Peer review of: Prolonged tau clearance and stress vulnerability rescue by pharmacological activation of (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

In this paper, the authors examine tau reduction via inhibition of mTOR signalling as a potential 
tau therapeutic in iPSC-neurons. The authors show that a single dose can lower tau protein levels 
for up to 12 days following treatment, and this reduces the susceptibility of neurons to stressors 
such as ABeta. Tau reduction is a promising avenue as a potential therapeutic, with increasing 
evidence that tau reduction can be protective against amyloid toxicity, and tau ASO are already in 
clinical trials. Thus, the data will be of broad interest to the field. The data are well presented and 
the manuscript is well-written and easy to follow. Some points minor points to be addressed: 

• The authors suggest an increase in components of the ALP machinery specifically in mutant vs. 
control neurons (Figure S1). However, it is hard to appreciate this as each line has a separate, 
spliced panel – and so difficult to assess whether they are different exposures etc. It would be 
useful to include samples from each line run on a single gel – e.g. n=3 from each line at 12 weeks 
on the same gel to allow the reader to appreciate the change and any variability. 

• The authors routinely include a side panel of recombinant tau ladder beside the tau western 
blots, but this is redundant as 1. It is a spliced lane and not part of the main gel 2. The samples 
are not dephosphorylated and so the molecular weight will be a reflection of many factors 
including phosphorylation state 3. As a recombinant tau ladder, one assumes the ladder itself is 
dephosphorylated and therefore wouldn’t be detected by the pTau 396 antibody? I would suggest 
removing the ladder throughout as it doesn’t add to the figures or help interpretation. 

• The banding patterns observed with the pTau 396 antibody are highly variable between lines, 
time points and experiments. For the quantification of these blots, the authors use some higher 
molecular weight bands - can they provide evidence these bands are tau species and not non-
specific – especially as they do not appear to be detected by the Tau5 antibody? 

• The prolonged decrease of tau levels following mTORi is a very interesting finding by the 
authors. However, the western blots presented in Figure S8 show some variability between 
compounds and experiments – e.g. pTau396 in the 10M AZD2014 appear to decrease at the later 
timepoints only, total tau in the OSI-027 treated cells fluctuates a little. I think it would help the 
reader to have an example western blot for each compound included the main figure, and all 3 
replicates as supplementary data. 

• Although the treatments do not appear to reduce neuronal viability, the cells used will still be in 
a neurodevelopment phase and I wonder if tau reduction could be having more subtle effects on 
neurodevelopment – for example reduced neurite outgrowth. It would be beyond the scope of the 
current manuscript to look at this, however it would be good to include this in the discussion and I 
think it would also be good to include western blot data with tau, actin, and the additional loading 
control of Beta-III-tubulin (or equivalent) in Figures 2 and 7 to show that it is a specific reduction 
in tau and not a general reduction in neuronal protein (which could be reflective of a reduction in 
neuronal content/complexity). 

Reviewer #2 (Remarks to the Author): 

This work explores the relationship between pathogenic tau and autophagy in iPSC derived 
neurons. This question has been extensively studied previously in cellular and animal models, but 
this work extends to iPSC-derived human neurons. It begins with a characterization of autophagy 
in a series of patient and unaffected iPSC-derived neural progenitor cells (NPCs) and differentiated 
neurons (6-8 weeks), showing that autophagy is elevated in the cells from diseased individuals 

(Fig 1). This baseline data is used to rationalize a focused chemical screen of molecules that are 
known to impact autophagy. The screen relies on LysoTracker and CYTO-ID and high content 
microscopy. After screening, four molecules were subject to dose dependence studies (Fig 2a) and 
then studied in the wider panel of iPSC-derived cells. The top compounds had stronger activity 
against insoluble tau (60-90%) than soluble tau (50%), which seems consistent with an 
autophagic mechanism. The authors then explore the effects of pharmacologic autophagy 
induction on the stress responsiveness of the iPSC-derived cells. Using Abeta and NMDA as 
stressors, they find that stress responsiveness was improved (as judged by rescue of cell viability). 
To confirm these findings, they conduct a series of studies in human ex vivo neurons to show that 
tau is recruited to the phagolysosomes/lysosomes by the mTor inhibitors. Target validation was 
also conducted using known mTorc1/2 substrates (p-Akt, p-beclin, etc.; Fig 6). This last point is 
important, as the inhibitors could be acting through other pathways – but the author’s rigorously 
address this issue. Finally, and perhaps most remarkably, the authors find that these effects are 
persistent for 12-16 days after washout. Initial PK studies (Fig 7) suggest that compound is 
retained in cells for a significant period, at near the therapeutic levels. Overall, this is an 
impressive study, which both confirms previous models of autophagic protection from tauopathy 
and extends those studies with some interesting PK-PD relationships. The manuscript is also well 
written and logically presented. Only very minor changes are suggested. 

1. In Figure 1F, it would be useful to add more quantitative criteria for triage. What percentage 
toxicity was allowed? What percentage of compounds passed each filter? A few more details would 
help the reader understand whether the ultimately identified compounds are unusual. 
2. Based on the dose- (Fig 2A-D) and time-dependence (Fig 7B-D), there seems to be a strong 
relationship between total tau and p-tau. In other words, both total and p-tau are reduced with 
similar drug effects. It seems worth pointing this out in the discussion, as the autophagy system in 
this model does not seem to prefer p-tau – just insoluble tau. 
3. Why didn't all of the autophagy related molecules in the screen show similar effects? What is 
special about AZD8055, AZD2014 and OSI-027?