Comment on the article “A Super-Resolved View of the Alzheimer’s Disease-Related Amyloidogenic Pathway in Hippocampal Neurons”

In the discussion of Yu et al. [1], the authors indicate one of the main messages as follows:
“In this context we would like to point out that we used STED microscopy, which enables a resolution of around 20–30 nm in the x,y-plane and around 100 nm in the z-axis. Due to the lower resolution along the z-axis, it is important to avoid overlap along the x-axis. A previous study using STORM microscopy suggested that AβPP is present in both the pre- and post-synapse [50]. However, this is probably an artefact due the fact that the axons and dendrites were positioned on top of each other and the resolution along the z-axis is limited even with STORM microscopy. Another study, using SIM microscopy and pre-and postsynaptic markers separated in the x-y plane showed presynaptic localization [51], in line with our findings.”

Our concerns regarding the central idea on the selective localization of AβPP are obtained from Fig. 4 in the manuscript [1] are listed below:

• The most glaring issue is in Fig. 4, which connects to the central theme of the discussion arguing for the absence of full-length AβPP and CTF. AβPP-CT staining in panels A and B would indicate the difference in the use of non-uniform scaling where the dynamic ranges of AβPP-CT imaging is quite different in both panels. This is very much evident since the frequency of occurrence of AβPP-CT when colocalized with synaptophysin is many-fold more than PSD95 in the same neuronal processes. The images in the panels indicate that there was either an issue with staining or an issue with segmentation and choosing of synapses since the density of detected AβPP-CT staining is very different from the upper to lower panel. This is very important since this qualitative analysis forms the crux of the message.
• On analyzing the colocalization of AβPP-CT, it could be observed that a lot of independent AβPP-CT staining is aligned laterally opposite to the synaptophysin tag further confirming the presence of AβPP in postsynaptic compartments. This strengthens the observation that is described in previous comments, which deals with selective dynamic scaling of the data to represent the authors hypothesis than what is present in the data.
• Standardization was not performed. Leica 3D STED can be operated in 2D mode with high lateral resolution and 3D mode with improved axial and lateral resolution. In the 2D mode, the expected experimental resolution is 50-60 nm at best, and in the 3D mode, X, Y, and Z resolution changes to a near isotropic PSF of around 90-100 nm in 3D. Resolution should be confirmed by using a marker for Point Spread Function (a fluorescent object less than 30 nm) when they compare it with techniques of comparable resolution.
• The paper should inform the readers about segmentation protocols or how/from where these spines were selected/annotated without a bias.
• A simple follow-through on Kedia at al. [2] shows that they have used several super resolution paradigms and exhaustive quantification to rule out this artefact occurring. They have relied on multiple super resolution imaging paradigms like PALM, STORM, and UPAINT along with both 2D and stereographic analysis of 3D STED in the cited paper [2]. Additionally, Kedia et al. [2] observed both endogenous and ectopically expressed molecules to interpret data in the same manuscript. The PALM studies show that ectopically expressed AβPP molecules are laterally diffused into morphologically characterized spines (Fig. 2 and Figure S4 of [2]). It was also supplemented by UPAINT data where they label only the AβPP present on the surface (Figure S5). Secondly, Kedia et al. [2] have used both 2D STED and 3D STED (Figure S9) to show that AβPP counter labelled by C Terminal antibody is present in postsynaptic density and other compartments of the excitatory synapse. In line with observation of STORM data in Kedia et al. [2], other paradigms confirm the presence of AβPP in the spine and functional zones of the synapse. Lastly semiautomated sampling and thresholding from 1000s of synapses Kedia et al. [2] used is robust and unbiased.
• The argument from the Rice et al. [3] paper is that there is less AβPP in post- compared to presynapse (Fig. S1 of [3]). Rice et al. [3] indicate differential expression of AβPP in pre-and postsynapse but not absence of the same.
• Presence of AβPP in postsynaptic compartment is in consensus with several biochemical, electrophysiological, and microscopic observations putting full-length AβPP in both pre- and postsynaptic compartments [4-17]. Of note, the seminal works put full length AβPP in dendritic microdomains, endosomes and excitatory synapses, presynaptic compartment, and dendritic spines [18-21]. The most rigorous set of studies by Helm et al. and Wilhelm et al., to date, confirm the presence of full-length AβPP in both mushroom and stubby spines as well as the synaptic membrane of presynaptic compartment [20, 21]. Helm et al. [21] and Wilhelm et al. [20] used a combination of ensemble based super resolution microscopy (STED), quantitative biochemical methods, electron microscopy, and molecular modelling to extract copy numbers of AβPP in neuronal cells and different kinds of spines. The work from Helm et al. [21] follows up their seminal work in quantifying AβPP numbers in the presynaptic compartment where they showed full length AβPP on the surface of the presynaptic membrane. Additionally, several layers of additional evidence that cannot be summarized in this letter puts full length AβPP in pre/post synapses, and we should also consider observations that highlight interaction between pre/post synaptic AβPP in synapses, surface staining assays, and how expression of detrimental forms of AβPP affected AMPA receptor recruitment and diffusion, linking their post synaptic localization and function.

The evidence we list here, along with the issues observed in Fig. 4, point out that the conclusion that “neither full-length AβPP nor APP-CTF was present at the post synapse” cannot be confirmed with the paradigm Yu et al. [1] used in the paper nor does it confirm the observations in Kedia at al. [2] is an artefact nor their statement about Rice et al. [3] confirming the absence of AβPP or AβPP-CTF in post synapse.

Deepak Nair
Centre for Neuroscience
Indian Institute of Science
deepak@iisc.ac.in

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