in people with Parkinson’s; they must originate from within cells. She and Tran hypothesized that neurons release pathological α-synuclein at the synapse, whereupon the next neuron rapidly takes it up. For the most part, antibodies do not enter neurons, but would have the opportunity to grab the synuclein during that brief time it was in the synapse.

Synuclein Transmission.

In a triple-chamber culture dish, neurons send axons across small grooves allowing synuclein aggregates (blue) to cross to adjacent chambers. Image courtesy of Cell Reports, Tran et al., Figure 5.

The researchers mimicked cell-to-cell transfer of synuclein with a three-chamber microfluidic culture system. Neurons forge connections between chambers by sending axons through one-way channels (Taylor et al., 2005, and Apr 2009 news story). When Tran added myc-tagged preformed synuclein fibrils to one end of the device, she observed myc-free synuclein aggregates appearing in the neurons in the adjacent chamber one week later, and in next chamber down the line a week after that. The lack of myc tags indicated that those aggregates were made of endogenous synuclein, not the original seeds. The authors concluded that the preformed fibrils seeded new inclusions, which transferred the pathology to neurons in the next chambers.

To test whether immunotherapy prevents this kind of cell-to-cell transmission of endogenous synuclein, Tran made synthetic synuclein seeds lacking the Syn303 epitope. The antibody still blocked the appearance of synuclein inclusions in the downstream chambers, confirming the antibody prevented direct cell-to-cell transfer of native synuclein, not just uptake of artificial seeds.

The authors noted the antibodies could work in their animal model either by blocking original seed uptake, or by interfering with neuron-to-neuron transmission later on, or both. “The mechanism by which their antibody is working in vivo remains uncertain,” agreed Marc Diamond of Washington University in St. Louis, who was not involved in the paper. He and Lee both suggested that the antibodies, once bound to synuclein, might promote its uptake by microglia.

Jan Stoehr of the University of California, San Francisco, praised the paper but pointed out that so far, all immunotherapies on neurodegenerative diseases have failed. However, Stoehr suggested Lee’s cell culture and animal models would be an ideal setting in which to compare antibodies and pick the best one to take into the clinic. Lee said her antibody’s affinity for α-synuclein was too low for human trials, but the lab is working on better ones. Others have already made the leap: The biotech company Neotope Biosciences, in collaboration with Roche, initiated a Phase 1 study of anti-synuclein antibodies in April.—Amber Dance.

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References

News Citations

  1. Toxic Synuclein Corrupts Native in Wild-Type Mice
  2. Are Synuclein Seeds Non-Starters?
  3. How Cells, and Drugs, Try to Control Glutamate in the Synapse

Paper Citations

  1. Bae EJ, Lee HJ, Rockenstein E, Ho DH, Park EB, Yang NY, Desplats P, Masliah E, Lee SJ. Antibody-aided clearance of extracellular α-synuclein prevents cell-to-cell aggregate transmission. J Neurosci. 2012 Sep 26;32(39):13454-69. PubMed.
  2. Masliah E, Rockenstein E, Mante M, Crews L, Spencer B, Adame A, Patrick C, Trejo M, Ubhi K, Rohn TT, Mueller-Steiner S, Seubert P, Barbour R, McConlogue L, Buttini M, Games D, Schenk D. Passive immunization reduces behavioral and neuropathological deficits in an alpha-synuclein transgenic model of Lewy body disease. PLoS One. 2011 Apr 29;6(4):e19338. PubMed. RETRACTED
  3. Yanamandra K, Kfoury N, Jiang H, Mahan TE, Ma S, Maloney SE, Wozniak DF, Diamond MI, Holtzman DM. Anti-Tau Antibodies that Block Tau Aggregate Seeding In Vitro Markedly Decrease Pathology and Improve Cognition In Vivo. Neuron. 2013 Oct 16;80(2):402-14. PubMed.
  4. Braak H, Del Tredici K, Rüb U, De Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging. 2003 Mar-Apr;24(2):197-211. PubMed.
  5. Volpicelli-Daley LA, Luk KC, Patel TP, Tanik SA, Riddle DM, Stieber A, Meaney DF, Trojanowski JQ, Lee VM. Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron. 2011 Oct 6;72(1):57-71. PubMed.
  6. Taylor AM, Blurton-Jones M, Rhee SW, Cribbs DH, Cotman CW, Jeon NL. A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods. 2005 Aug;2(8):599-605. PubMed.

External Citations

  1. Phase 1 study

Further Reading

Papers

  1. Lindström V, Ihse E, Fagerqvist T, Bergström J, Nordström E, Möller C, Lannfelt L, Ingelsson M. Immunotherapy targeting α-synuclein, with relevance for future treatment of Parkinson's disease and other Lewy body disorders. Immunotherapy. 2014 Feb;6(2):141-53. PubMed.
  2. Yu YJ, Watts RJ. Developing Therapeutic Antibodies for Neurodegenerative Disease. Neurotherapeutics. 2013 Apr 3; PubMed.
  3. Schneeberger A, Mandler M, Mattner F, Schmidt W. Vaccination for Parkinson's disease. Parkinsonism Relat Disord. 2012 Jan;18 Suppl 1:S11-3. PubMed.
  4. Valera E, Masliah E. Immunotherapy for neurodegenerative diseases: Focus on α-synucleinopathies. Pharmacol Ther. 2013 Jun;138(3):311-22. PubMed.

Primary Papers

  1. Tran HT, Chung CH, Iba M, Zhang B, Trojanowski JQ, Luk KC, Lee VM. Α-synuclein immunotherapy blocks uptake and templated propagation of misfolded α-synuclein and neurodegeneration. Cell Rep. 2014 Jun 26;7(6):2054-65. Epub 2014 Jun 12 PubMed.

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