Cytotoxic Effect of Amyloid-β1-42 Oligomers on Endoplasmic Reticulum and Golgi Apparatus Arrangement in SH-SY5Y Neuroblastoma Cells

Round 1

Reviewer 1 Report

Il manoscritto è ben scritto ma ci  sono alcune condizioni sperimentali che devono essere meglio controllate e verificate.

Line 86 - How was oligomer formation assessed?

What the authors reported is not sufficient "The 86 AβO products were separated and analyzed using SDS-PAGE and Western blotting" . Since Aβ toxicity is related to the degree of polymerization

The quantity and quality of oligomers (monomers, oligomers, protofibrils or fibrils) formed during the rapid aggregation reaction can be measured as a function of time. Aggregation kinetics can be followed using an in situ ThT fluorescence assay or by TEM morphological analysis.

 Because size and conformation influence the toxicity and internalization of different Aβ oligomers, it is necessary to use biophysical methods to assess the assembly conditions of these oligomers.

In addition, it might be useful to assess whether the different toxicity is related to different internalization of oligomers (monomers, oligomers, protofibrils or fibrils).

It is necessary to better describe how the quantifications shown in the figures were made.

Neurons derived from human iPSCs with AD accumulated AβO and had increased production of reactive oxygen species (ROS). AβO triggers excess intracellular ROS and calcium influx into the cytosol. The authors should evaluate, to confirm treatment toxicity, whether treatment with The manuscript needs extensive revision and further monitoring of oligomer formation. leads to increased ROS production in SH-SY5Y cells.

The manuscript needs extensive revision and further monitoring of oligomer formation.

English language is good

Author Response

Reply:  We appreciate your valuable comments on our work and we are agree that it is important to continue investigating the cytotoxic mechanisms of each one of  AβOs involved in early stages of AD. It should be noted that the work showed here only represents the beginning of the project with which we are currently working as a research group. As perspectives, we have objectives to complement this work with the analysis of internalization of various types of oligomers, their differential toxicity as well as possible treatments to avoid these conditions.

On the other hand, as analysis, we refer to the indication of the appearance of heavy bands that arises parallel to the disappearance of the band that represents the monomer, which was demonstrated by means of quantifications of the IR intensity of the WB according to time (we complemented the information in materials and methods section)*. Although on this occasion we didn´t use another methodology to confirm ABOs formation, we want to mentioned our experience in protein aggregation formation and the morphological characterization with TEM (Jarero-Basulto et al., 2013), although in this occasion it was not possible to use it. 

*At the complementary material solicited by the journal, we exemplify how the densitometries were performed.

Reviewer 2 Report

Alzheimer's disease (AD) is a neurodegenerative disease that is currently incurable. According to the most popular theory, its multifactorial cause is mainly the pathological deposition of β-amyloid deposits in the brain tissue in the form of senile plaques and excessively phosphorylated tau protein as neurofibrillary tangles. These structures promote subcellular changes that cause synaptic dysfunction, loss of cell communication, and even cell death, causing cognitive deficits. Great hopes for the cure of patients suffering from Alzheimer's disease are provided by ongoing, intensive research into the cause of its development. The authors of this manuscript aimed to investigate the cytotoxic effect of amyloid-β1-42 oligomers (AβO) on membrane organelles involved in protein processing, namely the endoplasmic reticulum (ER) and the Golgi apparatus (GA). Both intracellular organelles show changes in Alzheimer's disease and other neurological disorders.

SH-SY5Y neuroblastoma cell lines were selected in this study to further understand early changes in aberrant protein processing associated with the initial pathogenesis of AD. Various research methods and techniques were used in the work. The authors showed that the toxic effect of amyloid oligomers is time-dependent, suggesting that this process may be slow and long-lasting and may mark the onset of intrinsic dysfunction and disorganization in the ER and GA. The consequence of exposure to AβO in SH-SY5Y cells is cell death. The results presented by the authors of this paper constitute an important contribution to deepening our knowledge about the mechanism of the toxic effect of AβO on SH-SY5Y cells. These studies indicate that membrane organelles are susceptible to structural and functional changes, hence the importance of further research in this area to confirm these findings.

Comments

The results presented by the authors of this paper constitute an important contribution to deepening our knowledge about the mechanism of the toxic effect of AβO on SH-SY5Y cells. These studies indicate that membrane organelles are susceptible to structural and functional changes, hence the importance of further research in this area to confirm these findings.

The authors should slightly shorten the Results and Discussion section. I suggest a minor revision.

Author Response

Reply: We appreciate your valuable comments on our work and we are agree that it is important confirming these and other data in different experimental models as well as  continue investigating the cytotoxic mechanisms of ABOs involved in early stages of AD.

Both the results and the discussion sections were reduced.

Reviewer 3 Report

Comments to authors:

This manuscript provides evidence for amyloid-β1-42 oligomer-mediated disorganization of the endoplasmic reticulum and Golgi apparatus providing a potential mechanism for the pathology associated with AD.

Introduction.

Page 1, lines 39-43.

This is rather confusing. Normally, APP is processed on the cell membrane with the amyloid peptides being released into the extracellular medium. Lines 39-41 implies that processing occurs in ‘several membraneous organelles’ within the cell to produce the amyloid peptides. This may be the case when the ER is stressed but under normal situations, extracellular release is the primary mechanism. This should be clarified.

Page 2, lines 56-59.

To induce organelle alterations, Aβ oligomers have to be taken up from the extracellular medium. Please provide references that this occurs.

Page 2, lines 61-66.

Please explain how the extracellular accumulation of AβO can lead to damage to intracellular organelles.

Materials and Methods.

Page 2, lines 96-97.

What was the average molecular weight of your generated AβO allowing you to calculate the value of 10µM ?

Results.

Page 5, Fig 1A

The intensity of the AβM band  at 0 hours is low compared to the intensity of the AβO bands at later times. Is this due to greater interaction of the anti-Aβ1-42 with the oligomeric forms?

Page 5, lines 197-198.

Explain briefly how a concentration of 10µM was derived.

Discussion:

Page 12, lines 963-967.

Can you comment on the source of the Aβ observed intracellularly on exposure to AβO in Figure 3? Is this likely to be due to uptake of AβO from the extracellular compartment or does extracellular AβO lead to ER stress, leading to APP processing in the ER and other organelles?  

Author Response

Reply 1: Although it has been considered by many time that APP is processed mainly on the cell membrane with the amyloid peptides being released into the extracellular medium. Many evidences indicate that Aβ can be produced intracellularly in a variety of subcellular compartments, including the endoplasmic reticulum (ER), trans-Golgi network and the endo-lysosomal system. This data may be corroborated in the papers cited below.

1. Chafekar, S.M.; Zwart, R.; Veerhuis, R.; Vanderstichele, H.; Baas, F.; Scheper, W. Increased abeta1-42 production sensitizes neuroblastoma cells for er stress toxicity. Curr Alzheimer Res 2008, 5, 469-474.
2. Greenfield, J.P.; Tsai, J.; Gouras, G.K.; Hai, B.; Thinakaran, G.; Checler, F.; Sisodia, S.S.; Greengard, P.; Xu, H. Endoplasmic reticulum and trans-golgi network generate distinct populations of alzheimer beta-amyloid peptides. Proc Natl Acad Sci U S A 1999, 96, 742-747.
3. Zhang, Y.W.; Thompson, R.; Zhang, H.; Xu, H. App processing in alzheimer's disease. Mol Brain 2011, 4, 3.
4. Hartmann, T.; Bieger, S.C.; Bruhl, B.; Tienari, P.J.; Ida, N.; Allsop, D.; Roberts, G.W.; Masters, C.L.; Dotti, C.G.; Unsicker, K., et al. Distinct sites of intracellular production for alzheimer's disease a beta40/42 amyloid peptides. Nat Med 1997, 3, 1016-1020.

Reply 2: Although in this work we don´t address the mechanism of internalization of Aβ, equal to other research groups, we  are convinced that to generate subcellular alterations, the internalization of Aβ oligomers is not the only way, if not  that these structures also can be toxic from their external cellular location, as many evidences suggested. Nevertheless, some references where Aβ internalization have been reported are the following:

1. Wirths, O.; Multhaup, G.; Bayer, T.A. A modified beta-amyloid hypothesis: Intraneuronal accumulation of the beta-amyloid peptide--the first step of a fatal cascade. J Neurochem 2004, 91, 513-520.
2. Ida, N.; Masters, C.L.; Beyreuther, K. Rapid cellular uptake of alzheimer amyloid betaa4 peptide by cultured human neuroblastoma cells. FEBS Lett 1996, 394, 174-178.
3. LaFerla, F.M.; Green, K.N.; Oddo, S. Intracellular amyloid-beta in alzheimer's disease. Nat Rev Neurosci 2007, 8, 499-509.
4. Rosales-Corral, S.; Acuna-Castroviejo, D.; Tan, D.X.; Lopez-Armas, G.; Cruz-Ramos, J.; Munoz, R.; Melnikov, V.G.; Manchester, L.C.; Reiter, R.J. Accumulation of exogenous amyloid-beta peptide in hippocampal mitochondria causes their dysfunction: A protective role for melatonin. Oxid Med Cell Longev 2012, 2012, 843649.
5. Nazere, K.; Takahashi, T.; Hara, N.; Muguruma, K.; Nakamori, M.; Yamazaki, Y.; Morino, H.; Maruyama, H. Amyloid beta is internalized via macropinocytosis, an hspg- and lipid raft-dependent and rac1-mediated process. Front Mol Neurosci 2022, 15, 804702.

Reply 3: The extracellular mechanism of damage could involve mainly the AβO interaction with specific receptors generating alterations in common signaling pathways affecting the intracellular homeostasis. Particularly, we think that possibility one way to generate organelle alteration indirectly due to previous alteration of cytoskeleton generated as response to AβO-receptors interaction. Another explanation would be that the AβO- NMDA receptors interaction cause an increase level of Ca2+ in the intracellular space. In addition, the AβO are capable to interact with the plasmatic membrane and generate pores that also contribute to increased level of this ion. By its part, Ca2+ increased levels can induced oxidative stress and this condition in turn generate the peroxidation of membrane lipids of different organelles; also can activate different proteolytic enzymes. In addition to describe before, exist many other options.

Reply 4: The molecular weight of our AβOs generated in this work was variable from 45 KDa to 250 KDa, but we don´t considered the average weight to them to calculate the value of 10µM. Instead, we considered the monomeric molecular weight of Aβ lyophilized (Eurogentes, Anaspec Peptide, Amino acid analyst, M.W 4514.4), as reported in Klein et al., 2002 or Arrazola Sastre et al., 2023. It is worth mentioning that in previous works we have carried out in vitro aggregations of other proteins following the same procedure.

1. Arrazola Sastre, A.; Luque Montoro, M.; Llavero, F.; Zugaza, J.L. Amyloid beta(1-42) oligomers induce galectin-1(s8) o-glcnacylation leading to microglia migration. Cells 2023, 12.
2. Klein, W.L. Abeta toxicity in alzheimer's disease: Globular oligomers (addls) as new vaccine and drug targets. Neurochem Int 2002, 41, 345-352.
3. Jarero-Basulto, J.J.; Luna-Munoz, J.; Mena, R.; Kristofikova, Z.; Ripova, D.; Perry, G.; Binder, L.I.; Garcia-Sierra, F. Proteolytic cleavage of polymeric tau protein by caspase-3: Implications for alzheimer disease. J Neuropathol Exp Neurol 2013, 72, 1145-1161.

Reply 5: The intensity of monomer band at 0 hours although it seems to be smaller compared to the intensity of the AβO bands at later times, it is not. The perception could be due to the saturation of the wb reaction in the multiple banding of the different types of oligomers.

Reply 6: Although it is known that the concentrations of Aβ in the brain under physiological conditions are very low (nano molar levels), the concentration in this same organ under pathological conditions is unknown, although it can be high (micro molar levels). Many studies have used different synthetic Aβ peptides (1-42, 25-35) at several concentrations (e.g., 0.1–10 µM) and it have been observed that the toxic effect is concentration depending. A final concentration of 10 µM monomer equivalent is frequently used. For that reason, we decided to use this concentration.

Reply 7: We cannot ensure that the Aβ oligomers are inside the cell; in the continuity of the work we will perform tests to confirm or rule out possible internalization. So far and based on our observations, we believe that the oligomers could be attached to the cell membrane but still outside the cell. We include an image (less time to exposition) where we can support this comment.

We appreciate your valuable comments on our work.

"Please see the attachment (include a figure)" 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors did not make the requested corrections regarding the monitoring of fibril aggregation status. The manuscript was revised only in the references. The part about the toxicity of fibrils in relation to the degree of aggregation (monomers, oligomers, protofibrils or fibrils) was not discussed.

 I understand that the Authors are at the beginning of the project they are working with, but at least in the discussions they should add something about the limitations of this study. It is never mentioned in the manuscript that this study is preliminary. Therefore, I would like to ask the Authors to make an extra effort to review the manuscript and point out the lack of data on fibril morphology.

Enghish Launguage is good

Author Response

Reply 1:  We appreciate your valuable comments on our work. We insist that we demonstrated the monitoring of AβO aggregates formation with WB and its repetitions. This is a technique that is regularly used to demonstrate the formation of aggregates in reference to time (we included references in materials and methods about it). As we mentioned before, it is not the only methodology, but it is the only technical that we have available. Currently we don´t have enough time or resources to include the monitoring requested by you (ThT or TEM) for further validation of the current work. In addition, we included a paragraph in the discussion section where we address the fibrils and various types of aggregates toxic importance. We decided not to delve further into this point because it is not the priority objective of the work.

Reply 2: During the discussion section we repeatedly mentioned the limitations of the study (highlight in the text) and stressed that more research should be done in this regard. On the other hand, we believe that it is not necessary to mention that it is a preliminary study. We revisited the manuscript and made the suggested changes.