Peer review of: RNA-binding proteins Musashi and tau soluble aggregates initiate nuclear dysfunction (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

-The premise of the manuscript is the modulation of tau neurotoxicity by Musashi (MS) proteins. 
The finding of the role of RNA-binding proteins Musashi on tau soluble aggregates is novel. Indeed, 
aggregate Tau-mediated toxicity is relevant to understand the pathogenies of AD, and therefore, 
the groups finding of MS-initiated nuclear dysfunction in tauopathies assumes significance. 
-Comparing MSI protein accumulation between different tauopathies allows for the possible 
identification of distinctive and/or common pathogenic mechanisms between AD and other 
neurodegenerative diseases, and hence they will be of interest to others in the community and the 
wider field. 
-In synopsis, the present work is a well-designed and well-executed study. The manuscript is well-
written, and presentations of different points and discussion would move the field forward. 

Criticism: 
- Fig. 1-I- MS1- How many immunoreactive bands did appear in WB when probed with anti-MS1 
antibody? A full blot picture of anti-MS1 with HEK cell lysate and brain extracts should be shown. 
The Suppl fig shows only the activity of the secondary antibody alone. 
-Validation of the specific anti-MS1 band(s) by siRNA-type experiment would be more specific and 
is advisable, if possible. 
-Fig. 1-J- MS2- the same criticisms as above exist. 

-Fig. 2: Authors should be more specific about the human brain region tested. Which Broadmann 
area (number) was used? This may be relevant/ important for studying human brain disorders 
(different than animal work). 
-Fig. 2-F &G- Number of human brain tissues are very limited. This reviewer understands the 
difficulty in procuring autopsied samples from well-matched AD and control subjects; however, the 
study seems underpower, particularly from the context of several confounding factors. 
- I think there are inconsistencies regarding human brain samples. Table 1 shows 11 samples, ALS 
n=3, FTD n=2, AD n=3, control n=3. The blots indicate 12 samples, n=3 for each condition. 

-Fig. 6D- In addition to “Control”, it would be better to show “Control+Tet” treatment in parallel. 
-Supplemental Figure 1. Which region of the “brain” in brain homogenates were used? 

-Further, have the authors anything to say about the stark difference between AD and other 
samples regarding A11 blot responses? 

-The cytoplasmic/nuclear blots require a bit more clarity. Do the authors have any probings that 
show a response in the cytoplasmic samples for any protein at all? 

General comments: Is PCC abbreviation, used in the text, standard?. Readers may confuse it with 
Posterior Cingulate Cortex over Pearson’s Correlation Coefficient. 

Reviewer #2 (Remarks to the Author): 

Summary 
The submitted article by Montalbano et al. observes the interaction of oligomeric tau with the RNA 
binding proteins mushashi 1 and 2 in several neurodegenerative diseases, mouse models and 
stable transgenic cell lines. There are the makings of a very interesting finding here. However, at 
present there is disjointed flow to the data and some over-reach in the interpretation of the result 
meaning the conclusions are a little premature. 
It is interesting to see that MSI1 and 2 are elevated in neurodegenerative disease – but it is not 

clear how this is related to “tauopathies”. The strength of the manuscript suffers because the 
colocalization with tau oligomers appears to be low and the authors appear to have similar 
elevation of MSI1 and 2 in ALS and FTD without tau tangles, perhaps suggesting this elevation is a 
non-specific effect of neurodegenerative disease. It is interesting to see that there are nuclear 
membrane and chromatin modelling deficits in the inducible tau cell line – but it is not clear that 
this is directly related to formation of “mushashi and tau soluble aggregates”. 
Major comments 
The rationale for studying MSI 1 and 2 in tauopathies appears to be supported by the previous 
work of this group and others. However, the rationale for studying tau oligomer/MSI interaction in 
ALS and FTD is less apparent. This issue is particularly relevant to the question of whether 
mushashi dysfunction occur specifically in response to tau pathology or non-specifically in 
response to neurodegenerative disease. The FTD cases used in this study are poorly defined. Table 
1 indicates that the FTD cases are clinically diagnosed but have pathological observation of 
minimal Braak NFTs (less in fact than the control individuals) and little tau accumulation by 
immunoblot (Supp Figure 1B), which would suggest these FTD cases present with TDP-43 
pathology. There should be a neuropathological diagnosis of either FTLD-tau or FTLD-TDP. 
Furthermore, tau pathology is only associated with a small minority of ALS cases. This naturally 
raises the question of whether MSI1 and 2 colocalize and interact with the major pathology of ALS 
and FTLD-TDP, which is TDP-43. This seems particularly pertinent as there appears to be minimal 
colocalization of TOMA2 oligomeric tau and MSI1 and 2 (Figure 1G, H). 
It appears that the microscopic images of TOMA2 and MSI1 and 2 distribution do not clearly 
support the author’s assertion that TOMA2 oligomeric tau and MSI1 and 2 colocalise in AD, ALS 
and FTD. In figure 1G, H, in figure 2C, D, in supplemental figure 2, in supplemental figure 7 the 
vast majority of pixels are either red (TOMA2) or green (MSI1 or 2) but very little is yellow = 
colocalization. Indeed, the TOMA 2 signal in control tissue (supplemental figure 2) appears to be 
minimal, which may explain why an increase in colocalization between TOMA2 and MSIs is 
observed in the affected patient tissue. But it doesn’t follow that the TOMA2 and MSIs actively 
associate. In the patient samples (particularly the ALS and FTD samples) there appears to be 
accumulation of both TOMA2 and MSIs, but these appear to be in distinct puncta. 
On the other hand, in figure 2B there does appear to be a higher degree of colocalization of TOMA2 
and MSI1, and this is supported by the biochemical association of tau MSI1 and 2 with tau in the 
AD insoluble fractions (something which does not occur in the ALS and FTD cases). Line 454-455 – 
“…colocalization of MSI proteins and tau oligomers play a crucial role in AD, ALS and FTD…” 
The authors indicate (line 573 to 575) that higher mag images of tau transgenic mouse tau13/MSI 
PLA “allows us to appreciate… extracellular speckles, present in CTX, DG, CA1 and CA3” - but the 
panels in figure 5C to J are too low magnification to distinguish intra and extracellular distribution. 
In addition, the double labeling for TOMA2/MSI presented in Suppl. Fig. 7 (note that the text 
wrongly refers to Suppl. Fig. 6), does not present convincing evidence of double labeling, contrary 
to what is stated in the results section. 
There appears to be a somewhat hard transition to the data of figure 6. Whereas the prior figures 
focused on mouse brain tissues, figures 6 and 7 focus only on iHEK cells, with no reference to or 
validation in brain tissues. Thus, the flow of the article seems to revert to additional figures from 

the author’s previous publication “Tau oligomers mediate aggregation of RNA‐binding proteins 

Musashi1 and Musashi2 inducing Lamin alteration” (Aging Cell, 2019). While this is excellent data 
it would need to be validated in mouse brain tissue to represent a significant conceptual advance 
from the previous work. At the very least, it would be prudent to test whether tau and MSI 
oligomers cause nuclear membrane defects: show interaction or tau oligomers and/or MSI 
oligomers with nuclear membrane components, show that addition of MSI oligomers alone induce 
nuclear membrane defects or show that tau-oligomer induced nuclear membrane defects are 
blocked by modifying MSIs. Indeed, the from the IPs of MSI1 and 2 show mostly interaction with 
histones, which is a really interesting finding with perhaps significant effects on gene expression. 
However, again the work is about mushashi and tau soluble aggregates [initiating] nuclear 
dysfunction”, but there is little evidence herein that it is specifically soluble oligomers that are 
exerting these effects in the iHEK cells. 
Minor comments 

References 27 and 28 are duplicated. Line 111. 
Line 132 – Would researchers in the field often refer to ALS and FTD without NFTs as tauopathies? 
Miss-spelling of Histone3. Line 246 
Line 442 – “Figure 1F-1G” appears to be incorrect 
Please include high mag images from TOMA2 and MSI1 staining of control tissue in main figure 2. 
Line 488 – the authors presumably mean “aged P301L mice.” 
Line 492 – there appears to be no figure 3S (perhaps this is a typo). 
Figure 3H and K – could the authors make the white/red arrows a little easier to see. 
Figure 3 legend – several miss-spellings of “month”. Lines 515, 523 and 525. 
Line 540 – “we detected a cell specific staining in 7-month old mice”. Which specific cell types are 
the authors referring to? Was staining with cell-type markers performed? 
The authors might discuss why MSI2 shows reduced signal and cytoplasmic distribution in the 7 
month old P301L mice (CA1, CA3, CTX) (line 543-544), but increased signal and perhaps nuclear 
distribution in AD (line 397). 
There is no discussion of supplemental figures 4 and 5 in the main text. Please discuss how aging 
in these WT and tauKO mice effects MSI1 and 2; and whether the changes in MSI1 and 2 in figure 
4 are specific to the tau expression. As such there it is unclear that changes in MSI are tau-
dependent (line 748). 
Line 566 to 568 – Contradicts figure 4 showing increased nuclear signal from MSI1 in 2 month old 
P301L mice? Line 532 “In general, we detected in P301L 2-months old mice a nuclear and axonal 
MSI1 distribution in hippocampi and cortex”. 
Please include secondary only controls to confirm specificity of antibodies for IHC (and either a 
citation or use of knockout tissue to confirm TOMA2 specificity). 
Figure 5 – in 2month old P301L mice, the low magnification image panel A shows no MSI1/TOMA2 
PLA signal in DG. Higher mag image panel D shows robust MSI1/TOMA2 PLA signal in DG nuclei. 
Could the authors explains this apparent mismatch. 
Supplemental Figure 6: The text states that the TOMA2/MSI immunofluorescence is in 
Supplemental figure 6, but it is actually in Supplemental Figure 7. 
Line 648 – It appears that removal of tet does lead to cells without nuclear membrane defects. The 
24hr on, 48hr off experimental condition leads to much fewer cells. Is it possible that those cells 
that had nuclear membrane dysregulation are dying, perhaps indicting poor recovery even after 
removal of tet? 
Line 670 – “For euchromatin, we used H3K4me3 and H3K9me3 for heterochromatin”. This 
phrasing is confusing, which marker was used for which chromatin state? 
Lines 698 to 700 – it is hard to follow the meaning of: “Furthermore, TTC implement interaction 
(after Tet induction) with LaminB1, while MSI1, as well as for the Histones, showed a marked 
reduction of interaction (Figure 7I).” 
There is often use of terms that are somewhat unconventional. The authors should more clearly 
define their use of the following terms: 
- What is a “slot plot”. Line 458. Used in figures 2F, G and supp figure 3C. 
- “LUT”. Used in figures 3H, 7D 
- “increment” and “decrement” (throughout the manuscript, examples: line 671, 673, 684, 696 
etc) 
o Usually defined at the “quantity of an increase” 
- Line 696 - “TTC35” – no indication in text as to what this is. 
- “membrane bumping” line 647. 

Reviewer #3 (Remarks to the Author): 

The study by Montalbano et al. demonstrates convincingly that tau oligomers can interact and 
form soluble aggregates with MSI1 and MSI2 proteins in vitro (HEK cells) and in the mature 
neurons. 

Authors suggest that the pathogenic tau forms such aggregates in the neuron nuclei of the AD, 
ALS and FTD human and mouse brains that leads to the remodeling of chromatin packing and 
nuclear membrane structure. Co-localization of MSI1 and MSI2 with tau bundles is also observed 
in iHEK cells or mouse brain, where the expression of the P301L tau mutant was induced. Latter is 
known to form similar to the pathogenic tau aggregates. 

The results show also that MSI1 is accumulating in nuclei of the aged P301L mice (7 month), but 
not associated to nucleoli and is not homogeneously distributed. MSI2 is low expressed in 2-
month-old mice, but increases with age and detected in neuronal progenitor stem cells and mainly 
cytoplasmic. 

After the performing different co-IPs and the mass-spectroscopic analysis of the nuclear and 
cytoplasmic fraction (see Fig. 7), the authors conclude that folded nuclear tau and MSI1 regulate 
the nuclear activity and stabilize chromatin distribution. In contrast, toxic tau conformers interact 
with MSI2 and destabilize the nuclear envelope, nucleoporins and chromatin localization. 

They suggest also that the pathogenic tau interaction with MSI1 and MSI2 proteins in the cell 
nuclei of the mature neurons can impact nucleocytoplasmic transport and cause lamina 
disfunction. 
All these observations are novel and would be highly interesting for the community focused on the 
neurodegenerative diseases. This work might be of the great interest for the wider field of scientist 
as well. 

Particularly, I appreciate their extensive mass spectroscopic analysis combined with co-IPs and IF 
that provides valuable information on how different nucleoporins of nuclear pore complexes 
(NPCs), importins and NLS-cargos (e.g. different histones, laminA, laminB1, emerin, LAPs etc.) are 
affected by the accumulation of the toxic/pathogenic tau in the cells. 
As shown in Fig. 6 F-H or in Fig. S8 D most of the cargo proteins that bare nuclear localization 
signal (NLS) are significantly reduced in the nucleus after the Tet-induced expression of toxic 
P301L tau. It might indicate that the toxic tau affects most likely a nucleocytoplasmic import of 
these NLS-cargos by preventing their nuclear entry through NPC and release into the nucleus that 
requires RanGTP. As it was shown previously (Eftekharzadeh et. al, 2018, 2019) toxic tau can co-
aggregate with some FG-nucleoporins, e.g. with Nup98 or Nup62, and as such impact NPC 
permeability. On the other hand, toxic tau is also compromising RanGTP/RanGDP gradient 
(Eftekharzadeh et. al, 2018, 2019) that would also result in the reduced amount of the NLS-cargo 
in the cell nuclei. Consequently, an import of LaminA, LaminB1 and other lamina binding proteins 
into the nucleus might also be impaired and result in the mis-formation of the proper lamina. 
Nup50 or Nup153 (both bare NLS-signal and are delivered into the nucleus by importins during 
NPC assembly with following incorporation into the nuclear basket) are also reduced in the nuclear 
fraction and significantly elevated in the cytoplasm that might mean that their delivery into the 
nucleus is also hindered (but not cellular expression level). It would be beneficial for the paper, if 
these considerations are also taken into account in the discussion part. 

I would highly recommend this paper for the publication in Nature Communication after the minor 
revisions. 

Minor: 

1. Lane 111: I guess here instead ‘…through binding of the nuclear pore complex (NUP), Nup98.’ It 
should be written: ‘…through binding of the nuclear pore complex (NPC), e.g. to Nup98’. 

2. Lane 128: does it mean here ‘P301L’ tau mutant (instead of ‘P310L’)? 

3. Lane 376: and Fig. 1 (G and H) aggregates of tau and MSI 1 and MSI 2 are shown respectively. 
Authors claim that tau and MSI protein aggregates are co-localizing that is not really obvious from 

these IF figures. 

4. Fig. S3 D and F: should it be ‘MSI2’ written in green in the IF headings? 

5. Fig 5: when comparing panels C and G; D and H; E and F; and F and G, it seems logic to 
conclude that the difference in PLA staining between upper and lower panels (e.g. in C) is due to 
the tau-13 and TOMA-2 staining, because second component (MSI1 antibody is present in both 
panels). Therefore, it is expected that the same difference in staining should be observed in PLA 
panels (low versus top) in G (same age, same mice model; same brain area) with extra signal that 
could be observed (or not) for MSI2 in this case instead of MSI1 like in C. But it seems PLA signal 
is much weaker here. Why? And why it is interpreted as difference in the MSI1 and MSI2 
localization? Same question, when comparing panels D and H in this figure. 

6. Fig. S7: it is shown that tau is enveloping nuclei, but not really present inside of nuclei as much, 
but MSI1 is mainly nuclear localized in P301L mice brain. Why is it concluded that they are co-
localized in the nucleus? 

7. Lane 627: LaminB1 and histone 3 level is decreased in the 7 months old P301L mice. Could it be 
connected with the disruption of NCT (nucleocytoplasmic transport), blockage of NPCs upon 
oligomeric toxic tau accumulation in the aged mice? If yes, this assumption would be then 
consistent with the mass spect. data shown in Fig. 6. It seems that nuclear import of NLS-type 
cargos is impaired and LAPs, LaminA and B and some basket NPC proteins like Nup153, Nup50 are 
reduced in the nuclear fraction and their amount elevated in the cytoplasm This could be really an 
indication that their expression level is not much affected, but their nuclear import is partly 
inhibited by the aggregating tau. 

8. Lane 635: might be better to write ‘nuclear proteins’ instead of ‘receptors’; 

9. Lane 638: ‘nucleoporins’ instead of ‘Nucleoporin’ in singular. 

10. Fig. 7: IP in Fig. 7G shows that toxic tau interacts with histones directly or via MSI2 (if 
considering Fig.7 E and G). For stronger conclusion (shown in Fig. 7 J,K) a direct interaction of 
toxic tau (e.g. P301L mutant tau) with the recombinant MSI1 and MSI2 or with histones (e.g. 
histone 3) and lamins would be a plus. It would help to clarify whether toxic tau reduces 
interaction of MSI1 with histones by binding to them in the nucleus (Fig. 7 H) or less histones are 
detected in the nuclear fraction of this IP only because histones less efficiently delivered into the 
nucleus after Tet-induced P301L tau expression (due to change in pore permeability, interaction 
with tau at the nuclear pores etc.).