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  • Reply to: Comment on the article “A Super-Resolved View of the Alzheimer’s Disease-Related Amyloidogenic Pathway in Hippocampal Neurons”   1 week 5 days ago

    We are grateful to the editor and authors for clarifications and corrections included in the final version of the paper in response to our comments. We thank the authors for the edits on Paragraph 6 of the Discussion section in the final version and their response to our comments.

    Deepak Nair

    Centre for Neuroscience
Indian Institute of Science


  • Reply to: Comment on the article “A Super-Resolved View of the Alzheimer’s Disease-Related Amyloidogenic Pathway in Hippocampal Neurons”   1 month 1 week ago

    Dr. Deepak Kumaran Nair, we are proud of our study, which has been carefully and thoroughly conducted and we stand firmly behind all the data presented. Despite your aggressive tone and several incorrect statements, we thank the Editors for giving us the opportunity to clarify the points raised. In fact, we believe that these points are very important to highlight.

    Regarding the performance of the STED microscope used, we slightly modified the 6th paragraph in the discussion and specified the calibrated resolution upon correction of the proofs. This, however, does not change our conclusions, if anything, it rather strengthens them.

    One important point emphasized in this study [1], as well as in two of our previous studies [2, 3], is the need to take into consideration the biological 3D direction of the synapse in order to separate the pre- and postsynaptic sides, even with the super-resolution microscopy techniques used here. We have spent several years imaging neurons with synaptic markers and actin staining by super-resolution microscopy and know how difficult it can be to find synapses in the preparations where the pre- and postsynapse can be visualized clearly separated, without overlap along the z-axis. In the primary hippocampal neuron cultures, axons are intimately surrounding the dendrites in an intricate manner to form synaptic contacts, where the synaptic cleft is only around 25 nm wide. The synapses in these cultures are present in various directions. Thus, in order to determine the pre- versus postsynaptic side, we select synapses for which the pre- and postsynapse are separated in the x,y plane and use 2D STED, the mode for which we obtain the best possible resolution in the x,y plane (which is not possible to obtain along the z-axis even with 3D STED) to image them. Notably, using super-resolution microscopy techniques with even higher resolution, such as MINFLUX and iPALM, we will be able to overcome such issues in future approaches.

    Importantly, as specified in our article, synapses were selected in an unbiased manner:
    Actin staining was used to visualize the morphology of the neurons, enabling selection of synapses where the pre- and postsynaptic sides are located side-by-side with the spine extended in the xy plane (to avoid the risk of false positive overlap along the z-axis). Healthy neurons were selected from the actin staining in confocal mode”.

    On top of that, we would like to emphasize that the synapses were selected with the AβPP channel off in order to perform the selection in an unbiased manner with respect to the AβPP staining.

    We believe that, in order to determine the synaptic side, our approach is more exact than approaches using automatic detection of thousands of synapses where the resolution is not sufficient or the direction of the synapse is not known. This is verified by the clear-cut data we obtained from 60 synapses in Fig 4D. We could have calculated a higher number of synapses if we deemed it to be necessary. However, there is such a clear difference between the AβPP C-terminal staining in the pre- versus postsynapse, that the statistical evaluation is very certain with n=60. Please note that these data were from three different experiments (three different batches of mice) stained and imaged on different days.

    Regarding Fig. 4, you seem to have missed the fact that the A and B panel were taken from different images from different staining combinations. Scaling and dynamic ranges were carefully set. The reason that there is more AβPP-CT staining in the A-panel is that the level of axons and presynapses is higher in this panel than in the B-panel. In images where we wanted to determine the synaptic localization, we used only two STED channels, since it is increasingly difficult to obtain high resolution when more STED channels are added. The actin staining helps to outline the shape of the dendrites, but the staining is much weaker in the axons [3]. Thus, it is easier to ensure the direction of the synapse in cases where a presynaptic marker and actin staining are combined than when actin staining and postsynaptic marker are combined. In panel 4B, we point at regions where we are sure of the direction since there is not such a high density of axons in this region. Figure 4A and B are two original images out of several showing the same localization. We have imaged many samples and taken many images and can assure that these ones are representative. As you can see in Fig. 4D, we analyzed 60 synapses from each staining combination and the results are very clear.

    As we have written in the paper, and as you correctly noted, not all AβPP-CT colocalized with the synaptic vesicle marker synaptophysin in Fig. 4A. Some colocalized with other types of vesicles. We also found AβPP-CT in other parts of neurons (axons, soma, and dendrites) but not in the dendritic spines. It is obvious that AβPP-CT did not colocalize with dendritic spines, since the spines are clearly stained by actin. We believe that most of the AβPP-CT not colocalizing with synaptophysin are still in axons but some could also be in dendrites but not the postsynapse.

    We cited the studies by Kedia et al. [5] and Rice et al. [4] and compared them to our study due to the fact that these studies are the ones we are aware of that have used super-resolution microscopy to study the pre- versus postsynaptic localization of AβPP-CT in fixed primary hippocampal neurons. Two of these studies ([1] and [4]) show similar results, but not [5]. Figure S1 in [4] shows a SIM image with the pre- and postsynaptic markers clearly separated. The SIM image clearly shows AβPP overlapping with the pre- but not the postsynaptic marker.

    Having said that, we are well aware of the fact that there is controversy with regards to the synaptic localization of AβPP. As summarized in our recent review article, studying the subcellular localization of AβPP and its fragments in neurons is highly challenging for several reasons [6]. The subject is extensive, and it is not possible to refer to all excellent articles available in this field, which are plenty-fold more than the ones cited by Dr. Nair in his Letter to Editor. There may be differences between different model systems used (e.g., different cell types and different regions in the brain). AβPP and its fragments play many roles in neurons, and they are certainly not only present inside the neuron but also released and bind to receptors on the neuronal membrane, reportedly both in the pre- and postsynaptic region, and can be transferred from neuron to neuron and thus affect synaptic functions in many ways. Our study focused on intracellular AβPP with the purpose of elucidating AβPP processing along the amyloidogenic pathway. In summary, our results are valid for the model system we used, i.e., mouse primary hippocampal neurons.

    On behalf of all authors,
    Sophia Schedin Weiss

    REFERENCES

    [1] Yu Y, Gao Y, Winblad B, Tjernberg L, Schedin Weiss S (2021) A super-resolved view of the Alzheimer's disease-related amyloidogenic pathway in hippocampal neurons. J Alzheimers Dis, doi: 10.3233/JAD-215008.

    [2] Schedin-Weiss S, Caesar I, Winblad B, Blom H, Tjernberg LO (2016) Super-resolution microscopy reveals gamma-secretase at both sides of the neuronal synapse. Acta Neuropathol Commun 4, 29.

    [3] Yu Y, Jans DC, Winblad B, Tjernberg LO, Schedin-Weiss S (2018) Neuronal Abeta42 is enriched in small vesicles at the presynaptic side of synapses. Life Sci Alliance 1, e201800028.

    [4] Rice HC, de Malmazet D, Schreurs A, Frere S, Van Molle I, Volkov AN, Creemers E, Vertkin I, Nys J, Ranaivoson FM, Comoletti D, Savas JN, Remaut H, Balschun D, Wierda KD, Slutsky I, Farrow K, De Strooper B, de Wit J (2019) Secreted amyloid-beta precursor protein functions as a GABABR1a ligand to modulate synaptic transmission. Science 363, eaao4827.

    [5] Kedia S, Ramakrishna P, Netrakanti PR, Jose M, Sibarita JB, Nadkarni S, Nair D (2020) Real-time nanoscale organization of amyloid precursor protein. Nanoscale 12, 8200-8215.

    [6] Lin T, Tjernberg LO, Schedin-Weiss S (2021) Neuronal trafficking of the amyloid precursor protein-What do we really know? Biomedicines 9, 801.

  • Reply to: The FDA Approves Aducanumab for Alzheimer’s Disease, Raising Important Scientific Questions   2 months 4 weeks ago

    When Jack asks “Who was able to buy low-cost shares from Biogen stock prior to share prices zooming 38% after June 7, adding $16 billion to the company’s market value?” I say “the insiders” who somehow knew Aduhelm was going to be approved by the FDA. In short, “sell high buy low”, that is how the stock market operates. With the price of $56,000 per patient-year, Aduhelm can easily be a $56 billion drug which does not work, and does not help anybody except Biogen and investors. Unless . . .

  • Reply to: Kristian Steen Frederiksen   4 months 3 days ago

    No comments

  • Reply to: Levels of Retinal Amyloid-β Correlate with Levels of Retinal IAPP and Hippocampal Amyloid-β in Neuropathologically Evaluated Individuals.   5 months 1 week ago

    The paper is interesting because, consistent with the recent scientific literature, it contributes to the characterization of the different Aβ isoforms deposited in the retina of AD patients, with the aim of identifying new in vivo approaches and tools that allow an early diagnosis of Alzheimer's disease.

  • Reply to: Neurobiology of COVID-19.   5 months 1 week ago

    Comprehensive review concerning a topic still largely unknown at the time of publication in JAD (2020) and concerning the issues of Covid in relation to the CNS-