Induction of Brain Insulin Resistance and Alzheimer’s Molecular Changes by Western Diet

The term Western diet (WD) describes the consumption of large amounts of highly processed foods, rich in simple sugars and saturated fats. Long-term WD feeding leads to insulin resistance, postulated as a risk factor for Alzheimer’s disease (AD). AD is the main cause of progressive dementia characterized by the deposition of amyloid-β (Aβ) plaques and neurofibrillary tangles consisting of the hyperphosphorylated tau (p-Tau) protein in the brain, starting from the entorhinal cortex and the hippocampus. In this study, we report that WD-derived impairment in insulin signaling induces tau and Aβ brain pathology in wild-type C57BL/6 mice, and that the entorhinal cortex is more sensitive than the hippocampus to the impairment of brain insulin signaling. In the brain areas developing WD-induced insulin resistance, we observed changes in p-Tau(Thr231) localization in neuronal subcellular compartments, indicating progressive tauopathy, and a decrease in amyloid precursor protein levels correlating with the appearance of Aβ peptides. These results suggest that WD promotes the development of AD and may be considered not only a risk factor, but also a modifiable trigger of AD.

Introduction
Alzheimer's disease (AD) is an aging-dependent, irreversible progressive neurodegenerative disorder and the most common cause of dementia, accounting for an estimated 60-80% of cases worldwide [1,2]. The lack of efficient AD treatments stems from the still incomplete understanding of AD causes and factors modulating AD progression. The prevailing AD hypothesis points to the central role of altered cleavage of the amyloid precursor protein (APP) with an accompanying pathology of the tau protein. While elucidating the role of various factors in the pathological processes associated with these two proteins is still in focus for the identification of novel therapeutic targets, the role of lifestyle factors, including unbalanced nutrition, in the development of AD is now beginning to attract considerable attention.
Over 95% of AD cases are sporadic and late onset, with a complex etiology. Studies indicate that sporadic AD can be triggered by external factors (e.g., environmental and metabolic) and may be associated with other chronic comorbidities, especially those derived from bad lifestyle. Sporadic AD is often accompanied by disorders such as hypercholesterolemia, dyslipidemia, impaired insulin signaling, or impaired cellular energy metabolism [3,4] Furthermore, an increased risk of sporadic AD is associated with the presence of the ε4 allele of apolipoprotein E (APOE ε4) [5], involved in lipid metabolism.
Typical AD brain lesions are senile extracellular plaques composed of Aβ peptides and neurofibrillary tangles (NFTs), which are degenerating neurons filled with over- 2 of 36 phosphorylated, aggregated tau protein (p-Tau) [2]. Tau pathology begins in the temporal lobe region of the intraparietal (entorhinal) cortex and gradually progresses, involving structures such as the hippocampus and association cortex. Senile Aβ plaques initially localize in the frontal area of the brain followed by occurrence and deposition in the temporal lobe and later spread into further areas of the brain. Intracellular accumulation of Tau impairs basic neuronal functions essential for survival and leads to a path of degeneration. Tau pathology correlates with progressive gray matter loss and the appearance of the first typical clinical symptoms. In contrast, the initial stage of Aβ plaque formation does not necessarily produce clinical symptoms and is also often observed in patients without symptoms of dementia [6].

p-Tau231 as One of the Earliest AD Biomarkers
Tau belongs to the family of microtubule-associated proteins (MAPs). It is found mainly in the central nervous system (CNS) in glial cells (astrocytes and oligodendrocytes) and in neurons. Tau undergoes numerous post-translational modifications of which the best known is phosphorylation. The balance between tau phosphorylation and dephosphorylation plays a key role in tau function in the cell. Excessive phosphorylation of tau at the threonine 231 residue (Thr231) lowers the ability of tau to bind to microtubules, and may lead to neuronal cytoskeleton disruption; this modification is associated with p-tau pathological aggregation in the cell and is identified in NFTs in AD brains [7][8][9][10][11] In line with this, phosphorylation at Thr231 has been shown to be one of the consistently increased post-translational modifications of tau in the brain that distinguishes neuropathological Braak stages 0-I from III-IV [12,13]. P-Tau(Thr231) is also present in pre-neurofibrillary tangles, prior to overt filament formation, and is described as one of the earliest AD biomarkers in the cerebrospinal fluid (CSF) and plasma of AD patients [10,14]. Plasma p-Tau(Thr231) demonstrates high diagnostic accuracy in identifying subjects at risk for AD and correlates strongly with Aβ-PET and tau-PET, segregating Aβ-PET quartiles better than plasma p-Tau(Thr181) and CSF p-Tau(Thr217) [12]. Furthermore, plasma p-Tau(Thr231) corelates strongly with the tau pathology occurrence in the entorhinal cortex (Braak I-II), one of the earliest regions affected in AD [15].

Western Diet
The term Western diet (WD) describes the consumption of highly processed foods containing simple sugars and saturated fats as the primary components [16,17]. In fact, the "Western diet" defines a broad spectrum of combinations and sources of macronutrients and micronutrients [18,19]. In the exemplary WD, more than half of the supplied energy comes from carbohydrates (51.8%), about 32.8% from fats and 15.4% from protein. In contrast, in the balanced diet, energy provided from protein should account for 19-35%, 22-40% from carbohydrates, and up to 30% from fats. WD consumption is characterized by a high insulin index value and a high glycemic load value [16]. Continuous consumption of high glycemic index foods results in higher average daily blood glucose levels and greater pancreatic insulin secretion [20]. Chronic hyperglycemia and hyperinsulinemia can induce hormonal and physiological changes that promote insulin resistance and the development of other diseases in modern societies [16]. There is ample evidence supporting the utility of appropriate animal models in rodents that mimic the human spectrum of metabolic disorders resulting from WD dietary intake, such as hyperglycemia, hyperinsulinemia, and insulin resistance, among others. Of the models tested, strain C57BL/6 appears to be the most sensitive to WD-induced changes [21][22][23][24][25].
In WD there are also significantly reduced amounts of vitamins and minerals, which promotes avitaminosis and malnutrition of the body despite excessive dietary caloric supply [26][27][28].

Insulin and Insulin Resistance in the Brain
Insulin is the major anabolic peptide hormone produced not only in the β cells of the pancreatic islets of Langerhans, but also by brain cells. The main metabolic stimulus for insulin secretion is an increase in blood glucose levels. The insulin signaling pathway is activated by insulin secretion, which is subject to hormonal, substrate, and neuronal regulation. Insulin is recognized and attached in the body to the insulin receptor (IR) [29,30]. The IR response to insulin is mediated primarily by the insulin receptor substrates (IRS) [31]. Under physiological conditions, insulin attachment to its receptors leads to the activation of the protein kinase B (Akt/PKB) pathway, among others [32]. For example, upon insulin activation, the autophosphorylation of IRS results in the activation of phosphoinositide 3-kinase (PI3K) followed by the downstream activation of phosphorus-dependent protein kinases 1 and 2 (PDK1 and PDK2), which initiates Akt/PKB activation. Upon activation, Akt regulates cell survival, metabolism, and proliferation; affects the organization of the cell cytoskeleton; and leads to the movement of glucose transporters (e.g., GLUT2/4) from the cell interior to the cell membrane, resulting in glucose uptake. Akt is also involved in the inactivation of glycogen synthase kinase-3 (GSK-3). The active beta isoform of GSK3 (GSK-3β) is responsible for the pathological phosphorylation of the tau protein. In turn, normal insulin activity leads to a decrease in GSK-3 activity, which results in less phosphorylation of the tau protein [29][30][31][32][33]. Until recently, it was thought that the brain was not sensitive to insulin. It is now known that binding of the insulin molecule to the IR regulates glucose uptake into the brain through the translocation of glucose transporters. One source of brain insulin is the periphery, where blood insulin enters the brain through the blood-brain barrier (BBB). Insulin transport by capillary endothelial cells in the BBB is affected by many systemic factors, including those related to metabolic disorders, i.e., obesity, systemic inflammation, or diabetes. The second confirmed source of insulin in the brain is its de novo synthesis in brain cells [34][35][36][37]. Insulin in the brain plays many important functions, such as brain-body communication, in which it acts in a counter-regulatory manner to its functions played in the periphery. Brain insulin can participate in the responses to the hypoglycemic state by increasing glucose levels and decreasing serum insulin levels. Insulin in the (CNS) is also a growth factor participating in synaptogenesis and nerve growth, in control of the energy metabolism, neuronal plasticity, neuronal survival under oxygen and glucose deprivation, dynamics of dendrite formation, and increased neuronal activity. Many of these processes play key roles in consolidation of short-term and long-term memory and cognitive function.
Insulin resistance is defined as the lack of sensitivity of target tissues to insulin, even at high insulin concentrations. Insulin resistance is associated with the development of metabolic syndrome including hypercholesterolemia, triglyceridemia, dyslipidemia, obesity, and non-alcoholic fatty liver disease (NAFLD) and results in diseases such as type 2 diabetes (T2D) [38]. In the early stages, the pancreas compensates for the insensitivity of IRs to insulin with increased release of insulin into the bloodstream by pancreatic β-cells. Long-term increased insulin production results in pancreatic cell hypertrophy and hyperinsulinemia [39]. Key players in IR desensitization are substrates for the insulin receptor, specifically IRS-1, whose phosphorylation at serine residue 616 or 307 prevents the insulin molecule from binding to the IR and is a major mechanism leading to insulin resistance [40,41]. Blockade of insulin signaling prevents glucose transport into cells and gradually leads to accumulation of glucose (hyperglycemia) when continuously supplied with food. With feedback, rising blood glucose levels signal an overproduction of insulin in pancreatic cells (hyperinsulinemia), which can result in pancreatic dysfunction and consequently the development of diabetes. The main factor inducing excessive phosphorylation of IRS-1 at serine residues is proinflammatory cytokines. Another factor known to promote this process is the excessive storage of free fatty acids in the obese state [39][40][41][42].
The aim of this study was to verify the hypothesis that WD, by inducing insulin resistance in the brain, can trigger the initiation and propagation of major neuropathological features of AD, such as Aβ plaques and neurofibrillary tangles. To this aim, male C57BL/6J wild-type (WT) mice were fed a WD or standard diet (SD; CTR) from 3 months of age. The effects of WD on brain levels of the insulin signaling pathway components, p-IRS-1(Ser616), p-Akt(473), and p-GSK-3β(Ser9), and on neuropathological AD biomarkers, p-Tau(Thr231) and APP/Aβ levels, were analyzed in animals at 4, 8, 12, and 16 months of age (animal groups 4M, 8M, 12M, and 16M). Levels of these proteins were assessed in two brain structures characteristic of the preclinical, pre-symptomatic stages of AD development-the entorhinal cortex and the hippocampus.

WD Evokes Hyperinsulinemia, Hyperglycemia, and Systemic Insulin Resistance
Blood glucose levels are dependent on the quantity and quality of carbohydrates consumed in the diet and the functioning of insulin signaling. Elevated blood glucose levels (hyperglycemia) are observed in diabetes and may be indicative of insulin resistance or deficient insulin secretion. Therefore, in this study, blood plasma levels of glucose, insulin, and homeostatic model assessment for the insulin resistance (HOMA-IR) index were determined in all age groups of experimental animals fed a WD and compared to results in mice fed a standard diet (CTR) (Figure 1).

WD Evokes Hyperinsulinemia, Hyperglycemia, and Systemic Insulin Resistance
Blood glucose levels are dependent on the quantity and quality of carbohydrates co sumed in the diet and the functioning of insulin signaling. Elevated blood glucose lev (hyperglycemia) are observed in diabetes and may be indicative of insulin resistance deficient insulin secretion. Therefore, in this study, blood plasma levels of glucose, insul and homeostatic model assessment for the insulin resistance (HOMA-IR) index were d termined in all age groups of experimental animals fed a WD and compared to results mice fed a standard diet (CTR) (Figure 1).  Table S1). A significant increase in glucose levels in 4-month-old mice fed a WD reveals a hyp glycemic peak. At the same time point, we also observed a significant increase in insulin leve which translates into an increase in the HOMA-IR ratio indicative of a state leading to perip eral insulin resistance. These observations indicate a strong early response of the body to W Figure 1 shows that glucose levels decreased in the older groups to levels comparable to t control groups (4M WD vs. 16M WD), which is likely related to an increase in insulin secreti in response to the WD. In the 8-month-old animals, insulin levels reached a plateau and g cose levels decreased. This indicates a short-term achievement of homeostasis. However, s nificantly higher insulin levels in the WD group were again observed in the 12-and 16-mon old groups of animals, confirming a steady increase in insulin levels under the influence of t continued WD. The concomitant increase in HOMA-IR in the 12-month-old mice relative  Table S1). A significant increase in glucose levels in 4-month-old mice fed a WD reveals a hyperglycemic peak. At the same time point, we also observed a significant increase in insulin levels, which translates into an increase in the HOMA-IR ratio indicative of a state leading to peripheral insulin resistance. These observations indicate a strong early response of the body to WD. Figure 1 shows that glucose levels decreased in the older groups to levels comparable to the control groups (4M WD vs. 16M WD), which is likely related to an increase in insulin secretion in response to the WD. In the 8-month-old animals, insulin levels reached a plateau and glucose levels decreased. This indicates a short-term achievement of homeostasis. However, significantly higher insulin levels in the WD group were again observed in the 12-and 16-month-old groups of animals, confirming a steady increase in insulin levels under the influence of the continued WD. The concomitant increase in HOMA-IR in the 12-month-old mice relative to the control group indicates peripheral insulin resistance. It should be noted here that, in the groups from 8M to 16M, the response to the WD varied greatly among individuals, and as a result statistical significance was not obtained.
In summary, in animals maintained on the WD for the shortest period of 3 weeks (4M WD), significant differences between the WD and CTR groups may be related to a strong initial response to a novel adverse stimulus that disrupts homeostasis. After 5 months of WD feeding (8M WD vs. 8M CTR), the stabilization of peripheral insulin and HOMA parameters was observed, probably as a result of strongly interacting compensatory mechanisms and stabilizing homeostasis, despite constant WD stimulus. Subsequently, the results showed a gradual stabilization of the differences between the CTR and WD groups in mice maintained on WD for 9 months (12M WD vs. 12M CTR) and a gradual weakening of protective and compensatory mechanisms with aging, allowing for a worsening of adverse responses to WD-induced changes. The reference ranges of the analyzed parameters were established based on the extreme results for the control group in each age group of mice: for glucose 17.82-23.37 mM/L and for insulin 100.1-147.8 pM/L. Physiologically occurring mean baseline HOMA-IR scores in control mice were 13.22-21.83.

WD Differentially Impacts Insulin Signaling Pathway and Tau Phosphorylation in Entorhinal Cortex and Hippocampus
Next, we analyzed the effects of WD on markers of the insulin signaling pathway and of AD-type neuropathology in the two brain regions: in the entorhinal cortex and the hippocampus. Since AD is age related, we analyzed the influence of both the age factor (4M, 8M, 12M, and 16M) and the diet factor.
In addition, in both brain regions, immunofluorescence analysis of brain tissue sections was performed to assess the presence and location of neuropathological changes typical of AD. The subcellular localization of p-Tau(Thr231) was assessed, because during the initial accumulation of phosphorylated forms of the tau protein, the localization of hyperphosphorylated tau including p-Tau(Thr231) changes. Tau dissociates from microtubules and is progressively displaced from nerve fibers into the cell bodies, leading to destabilization of the cytoskeleton. Analogous brain tissue sections series were immunofluorescently stained to determine whether any changes in APP levels by the WD may be related to amyloidogenic proteolysis of this protein leading to the formation of pathological Aβ peptides in the brain. For this purpose, an antibody, 6E10, specifically recognizing the Aβ peptide was used.
2.2.1. WD Causes Brain Insulin Resistance but Does Not Impact the Level of p-Tau(Thr231) in the Entorhinal Cortex of WT C57BL/6J Mice Figure 2A,B presents the Western blotting results of mouse brain tissue lysates performed to analyze the insulin signaling pathway in entorhinal cortex of C57BL/6 mice under WD conditions. The data reveal a significant increase in p-IRS-1(Ser616) starting from 8 months of age, which indicates the development of insulin resistance in the entorhinal cortex after approximately 5 months of WD feeding. With respect to peripheral insulin resistance, which was observed in the young, 4M WD group, these data show a delayed response to WD in the brain. When a brief restoration of insulin homeostasis was observed peripherally in 8-month-old mice (Figure 1), data in Figure 2A demonstrate sustained insulin receptor (IR) insensitivity and insulin resistance in the brain at this time.   Tables S1 and S2). (C) Microphotographs showing representative preparations from the entorhinal cortex of C57BL/6 mouse brains from groups at 4-, 8-, 12-, and 16-monthsold, immunofluorescently labeled with an antibody recognizing a tau protein isoform phosphorylated at the threonine 231 site. Negative and positive controls to staining specificity were performed in 20-month-old mice of the transgenic mouse model of AD-Tg2576 (APPswe) and a diagnosed human AD patient. 400× magnification.
Analyzing further components of the intracellular insulin signaling pathway, we observed a trend toward decreased levels of the active form of p-Akt(Ser473) in 12-monthold animals fed the WD (Figure 2A,B), which correlates with the increase in the level of p-IRS-1(Ser616) in this group of animals. The pronounced individual variability observed in the levels of p-Akt(Ser473) may be due to the multifunctionality of this kinase and interactions with many other cellular factors.
The next component of the signaling pathway examined is GSK-3β ( Figure 2A,B). It was observed that, only in the 12-month-old group, there was a decrease in the level of the inactive form of GSK-3β (p-GSK-3β(Ser9)). This result correlated with a decrease in p-Akt(Ser437) activity and increased level of p-IRS-1(Ser616). In the oldest 16M mice there was a slight rise of the p-GSK-3β(Ser9) level, with a concomitant increase in the level of p-Akt(Ser473) observed, independently of significantly increased level of p-IRS-1(Ser616). The data obtained show that WD causes an insulin signaling pathway downregulation specifically in the entorhinal cortex of 12-month-old mice. However, these WD-induced changes did not affect the level of p-Tau(Thr231) in the entorhinal cortex.

WD Causes Changes in the Location of p-Tau(Thr231) in Neuronal Compartments in Entorhinal Cortex of C57BL/6J Mice
Consistent with the results obtained from Western blotting (Figure 2A,B), the immunofluorescence analysis of sections from the entorhinal cortex area using an anti-p-Tau(Thr231) antibody showed no significant differences under WD ( Figure 2C). Although there were no differences in the p-Tau(Thr231) levels, we observed accelerated changes in the localization of the p-Tau(Thr231) isoform in subcellular compartments. The analysis in the youngest groups of animals (4M WD and 4M CTR) revealed a typical pattern of tau protein labeling, both along the course of nerve fibers and in the layer of cytoplasm surrounding the cell nucleus. In 8-month-old mice, such a labeling pattern of p-Tau(Thr231) was observed only in control animals (8M CTR). In contrast, in the 8M WD group, disruption of p-Tau(Thr231) labeling in fibers and restriction of immunostaining mainly to neuronal cell bodies was observed, indicating impairment of normal tau function and dissociation of this protein from microtubules ( Figure 2C). In control mice, this phenomenon was age related and appeared in the group of 12-month-old mice (12M CTR) and later in 16-month-old mice (16M CTR).
This result indicates that WD accelerates the alteration of tau protein compartmentalization in neurons and the progressive destabilization of the cell cytoskeleton several months earlier than is observed in the age-dependent progression of tauopathy in control mice.

WD Does Not Cause Insulin Resistance or Changes the Level of p-Tau(Thr231) in the Hippocampus of WT C57BL/6 Mice
In contrast to the results from the entorhinal cortex, in the hippocampal area, we did not observe insulin resistance induction by WD ( Figure 3A,B). A significant increase in the level of p-IRS-1(Ser616) was observed only in the 8M WD group of mice in comparison to 4M WD mice. In the older 12M and 16M groups, the p-IRS-1(Ser616) levels decreased in control groups and similarly in WD groups. This indicates that this phenomenon in the hippocampus is dependent predominantly on age but is rather independent of WD. Such observations clearly confirm the different sensitivity of entorhinal cortex and the hippocam-pus to diet conditions and indicate a specific sensitivity of entorhinal cortex neurons and their quick response to WD-dependent processes resulting in insulin signaling changes. and their quick response to WD-dependent processes resulting in insulin signaling changes.
In the hippocampus only in the 8M WD group, the levels of two assessed kinases, the active form of Akt and the inactive form of GSK-3β, showed a decreasing trend compared with age-matched controls. This is consistent with an increase in p-IRS-1(Ser616) levels in this group. In the other age groups, no differences were observed between animals fed the SD (CTR) and the WD. At the same time, the subtle changes observed in the levels of p-IRS-1(Ser616), p-Akt(Ser473), and p-GSK-3β(Ser9) proteins in the hippocampus were not associated with significant changes in the levels of p-Tau(Thr231) in all assessed age groups. Statistical analysis revealed an aging-related increase only in the level of p-Tau (Thr231) between 8M control mice and 12M control mice.

The Comparison of Entorhinal Cortex and Hippocampus Areas in Terms of WD-Induced Amyloidopathy
The results analyzed so far show that WD specifically induces insulin resistance and impairment of tau protein function typical of progressive tauopathy, selectively in the entorhinal cortex area and not in the hippocampus. In the next step, we compared the effects of WD on both of these brain structures in terms of the induction of the amyloidopathy. To this end, we determined the level of a normal, full-length, amyloid precursor protein (APP) and performed immunofluorescence analysis of the products of pathological amyloidogenic proteolysis of APP-Aβ peptides.
WD Causes a Decrease of Full-Length APP Protein Level Specifically in Entorhinal Cortex but Not in the Hippocampus in WT C57BL/6J Mice Figure 4A,B shows a WD-induced decrease of the full-length APP protein only in the entorhinal cortex, in the group of deeply aged 16-month-old animals. This WD-induced decrease of APP levels may point to an increasing level of APP degradation as a result of induction of amyloidogenic proteolysis process and the formation of its products in the form of pathological Aβ.     Table S3. In the hippocampal area we did not observe such significant changes, apart from an apparent decrease in the 4M group fed the WD ( Figure 4A,B). In older WD groups, the APP levels reached parity with those observed in control groups. These data again confirm the greater sensitivity of the entorhinal cortex neurons to WD.
Since Western blotting data showed an effect of WD on full-length APP levels in the entorhinal cortex but not in the hippocampus, in the subsequent step, we performed an immunohistochemical analysis of Aβ peptide occurrence only in the sections from the entorhinal cortex ( Figure 4C). Regardless of age in all four control groups (4-, 8-, 12-, and 16-month-old groups), the intensity and pattern of Aβ labeling was similar, and only a very weak Aβ signal was observed in the cytoplasm of neurons. In contrast, in 16-monthold mice fed the WD (16M WD), the evaluation demonstrated an intensification of Aβ peptide staining in the cytoplasm of neurons in entorhinal cortex ( Figure 4C). This result is consistent with the decrease in APP levels in the 16M WD group of mice shown in Figure 4A. Additionally, in agreement with the results shown in Figure 4A,B, in 4-, 8-, and 12-month-old mice, the WD did not significantly affect either the level of the fulllength, normal form of APP, or the change in the labeling pattern of the Aβ peptide in the entorhinal cortex ( Figure 4C).
The results presented so far indicate that the first area where the molecular mechanisms leading to the neuropathological changes typical of Alzheimer's disease are disrupted is the entorhinal cortex. This result is consistent with the existing knowledge on the progression of amyloidopathy and tauopathy processes in the temporal lobe in humans. Additionally, consistent with results from studies in humans, we observed that tau protein-related disorders appeared much earlier than amyloidopathy.

Analysis of WD-Derived Insulin Signaling Impairment on AD Markers, Based on Individual Extreme Values
Next, we performed a detailed analysis of the studied parameters in single individuals. In order to assess the correlation between insulin resistance and tau pathology, we selectively analyzed the individuals with the highest and lowest levels of the p-IRS-1(Ser616) and p-Tau(Thr231). Supplementary Materials Figure S1 presents the extreme values for each individual in all experimental and age groups. Figure 5 shows that some molecular responses to the WD show individual variability and that the differences are the most common and higher in the young 4M animals and stabilize in the older 12M and 16M animals. Figure 5A presents analysis of extremes of p-IRS-1(Ser616) and p-Tau(Thr231) in the entorhinal cortex of mice in all age groups. After the initial 3 weeks of WD feeding, 4M animals showed the greatest variation in response to WD and revealed no direct relationship of developing cerebral insulin resistance to levels of p-Tau(Thr231) and APP ( Figure 5A, 4M). The individual with the highest level of p-IRS-1(Ser616) showed an increase in p-Akt(Ser473) and p-GSK-3β(Ser9) levels, with no increase in p-Tau(Thr231) and decreased APP levels relative to the mean value for the 4M WD group. In contrast, the highest value for p-Tau(Thr231) correlated with a decrease in APP levels, but these were not associated with impaired insulin signaling in this individual.

Analysis of the Entorhinal Cortex of C57BL/6J Mice Fed with WD
The most consistent linear runs and smallest relative min-max ranges for the group were observed in 12-month-old mice after 9 months of WD feeding (Figure 5A: 12M; Figure S1: 12M). In the 12M WD group, there is a stabilization of molecular parameters. In this group, we observed a significant decrease in the level of the active form of Akt, correlating with a decrease in the level of the inactive form of GSK-3β compared to animals on the standard diet (12M CTR, gray bars). However, the disruption of normal activity of both kinases does not translate into an increase in p-Tau(Thr231) and a decrease in APP, either for the range of values for the WD group (blue bars) or for animals maintained on the standard diet (gay bars). The highest level of p-Tau(Thr231) in the entorhinal cortex of 12-month-old mice did not correlate with changes in the levels of any of the other markers tested.
In the entorhinal cortex of the oldest group of 16-month-old C57BL/6J mice ( Figure 5A  16M), the increase in the p-IRS-1(Ser616) level correlated with a significant decrease in APP, but did not raise the level of p-Tau(Thr231).
These results confirm that WD influences both p-Tau(Thr231) and APP and may affect the insulin signaling pathway, but the changes are related to differential responses to experimental conditions in particular individuals. It is noteworthy that WD-induced changes in p-IRS-1(Ser616), p-Akt(Ser473), and p-GSK-3β(Ser9) do not necessarily occur simultaneously. A decrease in APP level and likely activation of amyloidogenic proteolysis processes occurs with long-term WD feeding and correlates with an increase of p-IRS-1(Ser616), but this process does not occur simultaneously with tau pathology.

Analysis of the Hippocampus of C57BL/6J Mice Fed with WD
In the hippocampus, the linear patterns of the dependencies of the individual markers assessed are contra-directional compared to those in the entorhinal cortex in all age groups of animals fed the WD ( Figure 5B).
In 4-month-old animals (4M WD), the highest level of p-Tau(Thr231) was observed in the individual with the lowest level of p-IRS-1(Ser616). In contrast, a decrease in APP level was observed with the highest level of p-IRS-1(Ser616) for the WD-fed group.
The WD in 12-month-old mice (12M WD) increases the p-IRS-1(Ser616) and decreases p-Akt(Ser473) and p-GSK-3β(Ser9), but neither p-Tau(Thr231) nor APP are significantly affected. In contrast, the highest level of p-Tau(Thr231) was not dependent on changes in the levels of all three insulin signaling markers and was also not associated with a decrease in APP level.
Only in the hippocampus of 16-month-old mice fed with WD (16M WD) was the highest level of p-IRS-1(Ser616) observed consistent with the highest level of p-Tau(Thr231), but unlike the observations from the entorhinal cortex, no reduction in APP levels was observed in these individuals.
affected. In contrast, the highest level of p-Tau(Thr231) was not dependent on changes in the levels of all three insulin signaling markers and was also not associated with a decrease in APP level.
Only in the hippocampus of 16-month-old mice fed with WD (16M WD) was the highest level of p-IRS-1(Ser616) observed consistent with the highest level of p-Tau(Thr231), but unlike the observations from the entorhinal cortex, no reduction in APP levels was observed in these individuals.  Figure S1).
From the comparison of the entorhinal cortex and hippocampus in Figure 5, based on the analysis of the extreme values for p-IRS-1(Ser616) and p-Tau(Thr231), it is again apparent that each of these brain structures presents a different sensitivity to the effects of the WD. These detailed analyses of single individuals within age groups consistently showed that the entorhinal cortex is more sensitive to the effects of the dietary WD than the hippocampus. As a result of diet-dependent metabolic abnormalities, the processes of  Figure S1).
From the comparison of the entorhinal cortex and hippocampus in Figure 5, based on the analysis of the extreme values for p-IRS-1(Ser616) and p-Tau(Thr231), it is again apparent that each of these brain structures presents a different sensitivity to the effects of the WD. These detailed analyses of single individuals within age groups consistently showed that the entorhinal cortex is more sensitive to the effects of the dietary WD than the hippocampus. As a result of diet-dependent metabolic abnormalities, the processes of progressive tauopathy and amyloidopathy begin in the entorhinal cortex, probably even before the manifestation of hippocampus-dependent clinical symptoms of AD. Another conclusion from the analysis of extreme values for p-IRS-1(Ser616) and p-Tau(Thr231) confirms that tauopathy and amyloidopathy both may be induced by metabolic disturbances induced by WD through independent cellular processes, not associated with the IRS-1/Akt/GSK-3 pathway.

Comparative Analysis of the Effect of WD on AD Pathology in Wild-Type C57BL/6J Mice and in the AD Mouse Model Tg2576 (APPswe)
Next, we compared the effect of WD on AD pathology in wild-type C57BL/6J mice and in the AD mouse model Tg2576 (APPswe) ( Figure 6A,B). In this comparison, we considered animals up to 12 months of age because this age range reflects the early, pre-amyloid stages of AD. Moreover, phenotypic characterization of the Tg2576 mouse model of AD indicates that Aβ plaques appear in these mice at the earliest around 12 months of age. In our previous study, we showed that the deposition of senile plaques in Tg2576 mice begins between 12 and 16 months of age [43]. In the present study, no significant changes in APP levels in the entorhinal cortex were observed in either mouse strain until 12 months of age. As was shown earlier in Figure 4, APP levels decreased only in the entorhinal cortex in 16-month-old C57BL/6J strain mice. As demonstrated in Figure 6A, the analysis of the entorhinal cortex of AD mouse model showed a decrease in p-IRS-1(Ser616) levels with age in the control (CTR) group, similarly as in the WD group. No significant differences in p-IRS-1(Ser616) levels were observed with a weak decreasing trend with age in 12-month-old Tg2576 mice. The exact inverse relationship of p-IRS-1(Ser616) levels with WD intake were observed in wild-type C57BL/6J mice, where we observed a significant increase in p-IRS-1(Ser616) levels in the entorhinal cortex of 8M WD and 12M WD animals compared to age-matched controls and the youngest 4-month-old animals.
The data presented in Figure 6A show that, in the wild-type model, without genomewide alterations, WD induces insulin resistance, whereas in the AD model, WD has a downregulatory effect on p-IRS-1(Ser616) levels, suggesting a protective role for the transgene, Swedish double mutation (K670N, M671L) in APP gene.
In contrast, when analyzing p-Tau(Thr231) levels, we observed no effect of WDdependent insulin resistance in wild-type C57BL/6J strain mice, and in the entorhinal cortex of AD Tg2576 mice, there was an increase in p-Tau(Thr231) levels with age, following integration of the transgene in a manner independent of insulin signaling. Only in the youngest group of Tg2576 mice did the analysis show a significant increase in p-Tau(Thr231) under WD compared to control animals, through mechanisms independent of insulin signaling.

The Levels of p-IRS1(Ser616), p-Tau(Thr231), and APP in the Hippocampus of Wild-Type C57BL/6J Mice and in the AD Mouse Model Tg2576 (APPswe)
As presented in Figure 6B, we did not observe any significant dependencies of the levels of studied markers under WD conditions in the hippocampus in both mouse strains. Eventual significant differences are dependent on individual variability and larger or smaller standard deviations shown by the statistical analysis. We revealed only a decreased tendency of p-IRS-1(Ser616) level in 12M Tg2576 mice, similarly as in wild-type 12M C57BL/6J mice. In the hippocampus, we also observed an increase of p-IRS-1(Ser616) in 4M WD group comparing to 8M WD both in WT and Tg2576 mice. Results obtained from p-IRS-1(Ser616) analysis are not related to the results from p-Tau(Thr231) in the hippocampus of either mouse strain. We observed no significant changes in p-Tau(Thr231) level under WD conditions. Full-length APP level in Tg2576 mice significantly increase with age in transgene-dependent manner ( Figure 6B). In C57BL/6J mice we didn't observe such growth in APP levels. Conversely, there is a noticeable decrease during WD feeding as an extreme reaction of C57BL/6J mice to toxic conditions revealed in the youngest 4M mice group. This difference disappears as the WD is applied in these mice (C57BL/6J), suggesting the activation of compensatory processes leading to a return to homeostasis.

WD Influence on p-Tau(Thr231) and Aβ Immunostaining in Entorhinal Cortex of Transgenic Mouse Model of AD
The immunohistochemical analysis of entorhinal cortex tissue section from Tg2576 transgenic mice (Figure 7) revealed a weak intensity of p-Tau(Thr231) staining but typical for its cytoskeletal localization in the cells, in 4M CTR and 4M WD mice. However, in the 4M WD group, a greater accumulation of p-Tau(Thr231) isoform around the cell nucleus was observed. A pattern similar to 4M WD pattern of staining was observed in 8M CTR animals (Figure 7). However, in the 8M WD group, a disappearance of fibers of p-Tau(Thr231) staining was seen, which indicates the start of the disruption of tau functions in microtubule stabilization under WD conditions. In 12M control Tg2576 mice, p-Tau staining only in the cytoplasm of neuronal cell bodies was observed. In 12M Tg2576 mice fed with the WD (12M WD), a significantly higher intensity of cell bodies p-Tau(Thr231) immunostaining was visible when compare to 12M CTR Tg2576 mice. It may additionally indicate a growing cellular production of this p-tau isoform in pathological conditions during WD nourishment.
Immunohistochemical analysis using a 6E10 antibody directed against Aβ peptide ( Figure 7) revealed an increasing trend of Aβ deposits around cell nuclei from 4 to 12 months of age in Tg2576 mice maintained on the WD, when compared to control Tg2576 groups. The intensification of Aβ labeling in the transgenic model of AD (Tg2576) is slightly dependent on age, but the WD increases Aβ levels and enhances its cytoplasmic labeling in all age groups of Tg2576 mice tested (4M WD, 8M WD, and 12M WD).

Discussion
The main research questions posed by this study were whether WD consumption can induce a state of insulin resistance in the brain, and whether this WD-induced insulin resistance can induce molecular mechanisms of AD without any AD-related mutations or AD genetic background in WT animals (C57BL/6J mouse strain).

Insulin Signaling Alteration in AD Human Brain
Disruption of normal insulin signaling and insulin resistance in brain cells (neurons and glial cells) has serious systemic consequences, but is also involved in many neurodegenerative processes, most notably in AD. It has been confirmed that de novo insulin synthesis occurs in the brain and insulin-related disorders in the CNS are not necessarily dependent on peripheral insulin [37,[44][45][46]. The highest levels of mRNA expression of the INS gene in humans have been confirmed, among others, in the areas of the brain that we study and present in this paper, namely in the temporal lobe region [47][48][49], where the propagation of neuropathological changes typical of AD begins. Reduced insulin expression and signaling mechanisms, expression of insulin mRNA, protein level, insulin

Discussion
The main research questions posed by this study were whether WD consumption can induce a state of insulin resistance in the brain, and whether this WD-induced insulin resistance can induce molecular mechanisms of AD without any AD-related mutations or AD genetic background in WT animals (C57BL/6J mouse strain).

Insulin Signaling Alteration in AD Human Brain
Disruption of normal insulin signaling and insulin resistance in brain cells (neurons and glial cells) has serious systemic consequences, but is also involved in many neurodegenerative processes, most notably in AD. It has been confirmed that de novo insulin synthesis occurs in the brain and insulin-related disorders in the CNS are not necessarily dependent on peripheral insulin [37,[44][45][46]. The highest levels of mRNA expression of the INS gene in humans have been confirmed, among others, in the areas of the brain that we study and present in this paper, namely in the temporal lobe region [47][48][49], where the propagation of neuropathological changes typical of AD begins. Reduced insulin expression and signaling mechanisms, expression of insulin mRNA, protein level, insulin receptors, and downstream signaling elements have been demonstrated in patients with sporadic AD. Impaired insulin signal transduction with reduced tyrosine-kinase activity of the IR has also been reported in patients [50][51][52]. Insulin resistance is associated with reduced responses to insulin signaling in the IR/IRS-1/PI3K signaling pathway. In the brain, reduced insulin response is associated with elevations of IRS-1 phosphorylated at serine 616 (p-IRS-1(pS616)) from mild cognitively impaired (MCI) cases to AD cases. The protective role of this hormone against Aβ accumulation is reduced, at the same time as the expression and function of insulin are downregulated by Aβ deposits. The alterations of these effects in AD patients interfere with the neuroprotective actions of insulin, facilitating the brain's susceptibility to neurodegeneration. Brain insulin resistance is considered an early and common feature of AD, which seems to be closely associated with the IRS-1 dysfunction [31,53].
When considering the effect of WD on impaired insulin signaling in the brain in AD patients, it is important to consider the effect of dietary components such as simple sugars and cholesterol. Studies in AD patients have shown that reduced cerebral glucose and oxygen utilization occurs [54]. Glucose metabolism in neurons in relation to insulin is particularly important for normal brain function. Glucose is critical for brain energy metabolism, and transient or sustained changes in glucose supply can be crucial for neuronal circuit function. Cognitive deficits are associated with insulin resistance, and impairment of insulin-dependent glucose uptake mechanisms can lead to impaired energy metabolism [49]. Using PET technology, reduced glucose metabolism in the temporoparietal cortex, posterior cingulate cortex, and frontal regions in patients has been confirmed [55], which is associated with the occurrence of cognitive impairment typical of AD. Moreover, elevated levels of circulating diet-derived cholesterol increase the risk of AD because cholesterol modulates Aβ synthesis, inhibits the clearance of Aβ, and potentiates the interaction of Aβ with neuronal membranes. Aβ binds to cholesterol to catalyze the formation of oxysterols, which alter the insulin signaling pathway by inhibiting the phosphorylation of the ERK/Akt route [31,56]. Studies on autopsied human frontal cortices have found that the protein content and activity of the brain insulin/PI3K/Akt signaling pathway are significantly decreased in AD patients. Such impairment of the brain insulin/PI3K/Akt pathway leads to the overactivation of GSK-3β, which promotes altered tau hyperphosphorylation and neurodegeneration [57].
The results obtained in this work substantiate observations of the WD's role in humans. First, we confirmed that the WD indeed resulted in the peripheral metabolic disturbances in WT mice as in the AD mice. Next, we found that the peripheral metabolic disorders in WT mice were followed by induction of an aging-dependent insulin resistance in the brain. In aged WT C57BL/6J mice, the WD led to enhanced amyloidogenic proteolysis of APP, in similar fashion to the APP mutant mice. Despite significant individual variability in wild-type C57BL/6J mice in response to the WD observed in analyses of all parameters examined, these results clearly show that the WD may be considered not only a risk factor, but also a trigger of AD in the absence of AD genetic causes.
In addition, our data revealed the different WD sensitivity of the first brain structures affected in AD, the entorhinal cortex and hippocampus, which are located in the medial part of the temporal lobe of the brain. We found that the entorhinal cortex is more sensitive than the hippocampus to the impairment of brain insulin signaling.
In the entorhinal cortex, WD-induced insulin resistance was followed by progressive tauopathy indicated by changes in p-Tau(Thr231) presence and changes in localization in neuronal subcellular compartments. This result confirms the hypothesis that the PI3K/Akt/GSK-3 signaling pathway underlying insulin resistance in the brain contributes to tau phosphorylation and tauopathy. However, we also observed a WD-induced decrease in APP levels correlating with the appearance of Aβ peptides in the entorhinal cortex of WT animals. Very little data indicate that insulin resistance can increase levels of Aβ peptide in the brain during AD [58]. Our results demonstrated the development of amyloid pathology in parallel to tauopathy in the course of WD feeding in relation to the aging process. The analysis of extreme values for p-IRS-1(Ser616) and p-Tau(Thr231) in our study confirms that tauopathy and amyloidopathy both may be induced by metabolic disturbances induced by the WD through independent cellular processes.
The involvement of particular signaling proteins in the WD-induced brain insulin resistance and the relevance of AD brain biomarkers analyzed in this study for the pathology are discussed below in the light of the existing literature.

The Effect of the Western Diet (WD) on Inducing an Insulin Resistant State in the Brain
In the present study, the areas of the entorhinal cortex and hippocampus from mice brains were analyzed, in order to assess the effect of the WD on the disruption of the insulin signaling pathway. Our results are congruent with the body of reports showing an increase in IRS-1 phosphorylation at the serine (Ser) 312 and 616 sites in AD pathogenesis, whereas under normal signaling conditions, IRS-1 is phosphorylated at the tyrosine site. We observed a WD-derived increase in the level of p-IRS-1(Ser616) in the entorhinal cortex, which indicates the development of insulin resistance. In contrast, the results obtained from our analysis of the hippocampus showed that this structure is insensitive to the WD, where a decrease in p-IRS-1(Ser616) level was observed. There is a report that showed increased levels of p-IRS-1(Ser616) phosphorylation in the hippocampus when C57BL/6J mice were fed with a high-fat diet (HFD) [59]. It is likely that this difference compared to the results of this study is due to the different composition of the diets used. The diet used in the Arnold et al. study did not contain a simple carbohydrate component, and thus the result appears to be due to the effects of only saturated fatty acids. Another study conducted on brain tissue from AD patients showed an increase in serine phosphorylation of IRS-1 in a group with concomitant T2D, indicating an important role of carbohydrate disturbance in these processes [60]. In line with our results, another report showed that 7 days of feeding rats with a WD-type of diet (high-fat-fructose diet, (HFFD)) resulted in a decrease in the level of IRS-1 protein phosphorylation at the tyrosine residue and a decrease in the level of the active form of Akt in the hippocampus [61].

The Effect of the Western Diet (WD) on Kinase Akt Activity
No significant WD-induced changes were observed in the levels of p-Akt(Ser473) in the hippocampus. However, in the entorhinal cortex, a WD-induced decrease in the active form of Akt was observed with aging, in 12-month-old animals. It correlates with an increase in the level of p-IRS-1(Ser616) in the entorhinal cortex in the 12M WD group. A similar correlation was reported in C57BL/6J mice in which a high-fat diet with sugary drinks (HFS) increased the levels of p-IRS-1 protein and caused a decrease in the level of the active form of Akt in lysates from the whole brain [62].

The Effect of the Western Diet (WD) on Kinase GSK-3β Activity
Thr231 is an important priming residue on tau and is a primary phosphorylation site for GSK-3β. The Thr231 residue is located in the assembly domain of tau and phosphorylation of this residue reduced tau affinity for microtubules [10]. Excessive phosphorylation of tau at Thr231 is associated with its pathological aggregation in the cell and is identified in NFTs in the brain of AD patients. Additionally, APP is implicated in regulation of GSK-3 kinase activity. In vitro studies demonstrated that the APP intracellular domain (AICD) interacts with GSK-3 and induces its activity [63]. Analysis of AD human brains showed a reduced level of the inactive form, p-GSK-3, in MCI and late-stage AD (LAD) individuals compared with controls without dementia. Additionally, the phosphorylation of APP at Thr668 was significantly increased in LAD compared to MCI and non-AD individuals, also implying a role for activation of GSK-3 in inducting this phosphorylation. Moreover, the Aβ peptide appearance induces aberrant activation of GSK-3, leading to APP and tau phosphorylation, impaired axonal transport, and microtubule destabilization observed in AD [64]. The present study evaluated the phosphorylation level of GSK-3β, which is directly involved in the hyperphosphorylation of the tau protein in the pathogenesis of AD. There was a decreasing tendency in the level of the inactive form of p-GSK-3β(Ser9) in the entorhinal cortex of 12-month-old animals. These results are consistent with abovedescribed increase in p-IRS-1(Ser616) and decrease of p-Akt(Ser473) levels in this age group of animals.

The Effect of the Western Diet (WD) on Tau Protein Phosphorylation
Increases in GSK-3β kinase activity were shown to result in increased levels of tau protein phosphorylation [62]. Calvo-Ochoa et al. observed increased levels of tau protein phosphorylated by GSK-3β in rats fed a HFFD [61]. Another study using homogenates from the frontal cortex of C57BL/6 mice showed approximately 60% higher levels of p-Tau(Thr231) in the group fed with a HFD diet compared to the control group [65]. In the present study, the quantitative analysis revealed the lack of the effect of WD on p-Tau(Thr231) phosphorylation levels in the entorhinal cortex, despite the confirmed insulin resistance status. Similar observations were made by Grautze et al., who examined the effects of a HFD, high-cholesterol diet (HCD), high-sugar diet (HSD), and a diet containing a combination of all these, on tau protein-related pathology. They conducted the experiment in the entorhinal cortex and hippocampus of hTau mice but did not observe any significant changes in p-Tau [66]. In contrast, in a neuropathological study, colocalization of NFTs and markers of insulin resistance was demonstrated in patients diagnosed with AD [35]. Patients diagnosed with both AD and T2D had more extensive deposition of tau in the new neocortex, as assessed by 11C-PBB3 PET [67]. A lower cerebral metabolic rate of glucose consumption (FDG-PET) has been demonstrated in AD patients and in middle-aged adults at risk for AD, and this parameter is now recognized as one of the neuroimaging methods useful for early AD risk diagnosis [68]. Moreover, peripheral insulin resistance was negatively correlated with gray matter volume in the medial prefrontal cortex, temporal lobe, and parietal areas in AD patients [69]. The divergent reports on this topic may be due to differences in diet composition, differences in the length of feeding, and different responses of individuals to experimental conditions. In addition, wild-type animals like C57BL/6J have high individual variability, which is also evident in results presented in this paper. It is also possible that WD, as a low-grade factor that acts chronically over a long period of time, may produce effects by inducing neuropathological changes that are different across time. Analyzing the proteostatic disequilibrium, pathway disruption, and developed insulin resistance in AD, we can also expect inhibitory activity of protein phosphatase 2A (PP2A) on NFT and Aβ deposition. PP2A contributes to the dephosphorylation of the tau protein, as well as p-IRS at serine residues, and may lead to a return to homeostasis and prevent neurodegenerative processes [33,70]. However, in the present study, the qualitative microscopic imaging analysis revealed a disruption of the compartmentalization of p-Tau(Thr231) inside the neurons in entorhinal cortex under WD conditions. In control animals, age-dependent impairment of p-Tau(Thr231) labeling was observed during aging in 12M and 16M mice. In contrast, in animals fed with WD, the same labeling pattern was observed already in 8M WD. This indicates WD-induced impairment of normal tau protein function and may be associated with progressive breakdown of the cellular cytoskeleton leading to degeneration of neurons in WT animals.

The Effect of the Western Diet (WD) on Amyloidogenic Proteolysis of APP
In WT C57BL/6J mice fed with the WD, we observed a reduction of the level of the fulllength form of APP in the entorhinal cortex in the oldest, 16M, WD mice. It may suggest the activation of the process of amyloidogenic proteolysis leading to the formation of the toxic Aβ peptide. Immunofluorescent microscopic analysis revealed an increase in the intensity of Aβ labeling in the cytoplasm in entorhinal cortex neurons also only in the 16M WD group. To compare, no Aβ was observed in any age groups in mice fed with standard chow. This is consistent with the existing knowledge that the mouse, as a species, does not develop Aβ plaques typical for AD in human. The appearance of Aβ labeling in the neuronal cytoplasm under WD in 16M mice indicates that WD can, however, induce the process of amyloidogenic proteolysis in aged WT animals even without genetic determinants.
The data obtained in our study contribute to the current knowledge on the sequence of progression of tauopathy and amyloidopathy in the human brain in the course of AD development. It is known that tauopathy starts in the area of the entorhinal cortex, and later spreads to the hippocampus, which is reflected in the grading of tau pathology propagation according to . In the advanced stages of AD, tau pathology spreads to the frontal and parietal lobes. Amyloidopathy starts from the frontal and parietal lobes, and then the pathology spreads to the temporal lobe, occipital lobe, and cerebellum in the most advanced stage of the disease [74,75]. The entorhinal cortex and hippocampus, which are located in the medial part of the temporal lobe of the brain, are the two brain areas primarily responsible for memory functioning, processing, and consolidation of short-term into long-term memory. The entorhinal cortex is directly adjacent to the hippocampus and the neocortex, so it mediates the transfer of information from the neocortex to the hippocampal structure [76,77]. In the entorhinal cortex and hippocampus, neuronal loss occurs at the early stage of AD development [78,79]. While it is known the first brain structures affected in AD are the entorhinal cortex and hippocampus, the comparison of these two structures in terms of the sequence of AD biomarkers' appearance upon WD or insulin resistance is less known. Our data suggest that WD induces both tauopathy and amyloidopathy first in the entorhinal cortex, and thus changes in this area probably precede the changes in the hippocampus.
Overall, our data support the view that AD can be initiated by metabolic impairment in the periphery [38,43]. It also strongly indicates that a healthy, balanced diet may be one of the most efficient AD prevention methods.

Animal Experimental Groups
Ten-week-old males of C57BL/6J wild-type strain mice were purchased from Medical Mice were divided into two experimental groups: (1) control (CTR) group fed a standard, balanced diet (SD) starting from 13 weeks of age until they reached 20 months; and (2) mice fed with a Western diet (WD) in the same scheme as the CTR group. Each experimental group was randomly divided into five age groups: 4-, 8-, 12-, 16-, and 20-month-old mice. Due to the increasing mortality with age, completed results are not available for all subgroups. Number of animals included in this article are shown in Table 1 for C57BL/6J mice and Table 2 for APPswe mice.

Diets: Western Diet (WD) and Standard Diet (SD)
The composition of the Western diet (EF R/M E15126-34) and balanced control diet (R/M-H V1534) was from the Ssniff Spezialdiäten GmbH ® Company (Soest, Germany) as previously published [43]. The percentage composition of the diets' ingredients is shown in Table 3.

Mouse Euthanasia and Tissue Collection
Laboratory animals were euthanized by intraperitoneal injection of a mixture of medetomidine at a dose of 1 mg/kg (ORION, Warsaw, Poland; Dexdomitor 0.5 mg/mL) and ketamine at a dose of 75 mg/kg (Biowet, Drwalew, Poland; Ketamine 100 mg/mL) dissolved in 0.9% sterile sodium chloride (NaCl). Blood was collected into heparinized tubes (Sarstedt, Nümbrecht, Germany) from the heart of anesthetized mice for further biochemical analyses, followed by trans-cardial perfusion with cold phosphate-buffered saline (PBS) with 0.1% heparin and 0.184% sodium orthovanadate (Sigma Aldrich, St. Louis, MO, USA). All brain tissue was divided into two hemispheres. Right hemispheres of the brain were used for immunofluorescence staining. Right hemispheres were fixed in 10% buffered formalin, then sections were obtained after embedding in paraffin. From the left hemispheres, the hippocampus and entorhinal cortex were separated to prepare brain tissue lysates for quantifying analysis by Western blot. Tissue was rapidly frozen in liquid nitrogen, then stored at −80 • C before homogenization.

Human Brain Tissue Source
The tissue was obtained from the resources of the Polish Brain Archive of the Institute of Psychiatry and Neurology in Warsaw, Poland. After autopsy, the tissue was stored in 10% buffered formalin until processed in a closed apparatus (Leica HistoCare Pearl Tissue Processor, Leica Microsystems, Wetzlar, Germany). Tissue was rinsed for 24 h in distilled water, then it was dehydrated in a series of increasing alcohol concentrations (6 replicates). The tissue was cleaned in xylene (4 replicates), then saturated with liquid paraffin (62 • C) in triplicate after 2 h and embedded in blocks. The tissue collected in paraffin blocks was cut into sections 8 µm thick using a rotary microtome.

Plasma Total Glucose and Insulin Concentrations
The blood glucose and insulin plasma concentration measurements were collected from mice after 5-6 h of fasting. Plasma was obtained by centrifugation of blood at 4065 rcf for 30 min at 15 • C. Plasma glucose concentration was determined with a biochemical analyzer (Fuji DriChem NX500 Automated Clinical Chemistry Analyzer, Fuji, Tokyo, Japan). Plasma insulin concentration was assayed using Ultrasensitive Mouse Insulin ELISA kit (Crystal Chem, Elk Grove Village, IL, USA; cat. #90080).  Figures A1a,b-A3a,b).

Histological Preparation and Immunofluorescence Staining of Brain Tissue Sections
Collected mouse brain tissues were subjected to a fixation process using 10% buffered formalin using an automated tissue processor (Spin Tissue STP 120, Thermo Scientific, Waltham, MA, USA). The tissue processor subjected the tissue to a series of increasing concentrations (50%, 70%, 80%, and 99.8%) of EtOH, three changes of 99.8% EtOH, three changes of xylene, and two changes to liquid paraffin at 60 • C. Using an embedding workstation (HistoStar, Thermo Scientific, Waltham, MA, USA), paraffin blocks were prepared from the brain fragments and then cut on a rotary microtome (Thermo Scientific, Waltham, MA, USA) to obtain 8 µm thick sections. Tissue was fixed in 10% buffered formalin. Before staining, the brain sections were deparaffinized in three changes of xylene and then rehydrated in decreasing ethanol concentrations (99.8%, 96%, 70%, and 50%) to water. The antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6) with 0.05% Tween in three cycles including a 5 min rinse under elevated microwave temperature set to 600 V, a 10 min cooling at room temperature (RT), and a 5 min cooling on ice. The sections were blocked with 5% normal goat serum with 1% bovine serum albumin in PBS with 0.025% Triton X-100 (PBS-TX) buffer for 1 h at RT. Next, brain slices were incubated overnight at RT with one of the following antibodies: rabbit polyclonal p-Tau (Thr231) 1:100 (Invitrogen, 44-746G) or β-Amyloid (Aβ) (Mouse Purified anti-β-Amyloid, 1-16 Antibody (BioLegend, San Diego, CA, USA, Previously Covance catalog #SIG-39320)). Incubation with a secondary goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 was performed for 2 h at RT. Cell nuclei were stained with Hoechst. The tissue sections after immunostaining were sealed with Moviol and cover slipped.

Statistical Analysis
Statistical analysis was performed in GraphPad Prism 9 (GraphPad, San Diego, CA, USA). Statistical analysis of the semi-quantitative Western Blot analysis included 3 to 5 patients per CTR or WD group. After checking the normality of the distribution using the Shapiro-Wilk test, a two-way ANOVA with multiple comparisons was performed. A p-value < 0.05 was considered a predetermined statistical threshold for significance (* p < 0.05, ** p < 0.01, *** p < 0.001). Completed statistical data are found in Supplementary Materials Tables S1-S4.

Microphotographs Collection
Microscopic analysis was performed using an automated Nikon Eclipse Ni-E microscope (Nikon, Tokyo, Japan) equipped with a Nikon DSFi1c camera (Nikon, Tokyo, Japan) in NIS-Elements Advanced Research 4.00.00 software (Nikon, Tokyo, Japan). Microphotographs were taken at 400× magnification.

Conclusions
The entorhinal cortex is more sensitive than the hippocampus to the development of WD-related brain insulin resistance and AD-type pathological changes in the brain of wild-type mice, without any genetic modifications.
WD-induced insulin resistance affects processes involved in amyloidogenic APP proteolysis and Aβ peptide formation in the entorhinal cortex.
The activation of molecular mechanisms leading to the neuropathological changes typical of Alzheimer's disease-Aβ and NFT-in the entorhinal cortex may precede the changes occurring in the hippocampus.
WD-related metabolic disturbances can induce tauopathy and amyloidopathy through independent cellular processes.
The observed progression of pathological processes leading to NFTs and senile Aβ plaques is age dependent and dependent on the continuity of exposure to a noxious agent.
In summary, these findings highlight the WD as a significant risk factor and a triggering factor for development of insulin resistance and of changes in AD-type pathological markers in the entorhinal cortex, which is believed to be the starting point of Alzheimer's disease in humans. They point to the possible role of well-balanced diet in the prevention of sporadic AD.


Mechanisms of SARS-CoV-2 and Male Infertility: Could Connexin and Pannexin Play a Role?

The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on male infertility has lately received significant attention. SARS-CoV-2, the virus that causes coronavirus disease (COVID-19) in humans, has been shown to impose adverse effects on both the structural components and function of the testis, which potentially impact spermatogenesis. These adverse effects are partially explained by fever, systemic inflammation, oxidative stress, and an increased immune response leading to impaired blood-testis barrier. It has been well established that efficient cellular communication via gap junctions or functional channels is required for tissue homeostasis. Connexins and pannexins are two protein families that mediate autocrine and paracrine signaling between the cells and the extracellular environment. These channel-forming proteins have been shown to play a role in coordinating cellular communication in the testis and epididymis. Despite their role in maintaining a proper male reproductive milieu, their function is disrupted under pathological conditions. The involvement of these channels has been well documented in several physiological and pathological conditions and their designated function in infectious diseases. However, their role in COVID-19 and their meaningful contribution to male infertility remains to be elucidated. Therefore, this review highlights the multivariate pathophysiological mechanisms of SARS-CoV-2 involvement in male reproduction. It also aims to shed light on the role of connexin and pannexin channels in disease progression, emphasizing their unexplored role and regulation of SARS-CoV-2 pathophysiology. Finally, we hypothesize the possible involvement of connexins and pannexins in SARS-CoV-2 inducing male infertility to assist future research ideas targeting therapeutic approaches.

INTRODUCTION
The discovery, virulence, and transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) took the world by surprise and have unfortunately led to the ongoing pandemonium. Although some coronaviruses have previously caused epidemics (Hamre and Procknow, 1966;McIntosh et al., 1967;Peiris et al., 2003;Woo et al., 2005), the emergence of SARS-CoV-2 as the virus SARS-CoV-2 and Male Infertility: Mechanisms SARS-CoV-2 impacts the male reproductive system potentially via angiotensin-converting enzyme two receptor (ACE2) utilization as a route to affect reproduction (Youni et al., 2020), similar to its entry to other host cells. Briefly, SARS-CoV-2has four main structural proteins: spike surface glycoproteins (S), small envelope proteins, matric proteins, and nucleocapsid proteins (Harmer et al., 2002;Stanley et al., 2020). The S proteins have two subunits (S1 and S2) responsible for receptor recognition and membrane fusion. SARS-CoV-2 binds its C-terminal domain of the S1 subunit to the host cell's ACE2 receptor (Illiano et al., 2020). Host proteases such as transmembrane serine protease 2 (TMPRSS2) then cleaves the viral S protein causing a conformational change, allowing permanent fusion of the viral and host cell membranes, and thus infection. SARS-CoV-2 infection elicits a pro-inflammatory cytokine storm, resulting in multi-organ damage such as the lungs, heart, and brain, which are also identified as the main target of SARS-CoV-2 mediated pathogenesis (Selvaraj et al., 2021). The testes of men with COVID-19 are often not exempted from the cytokine storm. The infection causes disrupted spermatogenesis, abnormal semen parameters, and orchitis in these men (Corona et al., 2020;Pan et al., 2020). SARS-CoV-2 may impair male fertility via altered ACE2 expression in the testes, elevated body temperature (fever), pro-inflammatory cytokines storm, hormonal disruption, oxidative stress, or possibly through the functional disruption of Cxs and/or Panxs.

ACE2, SARS-CoV-2, and Male Infertility
ACE2 is a transmembrane zinc metallopeptidase containing a single catalytic domain with homolog to angiotensin-converting enzyme (ACE), attached to the cell membrane of cells in different tissues, including the testis (Li et al., 2020b;Wang et al., 2020). The abundant expression of ACE2 in the different cells of the testes (spermatogonia, Leydig cells, and Sertoli cells) and some other components of the male genital tract such as the prostate gland and epididymis (Song et al., 2020b) have brought about the concept that SARS-CoV-2 can potentially affect male fertility negatively. Furthermore, ACE2 expression in the Leydig cells has been shown to aid testosterone production and steroidogenesis regulation, and it mediates local vascular regulation to balance the interstitial fluid volume. Ang1-7 and Mas, which are products of ACE2 catalyzation, were also observed in the seminiferous tubules and the interstitial component of the seminiferous tubules of men with normal spermatogenesis, but not in infertile men (Reis et al., 2010). Additionally, the protease that aids the binding and subsequently allows the entry of SARS-CoV-2 is abundantly expressed in the testes (Liu et al., 2020;Seymen, 2021). Sine ACE2 and TMPRSS2 are present in the male genital tract, including the testes, it can be speculated that upon viral entry, male fertility could be impaired.

SARS-CoV-2, Fever, and Male Infertility
Another concern about how SARS-CoV-2 may affect male fertility is sustained fever. Recent studies have indicated that high body temperature may induce a secondary cytokine storm (hyperinflammatory syndrome with fulminant and fatal hypercytokinemia with multiorgan failure) (Batiha et al., 2020;Swayne et al., 2020) which in turn harm spermatogenesis in COVID-19 patients (Batiha et al., 2020;Paoli et al., 2020). Hence, abnormal changes in the cytokine profile may affect male fertility, with a long-term impact.
Although the testis contains heat shock proteins (HSP) that protect against heat and chemical radiation, excessive exposure could harm male reproduction. Studies have revealed that spermatocytes are the most susceptible to heat shock, and apoptosis of these cells occurs upon exposure to heat (Rockett et al., 2001). Rockett et al. reported that specific proteins were upregulated following heat shock while others were downregulated. The subsequent effect of fever on male reproduction includes a temporary reduction in relative testicular weight and a brief period of partial or complete infertility (Rockett et al., 2001). In addition, a decrease in sperm motility, reduced in vitro fertilization (IVF) rate by sperm from heat shock (Jannes et al., 1998), increased rate of embryonic degeneration in rats (Setchell et al., 1988) and rams (Mieusset et al., 1992) were also reported.
Briefly, during heat shock, there is induction of HSPto protect against the damaging effects of heat. However, upon prolongation, the rate of cell cycle disruption is higher than the produced HSP, hence inducing apoptosis. Apoptosis ensues because there is a reduction in the expression of antiapoptotic genes such as Bag-1 to protect against apoptosis. The reduction of Bag-1 may also allow the inappropriate HSPmediated protein refolding during the cell recovery phase from stress. The abnormally folded proteins could, in turn, induce apoptosis of the affected cells. The following receptors were upregulated in response to heat shock: laminin receptor, laminin γ2, and HSP25 (Rockett et al., 2001). Laminin, along with collagen type IV, is a major component of the peritubular basal lamina of the seminiferous epithelium (Santoro et al., 2000). Interactions between Sertoli cells and the basal lamina are essential for maintaining the BTB.
Additionally, SARS-CoV-2 infection could enhance inflammatory response leading to fever. Human monocytes infected with SARS-CoV-2 exhibit increased production of chemokines (IL-8 and CXCL10), inflammatory cytokines (IL-6, TNFα and IL-10) and molecules that are generated primarily through an oxidative pathway from arachidonic acid (AA), including leukotrienes (the cysteinyl leukotriene -CysLT-and leukotriene B4 -LTB 4 -) (da Silva Gomes Dias et al., 2020). Other metabolites of AA, such as prostaglandin E2 (PGE2) are elevated in patients with COVID-19 disease and are associated with the disease severity and sex, with higher levels in men compared to women (Ricke-Hoch et al., 2021).
Moreover, in male zebrafish, PGE2 was demonstrated to reduce the proliferation of spermatogonia progenitors, although it increased the self-renewal proliferation of type A undifferentiated spermatogonia resulting in their accumulation (Crespo et al., 2020), with Cx43 serving as important portals for prostaglandin release (Riquelme et al., 2015). Therefore, the controlled prostaglandin levels released during COVID-19 could be a critical factor that induces the sperm alteration observed in some men infected with SARS-CoV-2.

SARS-CoV-2, Inflammation and Male Infertility
During a viral infection, there is an elevation in systemic inflammation. This inflammatory response induction is meant to protect the host; nevertheless, the prolonged or sustained response could potentially cause harm (Hassanin et al., 2018). The mediation of systemic inflammation on somatic cells is different from the immune response in the testes. This is due to the BTB near the basement membrane of the seminiferous tubules, where it divides the epithelium into the basal and adluminal compartments. The BTB elevates the immune status in the testes and preserves male gonads from uncontrolled immune responses. However, the overproduction of inflammatory cytokines induced by viral infections can lead to autoimmune responses and infiltration of leucocytes, which subsequently disrupt spermatogenesis and interfere with sexrelated hormones secretion (Hedger and Meinhardt, 2003). During a sustained inflammation state, there is an increase in the generation of inflammatory cytokines and transcriptional factors such as interleukins (IL)-1, IL-6, IL-8, IL-10, IL-17, TNF-α, and NF-kβ, which in turn leads to an increase in the production of reactive oxygen species (ROS) and concomitantly elevating polymorphonuclear leukocytes and granulocytes. Although, a specific timeline for the initiation of each inflammatory response is yet to be agreed upon; however, studies have shown that the intensity, and the time course of the observed inflammatory response (s) and or subsequent impact depends on the cell or tissue types, the concentration of the pro-inflammatory agent that the cell or tissue of interest were treated with, the duration of treatment. These responses consequently lead to the development of oxidative stress (OS). The occurrence of OS may cause impaired spermatogenesis by damaging the intactness of BTB or disrupting testicular cells functions (Haghpanah et al., 2021). In severe cases of COVID-19, it was shown that there is an increase in the level of expression of inflammatory cytokines and their receptors in testicular cells (Shen et al., 2020). This suggests that systemic inflammation may in part disrupt the integrity of the BTB. Thus, causing its permeability to inflammatory cytokines and consequently lead to the altered spermatogenesis seen in these cases.

SARS-CoV-2, Altered Hormone, and Male Infertility
SARS-CoV-2 can cross the blood-brain barrier (BBB) and dock with ACE2 expressing neurons and glial cells, thereby causing neuroinflammation and neuropathogenesis. The hypothalamus, which is the center that controls body temperature and hormonal balance, amongst other physiological functions, is therefore affected during SARS-CoV-2 infection . Studies have also shown that SARS-CoV-2 is able to cross the BBB through a transcellular pathway accompanied with matrix metalloproteinase 9 (MMP9) mediated basement membrane disruption without causing tight junction alteration (Zhang et al., 2021).
Under normal circumstances, the hypothalamus secretes the gonadotropic releasing hormone (GnRH), and it activates the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. The secreted FSH stimulates the Sertoli cells to nurture maturing germ cells, while LH stimulates Leydig cells towards testosterone production. This feedback loop is strictly controlled. However, any condition that can cause the malfunctioning of the hypothalamic-pituitary-gonadal axis (HPGA) may result in male fertility impairment. For instance, low GnRH levels cause a decrease in FSH and LH, resulting in impaired function of the Sertoli and Leydig cells. Although there was no significant difference in the serum testosterone and serum FSH, the ratio of testosterone to LH (T:LH) and the ratio of FSH to LH (FSH: LH) were significantly reduced in the sera of COVID-19 patients compared to the control group. This suggests that being infected with SARS-COV-2 may alter sex-related hormones (Ma et al., 2020). Additionally, infection with COVID-19 has been shown to cause histopathological changes in postmortem testicular tissues . Taken together, it is theoretically possible that the normal functioning of the HPGA is altered during SARS-CoV-2 infection, which subsequently could cause Leydig and Sertoli cell malfunctioning, thereby driving the pathogenesis of testicular dysfunction in COVID-19 patients.
Having discussed the already suggested pathways through which SARS-CoV-2 can negatively impact male fertility, the remaining section will highlight the potential role of Cx and Panx in SARS-CoV-2 induced male infertility.

Connexins and Pannexins: The Communicating Channels
Tissue homeostasis is dependent on signal transduction that regulates intimate molecular exchanges between extracellular, intracellular, and intercellular networks. Direct cell-to-cell contact or interaction between cells and their extracellular environment is crucial in maintaining proper cellular functions. Such communication is maintained via Cxs or gap junction (GJ) channels (Raghavan et al., 2021;Taylor et al., 2021) or via poreforming channels -Panxs. The functional properties of these channels are attributed mainly to different physiological and pathophysiological stimuli and their channel activity and localization, which are evidenced to be modulated by different post-translational modifications (Luu et al., 2021).
While no sequence homology exists between Cxs and Panxs, they share similar topologies. Both protein families display four α-helical transmembranes (TM) domains, two extracellular loops (EL), and one intracellular loop (IL), with their amino (NT) and carboxyl termini exposed to the cytoplasm (Wei et al., 2015;Waisman et al., 2015). Unlike Cxs, which mediate autocrine signaling by forming a 2-3 nm gap separation via the docking of two "connexon" hemichannels (Waisman et al., 2015), Panxs channels do not form gap junctions (GJs) (Figure 1). Panxs are integral large pore-forming channels that mediate paracrine signaling via ATP release into the extracellular milieu Draganov et al., 2015). Analogous to Cxs, Panxs also function as conduits of physiologically essential molecules and ions such as intracellular Ca2+ as being a secondary messenger, glucose uptake, and other small molecules (up to 1.5 kDa). Nonetheless, the mediated autocrine and paracrine cell signaling plays diverse roles in achieving proper regulation and coordination of different tissue functions. Regulatory mechanisms include vascular functions and permeability, smooth muscle proliferation, platelet aggregation, and leukocyte transmigration (Langlois et al., 2014;Ma et al., 2014;Makarenkova and Shestopalov, 2014). Of note, the role of Panx channels has been well implicated in the development of several tissues and cell types, including cell proliferation and differentiation (Oviedo-Orta et al., 2013;Pfenniger et al., 2013;Wang et al., 2013;Bond and Naus, 2014). Overall, these channels need to be tightly regulated to maintain cellular homeostasis.

Connexins and Pannexins in Inflammation: Relative to COVID-19
Inflammation is a disease hallmark, requiring immune system activation, accompanied by regeneration, apoptosis, or necrosis events. The immune response to an injury requires ATP release into the extracellular space for inflammasome maturation (Orellana et al., 2013), which is reflected by the production of inflammatory cytokines and chemokines (Orellana et al., 2013). Together, Cxs and Panxs play a critical role during the inflammatory response, highlighted in this section. Among the Panx protein family, Panx1 has been the best characterized protein channel in diseases for ATP release (Gao et al., 2013;Hung et al., 2013). Panx1 is believed to be a key regulator of the inflammatory process by forming a complex with the purinergic receptors (P2X1, P2X4, and P2X7), thus mediating ATP release that leads to pro-inflammatory cytokine processing through the maturation and release of IL-1α, IL-1β, and IL-18 (Penuela et al., 2012;Looft-Wilson et al., 2012;Lohman et al., 2012;D'hondt et al., 2013). Such protein complex formation has also been shown to generate ROS (Kerr et al., 2012). In addition to inflammasome activation, Panx1 mediated ATP release channels have also been attributed to immune functions through the "findme" signals that play a role in removing apoptotic cells (Seminario-Vidal et al., 2011;Cerami et al., 2012;Gulbransen et al., 2012;Kerr et al., 2012;Looft-Wilson et al., 2012). Furthermore, the Panx1-purinergic complex contributed to cell death under ischemic conditions and seizures. This was studied in different diseases, such as inflammatory bowel diseases, Crohn's, AIDS, and epilepsy (Santiago et al., 2011;Pointis et al., 2011;Ishikawa et al., 2011;D'hondt et al., 2011;Bond et al., 2011;Billaud et al., 2011;Bargiotas et al., 2011). Of note, Panx1 channel expression and activity were reported to increase significantly upon injury or inflammation (Swayne et al., 2010). Besides inflammation induction, the ATP released via Panx channels into the extracellular environment has also been associated with tumor growth and cancer metastasis, thus promoting cancer progression (Garré et al., 2010;Iwamoto et al., 2010;Riteau et al., 2010). This was addressed by Furlow et al., who showed that breast cancer cells expressing the mutant form of Panx1 (Panx11-89) increase ATP release, thus enhancing the metastatic potential of these cells (Scemes et al., 2009;Celetti et al., 2010;Chekeni et al., 2010;Furlow et al., 2015). Similarly, Panx2 and Panx3 have also been shown to be a conduit for ATP release; however, their functions were mainly attributed to cellular differentiation events (Penuela et al., 2009;Figueroa and Duling, 2009). Taken together, findings support the involvement of Panxs in inflammasome activation under "non-homeostatic" conditions. Several other studies also highlighted GJ-mediated cell communication's importance in regulating inflammation in different diseases, including atherosclerosis, hypertension, and diabetes (Brough et al., 2009;Dbouk et al., 2009). However, evidence also suggested that Cxs are involved in inflammation as independent functional hemichannels and not as GJs (Willebrords et al., 2016). It was evidenced that ischemic injuries induced inflammation results in the opening of Cx FIGURE 1 | Connexin and pannexin molecular structure. (A) A schematic diagram of Connexin and Pannexin protein structure. Both protein channels share similar topology by having four α-helical transmembrane (T) domains, two extracellular loops (EL), and one intracellular loop (IL), with their amino (NT) and carboxyl termini exposed to the cytoplasm. Both, Cxs and Panxs are shown to oligomerize into a hexamer to form single membrane channels at the cell surface, namely (B) A representation of Connexons and Pannexons in cellular communication. Connexons may mediate autocrine signaling as independent hemichannels or may form junctional plaque (or gap junction) via the docking of two connexons of adjacent cells. Connexons can be of homomeric hemichannels (single Cx isoform) or heterotypic hemichannels (multiple Cx isoforms), that can interact with each other in different ways to mediate intercellular communication. Pannexons are known to mediate paracrine signaling. These channels tend to mediate paracrine signaling, however, they do not form GJ plaque.
Frontiers in Physiology | www.frontiersin.org May 2022 | Volume 13 | Article 866675 hemichannels (Thompson et al., 2008). Changes in Cx expression and GJ coupling and functioning in response to systemic inflammation have also been evidenced. For example, in response to inflammation, Cx40 and Cx37 expressions were downregulated during pulmonary diseases (Pelegrin, 2008). In another study, the expression of Cx43 and Cx32 and their GJ coupling were inhibited in astrocytes and liver diseases. Such response was due to releasing the pro-inflammatory cytokine IL-1β (Kang et al., 2008;Pelegrin, 2008). Analogous to Panxs, Cxs are involved in ATP release. For example, Cx43, being the ubiquitously expressed hemichannel among the Cx protein family, has been shown to participate in ATP release in many cell types (Penuela et al., 2007;Yen and Saier, 2007). It is suggested that ATP release is modulated by the activity of Cx43 hemichannels (Locovei et al., 2006;Eugenín et al., 2007).
On the contrary, Cx43 as a functional GJ has protective roles, as seen in different types of cancer (Fischer et al., 2005;Eltzschig et al., 2006). Using in vivo and in vitro approaches, the enhancement of Cx43 expression and GJ coupling resulted in the regulation of several cytokines (Moreno, 2004;Söhl and Willecke, 2004). Therefore, understanding these channels' expression profiles and function in inflammatory conditions makes them an attractive therapeutic potential to help manage different diseases.

Connexins and Pannexins in COVID-19 Pathology
Despite the extensive research on understanding SARS-CoV-2 viral mechanism and vaccine evaluation, this pandemic is still spreading worldwide (Bennett and Zukin, 2004). Till date, there is still no clear evidence about the interaction between SARS-CoV-2 and the Cxs and Panxs protein channels. Since Panx1 channels are critical regulators of the inflammasome, playing a crucial role in lung vascular inflammation and edema (Baranova et al., 2004) and host responses to viruses (Sáez et al., 2003;Bao et al., 2004). It is crucial to consider Panxs' involvement in the SARS-CoV-2 inflammatory mechanism. A recent study by Luu et al. has shed light on SARS-CoV-2 inducing Panx1 channel activation. Interestingly, this was associated with ATP, and IL-1β increased mRNA expression (Bennett and Zukin, 2004). Although more investigations are required, these findings provide a rationale in supporting further investigations regarding the involvement of Panx channels in SARS-CoV-2. Nonetheless, Cxs involvement should also be addressed. It is observed that the expression of Cxs, specifically Cx43, in addition to adherents junction proteins, are significantly decreased upon infection with SARS-CoV-2. This was explained by Raghavan et al., who showed that the S protein of the virus induced degradation of GJ coupling, thus affecting endothelial barrier function, which resulted in vascular damage and loss of barrier integrity, as well as disrupted intercellular communication, as observed in COVID-19 individuals (Goodenough and Paul, 2003). Moreover, it is important to note that proinflammatory cytokines decrease Cx43 expression (González et al., 2002). Both Cxs and Panxs provide a promising approach for treating inflammation, as they are attractive targets to consider in understanding COVID-19 pathogenesis.
Although several pertinent queries remain to be elucidated regarding their induced disease mechanisms, particularly in SARS-CoV-2 induced male infertility, this study will hypothesize their plausible involvement.

SARS-CoV-2, Communicating Channels, and Male Infertility
As mentioned earlier, GJs are intercellular plasma membrane channels that create electric and metabolic coupling, mediating cell-to-cell communication in a wide variety of tissues. They are involved in embryogenesis, spermatogenesis, and other cellular processes (Cyr, 2011). In spermatogenesis, the development and differentiation of cells require strict regulated cell-to-cell interaction, rendering the GJs channels important to the process of spermatogenesis. The importance of junctional channels for male fertility cannot be overemphasized. For instance, loss of Cx43 in mouse fetus leads to a decrease in the number of germ cells (Kidder and Cyr, 2016), while loss of Cx43 in adults leads to the absolute inhibition of spermatogenesis. Cxs 26,32,33,36,45,46, and 50 are present in different testis cell types in various mammals, making them essential for differentiation and maturation. Loss of Cx46 leads to increased germ cell apoptosis and loss of BTB integrity (Gregory et al., 2011). Loss of Cx43 in the Sertoli cells leads to Leydig cell hyperplasia, suggesting a connection between the 2 cells (Sharpe et al., 2003;Lie et al., 2013). In the epididymis, Cxs, 26, 30, 3, 31.1, 32, and 43 are identified and involved in the epithelium differentiation. The decrease in Cx43 leads to decreased sperm motility (Dubé and Cyr, 2013). Different Cxs can form heteromeric connexons, and connexons of different connexin compositions can dock to form individual GJ. Building on these facts, the number of different types of connexin channels in the testis is likely to be larger than the number of Cxs present (Laird, 2006).
A study reported the decreased Cx43 expression in azoospermia (Adam and Cyr, 2016) and a decline in the mRNA Cx43 expression of men with non-obstructive azoospermia (Dubé et al., 2012). Another study reported a correlation between the severity of seminiferous tubule damage and the decrease in Cx43 immuno-staining (Kotula-Balak et al., 2007). Although these effects are seen in azoospermic men, no exact mechanism can be pinpointed as the original pathway affected, and it cannot be said that the symptoms of diseases resulted from the loss, decreased or mal expression of Cxs. However, what is valid is that these communicating channels are essential for spermatogenesis, either during fetal or adult life.
In addition to the importance of endocrine control and other maintained physiological processes, the testicular cell-cell interactions in spermatogenesis are also vital (Pointis et al., 2010). GJs are localized between adjacent Leydig cells, between Sertoli cells, and between Sertoli cells and specific germ cells. The cell-cell interaction is accomplished through the dependent junctional pathways necessary for regulating spermatogenesis and maintaining male reproduction (Mruk and Cheng, 2004). The junctions are composed of proteins implicated in cell adhesion (catenin and nectin), regulation of paracellular diffusion, establishing cell polarity, and cell attachment such as the BTB (occludin and claudin) and GJ (Cxs) (Pointis et al., 2010).
A study that investigated the effect of SARS-CoV-2 on BTB of patients who died from COVID-19 complications revealed an increase in testicular inflammation and the destruction of the BTB (Peirouvi et al., 2021). Significant adverse changes in the arrangement of testicular cells and decreased Sertoli cells number were observed. Furthermore, a significant reduction in testicular occludin, claudin-11, and Cx43 expression were also detected in the COVID-19 patients (Peirouvi et al., 2021).
On the other hand, Panx-1 and Panx-3 are expressed in the testis and epididymis (Cyr, 2011). Panx-1 is mainly expressed in the Sertoli cells at the level of the BTB, while Panx-3 is localized in the interstitial space, specifically found on Leydig cells. Panxs are also present in the epididymis and may mediate the release of ATP into the epididymal lumen to aid sperm maturation. Thus, the basal presence of Panx-1 in the epididymis may regulate principal cell function and mediate the secretion of ATP into the basal extracellular space (Cyr, 2011).
Although studies have shown that Panx-1 channels are inactive under healthy conditions, these channels are rendered actively open under diseased conditions, thus propagating the pathogenesis of the disease (Valdebenito et al., 2018;Gajardo-Gómez et al., 2020). This particular observation has not yet been reported in the testis and epididymis. Though what is known is that Panx-1 is strategically localized on the Sertoli and Leydig cells of the testis. This means that 1) they may play a role in the GJ complexes of the BTB, thereby allowing the influx of small ions, nucleotides, and proteins, which may subsequently aid the release of ATP. 2) Their localization in the Leydig cell may indicate their use for testosterone production. This is mainly explained by a study performed on orchidectomized rats. Studies on the FIGURE 2 | Mechanism of SARS-CoV-2 and male infertility. The S1 subunit of the SARS-CoV-2 binds the ACE2 receptor on the Sertoli cell membrane, which in turn activates TMPRSS2 and cause a conformational change to the complex, thus allowing the entry of the virus. Connexin 43 is abundant on the Sertoli cell membrane and has been shown to play a role in the Sertoli cell's functionality. In the state of inhibition or depletion of Cx43 on Sertoli cells due to infection or apoptosis, spermatogenesis was reported to be completely inhibited. Therefore, upon SARS-CoV-2 entry, there is a decrease in the expression of Cx43 and other junctional proteins, thus disrupting the blood-testis barrier, and subsequently impairing spermatogenesis. Additionally, the binding of SARS-CoV-2 to ACE2 receptor could plausibly cause the opening of the pannexin channel, a process dependent on furin (another protease that aids viral entry), which could cause the release of ATP into the cell. Excessive ATP leads to the formation of inflammasome and thus cytokine secondary storm ensues. Elevated inflammation inturn causes an increase in the formation of reactive oxygen species (ROS) and further leads to the development of oxidative stress (OS). Occurrence and sustained OS status causes apoptosis and subsequently impaired spermatogenesis. Persistent fever can also increase the generation of ROS and cause OS, due to the reduction of heatshock proteins (HSP) during viral infection. Since SARS-CoV-2 can cross the blood-brain barrier, upon infection, the function of the hypothalamus is impaired, thus resulting in reduced secretion of gonadotropin-releasing hormone and consequently reducing the generation of luteinizing (LH) and follicle-stimulating hormones (FSH). Altered endocrine control leads to impaired spermatogenesis and ultimately, reduced male fertility.
Frontiers in Physiology | www.frontiersin.org May 2022 | Volume 13 | Article 866675 epididymis have indicated that orchidectomy increases the number of Panx1 and Panx3 immunoreactive bands, as well as of total protein levels throughout the epididymis, while testosterone maintenance with orchidectomy reduces the number of bands in the caput and corpus; suggesting that testosterone is required for the post-translational function of Panx-1. Taken together, it is unclear whether the opening of the Panx-1 channel is detrimental to spermatogenesis and sperm maturation, as seen in other systems. Nevertheless, what is valid is that the activation of Panx-1 during infection may pose a threat to spermatogenesis and consequently male fertility. In summary, since ACE2, Panxs, and the purinergic receptors are present in the testis and epididymis Illes et al., 2020), upon SARS-CoV-2 infection; theoretically, Panx-1 present in the Sertoli and Leydig cells would be opened and cause the activation of the purinergic receptors; which could propagate the inflammation signaling. When inflammation is prolonged, male fertility is thus impaired. The mechanisms through which SARS-CoV-2 may affect male fertility are summarized in Figure 2.

Connexins and Pannexins as Therapeutic Targets in Male Infertility and SARS-CoV-2: A Commentary
Considerable insights continue to emerge regarding Cxs and Panxs molecular mechanisms in different diseases; however, developing new intervention strategies using drug therapy that targets Cx and Panx channels may prove beneficial. This will also bridge the gap between Cx and Panx therapeutic strategies with COVID-19 management and treatment response. Drug targets are available to inhibit channels of Cx and Panx proteins. These include alcoholic substances, glycyrrhetinic acid, anaesthetics, and fatty acids. It is important to note that such pharmacological tools may not differentiate between GJ or hemichannels and, therefore, might require the need of a specific targeting inhibitor. In this case, using mimetic peptides, which reproduce specific amino acid sequences in either Cx or Panx, might serve as a beneficial approach (L. Harris, 2001;Ware and Matthay, 2000), yet novel therapies are urgently required. In the context of male infertility, therapeutic strategies have to focus on either the re-expression of the Cxs or on enhancing Cx hemichannel activity to retain its proper junctional coupling, which restores disrupted intercellular communication (Panchina et al., 2000). Also, targeting strategies should not exclude the Panx protein channels. Probenecid, a potent Panx-1 inhibitor, has proven to be a promising target of inflammation in different diseases (Beyer et al., 1995;Goodenough et al., 1996;Duffy et al., 2000), including COVID-19 pathogenesis, where Panx-1 mRNA expression was responsible for increased ATP and IL-1β levels in human lung epithelial cells infected with SARS-CoV-2. Another FDAapproved drug, spironolactone, has also proven to be a potential Panx-1 blocker in COVID-19 patients. Knowing that the drug is meant for hypertension patients, spironolactone could be beneficial as a Panx-1 blocker in the context of COVID-19 (Aoumari et al., 1990;Bennett and Zukin, 2004).

CONCLUSION
The current evidence does not allow to assure the role of Cxs and Panxs in the adverse reproductive effects observed in individuals with COVID19, however, there are some indications of this relationship, providing a justification that supports the need for new studies evaluating the role of these protein channels, with the purpose to understand and perhaps avoid, through the use of blockers of these proteins as treatments, guaranteeing that human fertility will not be affected due to SARS-COV-2; an infection that is here to stay.
Based on current evidence, plausibly SARS-CoV-2 is not present in semen. Nevertheless, it exerts a negative effect on gametogenesis, a process that subsequently affects sperm parameters, guaranteeing that the testicular architecture could be altered. Therefore, it is essential to understand the role of these protein forming hemichannels in the pathogenesis of SARS-CoV-2 induced male reproductive impairment, which could help avoid these harmful effects.
Finally, infertility cases due to male factors continue to increase worldwide, and after the pandemic, this might further increase. Therefore, understanding these possible interactions will allow bridging critical knowledge gaps in our perception of the molecular mechanisms involved in male infertility.

ACKNOWLEDGMENTS
This study was in part supported by the Al Jalila Foundation and the MBRU Publication Fund. Figures were created on BioRender.