Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration

Essential revisions:

Since it is unrealistic for the authors to add therapeutic testing to this paper, they should acknowledge the need to test their ideas in existing animals’ models and/or develop appropriate models to test whether inhibition of mTOR or protein synthesis are universally beneficial and assess the mechanisms involved in vivo. Reviewer #1: My main question about this study is the extent to which these observations are relevant in a whole animal or person. It is not clear that ATP deficiency is a major driver of disease in mitochondrial disease, nor is it clear that rapamycin treatment significantly reduces protein synthesis in vivo. I can imagine that the high nutrient growth conditions used for cell culture create a somewhat artificial system in which protein synthesis and ATP consumption by protein synthesis are elevated relative to any physiologically relevant situation. In this regard, it may be worth noting that the Ndufs4 study by Johnson et al. did not detect a significant difference in ATP levels in KO compared to WT animals nor was a change in ATP levels in whole brain upon rapamycin treatment observed. Obviously, this negative result from Johnson et al. is not conclusive, but it would greatly enhance the relevance of this study if the authors could offer some direct experimental evidence that their proposed mechanism is important in a mouse model. At a minimum, the possibility that the importance of the translation/ATP mechanism proposed here is enhanced in cell culture relative to tissues in vivo should be addressed in the text. We appreciate and agree with the reviewer's point that the cell culture system may not fully reflect the in vivo situation. Going forward it will indeed be important to examine whether rapamycin treatment or reduction of protein synthesis is able to preserve neuronal ATP levels and show beneficial effects in animal models of mitochondrial diseases, but it would be hard to generate a model of MILS for mitochondrial DNA mutant, and we feel that this is beyond the scope of the present study. To acknowledge the limitations of the current study and highlight the issues raised by the reviewer, we have added the following paragraph in the Discussion.

"We emphasize that, whether or to what extent, our observations from a cell culture-based mitochondrial disease model reflect the in vivo situation needs further investigation using animal models with various mitochondrial deficiencies. […] An engineered ATP fluorescent biosensor is available for cell culture systems (Tantama et al., 2013), which could be adapted and introduced into animal models. "

Reviewer #2: Comments: In the subsection “Shutoff of aerobic glycolysis during neuronal differentiation exposes ATP synthesis deficiency in T8993G MILS neurons”: "Without LDHA and HK2, neurons cannot[…]" – cannot seems too strong. If the authors reintroduce LDHA and/or HK2 and this rescues ATP levels the statement would be justifiable. As it stands "cannot" would be better stated something like "appear unable". The reintroduction would be an interesting experiment to perform.

We would like to thank the reviewer for highlighting the importance of our work.

We were also intrigued by the disappearance of HK2 and LDHA during neuronal differentiation and the functional significance of their loss. In a separate project to investigate metabolic changes during differentiation of neural progenitor cells into neurons we analyzed the effect of constitutive expression of HK2 and LDHA during neuronal differentiation, and these data will be included in another manuscript describing the metabolic transition from neural progenitor cells to post-mitotic neurons. The data are shown in Author response image 1. In brief, we stably transduced neural progenitor cells (NPCs) with constitutively expressed HK2 and LDHA; ~80% of NPCs had discernible expression and proliferation appeared normal. However, constitutive HK2 and LDHA expression during neuronal differentiation led to extensive cell death and significantly increased GFAP-positive glial cells from 4% of total cells in the control NPC cells containing expression vector alone to ~40% of total cells. The LDHA signals were readily detected in GFAP-positive glial cells but not in MAP2-positive neurons. Importantly, the dead cells signified by condensed nuclei were associated with punctate staining of LDHA and MAP2, a neuronal marker, indicating that neurons were sensitive to HK2 and LDHA re-expression. At the moment, we do not fully understand the mechanism behind this phenomenon. We agree with the reviewer's point, and have changed the phrase according to their suggestion.

The effects of HK2 and LDHA re-expression on neuronal differentiation. (A) Immunoblotting analysis of HK2 and LDHA in NPCs and 3-week neurons constitutively expressing HK2 and LDHA (Flag-tagged). (B) Immunostaining of NPCs with anti-FLAG antibody (green), and nuclear staining was done with Hoechst (red). The percentage of Flag-positive were quantified, and 100 cells were counted for each group. (C) Immunostaining analysis of MAP2 and GFAP in 3-week neurons. The percentage of GFAP and MAP2 cells were quantified, and 100 cells were counted for each group, and three times of neuronal differentiation were included. Bars are mean ± SD, n=3. The GFAP mRNA abundance in the RNA extracted from neuronal culture was quantified by real time PCR and normalized to β-actin, and presented by fold increase compared to neurons differentiated from control NPCs. Bars are mean ± SD, n=3. (D) Nuclear staining with Hoechst in NPCs and 3-week neurons. The percentage of condensed nuclear were quantified, and 50 cells were counted for each group. Bars are mean ± SD, n=3. (E) Immunostaining analysis of LDHA, MAP2 and GFAP in 3-week neurons differentiated from NPC constitutively expressing HK2 and LDHA. (F, G) Co-localization of irregular puntated staining of LDHA (green) with MAP2 (red, in F) or condensed nuclear staining with Hoechst (red, in G) in 3-week neurons differentiated from NPC constitutively expressing HK2 and LDHA.

In the first paragraph of the subsection “Rapamycin treatment significantly increases ATP level and decreases aberrant AMPK phosphorylation in T8993G MILS neurons”: T8993G mutant neuron lines all nicely show increased S6 phosphorylation (Figure 5A). Does rapamycin treatment inhibit this?

Rapamycin treatment effectively decreased S6 phosphorylation in MILS neurons. The new data were added into Figure 5B.