Effect of Letrozole on hippocampal let-7 microRNAs and their correlation with working memory and phosphorylated Tau protein level in an Alzheimer's disease rat model

Let-7 microRNAs may contribute to neurodegeneration, including Alzheimer's disease (AD), but, they were not investigated in Streptozotocin (STZ)-induced AD. Letrozole increases the expression of Let-7 in cell lines, with conicting evidence regarding its effects on memory. This study examined Let-7 microRNAs in STZ-induced AD, their correlation with memory and hyperphosphorylated Tau (p-Tau) and the effects of Letrozole on them. Seven groups of adult Sprague Dawley rats were used: Intact, Letrozole, Letrozole Vehicle, STZ (with AD induced by intracerebroventricular injection of STZ in articial CSF), CSF Control, STZ + Letrozole (STZ-L), and CSF + Letrozole Vehicle. Alternation percentage in T-maze was used as a measure of working memory. Let-7a, b and e and p-Tau levels in the hippocampus were estimated using qRT-PCR and ELISA, respectively. There were signicant decreases in alternation percentage and increase in p-Tau in the STZ, Letrozole and STZ-L groups. Expression levels of all studied microRNAs were signicantly elevated in the Letrozole and the STZ + L groups, with no difference between the two, suggesting that this elevation was due to Letrozole. Negative correlations were found between alternation percentage and the levels of all studied microRNAs, while positive ones were found between p-Tau concentration and the levels of studied microRNAs. These ndings support the theory that Letrozole aggravates pre-existing lesions and add to the evidence of Let-7 neurotoxicity.


Introduction
Alzheimer's disease (AD) is a neurodegenerative condition that causes aging-related cognitive impairment and dementia (Davey, 2017) and accounts for 50-75% of dementia cases, making it the chief cause of dementia (Lane et al., 2018). It is pathologically characterized by accumulation of beta-amyloid (Aβ) plaques, and neuro brillary tangles of aggregated hyperphosphorylated Tau protein (Liu et al., 2014).
The role of microRNAs (miRNAs) in numerous diseases is currently being heavily studied. miRNAs are approximately 22-nucleotide long, non-coding RNAs that function as gene expression regulators at the post transcriptional level. The Let-7 miRNAs are conserved in multiple species, and are abundantly expressed in the brain (Lee et al., 2016;Derkow et al., 2018). Let-7 miRNAs play roles in cellular differentiation, tumor suppression and neurodegeneration/ neurotoxicity (Lehmann et al., 2012;Shamsuzzama et al., 2016 andGu et al., 2017). Various members of the Let-7 family were found to be differentially expressed in the cerebrospinal uid of AD patients (Derkow et al., 2018).
Up to our current knowledge, levels of Let-7 miRNAs were not previously investigated in a Streptozotocin (STZ)-induced AD model, neither was the effect of Let-7 modulation on the memory of the animals in such model.
Multiple drugs were found to increase the expression of Let-7 in various cell lines (Lozier et al., 2015;Francess et al., 2018), an example of which is Letrozole (Shibahara et al., 2012;Cho et al., 2016), which is a potent aromatase inhibitors with an experimental evidence documenting its high permeability across the blood brain barrier (BBB) (Dave et al., 2013).
Memory changes are somewhat a common complaint in patients treated with Letrozole, however, there are several con icting studies examining the possible effects of aromatase inhibitors on memory. Some studies have shown that Letrozole administration resulted in memory impairment (Nayebi, Pourrabi and Hossini, 2014;Zameer and Vohora, 2017), while other studies have shown that Letrozole improved the acquisition of working memory and that inhibition of brain estrogen synthesis may have bene cial effects on spatial memory (Alejandre-Gomez, Garcia-Segura and Gonzalez-Burgos, 2007;Aydin et al., 2008).
Another question arises of whether the memory impairments observed with Letrozole treatment occur because of neurosteroidal de cits, or they become exacerbated in the presence of an additional insult associated with aging such as pathologic Aβ. Evidence of synaptic pathology and changes in mitochondrial morphology was found in hippocampal cultures treated with both Letrozole and Aβ, suggesting that Letrozole potentiates the pathological effects of Aβ (Chang et al., 2013). Therefore, this study was designed to examine Let-7 miRNAs levels in the hippocampi of STZ-induced AD rats, and their possible correlation with memory status and/or phosphorylated Tau levels and assess the effects of Letrozole on the memory and levels of Let-7 miRNAs in animals treated only with Letrozole as well as animals treated with Letrozole on top of having an existing STZ-induced AD, possibly providing an insight into another mechanism by which Letrozole could exert an effect on cognition via microRNA modulation.

Animals and grouping
A total number of fty-one adult Sprague Dawley rats of the same age and weighing 150-200 gm., were subjects of this study. Rats were supplied by the animal unit of the Medical Experimental Research Center (MERC), faculty of Medicine, Mansoura University. An ethical approval for this study was obtained from Group 2: Letrozole Group (n=9): Letrozole (Synthon, Netherlands ) was given at a daily dose of 2.5 mg/kg (Alejandre-Gomez et al., 2007) by oral gavage (Aydin et al., 2008). Letrozole was dissolved in 0.5 % carboxy methylcellulose (CMC) solution, and was given for 15 days (Zameer and Vohora, 2017 Group 4: STZ Group (n=9): In this group, sporadic Alzheimer's disease-like state was induced using intracerebroventricular injection (ICV) of 3 mg/kg of Streptozotocin (Sigma-Aldrich, USA) dissolved in arti cial cerebrospinal uid (aCSF).
Group 6: STZ + Letrozole (STZ-L) Group (n=9): In this group, sporadic Alzheimer's disease-like state was induced using ICV injections of 3 mg/kg of Streptozotocin dissolved in aCSF. Following the surgery, rats were allowed a recovery period of 7 days then, given 2.5 mg/kg Letrozole by oral gavage, for 15 days.

Induction of AD by ICV injection of STZ
Animals were anesthetized and the general guidelines and steps for stereotaxic surgery were followed as mentioned by Hau and Schapiro, (2002) and Langhans et al., (2016). A single dose of Streptozotocin (3 mg/Kg (Mehla et al., 2013)), in 10 μL aCSF (119 mM NaCl, 26.2 mM NaHCO 3 , 2.5 mM KCl, 1 mM NaH 2 PO 4 , 1.3 mM MgCl 2 , 10 mM glucose and 2.5 mM CaCl 2 in distilled water, then passed through a 0.22 µm lter to sterilize (Cold spring harbor, 2011)) was injected bilaterally into the lateral ventricles using coordinates according to the rat stereotaxic brain atlas (Paxinos and Watson, 2006), and the Waxholm Rat atlas via the scalable brain atlas (Papp et al., 2014;Bakker et al., 2015;Kjonigsen et al., 2015 andSergejeva et al., 2015): anteroposterior from bregma (AP)= -0.9 mm, mediolateral from the midline (ML)= ± 1.5 mm and dorsoventral from the skull (DV)= 3.6 mm. Animal were allowed a week to fully recover prior to administration of any further treatment. Typically, the effects of STZ on memory functions start to manifest 15 days following the injection (Mehla et al., 2013). Hematoxylin and eosin (H&E) staining of hippocampal sections from representative samples was done to further con rm the presence of neurodegeneration.
Testing of working memory: Spontaneous alternation in a modi ed T-maze A manually-run maze was constructed. The maze design was adapted from Deacon and Rawlins, (2006). No habituation to the maze was allowed, as it is the novelty of the maze that motivates the spontaneous exploration/ alternation. For each rat, one sample trial and ve choice runs were performed per day for two days, amounting to a total of 12 trials per rat, and a total of ten possible alternations. A percentage or proportion correct choice (alternation) per animal was calculated as follows (Deacon and Rawlins, 2006;Wu et al., 2018):

Animals and grouping
A total number of fty-one adult Sprague Dawley rats of the same age and weighing 150-200 gm., were subjects of this study. Rats were supplied by the animal unit of the Medical Experimental Research Center (MERC), faculty of Medicine, Mansoura University. An ethical approval for this study was obtained from the Institutional Animal Care and Use Committee (IACUC), Zagazig University, in line with the Guide for the Care and Use of Laboratory Animals 8th Edition 2011.
Rats were housed in plastic rodent cages (3-5/cage) under hygienic and environmentally controlled conditions i.e., ambient temperature of 25±2°C, and 12-hour light/ 12-hour dark cycle. They were allowed 14 days for acclimatization, during which, and throughout the entire period of the study, they were fed standard commercial rat chow and had free access to food and water.
After the acclimatization, rats were randomly divided into seven groups as follows: Group 1: Intact Control Group (n=6): No procedure or treatment was applied to this group. It served to establish basal levels of studied parameters (Cognitive function, hippocampal phosphorylated Tau level and Let-7a, b, e microRNA levels).
Group 6: STZ + Letrozole (STZ-L) Group (n=9): In this group, sporadic Alzheimer's disease-like state was induced using ICV injections of 3 mg/kg of Streptozotocin dissolved in aCSF. Following the surgery, rats were allowed a recovery period of 7 days then, given 2.5 mg/kg Letrozole by oral gavage, for 15 days.
Induction of AD by ICV injection of STZ Animals were anesthetized and the general guidelines and steps for stereotaxic surgery were followed as mentioned by Hau and Schapiro, (2002) and Langhans et al., (2016). A single dose of Streptozotocin (3 mg/Kg (Mehla et al., 2013)), in 10 μL aCSF (119 mM NaCl, 26.2 mM NaHCO 3 , 2.5 mM KCl, 1 mM NaH 2 PO 4 , 1.3 mM MgCl 2 , 10 mM glucose and 2.5 mM CaCl 2 in distilled water, then passed through a 0.22 µm lter to sterilize (Cold spring harbor, 2011)) was injected bilaterally into the lateral ventricles using coordinates according to the rat stereotaxic brain atlas (Paxinos and Watson, 2006), and the Waxholm Rat atlas via the scalable brain atlas (Papp et al., 2014;Bakker et al., 2015;Kjonigsen et al., 2015 andSergejeva et al., 2015): anteroposterior from bregma (AP)= -0.9 mm, mediolateral from the midline (ML)= ± 1.5 mm and dorsoventral from the skull (DV)= 3.6 mm. Animal were allowed a week to fully recover prior to administration of any further treatment. Typically, the effects of STZ on memory functions start to manifest 15 days following the injection (Mehla et al., 2013). Hematoxylin and eosin (H&E) staining of hippocampal sections from representative samples was done to further con rm the presence of neurodegeneration.

Testing of working memory: Spontaneous alternation in a modi ed T-maze
A manually-run maze was constructed. The maze design was adapted from Deacon and Rawlins, (2006). No habituation to the maze was allowed, as it is the novelty of the maze that motivates the spontaneous exploration/ alternation. For each rat, one sample trial and ve choice runs were performed per day for two days, amounting to a total of 12 trials per rat, and a total of ten possible alternations. A percentage or proportion correct choice (alternation) per animal was calculated as follows (Deacon and Rawlins, 2006;Wu et al., 2018): see formula 1 in the supplementary les.
Biochemical and molecular testing Animals were euthanized by cervical dislocation following The University of Texas at Austin (2013) guideline and the brain was immediately removed from the skull and washed with ice cold saline. The hippocampi were rapidly dissected following the method described by Spijker (2011) and weighed. Each hippocampus (weighing 50 mg) was submerged in either RNAlater RNA stabilization reagent (Qiagen, USA, Cat No./ID: 76104), or sterile Phosphate-buffered saline (PBS) and stored at a temperature of -80°C for biochemical analysis (Silva et al., 2004).
Total RNA, including microRNAs, was extracted from the hippocampus using the miRNeasy Mini Kit (Qiagen, USA, Cat No./ID: 217004) following the kit's manual, and the purity and concentration the extracted total RNA were spectrophotometrically determined using a NanoDrop (Desjardins and Conklin, 2010). Reverse transcription (RT) reaction was done for each sample using the TaqMan™ microRNA reverse transcription kit (Applied biosystems, USA, Cat no.: 4366596) following the kit's manual. A speci c TaqMan microRNA assay was used, following the manufacturer's instructions, in qRT-PCR to detect each of the three target microRNAs (Let7a, b and e) as well as U6 small nuclear RNA as an endogenous control (rno-let-7a-5p [Assay ID: 000377], rno-let-7b-5p [Assay ID: 000378], rno-let-7e-5p [Assay ID: 002406] and U6 snRNA [Assay ID: 001973], Applied biosystems, USA, Cat no.: 4427975). Changes of miRNA expression were calculated for each of the three tested miRNAs using the 2^(-∆∆Ct) method Schmittgen, 2001 andLivak, 2008).
Rat p-Tau (Ser396) protein ELISA kit (Bioassay technology laboratory, Shanghai, China) was used according to the manufacturer's instructions to measure p-Tau concentration in the hippocampus.

Statistical analysis
The statistical analysis was done using IBM SPSS Statistics for Windows, version 22 (IBM Corp., Armonk, N.Y., USA). For quantitative variables, mean and standard deviation were computed. Median and range were computed in case of data that were not normally distributed (Non-parametric data). One way analysis of variance (ANOVA) test was used for comparison of means of more than two groups in normally distributed data, and Kruskal Wallis test was used in case of data that were not normally distributed. Spearman's correlation coe cient was used as a measure of the strength of association between two variables. For all the mentioned statistical tests, P values of >0.05 indicated non-signi cant results, while P values of <0.05 indicated signi cant results.

Death and post-operative infection rates
Out of the initial 65 rats allocated for this study, 14 rats were eliminated due to either death or infections. A total of 10 rats died following the stereotaxic procedure, constituting a death rate of 15.38% (3 in the STZ group, 3 in the STZ-Letrozole group, 2 in the CSF group and 2 in the CSF-LV group). All rats died within the rst 24 hours following the stereotaxic injection. Four rats developed a localized infection, constituting an infection rate of 6.15% (2 in the CSF group and 2 in the CSF-LV group). The infection was either in the head wounds following the stereotaxic injection or in the abdomen at the site of the anesthesia injection. The remaining 51 rats were involved in the remainder experiments and subsequently the statistical analysis.

Veri cation of neurodegeneration in the STZ-injected rats via histological examination
Histological examination of H&E stained hippocampal sections from STZ-injected rats showed signs of degeneration as well as in ammatory changes (Fig. 1).
Comparing the state of working memory (in alternation percentage) and p-Tau levels among the different studied groups Tables (1, 2 and 3) show that there were statistically signi cant differences between different groups in both alternation percentage and p-Tau concentration.
Comparing Let-7 miRNAs expression (in fold change) among the studied groups Tables (4, 5, 6 and 7) and gures (2, 3 and 4) show that there were statistically signi cant differences between the different groups in the expression of Let-7 a, b & e. Using LSD to compare each two groups, it was found that there were no statistically signi cant differences between the Intact, CSF, LV, CSF-LV and STZ groups in the expression of any of the studied miRNAs.
However, there was a statistically signi cant elevation in the expression levels of all studied miRNAs in the Letrozole group compared with the Intact, CSF, LV, CSF-LV and STZ groups. There was also a statistically signi cant elevation in the expression levels of all studied miRNAs in the STZ-L group compared with the Intact, CSF, LV, CSF-LV and STZ groups. Finally no difference was found between Letrozole group and STZ-L group in the expression level of any of the studied miRNAs.

Discussion
AD causes aging-related cognitive impairment and dementia (Davey, 2017). Amnesic presentations as well as the presence of Aβ plaques and neuro brillary tangles of lamentous Tau proteins are required for diagnosis (Perl, 2010 andDeTure andDickson, 2019). Studies have shown that the cerebral glucose mobilization disorders are either preceding or accompanying the initial cognitive impairments in sporadic AD (De La Monte and Wands, 2005;Rivera et al., 2005).
Multiple studies provide evidence of the possible involvement of microRNA (miRNA) Let-7 family members in AD, and their neurotoxicity. These studies include models of Aβ 40 -incduced neurotoxicity in cell lines (Gu et al., 2017), injection of Let-7b into the CSF of wild-type mice (Lehmann et al., 2012), quanti cation of Let-7 miRNAs in the CSF of AD patients (Derkow et al., 2018), miRNA pro ling of the brains of transgenic AD mice (Wang et al., 2009 and, and studying the expression of miRNAs in the brains of a cholesterol-based rabbit model of AD (Liu et al., 2014).
Prior to this current study, levels of Let-7 miRNAs were not investigated in an STZ-induced sAD model, neither was the effect of Let-7 modulation on the memory of the animals in such model.
Results of the present work showed that STZ, Letrozole and STZ+Letrozole administration all caused memory impairments in rats, as evident by decreases in the alternation percentages of these groups compared to the control groups. The degree of memory impairment was more severe in the STZ+Letrozole rats, followed by the STZ group, then by the Letrozole group that showed the least degree of memory impairment.
These results are partially in agreement with Nayebi et al., (2014), whose results also showed a signi cant memory impairment in the STZ, Letrozole, STZ+Letrozole groups compared to the controls. However, while their results indicated that the Letrozole group had the least severe impairment among the three groups, there was no signi cant difference in the degree of memory impairment in the STZ group compared to the STZ+Letrozole group.
This discrepancy between our and their sets of results may arise from: 1) Differences in the animal strains used, as they used Wistar rats, 2) The dose of Letrozole given and the route of administration, as they used a 6 mg/kg dose administrated intraperitoneally, or 3) The use of a different memory test; the passive avoidance test. Passive avoidance and T-maze tests were shown to give different results while testing the effects of other drugs on memory processes (Wang et al., 2006).
Regarding the level of p-Tau (Ser396), results of our study showed a signi cant increase of p-Tau in the STZ, Letrozole and STZ+Letrozole groups compared to the control groups. While the administration of ICV-STZ leading to generation of hyperphosphorylated Tau and Tau pathology similar to that of AD was extensively documented in literature and previous studies (Grünblatt et al., 2007;Qu et al., 2011;Lee et al., 2013;Peng et al., 2013;Shi et al., 2017;Gupta et al., 2018;Mishra et al., 2018), the possible effect of Letrozole on hyperphosphorylated Tau levels is underexplored.
Hippocampal levels of p-Tau showed a signi cant negative correlation with the alternation percentages in the current study, which was postulated; as there is an evidence showing that both plasma and CSF levels of Tau correlate with the incidence of AD dementia in patients (Pase et al., 2019), and that CSF Tau levels have an inverse correlation with the short-term memory scores in patients with AD (Lin et al., 2009).
Regarding the expression levels of Let-7 a, b and e, miRNAs, we are rst to report the expression patterns of these three miRNAs in an STZ-induced sAD rat model. Let-7a, b and e miRNAs were not differentially expressed in the STZ group compared to the controls, as there was no signi cant differences in fold change of any of the three miRNAs between the STZ group and the controls.
Multiple studies have examined the expression levels of various Let-7 miRNAs in different models, samples and time intervals. For example, studies examining Let-7 members in the CSF of AD patients −of different subtypes− have found Let-7a-3p, b, e, f and i to be upregulated (Cogswell et al., 2008;Sørensen et al., 2016;Derkow et al., 2018 andLv et al., 2018), while Let-7a was found to be downregulated in the CSF of patients with late onset AD (Van Harten et al., 2015).
Studies examining Let-7 members in either the serum or plasma of AD and MCI patients have found Let-7f to be upregulated in AD (Hara et al., 2017), and Let-7b to be upregulated in MCI (Kenny et al., 2019), while Let-7d, g and i were downregulated (Kumar et al., 2013;Tan et al., 2014;Gámez-Valero et al., 2019).
Animal studies on different transgenic AD mice and cholesterol-induced AD rabbits have found Let-7a, b, c, d, e and f to be upregulated in the brain at different time intervals (Wang et al., 2009;Liu et al., 2014 and.
It is obvious that changes in the model, the sample, type of AD, Let-7 tested, and the age of the subject at which the samples were obtained yielded different results. Thus, it was not unexpected that the model chosen for the current study might show different results, since none of the previous studies, to date, have examined the levels of Let-7 in a similar STZ-induced AD rodent model. It may also be assumed that measuring expression levels of Let-7 in aged STZ-induced AD rats might yield different results than the ones obtained from this current study, as we only examined the hippocampal levels of selected Let-7 miRNAs after three weeks of STZ injection, i.e. during the acute impairment stage of this model.
Non-signi cant differences in the expression of Let-7 a, b and e in the STZ group does not necessarily mean that these miRNAs are not implicated in the disease process in this model. Let-7 activity was shown to be regulated by another miRNA, miR-107. miR-107 directly interacts with Let-7, suppresses its function and promotes its degradation (Chen et al., 2011). miR-107 was found to be downregulated in AD in multiple studies (Wang et al., 2008;Shamsuzzama et al., 2016;Herrera-Espejo et al., 2019;Swarbrick et al., 2019). It was also found to be deregulated in STZ-induced diabetic animals (Balbaa et al., 2017). Decreases in miR-107 levels or activity can lead to Let-7 stabilization and preservation of its action on the target genes (Jiang, 2019). However, further research is needed to elucidate the relation between Let-7 and miR-107 in AD.
On the other hand, Letrozole was successfully associated with Let-7a, b and e upregulation in the Letrozole and the STZ+Letrozole groups, which showed signi cant differences in Let-7a, b and e expression levels compared to all other groups. No signi cant difference was found in the expression of b or e between the Letrozole group and the STZ+Letrozole group, further serving to relate the observed upregulation to the action of Letrozole.
The observed effect of Letrozole on Let-7 miRNAs was not unexpected, as Letrozole was shown to be able to cross the BBB (Kil et al., 2009 andDave et al., 2013), and it was previously shown to be associated with increased expression of various Let-7 miRNAs, including Let-7f, b and a, in various cancer models (Shibahara et al., 2012;Cho et al., 2016;Isanejad et al., 2016).
Analysis of the current data showed signi cant correlations between Let-7a, b and e levels and both the alternation percentage and p-Tau level in the hippocampus. There was a signi cant negative correlation between the expression level of each of the Let-7 miRNAs tested (Let-7a, b and e) and the alternation percentage, and a signi cant positive correlation between the expression level of each of the Let-7 miRNAs tested and the hippocampal p-Tau level.
Studies exploring possible correlations between Let-7 members and either or both cognitive impairment and p-Tau levels are scarce, and majorly differ from the current work in regards to the type of sample, and the type of cognitive test/exam which is determined by the species to be examined.
For instance, Kenny et al., (2019) found no signi cant correlation between Let-7b expression and the cognitive decline in MCI patients, and Derkow et al., (2018) did not observe a signi cant correlation between amounts of Aβ 42 or total-Tau and Let-7b or Let-7e expression in the CSF of AD patients. Further research is needed to establish more evidence in regards to Let-7 correlation with memory status and p-Tau levels, preferably using larger samples and different AD models.
Based on the ndings of this current study, Letrozole alone was su cient to show a signi cant degree of memory impairment, but, the more severe impairment in the STZ+Letrozole group could further support the idea of a synergistic effect exerted by Letrozole in presence of an additional insult (Chang et al., 2013).
This current study cannot con rm whether this effect is due to the additional neurosteroidal de cit caused by Letrozole or its modulation of some neurotoxic miRNAs of the Let-7 family.
Upregulated Let-7 could play a role in the pathogenesis of AD by affecting multiple pathways that relate to insulin signaling and neuroin ammation, and are known to be disturbed by STZ administration as well as being regulated by estrogen.
STZ can accumulate via GLUT2 transporter uptake (Eleazu et al., 2013), and mRNA expression of insulin receptors (IR), and insulin-like growth factor-1 receptors (IGF-1R) and their protein levels in the membrane of hippocampal cells decrease after STZ administration (Salkovic-Petrisic and Hoyer, 2007). Insulin receptor substrates (IRSs) and Akt phosphorylation (i.e. activation) also decrease, resulting in disruption of the insulin signaling pathway, which was implicated in the process of learning and memory (Agrawal et al., 2011). An impairment of the insulin signaling pathway leads to activation of GSK3, ultimately resulting in hyperphosphorylation of Tau protein (Mishra et al., 2018) and promoting Aβ accumulation (Kamat et al., 2016). STZ also upregulates BACE1, which can result in increased cleavage of APP (Mishra et al., 2018). Let-7 was found to target the mRNAs of the insulin signaling pathway players, such as IR, IGF1R, IRS, PI3K and Akt (Frost and Olson, 2011;Zhu et al., 2011) and Let-7 transfection into a human cell line led to reduction IR and IRS (Zhu et al., 2011). We suggest that it is possible that the upregulation of Let-7 in the hippocampus could aggravate the toxic effects of STZ via further dysregulation and inhibition of the insulin signaling pathway. This suggestion can be supported by a recent study that found that LIN-28a, which is a powerful inhibitor of Let-7 biogenesis, prevented the toxic effects of STZ on pancreatic β-cells, and that LIN-28a downregulation of Let-7 was associated with increased phosphorylation of PI3K and Akt in a mouse insulinoma cell line (Sung et al., 2019).
Interestingly, estrogen binding to G-coupled estrogen receptor (GPER) could result in the activation of the PI3K/Akt pathway (Peixoto et al., 2017;Bian et al., 2019), which could lead to memory enhancement (Frick, 2019). In addition, the estrogen/ER-PI3K-Akt pathway has been shown to be a neuroprotective factor against toxic insults for neuronal cells (Honda et al., 2000). It might be possible that the estrogenreducing effect of Letrozole could have also added to the inhibition of the PI3K-Akt pathway already caused by the STZ toxicity and the upregulation of Let-7.
Another pathway by which upregulation of Let-7 could aggravate the effect of an STZ-induced lesion is through increasing neuroin ammation. STZ leads to increased expression of neuroin ammation markers such as NF-κB (Nuclear factor kappa light chain enhancer of activated B cells), and Glial brillary acidic protein (GFAP) positive astrocytes in the hippocampus (Islam et al., 2015;Mishra et al., 2018). Increased expression and activation of astrocytes for prolonged periods of time is evident in AD brain, in which severity of glial activation correlated with cognitive decline (Parachikova et al., 2007). Activation of astrocyte is accompanied by increased production of potentially neurotoxic factors including in ammatory cytokines, nitric oxide and reactive oxygen species (Zameer et al., 2019).
Microglia express Toll-like receptors (TLRs), which play an important role in the immune response as they recognize pathogen molecules. TLRs activation leads to subsequent activation of intracellular signaling cascades that ultimately cause the release of in ammatory molecules (Lehmann et al., 2012;Buon glioli et al., 2019). Let-7 is a potent activator of TLR7 signaling in microglia and neurons, leading to induction of NF-κB in microglia, an in ammatory response and initiation of neurodegeneration (Lehmann et al., 2012), which can add to the already existing pro-in ammatory effects of STZ, aggravating its neurodegenerative actions.

Declarations
Funding: The authors received no nancial support for the research, authorship, and/or publication of this article.
Con icts of interest/Competing interests: The authors declare that there is no con ict of interest.
Availability of data and material (data transparency): Data supporting the ndings of this study are available from the corresponding author upon reasonable request.
Code availability (software application or custom code): Not applicable.  Groups with different letters have statistically significant (P<0.05) differences.    Correlation between p-Tau concentration and the fold change of Let-7 miRNAs

Supplementary Files
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