Probiotics Fermentation Technology, a Novel Kefir Product, Ameliorates Cognitive Impairment in Streptozotocin-Induced Sporadic Alzheimer’s Disease in Mice

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by cognitive impairment. Gut microbiota dysfunction (dysbiosis) is implicated in the pathology of AD and is associated with several detrimental consequences, including neurotransmitter depletion, oxidative stress, inflammation, apoptosis, and insulin resistance, which all contribute to the onset of AD. The objective of this study was to assess the effectiveness of Probiotics Fermentation Technology (PFT), a kefir product, in alleviating AD symptoms via regulation of the gut microbiota using a streptozotocin- (STZ-) induced AD mouse model and to compare its activity with simvastatin, which has been proven to effectively treat AD. Mice received one intracerebroventricular injection of STZ (3 mg/kg). PFT (100, 300, 600 mg/kg) and simvastatin (20 mg/kg) were administered orally for 3 weeks. PFT supplementation mitigated STZ-induced neuronal degeneration in the cortex and hippocampus, restored hippocampal acetylcholine levels, and improved cognition in a dose-dependent manner. These effects were accompanied by reductions in oxidative damage, proinflammatory cytokine expression, apoptosis, and tau hyperphosphorylation. Moreover, PFT hindered amyloid plaque accumulation via the enhancement of insulin-degrading enzyme. These beneficial effects were comparable to those produced by simvastatin. The results suggest that PFT can alleviate AD symptoms by regulating the gut microbiota and by inhibiting AD-related pathological events.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder [1]. It is a devastating disease that is characterized by progressive cognitive impairment and memory loss [2]. The fundamental pathological hallmarks of AD are amyloid plaques (extracellular accumulations of abnormally folded amyloid beta protein (Aβ)) and neurofibrillary tangles that are composed of hyperphosphorylated tau protein [3]. The pathophysiological aspects of AD have not yet been fully investigated; however, synaptic dysfunction, membrane permeabilization, oxidative stress, inflammation, apoptosis, and a reduction in cerebral glucose utilization have been identified as risk factors of AD progression [4, 5]. Interestingly, it has recently been proposed that gut microbiota dysfunction (dysbiosis) correlates with the onset of AD [6, 7]. This hypothesis is based on many lines of evidence. First, the gut microbiota composition is influenced by aging, the main risk factor for AD [8]. Second, alterations in the microbiota lead to the release of significant quantities of amyloids and lipopolysaccharides, which modulate signaling pathways, increase the permeability of the intestine and the blood-brain barrier, and produce proinflammatory cytokines, which are all related to AD pathogenesis [7, 9, 10]. Third, dysbiosis generates oxidative stress, which is also associated with AD [11]. Furthermore, disturbance of the gut microbiome confers insulin resistance, which has also been linked with AD [8]. Therefore, AD is intricately interrelated with gut microbiota imbalance and may initiate from the gut. To this end, the modulation of the gut microbiota has become of increasing interest in the quest for new AD therapeutic agents.

Probiotic fermentation technology (PFT), a kefir grain product, is extracted from kefir (fermented milk) [12, 13]. Kefir is a health-endorsing probiotic drink that is formed by the fermentation of milk with kefir grains and is composed of mainly Lactobacillus kefiri. Earlier studies have demonstrated the numerous health benefits of kefir-derived Lactobacillus kefiri: it improves gut health by preserving the probiotic bacteria balance and reduces oxidative stress, inflammation, and insulin resistance [12, 14–17]. Interestingly, PFT has recently been examined for its ability to exert anticancer effects in animals with Ehrlich ascites carcinoma and in human gastric cancer cells in vitro [18, 19]. Since the majority of AD patients are elderly, the development of safe, well-tolerated drugs is important. Our recent study showed the effectiveness of PFT supplementation in modulating age-associated oxidative stress, suggesting that PFT may be a valuable therapeutic intervention for AD [20]. In addition, PFT has been shown to be a safe, nontoxic agent with no side effects [18].

In this study, we explore the potential effects of PFT in the treatment of AD, as well as comparing its activity with simvastatin, which has been proven to treat dementia, reduce cognitive decline in AD patients, and diminish the prevalence of AD [21, 22].

2. Materials and Methods

2.1. Animals

Adult male albino mice (25–30 g) were obtained from the animal facility of the Faculty of Pharmacy, Cairo University, Egypt. Mice were housed at a constant temperature (${25}^{°}\text{C}\pm 2^{°}\text{C}$) and humidity level ($60\%\pm 10\%$) under a 12/12 h light/dark cycle and were allowed a standard chow diet and water ad libitum. The study was approved by the Institutional Animal Care and Use Committee of Cairo University (CU-IACUC) (Permit Number: CU-III-F-35-20) and complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 2011).

2.2. Chemicals and Drugs

PFT kefir grain product is a mixture that primarily (~90%) consists of a heat-killed freeze-dried form of L. kefiri P-IF, whose characteristics have been reported previously [12, 13]. PFT also contains ~2%–3% of the following: one bacterial strain (L. kefiri P-B1) and the yeast strains Kazachstania turicensis, Kazachstania unispora, and Kluyveromyces marxianus. Positron-emission tomography scans showed 99.6% homology with regular kefirs. The yeast strains are not intentionally added but are present in large amounts when the product is obtained from the Caucasus mountains and are filtered out in order to maximize the Lactobacillus kefiri levels. PFT was provided by Paitos Co. Ltd. (Yokohama, Kanagawa, Japan).

Streptozotocin (STZ) and simvastatin were purchased from Sigma–Aldrich (St. Louis, MO, USA). Other chemicals and reagents, unless otherwise specified, were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA).

2.3. Induction of AD

Intracerebroventricular (ICV) injection of STZ, first described by Pelleymounter et al. and modified by Warnock, was used in the current study for the avoidance of cerebral vein penetration [23–25]. Mice were anesthetized with thiopental (5 mg/kg, i.p.), then the mouse’s head was stabilized using downward pressure above the ears, and the needle was inserted directly through the skin and skull into the lateral ventricle, which was targeted by visualizing an equilateral triangle between the eyes and the center of the skull to locate the bregma, allowing the needle to be inserted at the following coordinates from the bregma: 1 mm mediolateral, −0.1 mm anteroposterior, and−3 mm dorsoventral. Mice behaved normally approximately 1 min following the injection. The accuracy of the injection technique was established by injecting methylene blue dye that was detected in the lateral ventricles [26].

2.4. Experimental Design

Mice were randomly allocated into six groups, with 12 mice in each group. The study was conducted for a total of 21 days. Group I, the sham control group, received one ICV injection of 0.9% saline, in addition to oral administration of Tween 80 with 0.9% saline, daily for 21 consecutive days. Group II, the AD-model group, received one ICV injection of STZ (3 mg/kg) dissolved in 0.9% saline [27]. Groups III to VI received one ICV STZ injection (3 mg/kg), and group III was treated with simvastatin (20 mg/kg, p.o.) suspended in Tween 80 with 0.9% saline 5 h after STZ injection and then daily for a total of 21 doses [28]. Groups IV to VI were treated with PFT (100 mg, 300 mg, and 600 mg/kg, p.o., respectively) suspended in Tween 80 with 0.9% saline 5 h after STZ injection and then daily for a total of 21 doses (Figure 1) [29].

[figure omitted; refer to PDF]

On the first day of training in the MWM, there was no significant difference in the mean escape latency between the STZ and treated groups. From the second day until the fourth day, the mice in the treatment groups took a shorter period of time to reach the platform as compared with the STZ group. On the test day, animals treated with PFT (100, 300, and 600 mg/kg) displayed a substantial increase in the time spent in the target quadrant in which the platform was previously located (2.9-, 3.4-, and 4-fold, respectively) as compared with the STZ group ($F5,66=125$, $p<0.0001$). The effect of PFT was in line with that of simvastatin (2.5-fold increase as compared with the STZ group) (Figure 2).

3.2. PFT Reversed STZ-Induced Alterations in Ach and Amyloid β_1-42 Levels in the Hippocampus

Mice that received STZ showed a large decline in Ach levels and an increase in amyloid β_1-42 level in the hippocampus as compared to their sham control counterparts ($F5,30=21.9$ and 146, respectively, $p<0.0001$) (Figure 3). Improvements in cognition by PFT (100, 300, and 600 mg/kg) were reflected by augmentation of hippocampal Ach by 2.4-, 2.9-, and 3-fold, respectively, and suppression of amyloid β_1-42 by 34.1%, 45.3%, and 52.5%, respectively, as compared with the STZ group ($p<0.001$ and $p<0.0001$). These results reflected those exhibited by simvastatin (increase in Ach by 2.4-fold and decrease in amyloid β_1-42 by 43.4% as compared with STZ group).

[figure omitted; refer to PDF]

Cerebral cortex and hippocampus tissues were also examined for amyloid plaques, which were stained with Congo red. The results (Figure 10) showed that the sham control group had no amyloid deposition in the examined tissue sections. In contrast, the model group had a significantly high number of plaques in the cerebral cortex and hippocampus as compared with the sham control group ($\text{test}\text{statistic}=41.1$, $p<0.001$). However, PFT (100, 300, and 600 mg/kg) and simvastatin treatment conferred a marked reduction in the number of amyloid plaques as compared with STZ. The data regarding the amyloid plaques in the cerebral cortex and hippocampus are shown in Table 2.

[figure omitted; refer to PDF]

Table 2

The survival rate percent of intact neurons in the cerebral cortex, different hippocampal regions and amyloid plaques recorded in the brain of the mice.

Groups	Survival rate percent of intact neurons				Amyloid plaques
	Cerebral cortex	CA3	CA4	DG	Cerebral cortex + hippocampus
Sham control	95.3 ± 1.5	92.3 ± 1.5	89.0 ± 1.0	92.3 ± 1.5	—
STZ model	44.7 ±2.1${}^{\ast \ast \ast \ast }$	27.3 ± 1.5${}^{\ast \ast \ast \ast }$	31.0 ± 1.0${}^{\ast \ast \ast \ast }$	36.0 ± 1.0${}^{\ast \ast \ast \ast }$	12 (10-18)${}^{\ast \ast \ast \ast }$
STZ + simvastatin	89.0 ± 1.0${}^{\ast \ast }$####	82.3 ± 1.5${}^{\ast \ast }$####	71.0 ± 1.0${}^{\ast \ast \ast \ast }$####	81.0 ± 1.0${}^{\ast \ast \ast \ast }$####	2.5 (0-5)${}^{\ast }$##
STZ+ PFT (100 mg/kg)	73.3 ± 1.5${}^{\ast \ast \ast \ast }$####	77.7 ±2.1${}^{\ast \ast \ast \ast }$####	60.7 ± 1.5${}^{\ast \ast \ast \ast }$####	71.7 ± 1.5${}^{\ast \ast \ast \ast }$####	3 (2-6)${}^{\ast \ast }$#
STZ+ PFT (300 mg/kg)	83.0 ±2.0${}^{\ast \ast \ast \ast }$####	81.0 ± 1.0${}^{\ast \ast \ast }$####	66.3 ± 1.5${}^{\ast \ast \ast \ast }$####	71.7 ± 1.5${}^{\ast \ast \ast \ast }$####	3 (1-5)${}^{\ast \ast }$#
STZ+ PFT (600 mg/kg)	85.7 ± 1.5${}^{\ast \ast \ast }$####	85.7 ± 4.7${}^{\ast }$####	77.3 ± 1.5${}^{\ast \ast \ast \ast }$####	80.7 ± 1.5${}^{\ast \ast \ast \ast }$####	2.5 (0-4)###

Data of survival rate percent of intact neurons represents the mean ± SD of 3 mice per group and data of amyloid plaques represents the median and range of 3 mice per group; $\ast$ significantly different from sham control group at p <0.05, $\ast \ast$ significantly different from sham control group at p <0.01, $\ast \ast \ast$ significantly different from sham control group at p <0.001, $\ast \ast \ast \ast \ast$ significantly different from sham control group at p <0.0001, #significantly different from STZ group at p <0.05, ## significantly different from STZ group at p <0.01, ### significantly different from STZ group at p <0.001, #### significantly different from STZ group at p <0.0001 using one-way ANOVA followed by Tukey’s multiple comparisons test for statistical analysis of survival rate, and using Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparisons test for statistical analysis of amyloid plaque number.

Neurodegeneration was examined in the brain sections that were treated with Nissl stain, and the percentage of intact neurons was calculated as the survival rate. The results are summarized in Table 2. STZ resulted in a severe loss of neurons in the cerebral cortex and hippocampal regions CA3, CA4, and DG as compared with the sham control group ($F5,12=360.7$, 288.8, 696.6, and 592.6, respectively, $p<0.0001$). Treatment with PFT significantly conserved neurons in the cerebral cortex (1.6-, 1.8-, and 1.9-fold, respectively) and the hippocampus (CA3: 2.8-, 3-, and 3.1-fold, respectively; CA4: 2-, 2.2-, and 2.5-fold, respectively; DG: 2-, 2-, and 2.23-fold, respectively) as compared with the STZ group (Figure 11). The effects of PFT corresponded to those of simvastatin, which preserved neurons in the cerebral cortex 2-fold and in CA3, CA4, and DG 3-, 2.3-, and 2.3-fold, respectively.

[figure omitted; refer to PDF]

4. Discussion

The purpose of the current study was to address the role of gut microbiome modulation through the administration of the natural dietary product, PFT, in the prevention or improvement of AD symptoms in an STZ-induced AD mouse model. This was inspired by recent approaches that focus on the investigation of natural products present in diet as vital bioactive molecules against neurodegenerative diseases [37]. It was revealed that PFT could significantly improve cognitive impairment and dementia, prevent neuronal degeneration in the cortex and hippocampus, restore hippocampal Ach levels, and decrease the presence of amyloid plaques and disease biomarkers in a dose-dependent manner. Our experimental evidence suggests that PFT supplementation attenuates cognitive dysfunction by targeting oxidative stress and inflammatory and apoptotic pathways. Moreover, PFT enhanced the hippocampal level of IDE, a key Aβ-degrading enzyme, with a subsequent decrease in Aβ level in the hippocampus and an improvement in memory deficiency in the STZ-induced AD animal model. Furthermore, PFT significantly reduced tau hyperphosphorylation via suppression of ERK1/2, p38-MAPK, GSK-3β, and mTOR, the chief kinases that regulate tau’s hyperphosphorylation. Our results demonstrate that the remarkable effects of PFT in attenuating AD symptoms were comparable to those of simvastatin, a drug whose therapeutic effects have been empirically proven for AD. Notably, simvastatin has been shown in several studies to improve cognitive performance, reduce the levels of Aβ peptides, and prevent neuronal loss in AD through its ability to reduce oxidative stress, inflammation, and apoptosis along with the promotion of IDE secretion and modulation of the PI3K/Akt and MAPK/ERK1/2 pathways [38–41].

The ICV-STZ-induced AD model is considered to sufficiently mimic the progressive pathology of AD in the human brain [42]. In this context, STZ conferred most of the features of AD, with progressive deficits in learning, memory, and cognitive behavior, along with aggregation of Aβ and neuron loss in the cortex and hippocampus, and a massive reduction in hippocampal Ach level, in line with former studies [26, 43, 44].

Aβ accumulation and plaque development are the major biomarkers for the detection of AD, where Aβ_1-42 is the most abundant form of Aβ protein and is deposited early as plaques [45]. The molecular mechanisms that trigger the aggregation of Aβ in AD are not fully understood; however, dysbiosis is implicated [8]. Under conditions of dysbiosis, bacteria that inhabit the microbiome release mixtures of lipopolysaccharides, amyloids, and other microbial exudates into their proximal environment. These exudates may leak from the gastrointestinal tract due to increased gut permeability induced by dysbiosis and then accumulate in the brain [8, 9]. It is worth mentioning that aging makes the involvement of gut microbiota in amyloid development and propagation more significant, since both the gut and the blood-brain barrier become more permeable to small molecules during aging [46]. In this study, investigation of the Aβ_1-42 isoform revealed steep elevation of Aβ_1-42 in the hippocampi of STZ-treated mice, an effect that was reversed by modulation of the gut microbiota through supplementation with PFT. Additionally, dysbiosis is associated with alterations in the levels of certain neurotransmitters, including Ach [6]. Cognitive function was revealed to be regulated by Ach, which employs its effect on the striatum, hippocampus, and amygdala [47]. The hippocampus is the core brain region involved in memory and learning processes [48]. Thus, declined cholinergic function in the hippocampus causes cognitive impairments together with learning and memory insufficiencies [44]. In our results, we found that STZ treatment resulted in an obvious decrease in hippocampal Ach level, which was accompanied by spatial and short-term memory impairment. Conversely, the administration of PFT in mice with STZ-induced cognitive deficiency prevented this decrease in Ach concentration, with a subsequent improvement in memory and learning, as exemplified by the significant elevation in discrimination and preference indices in the NOR test and the increase in the time spent by mice in the target quadrant during the MWM test probe trial. This valuable effect could be attributed to the restoration of gut microbiota along with the ability of Lactobacillus, the main component of PFT, to yield Ach and to hinder acetylcholinesterase enzyme [49, 50]. The potential effect of PFT treatment was similar to that of simvastatin treatment in STZ-treated mice. The positive effect of simvastatin on cognition is attributed to the restoration of cholesterol homeostasis, since elevated cholesterol levels may result in Aβ formation and cognitive impairment [38]. In addition, the protective effects of simvastatin may also be related to its ability to diminish brain ischemia, prevent cholinergic neuronal loss, modulate brain-derived neurotrophic factor expression, and promote nitric oxide synthesis [28].

Oxidative stress is a chief contributor to aging and age-related diseases including AD [51]. According to hormesis (a dose-response phenomenon, characterized by low-dose stimulation and high-dose inhibition), temporary exposure of neurons to low levels of reactive oxygen species (ROS) has a protective effect, due to the activation of transcriptional regulators called vitagenes that endorse cell adaptive mechanisms to maintain homeostasis and to protect against more severe oxidative stress; nevertheless, chronic exposure to oxidative stress with massive generation of ROS leads to significant damage to cellular functions, which eventually results in the initiation of AD [52–54]. The absence of gut microbes is associated with oxidative stress. Aging has also been associated with an increase in gut permeability. Under normal circumstances, the gut microbiota produces antioxidants. However, during dysbiosis, lipopolysaccharides and amyloid dissemination cause an increase in ROS along with an induction of oxidative stress [6, 8]. This, in turn, leads to cognitive impairment since the hippocampus, which regulates cognitive function, is highly vulnerable to oxidative stress [55]. In the present study, STZ was associated with marked oxidative stress, which could possibly be attributed to its ability to induce ROS. This reduction in antioxidant capacity is associated with neuronal damage and consequential cognitive deterioration which was observed in STZ-treated mice. These findings are in agreement with a recent study [44]. Remarkably, PFT acted as a potent antioxidant, which was evidenced by a reduction in MDA content (indicator of lipid peroxidation) and activation of Nrf-2 transcription factor with subsequent induction of its target genes HO-1 and GSH in the hippocampi of STZ-treated mice. Notably, Nrf-2 plays a vital role in suppressing oxidative stress and inflammation through regulation of vitagene transcription, such as HO-1 and glutamate-cysteine synthetase that synthesize GSH, which display antioxidant activity and abate various forms of stress, thus maintaining redox balance and homeostasis [37, 56]. The antioxidant property of PFT was further confirmed by the protection of the brain from Aβ neurotoxicity and the restoration of memory. Numerous studies have suggested that the Lactobacillus in PFT produces exopolysaccharide, which displays high scavenging activity against ROS, increases the activity of antioxidant enzymes, and enhances the overall antioxidant capacity [57, 58]. The antioxidant activity displayed by PFT was equivalent to that exerted by simvastatin. In addition to its direct antioxidant effect, simvastatin has been proven to reduce circulating oxidized low-density lipoproteins and circulating markers of oxidation (such as nitrotyrosine), inhibit oxidant enzymes, and upregulate antioxidant enzyme activity [59].

Besides oxidative stress, inflammation also plays a fundamental role in the pathogenesis of AD [60]. Microglial activation and high levels of proinflammatory cytokines, such as interleukins and TNF-α, have been detected in the serum of AD patients [61]. Dysbiosis contributes to the pathogenesis of AD partly through the generation of a neuroinflammatory state [62]. It has been suggested that an increase in gut permeability following dysbiosis may lead to neuroinflammation and subsequent hippocampal damage [63]. After leaking from the gut, bacteria-derived polysaccharides and amyloids activate NF-κB signaling. NF-κB in turn stimulates proinflammatory microRNA-34a release, which reduces the expression of TREM2 (triggering receptor expressed on myeloid cells 2), resulting in phagocytosis dysfunction and accumulation of Aβ [64]. Furthermore, lipopolysaccharides and amyloids can endorse gut leakiness and trigger ROS in the brain with subsequent microglial activation that enhances proinflammatory cytokine release [65, 66]. It is worth mentioning that ROS and microglial activation along with dysregulation of redox homeostasis by suppression of Nrf-2 transcription results in stimulation of NF-κB signaling, which triggers elevation of TNF-α and upregulation of NLRP3 [37, 67, 68]. NLRP3 inflammasome activation contributes to the inflammatory events in AD pathogenesis, since it enhances the secretion of IL-1β, which further promotes microglial activation and accumulation of inflammatory and neurotoxic factors resulting in a vicious cycle that exacerbates neurodegeneration [67]. To this end, modulating the gut microbiome with probiotics may represent an effective strategy to diminish the level of chronic inflammation and Aβ associated with AD. In this context, STZ treatment resulted in marked neuroinflammation, which was efficiently inhibited by PFT supplementation as verified by the substantial decrease in the expression of hippocampal levels of NF-κB, NLRP3, IL-1β, and TNF-α in STZ-treated mice, with consequent amelioration of Aβ burden in the hippocampus. The anti-inflammatory effect of PFT corresponded to that of simvastatin, which has been shown to inhibit inflammation by reducing neutrophil infiltration, nitrotyrosine formation, NF-κB activation, and inducible nitric oxide synthase expression [69].

Immense neuronal loss is among the pathological features of AD. Apoptosis, a vital aspect of AD pathogenesis, is responsible for AD-associated neuronal death [70]. This phenomenon has been assumed to be due to the presence of apoptotic factors in AD brain tissue, in addition to the fact that the brain is highly vulnerable to apoptotic damage [71]. Caspase-3, the major source of apoptotic cell death in neurodegenerative diseases, is directly involved in AD apoptosis [72]. Remarkably, the microbiota can regulate apoptosis via gut-brain axis signaling [73]. In dysbiosis, harmful signals are propagated, leading to increased ROS production and inflammation with consequent mitochondrial dysfunction, which endorses cytochrome c release, thus triggering caspase-3 and inducing apoptosis and neuronal death [74]. In the present study, administration of PFT effectively attenuated the activity of caspase-3 in AD mice, an effect that was associated with neuronal preservation in the hippocampal structures and relevant restoration of attentional performance. PFT’s antiapoptotic activity was comparable to simvastatin’s effects in STZ-treated mice. Simvastatin has been shown to attenuate the pathology of AD due to its antiapoptotic effects in hippocampal cells [75].

Insulin has also been linked with AD pathology [76]. In this context, elevated insulin levels have been observed in AD brains, resulting in exaggerated inflammatory responses and accumulation of Aβ in the brain [77]. Besides stimulating Aβ secretion, insulin inhibits Aβ degradation by competing for IDE, the key regulator of Aβ in neurons [78]. Insulin not only exerts a direct effect on Aβ metabolism but also promotes mitochondrial dysfunction, oxidative stress, and apoptosis, all of which contribute to the development of AD [79]. In particular, the absence of gut microbes may lead to the development of insulin resistance and the suppression of IDE, which are both found in AD [80]. In this study, restoration of the gut microbiota via PFT supplementation enhanced IDE and significantly decreased Aβ levels in STZ-treated rats. In AD mice, PFT mediated IDE secretion as effectively as simvastatin, which has been reported to affect IDE via an unconventional autophagy-based secretory pathway [40].

Tau hyperphosphorylation is greatly implicated in AD pathogenesis [81]. Tau is a microtubule-binding protein that is responsible for the assemblage and stabilization of microtubules [82]. Pathological tau protein is abnormally hyperphosphorylated and accumulated forming neurofibrillary tangles, a hallmark of AD [82]. Tau phosphorylation is regulated by multiple kinases and phosphatases. ERK1/2, p38-MAPK, GSK-3β, and mTOR are the main kinases that are involved in tau hyperphosphorylation, while protein phosphatase 2A (PP2A) is the chief phosphatase that dephosphorylates tau [83, 84]. It has been demonstrated that the aforementioned kinases are highly activated in AD brains along with the declined activity of PP2A [81, 84]. Of note, Aβ and oxidative stress have been shown to stimulate MAPK kinase (MEK), which activates ERK1/2 and p38-MAPK through phosphorylation, thereby resulting in tau hyperphosphorylation and prominent neurofibrillary tangle formation [85, 86]. Moreover, Aβ and insulin resistance suppress phosphatidylinositol-4,5-bisphosphate 3-kinase- (PI3K-) Akt signaling leading to activation of GSK-3β and mTOR, which in turn causes PP2A inhibition and subsequent tau hyperphosphorylation [77]. Importantly, there is evidence that activation of mTOR promotes Aβ deposition through the inhibition of autophagy and disposes insulin resistance, thus leading to further tau phosphorylation, resulting in a vicious cycle that aggravates AD [87]. It has been proposed that gut microbiota alteration is associated with tau hyperphosphorylation, based on the fact that factors that enhance tau hyperphosphorylation, including Aβ, oxidative stress, and insulin resistance, are precipitated by dysbiosis [88, 89]. In addition, dysbiosis can induce leucine metabolism disorder, which enhances mTOR activity [90, 91]. Consequently, gut microbiome restoration could dampen tau hyperphosphorylation. Our study shows that PFT decreased tau phosphorylation along with downregulation of ERK1/2, p38-MAPK, GSK-3β, and mTOR expression in the hippocampi of STZ-treated mice, and this favorable effect was accompanied by suppression of Aβ accumulation and enhancement of cognitive function. The action of PFT was parallel to that of simvastatin, which has been demonstrated to modulate the PI3K/Akt and MAPK/ERK1/2 pathways and halt neurodegeneration [39, 92].

5. Conclusions

This study reveals that the gut microbiota affects learning and memory via the microbiota-gut-brain axis and that AD is highly related to alterations in the gut microbiota composition. The delivery of Lactobacillus via PFT, a kefir product, displayed several benefits in STZ-induced AD mice. We have demonstrated that PFT not only improves cognitive function along with amelioration of histopathological markers but also attenuates oxidative stress, suppresses neuroinflammation, reduces apoptosis, and enhances IDE. Thus, PFT ameliorates multiple factors that underlie AD pathology. As a safe, nontoxic agent, PFT represents a useful potential therapy for AD.

Authors’ Contributions

N El Sayed, E. Kandil, and M Ghoneum planned the study and wrote the manuscript. N El Sayed designed and performed the experiments. All authors revised and approved the manuscript.

Acknowledgments

PFT was provided by Paitos Co., Ltd., Yokohama, Kanagawa, Japan. This work was funded by Paitos Co., Ltd., Yokohama, Kanagawa, Japan; Grant #T0099108.

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