. Amyloidogenic α-synuclein seeds do not invariably induce rapid, widespread pathology in mice. Acta Neuropathol. 2014 May;127(5):645-65. PubMed.

Recommends

Please login to recommend the paper.

Comments

Make a Comment

To make a comment you must login or register.

Comments on this content

  1. I think this is a very carefully done study, and the authors are to be congratulated. The authors confirm some essential observations that have been made by others regarding the injection of α-synuclein seeds into mouse brain.

    However, they have appropriately raised caution regarding the interpretation of some of the data regarding “prion-like” spread of protein seeds among neurons in neuronal circuits. Specifically, they find that a phospho-antibody used to detect synuclein accumulation can cross-react with neurofilament proteins enriched in white matter, and this may indicate that apparent spread of synuclein aggregates through white matter tracts could be due to an artifact. Further, they observe that reactivity to this antibody can be induced by non-amyloidogenic synuclein injection, which is not consistent with induction of endogenous synuclein fibrils.

    Finally, they raise the important caveat to all existing work with synuclein injection experiments that it is critical to rule out spread of injected seeds through circuits. This could occur by seed uptake from neuronal projections directly into projections to the injection site (e.g., axons) with retrograde transport to distant cell bodies, or it could occur by actual trafficking of injected aggregates into one cell, down axons, and across to connected cells, as happens with agents such as wheat germ agglutinin or viruses. The point is that in the future, in order to fully confirm “prion-like” spread, endogenous synuclein must be shown to move from cell to cell, spreading pathology, and the possibility of injected material doing this must be ruled out.

    On a final note, this study highlights the idea that using only one marker (in this case a phospho-antibody) to monitor protein accumulation is fraught with potential difficulties, and thus that it will be important to use multiple ways of determining induction of an amyloid state in brain proteins. This applies to studies of tau as well as synuclein.

  2. We were happy to see that the authors were able to recapitulate formation of α-synuclein Lewy body and Lewy Neurite-like inclusions by intracranially injecting synthetic fibrils made from recombinant protein into the hippocampus and cortex of both non-transgenic mice and mice overexpressing mutant human α-synuclein. However, these authors do not report spread of pathology as reported previously by our and other labs (Fujiwara et al., 2002; Luk et al., 2012b; Luk et al., 2012a; Rey et al., 2013), thereby claiming that the α-synuclein fibrils do not lead to a prion-like spread of pathology across brain areas. Additionally, Dr. Giasson’s manuscript claims that much of the pathology reported in previous publications largely results from cross-reactivity of the antibody mAB81A, which recognizes phosphorylated α-synuclein, a marker of inclusions. However, there are several methodological differences between Dr. Giasson’s manuscript and the previous studies (Luk et al., 2012b; Luk et al., 2012a).

    In this study, the protocol used to generate fibrils differs significantly from that used by other groups. For example, Sacino and colleagues only formed fibrils for two days. However, if using wild type α-synuclein, it takes at least five to seven days for optimal fibril formation (Wood et al., 1999). Furthermore, the fibrils used in their study were sonicated by mild-bath sonication. As we previously reported (Volpicelli-Daley et al., 2011Luk et al., 2012bLuk et al., 2012a), repeated sonication with a probe tip sonicator is crucial to initiate seeding and spreading of α-synuclein pathology. This is likely because it is necessary to generate small seeds that neurons can take up by macropinocytosis (Holmes et al., 2013). These differences in fibril preparations can easily be seen by comparing electron microscopy images from the Sacino et al. paper (Supplemental Figure 15) to those presented by Luk et al. (Figure 1C, Luk et al. 2012a).

    These methodological differences likely (as the images suggest) led to morphologically and functionally distinct species of α-synuclein being ultimately injected. Data from the labs of Virginia Lee (Guo et al., 2013) and Ronald Melki (Bousset et al., 2013) have demonstrated that vastly different conformations can be generated from identical protein preparations depending on the incubation conditions. Overall, it can be concluded that Dr. Giasson’s group did not obtain the same results because of these critical methodological differences.

    With respect to the mAB81A antibody, Dr. Giasson’s group is correct that when used at a high concentration, mAB81a and other antibodies that recognize phospho-α-synuclein have background staining both in primary neurons and in brain sections. It is indeed typical for most phospho-specific antibodies to have some background staining. However, when mAB81A is diluted to 1:10,000, there is minimal background staining in controls, and intense staining of inclusions in primary neurons treated with fibrils, and sections from fibril injected brains that is well above background. These inclusions can also be visualized with other p-α-synuclein-specific antibodies such as a rabbit polyclonal antibody developed by Virginia Lee’s lab (Volpicelli-Daley et al., 2011), an Epitomics rabbit antibody provided by the Michael J. Fox Foundation (MJFR-13), and antibodies Syn506 and Syn514 that specifically recognize pathologic inclusions (Waxman et al., 2008) and were first characterized by Dr. Giasson.

    In conclusion, we believe that severe methodological discrepancies have resulted in a failure to recapitulate the prion-like spread of α-synuclein observed by many other labs. This highlights the importance for a standardized protocol to be developed.

    References:

    . Structural and functional characterization of two alpha-synuclein strains. Nat Commun. 2013;4:2575. PubMed.

    . alpha-Synuclein is phosphorylated in synucleinopathy lesions. Nat Cell Biol. 2002 Feb;4(2):160-4. PubMed.

    . Distinct α-synuclein strains differentially promote tau inclusions in neurons. Cell. 2013 Jul 3;154(1):103-17. PubMed.

    . Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):E3138-47. PubMed.

    . Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive neurodegenerative α-synucleinopathy in mice. J Exp Med. 2012 May 7;209(5):975-86. PubMed.

    . Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science. 2012 Nov 16;338(6109):949-53. PubMed.

    . Transfer of human α-synuclein from the olfactory bulb to interconnected brain regions in mice. Acta Neuropathol. 2013 Oct;126(4):555-73. PubMed.

    . Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron. 2011 Oct 6;72(1):57-71. PubMed.

    . Characterization of antibodies that selectively detect alpha-synuclein in pathological inclusions. Acta Neuropathol. 2008 Jul;116(1):37-46. Epub 2008 Apr 15 PubMed.

    . alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson's disease. J Biol Chem. 1999 Jul 9;274(28):19509-12. PubMed.

This paper appears in the following:

News

  1. Are Synuclein Seeds Non-Starters?