Lee SH, Le Pichon CE, Adolfsson O, Gafner V, Pihlgren M, Lin H, Solanoy H, Brendza R, Ngu H, Foreman O, Chan R, Ernst JA, DiCara D, Hotzel I, Srinivasan K, Hansen DV, Atwal J, Lu Y, Bumbaca D, Pfeifer A, Watts RJ, Muhs A, Scearce-Levie K, Ayalon G. Antibody-Mediated Targeting of Tau In Vivo Does Not Require Effector Function and Microglial Engagement. Cell Rep. 2016 Aug 9;16(6):1690-700. Epub 2016 Jul 28 PubMed.
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New York University School of Medicine
NYU School of Medicine
New York University
New York University
We read this paper with interest. Because the authors reference our work critically, we would like to clarify the points raised by the authors, specifically neuronal uptake of tau antibodies and the presence of Fc receptors on neurons.
We do not dispute the authors’ assertion that their antibodies are effective without being internalized by neurons. Several groups have reported success in preventing the spread of tau using antibodies that do not enter neurons (Castillo-Carranza et al., 2014; d'Abramo et al., 2013; Yanamandra et al., 2013). We have seen this mechanism of action in our own work as well (Congdon et al., 2016; Congdon et al., 2014; Congdon et al., 2015, SFN Abstr. Chicago, IL 579.13/C43). Blockage of neuronal uptake of tau certainly represents a valid avenue for the development of therapeutics.
However, we take issue with the suggestion that such uptake detected by us in brain slice cultures may be explained by mechanical damage to cells and membranes during sectioning. As detailed in one article referred to (Congdon et al., 2013), we performed control experiments to ascertain whether uptake was solely on the surface level, or distributed within the tissue. We retained some of the slices that had been incubated with radiolabeled antibody, and further sectioned the tissue. The radioactivity of each section was then assessed using a liquid scintillation counter. We observed similar levels of radioactivity throughout the tissue, indicating that the antibody uptake was not limited to the surface (Congdon et al., 2013). Additionally, when preparing the slice cultures for immunohistochemistry, the tissue was fixed and sectioned (Congdon et al., 2013; Gu et al., 2013). The sections that were stained came from throughout the slice.
Furthermore and as reported, we have seen internalization of tau antibodies in multiple model systems. In our initial report on the efficacy of active immunization to target tau pathology, we found that antibodies purified from immunized animals entered the brain and co-localized intraneuronally with tau markers (Asuni et al., 2007). We further confirmed these findings in whole animals given intracarotid or intravenous injections of either whole antibody or its scFv fragment. Both the whole antibody and the fragment were able to cross the blood brain barrier, and co-localized with tau and endosomal markers inside neurons (Krishnaswamy et al., 2014). We have also observed antibody uptake in brain slices, primary neuronal cultures, and human neuroblastoma cells lines, as detected by multiple methodologies including confocal imaging, western blotting, fluorescence measurement/flow cytometry, and radioactivity (Congdon et al., 2013; Gu et al., 2013; Shamir et al., 2016; Krishnamurthy et al., 2011). Other groups have also shown neuronal uptake of antibodies against tau as well as antibodies recognizing Aβ and α-synuclein (Masliah et al., 2005; Masliah et al., 2011; Tampellini et al., 2007; Collin et al., 2014; Kondo et al., 2015; Gustafsson et al., 2016).
Collectively, these data clearly demonstrate that antibody uptake into neurons is not dependent on broken cell membranes. It is likely that differences between studies in neuronal uptake of antibodies are primarily related to antibody properties. As we have repeatedly discussed, not all antibodies are taken up into cells to the same extent, which may in large part be explained by differences in antibody charge (for example see review, Pedersen and Sigurdsson, 2015).
With respect to Fc receptors on neurons, the authors state that they do not observe detectable levels of expression in neurons, and posit that our findings stem from non-specific antibody reactivity in our experiments. Based on their RT-PCR data (Figure 5A), it appears that detectable neuronal FcR levels were observed in their samples because otherwise ΔCt values could not be calculated. The values presented are relative to housekeeping genes and show that the FcR subtypes have higher expression levels in microglia compared to neurons, which is as expected. Likewise, the confocal images in Figure 5C are qualitative and may just indicate higher expression levels in microglia than in neurons.
The authors refer to publically available mouse transcriptome as further evidence that these receptors are not present but the database indicates detectable levels of Fc receptor expression in the purified neurons (Zhang et al., 2014), which again is much less than in microglia, as expected. Finally, it is known that expression levels can change depending on various factors such as the developmental stage of cells under study, so it is impossible to generalize what is or is not expressed in different models/tissue.
With regard to non-specific binding, we agree that it is always a factor to consider, but ours is not the only report of Fc receptors on neurons. Several other groups have published such findings using a variety of methods (Andoh and Kuraishi, 2004; Andoh and Kuraishi, 2004; Fernandez-Vizarra et al., 2012; Kam et al., 2013; Mohamed et al., 2002; Nakamura et al., 2007; Qu et al., 2011; Suemitsu et al., 2010; van der Kleij et al., 2010; Fuller et al., 2014; Okun et al., 2010). Finally, we obtained similar results with an Fc blocker as we did with an inhibitor of receptor-mediated endocytosis (Congdon et al., 2013).
Having addressed these critical points, we do agree with the authors’ overall conclusion that an antibody without an effector function is worthy of further development. Similar findings have been reported in the Aβ field although there are conflicting results, which may be explained by differences between antibodies, models, and experimental design (Bard et al., 2003; Das et al., 2003; Bacskai et al., 2002). Both the authors' antibodies are effective but the two are not always directly compared, and it is not clear at which dose because it is not appropriate to use a t-test for post-hoc analysis of ANOVA data as done throughout the article. Perhaps they would be more efficacious if they could enter neurons where most of pathological tau resides. These investigators are ideally suited to engineer these antibodies to improve their uptake into neurons to assess this possibility, as is routinely done in the cancer immunotherapy field to modulate uptake of antibodies into cells.
We appreciate the opportunity to address the issues raised in the article, and look forward to seeing additional work from the authors.
J. Gu, S. Krishnaswamy of the Departments of Neuroscience and Physiology of the New York University School of Medicine and P.K. Krishnamurthy Oligomerix Inc. in New York contributed to this comment.
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