5-ALA/SFC Mitigates Tau Toxicity via Lowering Oxidative Stress in a Drosophila Model of Tau Toxicity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the effects of 5-ALA combined with SFC on mitochondrial dysfunction and neurodegeneration in a Drosophila model. The authors report that tau expression induces mitochondrial abnormalities, including reduced ATP levels, altered mitochondrial distribution, and increased ROS production. Treatment with 5-ALA/SFC attenuated oxidative damage and tau phosphorylation, leading to mitigation of neurodegeneration, although it did not restore OXPHOS activity or mitochondrial distribution. This manuscript can be suitable for acceptance; however, the authors are encouraged to address the following concerns before then;

1. How are the ROS changes the authors discovered related to tau levels?
2. The method for preparing and administering the food should be described in more detail to ensure reproducibility.
3. The rationale for using a combination of 5-ALA and SFC should be clarified. Why were the effects of each compound not examined individually?
4. The authors should explain why the 0N4R tau isoform was used instead of other major isoforms such as 2N4R or 2N3R.
5. The use of Student’s t-test for statistics in Fig. 3C and Fig. 6C appears inappropriate and should be reconsidered also by ANOVA.

Author Response

Comments and Suggestions for Authors

This manuscript investigates the effects of 5-ALA combined with SFC on mitochondrial dysfunction and neurodegeneration in a Drosophila model. The authors report that tau expression induces mitochondrial abnormalities, including reduced ATP levels, altered mitochondrial distribution, and increased ROS production. Treatment with 5-ALA/SFC attenuated oxidative damage and tau phosphorylation, leading to mitigation of neurodegeneration, although it did not restore OXPHOS activity or mitochondrial distribution. This manuscript can be suitable for acceptance; however, the authors are encouraged to address the following concerns before then;

Response: Thank you for your review and comments.

 

1. How are the ROS changes the authors discovered related to tau levels? ->

Response: Thank you for this important comment. Treatment with 5-ALA/SFC reduced ROS levels in tau-expressing flies (Fig. 4) and was accompanied by a significant decrease in tau phosphorylation at disease-associated sites (Ser202 and Ser262; Fig. 7). In contrast, total tau protein levels were not altered by 5-ALA/SFC treatment (Fig. 7). These results suggest that ROS primarily influences tau post-translational modification, rather than total tau abundance, in this model.

We have revised the manuscript accordingly:

Line 363- 365:  ‘Western blotting of head lysates with a tau antibody and phospho-specific antibodies showed that 5-ALA/SFC feeding did not alter total tau levels, while phosphorylation at Ser202 and Ser262 was significantly reduced (Fig. 7B).’

Line 441-442: 5-ALA/SFC did not affect total tau levels, but it lowers tau phosphorylation at disease-associated sites (Fig. 7B).

 

2. The method for preparing and administering the food should be described in more detail to ensure reproducibility.

Response: Thank you for pointing it out. We revised the method section to include more detail of food preparation as:

Line 82-89

‘2.2. 5-ALA/SFC feeding

Flies were raised on cornmeal food (10% glucose, 9% cornmeal, 4% yeast, 0.8% agar, 1% nipagin, 0.3% propionic acid [w/v]) without 5-ALA (hydrochloride)/SFC or with the indicated concentrations of 5-ALA (hydrochloride)/SFC. 5-ALA/SFC solution was prepared by combining 5-ALA dissolved in double-distilled water and SFC dissolved in 10% HCl at a 1:0.05 ratio, then diluted with double-distilled water to a concentration of 10 times higher than the indicated final concentrations. Then, 1 volume of 5-ALA/SFC solution was combined with 9 volumes of cornmeal food, vortexed thoroughly, and left to solidify. Food added with the double-distilled water was used as a control.’

 

3. The rationale for using a combination of 5-ALA and SFC should be clarified. Why were the effects of each compound not examined individually?

Response: Thank you for this important comment. We used a combination of 5-ALA and SFC, based on previous reports showing that 5-ALA enhances mitochondrial function more effectively when administered with SFC, which serves as an iron source to promote heme synthesis. In particular, the combination—but not either compound alone—has been shown to increase OXPHOS subunit expression, oxygen consumption rate, and ATP production (M. Shimura et al., “Effects of 5-aminolevulinic acid and sodium ferrous citrate on fibroblasts from individuals with mitochondrial diseases,” Sci. Rep., vol. 9, no. 1, p. 10549, Jul. 2019, doi: 10.1038/s41598-019-46772-x).

We agree that examining each compound individually would provide additional insight into its respective contributions. However, the primary aim of this study was to evaluate the effect of the clinically relevant combination treatment on tau-induced pathology. Dissecting the individual effects of 5-ALA and SFC will be an important subject for future investigation.

We have revised the manuscript to clarify the rationale for using the combination:

Line 56-58: ‘A combination of 5-ALA and SFC, but not 5-ALA or SFC alone, increases expression of OXPHOS subunits, oxygen consumption rate, and ATP production in fibroblasts [18].

 

4. The authors should explain why the 0N4R tau isoform was used instead of other major isoforms such as 2N4R or 2N3R.

Response: Thank you for this important comment. While multiple tau isoforms (e.g., 2N4R, 2N3R) are expressed in the adult human brain, all six isoforms are known to be present in pathological aggregates in Alzheimer’s disease. Moreover, previous studies have shown that different tau isoforms can induce neurotoxicity when expressed in Drosophila.

In this study, we used the 0N4R tau isoform because this model has been extensively characterized and widely used across multiple laboratories (Wittmann et al., 2000). The 0N4R Drosophila model exhibits robust and reproducible neurodegenerative phenotypes, making it well suited for mechanistic and comparative studies. The use of this established model also facilitates comparison with a large body of existing literature.

Line 384-388: ‘Six isoforms are expressed in the adult human brain (2N4R, 1N4R, 0N4R, 2N3R, 1N3R, 0N3R) [41], and all tau isoforms are found in aggregates in the AD brain [42]. We used a Drosophila model expressing 0N4R [25], [43], [44], [45], [46], [44], [47], [48], [49], [50], [51], in which tau-induced neurodegeneration is well characterized and widely used.’

 

5. The use of Student’s t-test for statistics in Fig. 3C and Fig. 6C appears inappropriate and should be reconsidered also by ANOVA.

 

Response: Thank you for pointing this out, and we apologize for the confusion.

For Fig. 6C, which includes three groups, the data were analyzed using one-way ANOVA followed by Dunnett’s post hoc test. The figure legend was incorrect, and we have revised it accordingly.

For Fig. 3C, comparisons were made between two groups (control and tau), and therefore, Student’s t-test was applied. However, in the previous version, data for multiple genes were presented within a single graph, which may have caused confusion. We have now separated these into individual graphs to improve clarity.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

A very interesting paper by Tamura et al. with an in-depth depiction of the Tau-induced abnormalities of mitochondrial functions and the amelioration of these abnormalities by 5-ALA/SFC. The mechanisms for most part are clear, however there are a few suggestions for further improvement.

1. In Methods section 2.14 please state the post-hoc tests for statistical analysis
2. Can you provide a better image for Figure 1B ?
3. Fig.3D needs a better representation. The mitochondria in the neuropil appears significantly low in tau overexpressing flies compared to controls but this is not evident from the image.
4. In Fig.4 is the 4-HNE staining similar in control and Tau overexpressing lines ? How do you justify this ?
5. In Fig.7 the alteration of the vacuolation of the retina is not very clear. Can this be measured by phalloidin staining of photoreceptor neurons ?

Author Response

Comments and Suggestions for Authors

A very interesting paper by Tamura et al. with an in-depth depiction of the Tau-induced abnormalities of mitochondrial functions and the amelioration of these abnormalities by 5-ALA/SFC. The mechanisms for most part are clear, however there are a few suggestions for further improvement.

Response: Thank you for your review and support.

 

1. In Methods section 2.14 please state the post-hoc tests for statistical analysis

Response: Thank you for your comments. We revised the method section as:

Line 227 ‘For multiple comparisons, data were analyzed using one-way ANOVA with Dunnett’s post-hoc test….’

 

2. Can you provide a better image for Figure 1B ?

Response: Thank you for your comment. We replaced the image.

 

3. 3D needs a better representation. The mitochondria in the neuropil appear significantly low in tau overexpressing flies compared to controls, but this is not evident from the image.

Response: Thank you for pointing it out. We replaced them with representative images.

 

4. In Fig.4 is the 4-HNE staining similar in control and Tau overexpressing lines ? How do you justify this ?

Response: Thank you for pointing this out, and we apologize for the lack of clarity. As the reviewer noted, the 4-HNE signal intensity was not significantly different between control and Tau-expressing flies, although there was a trend toward an increase in the Tau group (Figure 4).

At first glance, this appears inconsistent with the results obtained using the mitochondrial redox sensor mito-roGFP2-Grx1 (Figure 1), which showed a significant increase in ROS levels in Tau-expressing fly brains. However, this discrepancy likely reflects differences in the specificity and sensitivity of the detection methods.

4-HNE is a secondary product of lipid peroxidation and is therefore an indirect marker of oxidative stress. Its accumulation depends not only on ROS levels but also on lipid composition, turnover, and detoxification processes, which can reduce its sensitivity to moderate changes in ROS. In contrast, mito-roGFP2-Grx1 directly reports the glutathione redox state within mitochondria and is expressed specifically in neurons, providing a more sensitive and compartment-specific readout of redox changes.

Therefore, the increase in ROS induced by Tau expression may be sufficient to alter mitochondrial redox status but not large enough to produce a statistically significant increase in 4-HNE staining. In contrast, the reduction of oxidative stress by 5-ALA/SFC treatment may be more robust, allowing it to be detected by both assays, including 4-HNE staining.

We revised the manuscript and included this discussion as:

Line 307-311 ‘Tau expression tends to increase 4-HNE signals, although the difference did not reach statistical significance (p=0.27, One-way ANOVA with Dunnett’s post-hoc test). 5-ALA/SFC feeding reduced 4-HNE signals significantly (Fig. 4). These results indicate that 5-ALA/SFC feeding lowers oxidative stress in tau-expressing neurons.’

Line 393-403 ‘We found that tau expression increases mito-roGFP2-Grx1 signal, indicating that mitochondrial ROS production is increased (Fig. 1). In contrast, 4-HNE staining was not significantly different between control and tau-expressing flies, although a trend toward an increase was observed (Fig. 4). This discrepancy likely reflects differences in the specificity and sensitivity of these detection methods. 4-HNE is a secondary product of lipid peroxidation and therefore represents an indirect measure of oxidative stress. Its accumulation depends not only on ROS levels but also on lipid composition, turnover, and detoxification pathways, which may limit its sensitivity to moderate ROS changes. In contrast, mito-roGFP2-Grx1 directly reports the glutathione redox state within mitochondria and is expressed specifically in neurons, providing a sensitive and compartment-specific readout. Thus, tau-induced ROS elevation may be sufficient to alter mitochondrial redox status without producing a statistically detectable increase in 4-HNE.’

 

5. In Fig.7 the alteration of the vacuolation of the retina is not very clear. Can this be measured by phalloidin staining of photoreceptor neurons ?

Response: Thank you for this helpful suggestion. To improve the clarity of vacuolation in the retina, we have added thresholded (gray-scale) images to better highlight vacuole structures and facilitate their visualization. In addition, we indicated the lamina area in which vacuoles are quantified.

Figure 7 legend has been revised as follows:

"Tau-induced neurodegeneration is observed as vacuoles in the lamina (arrows). (Left) Representative images (top: hematoxylin–eosin-stained sections; bottom: corresponding thresholded images used for quantification of vacuoles within the lamina, indicated by dashed lines). (Right) Quantification of vacuole area, normalized to the total lamina area."

Regarding the reviewer’s suggestion to use phalloidin staining of photoreceptor neurons, we agree that this approach can provide structural information on photoreceptor organization. However, in this study, we focused on vacuolation in the lamina as a well-established histopathological readout of neurodegeneration in this model. We believe that the improved visualization and quantification presented here adequately address the reviewer’s concern.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have appropriately responded to the referee's concerns. I have no further comments. Great works.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have successfully addressed all my suggestions.