Nanotube‐like processes facilitate material transfer between photoreceptors

Abstract Neuronal communication is typically mediated via synapses and gap junctions. New forms of intercellular communication, including nanotubes (NTs) and extracellular vesicles (EVs), have been described for non‐neuronal cells, but their role in neuronal communication is not known. Recently, transfer of cytoplasmic material between donor and host neurons (“material transfer”) was shown to occur after photoreceptor transplantation. The cellular mechanism(s) underlying this surprising finding are unknown. Here, using transplantation, primary neuronal cultures and the generation of chimeric retinae, we show for the first time that mammalian photoreceptor neurons can form open‐end NT‐like processes. These processes permit the transfer of cytoplasmic and membrane‐bound molecules in culture and after transplantation and can mediate gain‐of‐function in the acceptor cells. Rarely, organelles were also observed to transfer. Strikingly, use of chimeric retinae revealed that material transfer can occur between photoreceptors in the intact adult retina. Conversely, while photoreceptors are capable of releasing EVs, at least in culture, these are taken up by glia and not by retinal neurons. Our findings provide the first evidence of functional NT‐like processes forming between sensory neurons in culture and in vivo.


1st Editorial Decision
Dear Dr. Pearson, Thank you for transferring your manuscript to EMBO Reports, which was previously reviewed at The EMBO Journal. Having looked at everything, I would like to invite you to submit a manuscript with minor revisions as outlined below.
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Thank you again for giving us to consider your manuscript for EMBO Reports, I look forward to your minor revision.
Kind regards, Deniz Senyilmaz Tiebe --Deniz Senyilmaz Tiebe, PhD Editor EMBO Reports Response to specific points raised by Reviewer 2 1.) Major: The major point of contention remains in that the current manuscript still does not depict any evidence that the nanotubes mediate "intercellular communication" between photoreceptors. In their response, the authors state that they believe that "intercellular communication" does not require the material being exchanged to be functionally relevant. This makes no sense and would be incredibly misleading to the readers. The authors point out that several previous papers describe nanotubes as mediating "intercellular communication". In some cases, this is warranted (e.g. demonstration of active propagation of Ca2+ signals within nanotubes). In many cases, this is absolutely not warranted (e.g. when the authors merely demonstrate the transfer of a GFP reporter). Using the overinterpretations of other papers as justification to repeat a similar overinterpretation is not appropriate.
We accept the reviewer's concerns. We have revised the manuscript to remove the use of the term "intercellular communication" beyond the introduction and discussion, where nanotubes and EVs are discussed in broader context. We have replaced this with our original term "material transfer".
2.) Major: I must stand by the point that there is no evidence in this manuscript that extracellular vesicles (EVs) mediate intercellular communication between photoreceptors and Muller glial cells. While photoreceptors may contain multivesicular bodies, this is not a sufficient evidence that photoreceptors produce exosomes in vivo and, in fact, the authors recognize this weakness of their argument. While Muller glia can take up injected EVs (or apparently any type of membrane material as shown in a revised Figure 2), there is no evidence that this is a normally occurring phenomenon. In fact, the authors admit that in their own chimera experiments, they do not observe any such event. Yet, they claim that: "we show for the first time that sensory neurons can engage in intercellular communication in vivo by both EVs and open-end NT-like processes, each targeting different cell populations." Please revise your conclusions here and elsewhere accordingly.
As noted above, we have amended the manuscript to remove any assertions regarding intercellular communication and have revised the descriptions and conclusions around photoreceptor derived EVs to state that we observed their uptake by Muller glia, and not photoreceptors, following injection in vivo and that this shows EVs are not the mediators of material transfer between photoreceptors.
3.) Major: Please show the images for the UV-treated transplanted photoreceptors (Figs. 2G and 5E). This is essential to control for the non-specific Muller glia uptake of membranous debris.
It is unfortunate that this oversight was not picked up by any of the reviewers in the first submission, as we had this information available. Full quantification of these experiments was included in two figures and no transfer was seen in any UV-treated transplantations, but we agree that inclusion of an image would support this quantification. We have included an additional image of UV-treated Nrl.Cre +/-transplants in Expanded View We consider this assertion to be unfair, given the recent acceptance of the co-submitted manuscript by Ortin-Martinez/Wallace on exactly the same topic. Both groups show very similar data, and both conclude the role of nanotube-like structures mediating material exchange between photoreceptors. To say that one, but not the other, is novel raises significant concerns.
We are happy to include a new sentence that makes specific reference to findings from not only Ortin-Martinez and colleagues, but also other papers at that time, that there were indications that material transfer might involve a physical interaction (page 9, start of second paragraph). However, it must be emphasised that this is different to actually elucidating the cellular mechanisms, as both we, and Ortin-Martinez and colleagues themselves, have now gone on to show. Indeed, Ortin-Martinez and colleagues concluded in their 2016 paper that "Donor/host DNA and mitochondrial transfer, and intercellular exchange via microvesicle/exovesicle GFP, tunneling nanotubes have been described in various systems, offering prospective cellular transfer mechanisms." 5.) Minor: As another over-emphasis of the "communicative" capacity of EVs, the authors state that: "Previously regarded as part of the cell's 'garbage disposal system', EVs are now recognized as lipidencapsulated carriers of bioactive material, including cytosolic and membrane proteins and genetic material, which can alter acceptor cell function in culture and in vivo". While EVs can indeed have functional significance, they are not always "carriers of bioactive material". Please soften this line.
We have revised the sentence as requested (see page 3, start of first paragraph), which now reads: "Previously regarded as part of the cell's 'garbage disposal system', EVs can carry cytosolic and membrane proteins and potentially even genetic material, which have been reported to alter acceptor cell function in culture and in vivo". 6.) Minor: Actin does not seem to be involved in material transfer through nanotubes while being important for nanotube formation. Indeed, the transfer was decreased by ~2-fold in the presence of actin inhibitors, which exactly corresponds to the observed ~2-fold reduction in nanotube formation. This should be made more clear in the text.
Thank you for pointing out this additional observation. We have added a sentence stating this to the relevant section (page 13, end of paragraph 1). Figure 1g. Scale bars for top panels should all be the same. No comparison between transducin, recoverin, and rhodopsin can be made as currently shown. We have amended the scale bars in the recoverin, rhodopsin and Crx graphs to include the split scale, as used for transducin -see Fig. 1g.   At the end of this email I include important information about how to proceed. Please ensure that you take the time to read the information and complete and return the necessary forms to allow us to publish your manuscript as quickly as possible.

7.) Minor:
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