Monascus purpureus Fermented Product Ameliorates Learning and Memory Impairment in the Amyloid Precursor Protein Transgenic J20 Mouse Model of Alzheimer’s Disease
Round 1
Reviewer 1 Report
In this manuscript, the authors investigated the effect of MPFP, a bioactive compound derived from traditional fermented food, on Alzheimer’s disease like pathologies using J20 mouse. The authors identified that the treatment of MPFP can improve memory function assessed in passive avoidance and MWM test and can improve Aβ pathologies. It is interesting that this study evaluated the function of traditional food-derived compound, but some major revisions should be done before the manuscript is published.
In major point, EPFP may improve AD pathologies via activation of PPARγ, but the results obtained by this study are lacking information to support the hypothesis. The comparison to rosiglitazone will not suggest any mechanisms. I think that the major mechanism of PPARγ activation is regulation of microglia. Did the authors investigate any effect of EPFP on microglia? Any in vivo, ex vivo, or in vitro experiments would be fine. For example, how about the immunohistochemical staining on IBA-1, a marker of activated microglia. This kind of information is needed to discuss PPARγ based mechanisms.
Another major point: In statistics, did authors use t-test to compare the difference between J20 and treated groups? The authors should use multiple comparison test in this experimental design over the study. In addition, please clarify the values of statistical analysis. I think it important to clarify statistical information in the field of neuroscience. The validities of these results can not be discussed in the present form.
In minor points, please add approval numbers of animal experiments.
In the morris water maze method, did authors use colored water to hide the platform? Did authors put any visual cues around the pool? How was the condition of the experimental room? These behavioral tests are often influenced by such subtle changes, so please clarify these experimental conditions in detail so that the readers can reproduce this study. This was same in the passive avoidance test.
On what day after the start of treatment did the behavioral tests performed?
The Figure 1D was not referred in the main manuscript. Please refer any figures in the main text. Also, I don’t understand what difference these asterisks mean.
Author Response
JOURNAL TITLE: Fermentation
MANUSCRIPT TITLE:
Monascus purpureus fermented product ameliorates learning and memory impairment in the amyloid precursor protein transgenic J20 mouse model of Alzheimer's disease
Dear Editor:
I wish to re-submit the manuscript titled “Monascus purpureus fermented product ameliorates learning and memory impairment in the amyloid precursor protein transgenic J20 mouse model of Alzheimer's disease” The manuscript ID is fermentation-1652949 to Fermentation.
We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. I look forward to working with you and the reviewers to move this manuscript closer to publication in Fermentation. The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. We have responded accordingly. Please find our detailed point-by-point responses below:
Responses to the comments of Editor and Reviewer
Reviewer 1:
- In this manuscript, the authors investigated the effect of MPFP, a bioactive compound derived from traditional fermented food, on Alzheimer’s disease like pathologies using J20 mouse. The authors identified that the treatment of MPFP can improve memory function assessed in passive avoidance and MWM test and can improve Aβ pathologies. It is interesting that this study evaluated the function of traditional food-derived compound, but some major revisions should be done before the manuscript is published. In major point, EPFP may improve AD pathologies via activation of PPARγ, but the results obtained by this study are lacking information to support the hypothesis. The comparison to rosiglitazone will not suggest any mechanisms. I think that the major mechanism of PPARγ activation is regulation of microglia. Did the authors investigate any effect of EPFP on microglia? Any in vivo, ex vivo, or in vitro experiments would be fine. For example, how about the immunohistochemical staining on IBA-1, a marker of activated microglia. This kind of information is needed to discuss PPARγ based mechanisms.
Ans: Thank you for your comments and reminder. We have added supplementary data (Figure 2a-2d) to discuss MPFP improves AD-related gene expression (Lines 373-395). In this study, however, we used MPFP lacking lovastatin, which still significantly decreased Abβpathology and markedly improved learning and memory in J20 APP transgenic mice. As MPFP lacks lovastatin, we hypothesized that other factors may account for its bioactivity. Monascus species produce several functional bioactive pigments, including the yellow pigments AK and MS, the orange pigments monascorubrin and rubropunctanin, and the red pigments monascorubramine and rubropuctamine, of which AK and MS are the major components [23]. Admittedly, our study is potentially limited by the lack of purified MS and AK to verify this hypothesis, and this remains a goal for future investigation. Nonetheless, in previous supporting studies, MS and AK were found to act as PPAR- g agonists [11,12], and numerous studies demonstrated efficacy of PPAR- g agonists in ameliorating disease-related pathology and improving learning and memory in AD animal models [24]. Consistent with this, Ros also improved cognition and spatial memory in AD mice by reducing Ab and tau pathology [25]. While it remains unknown how exactly PPAR- g ameliorates AD pathology, PPAR- g(and presumably MPFP/MS and AK) probably acts through multiple mechanisms [26]. As PPAR- g activation may be critical to MPFP/MS and AK bioactivity, we included Ros, a high-affinity thiazolidinedione PPAR- gagonist, in our study for comparison. Ros is widely used in diabetic therapy which markedly improves insulin sensitivity through activation of PPAR- gand retinoic X receptor (RXR) and downstream modulation of PPAR response element (PPRE) binding to gene promoter regions to alter gene transcription. Given the overlap between metabolic disease and neurodegeneration, Ros has been a therapeutic target of interest in AD. In APP transgenic mice, Ros has been shown to improved behavioral performance, activate Wnt signaling, normalize brain glucocorticoid receptor levels, and decrease insoluble Aβ42 levels [25, 27,28]. Furthermore, Insulin Receptor Substrate-1/2 in the insulin signaling pathway have been implicated as an important pathogenic step in AD; Ros could potentially replenish brainIRS-1/2 function, which could be beneficial in this regard. MPFP (AK and MS) may act in parallel to Ros in these respects.
- Another major point: In statistics, did authors use t-test to compare the difference between J20 and treated groups? The authors should use multiple comparison test in this experimental design over the study. In addition, please clarify the values of statistical analysis. I think it important to clarify statistical information in the field of neuroscience. The validities of these results can not be discussed in the present form.
Ans: Thank you for your comments and reminder. We have revised the statistical methods in the manuscript. (Lines 202-207).
- In minor points, please add approval numbers of animal experiments.
Ans: Thank you for your comments and reminder. We have added IACUC approval numbers 109029 in methods section. (Lines 94).
In the morris water maze method, did authors use colored water to hide the platform? Did authors put any visual cues around the pool? How was the condition of the experimental room? These behavioral tests are often influenced by such subtle changes, so please clarify these experimental conditions in detail so that the readers can reproduce this study. This was same in the passive avoidance test. On what day after the start of treatment did the behavioral tests performed?
Ans: Thank you for your comments and reminder. We have revised the methods section in the manuscript. (Lines 111-143). To assess memory and learning, the Morris water maze (MWM) test was performed with mice placed in a water-filled circular pool of 1.1-m diameter with four quadrants each marked by geometric patterns, poor Water temperature 25 oC and conducted in three parts: pre-training, hidden platform test, and probe test. During training, it was must be exposed to setting one inch above the water. This teaches the rat that there is a platform, and that it is the way to get out of the water. Later, after the animal is trained and ready for testing, the escape platform will be just below the surface of the water, and will not be visible because the water will be made opaque with milk or non-toxic paint. On the first pre-training day, the platform was set 0.5 cm above the water surface, and mice were allowed 90 seconds to search for the platform. Those unable to locate the platform in time were gently guided and allowed to rest on it for 1 minute. In the hidden platform test, conducted over days 2-5, the platform was submerged 1 cm below the water surface, and mice were randomly positioned at one of four starting points during each daily session, and allowed 90 seconds to locate the platform. Mice unable to locate the platform were again gently guided. Next, each mouse underwent 4 trials. In the probe test, performed on day 6, the platform was removed, and using a video camera mounted on the ceiling directly above the pool, randomly placed mice were assessed for swimming tracks and latency times to the 4 quadrants. All trials were recorded and analyzed using EthoVision software (Wageningen, the Netherlands).
The Passive Avoidance task is a fear-aggravated test used to evaluate learning and memory in rodent models of CNS disorders. In this test, subjects learn to avoid an environment in which an aversive stimulus (such as a foot-shock) was previously delivered. The chamber is divided into a light compartment and a dark compartment, with a gate between the two. Animals are allowed to explore both compartments on the first day. On the following day, they are given a mild foot shock in one of the compartments. Animals will learn to associate certain properties of the chamber with the foot shock. In order to test their learning and memory, the mice are then placed back in the compartment where no shock was delivered. Mice with normal learning and memory will avoid entering the chamber where they had previously been exposed to the shock. This is measured by recording the latency to cross through the gate between the compartments. The Passive Avoidance task is useful for evaluating the effect of novel chemical entities on learning and memory as well as studying the mechanisms involved in cognition.
The Figure 1D was not referred in the main manuscript. Please refer any figures in the main text. Also, I don’t understand what difference these asterisks mean.
Ans: Thank you for your comments and reminder. Using a video camera mounted on the ceiling directly above the pool, platform was removed. In figure 1d showed the probe test latency times (total 90s) in all zone analysis, performed on week 7 day 6, It was randomly placed mice were assessed for swimming tracks to measured how long the test subject latency in the target quadrant. All trials were recorded and analyzed using EthoVision software (Wageningen, the Netherlands).
The manuscript has been resubmitted to your journal. We look forward to your positive response.
Chien-Li Chen
Author Response File: 
Author Response.docx
Reviewer 2 Report
Minor revisions:
1 - Revise sentences and re-phrase:
Lines 46-48
Lines 75-79
Line 103
Line 110
Line 115
Line 153
Line 201
Lines 214-215
Lines 222-223
Line 240
Line 285
Line 305
Line 311
Line 390
Line 396
Line 405
Line 416
Line 429
2 – Repetition of already mentioned statements:
Lines 99-101
3 - Justify why only male mice was used in the study.
4 – Figure 2 c
- There is no “C” reference in the figure
- Change the images so they can shoe the same areas of the brain layers in order to compare them.
Major revisions:
5 – Justify the results stated in paragraph (3.4) “Effect of MPFP on TNF-aand neuroinflammation”. Specifically, in the assumption of the whole brain neuroinflammation (Lines 363-364).
6 – Authors must include more references in order to sustain the discussion statements.
7 – Authors must include image data to demonstrate the “neuroinflammation modulation effect” that is stated in the text.
8 – Discussion paragraph should be extended and compared with related previous studies in the field.
Author Response
JOURNAL TITLE: Fermentation
MANUSCRIPT TITLE:
Monascus purpureus fermented product ameliorates learning and memory impairment in the amyloid precursor protein transgenic J20 mouse model of Alzheimer's disease
Dear Editor:
I wish to re-submit the manuscript titled “Monascus purpureus fermented product ameliorates learning and memory impairment in the amyloid precursor protein transgenic J20 mouse model of Alzheimer's disease” The manuscript ID is fermentation-1652949 to Fermentation.
We thank you and the reviewers for your thoughtful suggestions and insights. The manuscript has benefited from these insightful suggestions. I look forward to working with you and the reviewers to move this manuscript closer to publication in Fermentation. The manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. We have responded accordingly. Please find our detailed point-by-point responses below:
Responses to the comments of Editor and Reviewer
Reviewer 2:
Comments and Suggestions for Authors
Minor revisions:
1 - Revise sentences and re-phrase:
Lines 46-48, Lines 75-79, Line 103, Line 110, Line 115, Line 153, Line 201, Lines 214-215, Lines 222-223, Line 240, Line 285, Line 305, Line 311, Line 390, Line 396, Line 405, Line 416, Line 429
Thank you for your comments and reminder. We have revised sentences and re-phrase in the manuscript. (Lines 32-486)
2 – Repetition of already mentioned statements: Lines 99-101
Ans: Thank you for your comments and reminder. We have removed repetitions statements in the manuscript.
3 - Justify why only male mice was used in the study.
Ans: Thank you for your comments and reminder. The animal test used in this experiment was a transgenic mouse instead of an aging mouse, in which the ovarian physiology of the mother mouse was hindered and the mother susceptible to environmental influences. Even under constant environmental conditions, it is still affected by the cyclical changes in ovarian endocrine. For the overall physiological and genetic performance, male animals are used.
4 – Figure 2 c. There is no “C” reference in the figure. Change the images so they can shoe the same areas of the brain layers in order to compare them.
Ans: Thank you for your comments and reminder. Change the images showed the hippocampal areas of the brain layers. (Line 331)
Major revisions:
5 – Justify the results stated in paragraph (3.4) “Effect of MPFP on TNF-aand neuroinflammation”. Specifically, in the assumption of the whole brain neuroinflammation (Lines 363-364).
Ans: Thank you for your comments and reminder. We have revised the results and discussion section in the manuscript (Lines 348-353, 458-466). However, our compound also reduced the secretion of harmful pro-inflammatory cytokines such as TNF-α, which has previously been shown to inhibit Ab clearance in AD; that do convince of a part of anti-inflammatory effect of the compound.
6 – Authors must include more references in order to sustain the discussion statements.
Ans: Thank you for your comments and reminder. We have revised the results and discussion section in the manuscript (Lines 208-466).
7 – Authors must include image data to demonstrate the “neuroinflammation modulation effect” that is stated in the text.
Ans: Thank you for your comments and reminder. We have added supplementary data (Figure 3a). The red lines indicate the possible mechanism of Monascus purpureus fermented product ameliorating Alzheimer's disease.
8 – Discussion paragraph should be extended and compared with related previous studies in the field.
Ans: Thank you for your comments and reminder. We have revised the discussion section in the manuscript (Lines 397-466).
The manuscript has been resubmitted to your journal. We look forward to your positive response.
Chien-Li Chen
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Thank you for your answers to my questions, you have addressed my comments and concerns.
