MiRNA Biomarkers for Alzheimer's Disease PDF

+-----------------------------------+-----------------------------------+ | **Reviewer Comments** | **Author's Reply** | +===================================+===================================+ | **Reviewer 1** | | +---...

+-----------------------------------+-----------------------------------+ | **Reviewer Comments** | **Author's Reply** | +===================================+===================================+ | **Reviewer 1** | | +-----------------------------------+-----------------------------------+ | Comment 1: | Several clinical evidences | | | suggests that miR-146a and | | Clearly define the study\'s | miR-125b are auspicious | | objectives in both the abstract | biomarkers for early Alzheimer\'s | | and introduction to ensure that | disease (AD) detection due to | | the purpose of using miRNA | their roles in regulating | | biomarkers in Alzheimer's disease | inflammation, oxidative stress, | | detection is well communicated. | pyroptosis and apoptosis in brain | | | regions such as hippocampus and | | | temporal lobe, that are prone | | | areas to develop to AD | | | pathogenesis. Therefore, the | | | current study focuses on the | | | spotlight these miRNA-based | | | biomarkers to identify early | | | molecular changes produces | | | clinical symptoms for early AD | | | detection which will facilitate | | | timely intervention. By marking | | | their roles in AD development, we | | | will delve the potential of | | | miR-146a and miR-125b as | | | non-invasive, cost-effective | | | biomarkers for early AD | | | diagnosis. | +-----------------------------------+-----------------------------------+ | Comment 2: | Numerous evidences regarding the | | | level of miR146a has been | | Expand the introduction by | reported among different stages | | incorporating more recent studies | of AD patients as well in | | or reviews on the role of miRNAs | preclinical animal models of AD. | | as biomarkers to emphasize the | Ansari et al. studied the level | | novelty and relevance of your | of miR146a among 45 MCI patients. | | work | 45 population of divided in | | | progressive mild cognition | | | impairment patients (pMCI) and | | | later stage AD patients further | | | indicated as stable mild | | | cognition impairment patients | | | (sMCI). Results of RT-qPCR | | | analysis of blood samples | | | indicates increased level of | | | miR146a in pMCI patients was | | | found as compared to sMCI, which | | | later develop AD. Increased level | | | of miR146a in pMCI patients was | | | supposed to be increased in | | | attempt to improve clearance of | | | Aβ in early stage of AD. | | | Interestingly, it was found that | | | miR146a is negatively related to | | | Aβ and Tau phosphorylation. | | | Whereas, miR1446a was reported to | | | directly linked with ApoE ε4 | | | allele, gene responsible for | | | later phosphorylation of Tau | | | protein and Aβ accumulation | | | further progression of AD (Ansari | | | et al., 2019). Similarly, in | | | another study of 60 population | | | containing 30 AD patients and 30 | | | healthy volunteers, the plasma | | | level of miR146a in AD patients | | | was reported significantly lower | | | than healthy volunteer (Yashooa | | | and Nabi, 2022). Further Lei et | | | al., provides more clear | | | statement regarding this | | | increased level of miR146a during | | | AD development. Serum sample of | | | 101 healthy and 112 AD patients | | | was analysed for miR146a level. | | | It was found that miR146a | | | directly linked with NF-κB | | | pathway which is associated with | | | increased oxidative stress, | | | neuronal pyroptosis and leads to | | | AD progression (Lei et al., | | | 2021). Further, Yan et al. | | | reported that increased Aβ | | | concentration was associated with | | | upregulation of miR146a during AD | | | progression (Fan et al., 2021). | | | Moreover, another preclinical | | | evidence supports this hypothesis | | | of increased miR146a level with | | | AD progression. It has been | | | reported that upregulation of | | | miR146a results in increased | | | neuronal oxidative stress through | | | MAPK signaling and further leads | | | to neuroinflammation and develop | | | AD (Zhan-qiang et al., 2023). To | | | overcome this confusion related | | | to level of miR146a in AD, Gonj | | | et al., evaluated the level of | | | miR146a in both AD patients as | | | well as in animal model of AD. | | | Clinical case study of 40 | | | population including 20 AD | | | patients and 20 non-AD patients, | | | showed that as compare to control | | | group level of miR146a was lower | | | in plasma of AD group. However, | | | in CSF miR146a level was | | | increased in AD group as compare | | | to control. Similarly, in animal | | | experiment of AD model lower | | | level of miR146a was found in | | | treatment group as compare to | | | disease group (Gong and Sun, | | | 2022). These all preclinical as | | | well as clinical evidences | | | strongly indicates towards the | | | increased level of miR146a | | | further leads to AD progression | | | and in early stage of AD miR146a | | | level significantly increases | | | that further lower in stable AD. | | | This differential level of | | | miR146a in different stage of AD | | | could be a plausible strategy to | | | detect early progression of AD as | | | well as could provide an idea | | | regarding the stage of AD. | | | | | | It has been reported that | | | expression of miR125b | | | significantly increased in | | | transgenic animal model of AD. | | | Whereas, after treatment miR125b | | | level significantly downregulates | | | in treatment group as compare to | | | disease group (Hong, Li, and Su, | | | 2017). In line with the previous | | | study, Jin et al. reported the | | | level of miR125b significantly | | | increased in 24 AD patients as | | | compare to 24 healthy volunteers. | | | Additionally, similar results | | | were obtained in their in-vitro | | | study of AD model using mouse | | | neuroblastoma Neuro2a APPSwe/Δ9 | | | cells. Results demonstrated that | | | elevated level of miR‑125b | | | restricted cell proliferation, | | | promote apoptosis, increase | | | inflammation and oxidative | | | stress. Moreover, miR-125b | | | overexpression was linked with | | | precipitation of amyloid | | | precursor protein, β-secretase 1 | | | and β-amyloid peptide expression, | | | and inhibition of sphingosine | | | kinase 1 (SphK1) protein | | | synthesis. These all cascade of | | | changes are the hallmark of AD | | | pathogenesis (Jin, Tu, and Liu, | | | 2018). Similarly, another study | | | reported that overexpression of | | | miR-125b promotes conversion of | | | t- tau to p-tau and an increase | | | production of p35, cdk5, and | | | p44/42-MAPK signalling. Further, | | | inhibition of miR-125b in neurons | | | reduces tau phosphorylation and | | | kinase expression as well as | | | activity, enzyme responsible for | | | tau phosphorylation. Moreover, in | | | mice intra-hippocampus injection | | | of miR-125b destruct associative | | | learning followed by increased | | | tau phosphorylation | | | (Banzhaf‐Strathmann et al., | | | 2014). Duan et al., using | | | clinical study of 71 AD and 71 ND | | | patients, suggested that | | | dysregulation of serum level of | | | miR-125b in AD patients may | | | promote the early diagnosis of AD | | | (Duan et al., 2024). Overall | | | reports strongly indicates that | | | miR-146a and miR-125b might be a | | | potential target gene involved in | | | the progression and development | | | of AD through different pathways. | +-----------------------------------+-----------------------------------+ | Comment 3: | miRNAs are mostly 22 nucleotides | | | containing endogenous molecules | | Ensure that the methods section | that play a pivotal role in | | provides a detailed explanation | regulation of gene expression at | | of how miR-146a and miR-125b were | the post-transcriptional level | | selected and analyzed, offering | for maturation of mRNA and | | enough information about the | further transcription of desired | | experimental design for | protein. Recent researches | | reproducibility. | signify that miRNAs releases into | | | body fluids such as cerebrospinal | | | fluid (CSF), plasma, serum, and | | | urine; these could be considered | | | as novel biomarkers for the | | | diagnosis of AD, Parkinson's | | | disease as well as cancer. | | | Numerous studies reported that | | | miRNA has correlated to the | | | biogenesis of AD, signifying | | | their potential as promising | | | biomarkers for early disease | | | prediction, even though direct | | | analysed from circulation. | | | microRNAs direct physiological | | | processes such as inflammation, | | | apoptosis, metabolism, | | | proliferation, development and | | | neural plasticity. Further, | | | previous studies have revealed | | | that microRNAs expressed in the | | | brains of rodents and humans has | | | regulation potential in AD. **It | | | has been suggested that a set of | | | 10 potential miRNA biomarkers can | | | anticipate pathological changes | | | associated with Alzheimer\'s, | | | even up to two decades before | | | clinical manifestation. These are | | | the hsa-mir-107, hsa-mir-26b, | | | hsa-mir-30e, hsa-mir-210, | | | hsa-mir-146a, hsa-mir-34c, | | | hsa-mir-34a, hsa-mir-485, | | | hsa-mir200c, and hsa-mir-125b; | | | could be a promising candidate to | | | diagnose early AD**. On the basis | | | of literature review and few | | | clinical and preclinical | | | evidences we selected two of | | | important miRNAs for over review; | | | these are miR146a and miR125b. | | | The objective of this research is | | | to spotlight two pivotal | | | biomarkers crucial for early | | | Alzheimer\'s detection: miR-125b | | | and miR-146a. These microRNAs | | | exhibit both high sensitivity and | | | specificity towards AD. | +-----------------------------------+-----------------------------------+ | Comment 4: | By writing this review our aim is | | | to recapitulate previous standing | | Include a justification for the | research on miR-146a and miR-125b | | chosen sample size or explain the | in early Alzheimer\'s disease | | statistical power of the study to | (AD) prediction and we have not | | address any potential limitations | conducted any new research | | in data interpretation. | experiment. Therefore, a definite | | | sample size or statistical | | | analysis is not applicable. | | | Though, the swotted studies were | | | carefully chosen based on their | | | methodological accuracy, sample | | | sizes, and statistical validity | | | to ensure complete and reliable | | | inferences. This review | | | highpoints the collective | | | conclusions to emphasize the | | | potential of miR-146a and | | | miR-125b as biomarkers for early | | | AD prediction. | +-----------------------------------+-----------------------------------+ | Comment 5: | microRNAs are a class of short | | | RNA fragments that primarily | | Enhance the discussion by | regulate gene expression | | elaborating on how the biomarkers | post-transcriptionally and are | | can be integrated into clinical | involved in several biological | | practice, particularly | processes, including Alzheimer's. | | emphasizing their diagnostic | Previous studies have been | | potential in the early stages of | reported that microRNAs are | | Alzheimer's disease. | crucial epigenetic regulator and | | | assist as a biomarker for several | | | disease like AD, PD, cancer etc. | | | Furthermore, many miRNAs | | | recognized in the nervous system | | | have actively involved in | | | neurogenesis, dendritic spine | | | regeneration, dendritic | | | outgrowth, etc., through | | | moderating the miRNAs encode the | | | deadly proteins, prompting neural | | | cell apoptosis and proliferation; | | | dysregulation of these miRNAs | | | could contribute to pathogenesis | | | of AD. The stability of miRNAs in | | | bodily fluids, such as blood and | | | cerebrospinal fluid, makes them | | | attractive candidates for | | | non-invasive biomarkers. | | | Interestingly, miR-146a and | | | miR-125b are shown to be | | | dysregulated in Alzheimer\'s | | | disease. They are involved in | | | regulating the inflammatory | | | response and neuronal function in | | | the brain and modulating the | | | immune system, which are the | | | prominent features of | | | multifactorial AD. It has also | | | been suggested that they may | | | contribute to regulating | | | amyloid-beta (Aβ) metabolism and | | | tau phosphorylation. In addition, | | | studies have revealed that | | | miR-146a and miR-125b can be | | | easily detected in serum and | | | other biological fluids, making | | | them possible non-invasive | | | biomarkers of AD diagnosis. Their | | | transcription levels are linked | | | with disease development, | | | indicating potential of miR-146a | | | and miR125-b in detecting | | | pre-symptomatic AD. | +-----------------------------------+-----------------------------------+ | Comment 6: | Current established biomarkers of | | | AD such as tau phosphorylation | | Incorporate a comparative | proteins and Aβ plaques are the | | analysis of miR-146a and miR-125b | hallmark AD, with increased | | with other known Alzheimer's | ration of p-tau and t-tau, and | | biomarkers, such as tau proteins | senile plaques in indicative of | | or amyloid-beta, to position your | neuronal degeneration. However, | | findings within the broader | these could be seen only in later | | landscape. | stage or after severe AD. | | | Additionally, these biomarkers | | | could not explain sufficiently | | | mechanistical pathogenesis behind | | | the development of AD. Whereas, | | | miR-146a and miR-125b might | | | detect early stage of AD as well | | | as it provides a novel dimension | | | to diagnosis and management of AD | | | by signifying exact primary | | | molecular patterns involved in | | | disease development. Moreover, | | | miR-146a is well reported to | | | regulate inflammatory mediators | | | via modifying NF-κB pathways, | | | TRAK1, CFH, TSPAN12, ROCK1, LRP2, | | | SHANK1 etc. pathways which are | | | critically dysregulated in AD. | | | Similarly, miR-125b is involved | | | in the regulation of neuronal | | | differentiation, synaptic | | | plasticity, and oxidative stress | | | response, via 15-LOX, VDR, CFH, | | | TREM2, IKBKG, SYN-2 etc. | | | pathways, all are vital for | | | neuronal health, function and | | | integrity. Additionally, miRNAs | | | like miR-146a and miR-125b are | | | stable, can be sampled | | | non-invasively and can detected | | | in serum and other body fluids, | | | offering a non-invasive approach | | | for early detection and | | | treatment, unlike tau and Aβ | | | which crucially require | | | additional invasive measures such | | | as lumbar punctures for CSF | | | analysis. Alternatively, | | | combining miR-146a and miR-125b | | | into the analytical framework, | | | with tau proteins and Aβ, could | | | provide specific and accurate | | | progression stage of disease, | | | leveraging potential tool for | | | improved and early diagnosis and | | | therapeutic treatment strategy. | +-----------------------------------+-----------------------------------+ | Comment 7: | Thank you for your valuable | | | comment. I have streamlined the | | Reduce repetitive mentions of | introduction and results sections | | neuroinflammation, amyloid-beta | of the manuscript to reduce | | plaques, and other key concepts | repetitive mentions of | | in the introduction and results | neuroinflammation, amyloid-beta | | sections to streamline the | plaques, and other key concepts. | | manuscript. | In addition, I have incorporated | | | several recent studies that | | | further elucidate the role of | | | miR-146a and miR-125b as | | | biomarkers for Alzheimer\'s | | | disease diagnosis. These | | | revisions enhance the | | | manuscript\'s clarity and | | | coherence, ensuring that the | | | significance of these biomarkers | | | in early AD detection is | | | communicated effectively. I | | | appreciate your guidance in | | | improving the manuscript. | +-----------------------------------+-----------------------------------+ | Comment 8: | I have added a new figure to the | | | manuscript illustrating the | | Consider adding more visual | specific brain regions where | | elements, such as figures or | miR-146a and miR-125b are | | tables, to better represent | upregulated during Alzheimer\'s | | biomarker levels, correlations, | disease. This visual element | | or the impacts on various | enhances the clarity of the data | | pathways, which will make data | presentation, making it easier to | | presentation clearer. | understand the correlations and | | | impacts of these biomarkers on | | | various pathways. I believe this | | | addition will provide a more | | | comprehensive and accessible | | | representation of the findings, | | | aligning with your suggestion for | | | improved data visualization. | +-----------------------------------+-----------------------------------+ | Comment 9: | This review study does not create | | | new experimental data; rather, it | | Perform additional statistical | synthesises previous studies. | | analyses, such as multivariate or | Therefore, it is outside the | | correlation analyses, to | purview of this work to do | | strengthen the connection between | further statistical analysis on | | biomarker levels and Alzheimer\'s | the examined research. To | | progression. | emphasise the significance of | | | miR-146a and miR-125b in early | | | Alzheimer\'s disease prediction, | | | we have critically assessed the | | | methodology and statistical | | | rigour of the included research | | | and provided a thorough overview | | | of their results. | +-----------------------------------+-----------------------------------+ | Comment 10: | The variation occurs by sample | | | collecting methodologies is one | | Discuss the limitations of using | of the key limitations of | | miRNA biomarkers in more depth, | utilizing miRNAs as biomarkers. | | especially regarding variability | Biological sample such as blood | | due to sample collection methods | from AD patients, miRNA in | | or differences among patients. | cerebrospinal fluid, or brain | | | tissue, the process by which the | | | samples are handled and | | | processed, and the time of | | | collection are some of the | | | variables that might affect the | | | expression levels of miRNAs. Many | | | studies comparing these | | | biological fluids found little or | | | no difference in extracellular | | | miRNA quantification while higher | | | concentrations were regularly | | | found in sera (Martinez and | | | Peplow, 2019). Variations in | | | pre-analytical processes, | | | including freeze-thaw cycles, | | | storage temperatures, and | | | centrifugation rates, could lead | | | to alterations in miRNA levels, | | | which compromise the accuracy of | | | the diagnosis. Addition | | | limitation includes the inherent | | | variation that exists in miRNA | | | expression in patients such as | | | age, sex, comorbidities and | | | genetic background that affects | | | the miRNA levels but | | | patient-to-patient variability | | | may be decreased by combining | | | miRNA profiles with other | | | indicators such as tau and | | | amyloid-beta. The available | | | information will be able to | | | standardize procedures and | | | perhaps compare profiles across | | | various patient cohorts (Moldovan | | | et al., 2014). | +-----------------------------------+-----------------------------------+ | Comment 11: | Our research primarily | | | concentrated on miR-146a and | | Provide detailed suggestions for | miR-125b as biomarkers for | | future research, such as | Alzheimer's disease (AD). Future | | investigating other miRNAs or | studies may expand this by | | integrating machine learning | profiling additional miRNAs, | | models for predictive | including miR-29, miR-181, and | | diagnostics. | miR-34, which have shown | | | involvement in processes like | | | neuroinflammation, amyloid-beta | | | regulation, and synaptic | | | dysfunction. Such investigations | | | could cover various stages of AD | | | progression, from early onset to | | | advanced phases. | | | | | | In addition to investigating | | | other miRNA there is a growing | | | opportunity to integrate machine | | | learning approaches into | | | predictive diagnostics for AD. By | | | following various machine | | | learning models such as support | | | vector machines (SVM) and deep | | | learning algorithms, future | | | research can combine miRNA | | | expression profiles with clinical | | | and imaging data to develop | | | predictive tools. These models | | | could enhance early detection by | | | recognizing miRNA expression | | | alterations related to cognitive | | | impairment. Future work could | | | also aim to refine these | | | algorithms could focus on | | | improving accuracy, including | | | sensitivity and specificity, | | | within clinical settings. | +-----------------------------------+-----------------------------------+ | Comment 12: | I have thoroughly reviewed the | | | manuscript to ensure that all | | Make sure that all abbreviations | abbreviations and technical terms | | and technical terms are clearly | are clearly defined upon their | | defined when they first appear, | first appearance. Additionally, I | | and avoid overuse of jargon to | have taken steps to minimize the | | make the manuscript more | use of jargon, making the | | accessible to a broader audience. | manuscript more accessible to a | | | broader audience. These revisions | | | aim to enhance the clarity and | | | readability of the paper, | | | ensuring that the significance of | | | miR-146a and miR-125b as | | | biomarkers for early Alzheimer\'s | | | disease diagnosis is effectively | | | communicated. | +-----------------------------------+-----------------------------------+ | Comment 13: | The findings of therapeutic | | | interventions include the | | Expand on how the study's | modulation in the levels of | | findings can move from bench to | miR-146a and miR-125b presents a | | bedside, with a focus on the | potential therapeutic strategy to | | therapeutic applications of | alter disease progression. When | | modulating miR-146a and miR-125b | miRNAs are downregulated, miRNA | | levels. | mimics can be developed to | | | restore normal levels but when | | | they are overexpressed anti-miRNA | | | oligonucleotides (antagomirs) can | | | be used to inhibit their | | | activity. Thus, targeting these | | | miRNAs through nanoparticle-based | | | carriers or viral vectors, could | | | offer a targeted therapeutic | | | approach to reduce inflammation | | | or improve synaptic function. | | | Additionally, miRNA-based | | | therapies e.g., anti-amyloid | | | treatments, thus creating a | | | multi-targeted approach to AD | | | treatment. | +-----------------------------------+-----------------------------------+ | Comment 14: | I have reorganized the discussion | | | section to improve the logical | | Reorganize the discussion section | flow of the manuscript. I first | | to first summarize the main | summarize the main findings | | findings before moving on to | regarding the role of miR-146a | | their broader implications, | and miR-125b as biomarkers for | | improving the logical flow of the | early Alzheimer\'s disease | | manuscript. | diagnosis. Following this, I | | | delve into the broader | | | implications of these findings, | | | discussing the potential impact | | | on clinical practice and future | | | research directions. These | | | changes ensure a clear and | | | coherent narrative that | | | effectively communicates the | | | significance of our review. | +-----------------------------------+-----------------------------------+ | Comment 15: | By specifying the roles of | | | miR-146a and miR-125b in | | Strengthen the conclusion by | regulation of neuroinflammation, | | summarizing how the study | amyloid-beta plaque formation, | | advances understanding of | and tau phosphorylation, the | | Alzheimer's pathogenesis and the | review highlights their | | diagnostic utility of miRNA | importance as non-invasive | | biomarkers, with a clear | biomarkers in early diagnosis of | | statement on its impact. | AD. Moreover, we also compile the | | | all-possible pathways through | | | which miR146a and miR125b could | | | precipitate AD. Overexpression of | | | miR146a dysregulate TRAK1, CFH, | | | TSPAN12, ROCK1, LRP2, SHANK1 etc. | | | pathways which are critically | | | involved in pathogenesis of AD. | | | Similarly, miR125b overexpression | | | leads to dysregulation of 15-LOX, | | | VDR, CFH, TREM2, IKBKG, SYN-2 | | | etc. pathways; these all are | | | involved in AD pathogenesis. | | | Further, stability of miRNAs in | | | blood and other fluids and their | | | involvement in disease | | | progression make them a potential | | | marker for diagnosis as well as | | | treatment. This review thus | | | provides a strong framework for | | | participating miRNA biomarkers | | | into clinical practice, flagging | | | the way for novel and accurate | | | diagnostic tool and better care | | | of AD. | +-----------------------------------+-----------------------------------+ | **Reviewer 2** | | +-----------------------------------+-----------------------------------+ | Authors will provide the map of | Thank you for your valuable | | part of the brain and other | feedback. In response to your | | organs and/or tissues where these | suggestion, I have added a new | | miRNAs up or down regulated | figure to the manuscript that | | during Alzheimer disease or any | maps the specific regions of the | | other diseases particularly in | brain where miR-146a and miR-125b | | aging process | are upregulated during | | | Alzheimer's disease. This visual | | | aid provides a clearer | | | understanding of the localization | | | and regulation of these miRNAs in | | | the context of AD, offering a | | | comprehensive view of their | | | impact on the disease. This | | | addition aims to enhance the | | | manuscript\'s clarity and | | | effectively communicate the | | | significance of these biomarkers | | | in Alzheimer\'s research. | +-----------------------------------+-----------------------------------+