Increase in BACE1 Phosphorylation in the Rat Hippocampus and Phytosphingosine in Plasma After Chronic Administration of Scopolamine

Abstract

Alzheimer’s disease (AD) is characterized by progressive cognitive decline associated with the accumulation of amyloid-β (Aβ) peptides and dysregulation of β-site amyloid precursor protein-cleaving enzyme (BACE1) and its phosphorylation at T252 (P-BACE1-T252) as well to the kinase’s expression and activity. In this study, the effects of chronic scopolamine administration on Aβ_1-42 levels, BACE1 expression and activity, P-BACE1-T252, PKA expression and BACE1, and PKA activity were evaluated, along with the identification of some metabolites in plasma. Twenty-seven male Wistar rats were divided into control and scopolamine-treated groups (2 mg/kg/day, i.p.) for six weeks. Scopolamine increased hippocampal Aβ_1-42 2.1-fold (p < 0.0001) by ELISA, which correlates with the increase in BACE1 expression (85%) by Western blot and its activity (p < 0.05) relative to that in the control group. In addition, despite the fact that an increase in P-BACE1-T252 expression by Western blot was observed in hippocampus and prefrontal cortex, it was more in the hippocampus; notably, this result correlates with the PKA expression and activity which was reduced in the hippocampus (p < 0.05) but not in the prefrontal cortex. Identification of some metabolites that has been reported during the administration of Aβ and could be present in the scopolamine model were carried out by UHPLC-MS/MS, finding elevated plasma phytosphingosine and decreased acetylcarnitine, suggesting disrupted lipid metabolism associated with scopolamine-induced cognitive impairment.

1. Introduction

AD is a neurological disorder caused by the degeneration of brain cells, and its principal symptom is dementia, which is characterized by decreased cognitive function and autonomy in daily activities [1]. The hippocampus and prefrontal cortex play fundamental roles in memory and executive functions. The hippocampus is a primary region for learning and memory [2] whereas the prefrontal cortex plays a regulatory role in a variety of cognitive functions, including attention, working memory, and spatial and long-term memory [3]. However, in AD patients, the hippocampus is atrophied, and the prefrontal cortex presents deterioration in functional connectivity due to neuronal death. These structural and functional alterations result in deficits in memory, attention, and executive control and contribute to the progression of the disease [4].

There are several hypotheses related to AD physiopathology; two of the principals are the cholinergic and amyloidogenic hypotheses. In the second one, Aβ is produced in the brain from amyloid precursor protein (APP), a protein that is involved in two metabolic pathways: the nonamyloidogenic pathway and the amyloidogenic pathway. In the nonamyloidogenic pathway, APP is cleaved by α-secretase, releasing sAPPα and C83; the latter is subsequently cleaved by γ-secretase, preventing the formation of the Aβ peptide. In the amyloidogenic pathway, β-site amyloid precursor protein-cleaving enzyme (BACE1) cleaves APP, generating sAPPβ and C99; these are subsequently cleaved by γ-secretase to produce Aβ, and this peptide could have 39 to 43 amino acids residues of length. Aβ_1-42 is prone to aggregation and plays a crucial role in the pathogenesis of AD [5].

In addition, BACE1 is a transmembrane aspartic protease whose catalytic activity can be regulated by posttranslational modifications (PTMs) such as phosphorylation [6,7]. In 2015, Soong et al. demonstrated that BACE1 is phosphorylated at T252 (P-BACE1-T252) by cyclin-dependent kinase 5 (CDK5) and observed that this modification increases the proteolytic activity of P-BACE1 by up to 60% [8]. Recently, it was reported that the same BACE1 phosphorylation can be done by glycogen synthase kinase 3β (GSK3β), which is also related to tau phosphorylation in AD [9,10].

Furthermore, the importance of BACE1 in AD is not only due to the Aβ production; also, this protein regulates the synaptic functions due to its participation in the phosphorylation of cAMP response element binding protein (CREB), a transcription factor involved in memory formation. CREB is phosphorylated by Protein kinase A (PKA) employing cyclic adenosin monophosphate (cAMP); however, when BACE1 is overexpressed, the amount of phosphorylated CREB is reduced as well as the PKA activity and cAMP levels. It has been demonstrated that BACE1 interacts with the adenylate cyclase enzyme that catalyzes the adenosin trifosfato (ATP) conversion to cAMP by its transmembrane domain, avoiding cAMP production and consequently diminishing PKA activity and CREB phosphorylation, contributing to memory loss [11].

The mechanism and proteins involved in AD have been modulated in several experimental models of AD. For example, there are transgenic rodents that overexpress APP, as well as other models such as Drosophila and Caenorhabditis elegans [12,13,14]. AD models are increasingly being developed in rats, since the rat genome has been sequenced and rats are easily subjected to genetic manipulation, allowing researchers to overcome limitations inherent to mouse models [15]. Non-transgenic senescence-accelerated OXYS rats have also been described as a spontaneous AD-like model, showing cognitive impairment and transcriptome alterations in genes Aβ-associated, and have been used as a complementary approach to induced experimental models [16,17]. Additionally, metacognition, once considered exclusive to primates, has been observed in rats [18]. Moreover, several chemicals, such as aluminum, colchicine, streptozotocin, scopolamine, ethanol, and excitotoxin, can be used to induce AD in rodents [19].

Scopolamine is an antagonist of muscarinic receptors, which induces memory impairment by blocking the cholinergic nervous system, thereby mimicking the symptoms of AD. The amnesia caused by scopolamine is primarily due to the blockade of muscarinic receptor 1 (M1) [20]. This blockade inactivates the ACh–M1 synaptic signaling pathway, and scopolamine is also known to increase the production of endogenous reactive oxygen species and proinflammatory cytokines. Consequently, scopolamine administration causes severe damage to the nervous system, particularly in the hippocampus and prefrontal cortex. A decrease in the number of neurons in these brain regions induces alterations in spatial memory and contributes to some hallmarks related to AD [21,22].

In addition, the memory loss in the scopolamine model has previously been reported using the Passive Avoidance Test, revealing a relationship between cholinergic dysfunction and memory [23]. Therefore, the chronic administration of scopolamine during six weeks also demonstrated memory loss through the Passive Avoidance Test [24]. Also, scopolamine administration for 1 week (1 mg/kg per day; ip) induced impaired learning and memory due to the rats exhibiting longer latency and fewer crossing times when the platform was removed in the Morris Water Maze test, compared with the control rats [25]. As well, several studies demonstrated that repeated doses of scopolamine induce cognitive impairment.

Consistently, chronic scopolamine administration (2 mg/kg for 6 weeks) has been associated with increased β-amyloid (Aβ), Tau and GSK3β levels, accompanied by significant memory impairment, as evidenced by deficits in spatial learning and memory using the Morris Water Maze and Y-maze tests [26]. Similarly, in male Sprague-Dawley rats treated with scopolamine for 15 days, significant memory loss was observed using the Radial Arm Maze and the Morris Water Maze, confirming learning and memory impairment in this model [27]. In addition, scopolamine administration (3 mg/kg/day) in male Wistar rats induced a significant reduction in memory performance in the Novel Object Recognition test, further supporting the reproducibility of scopolamine-induced cognitive deficits across different strains and behavioral paradigms [28].

In addition, it has been corroborated that in the AD model with scopolamine, cholinergic dysfunction and amyloid production are interconnected due to scopolamine administration regulating Aβ–BACE1 levels [29,30,31]. Also, scopolamine administration for six weeks increased Aβ production identified by congo red staining [30]. The Aβ produced can suppresses acetylcholine synthesis, contributing to cholinergic dysfunction [32]. Furthermore, acetylcholinesterase (AChE) facilitates Aβ assembly into fibrils, forming the amyloid plaques [33,34]. Therefore, cholinergic alterations influence amyloid precursor protein metabolism and tau phosphorylation, promoting neurotoxicity [35] and therefore memory loss.

Consequently, all physiopathological changes that occurred during AD progression resulted in the deregulation of some metabolites [36]. Metabolomics studies from the plasma of 23 AD patients revealed deregulated metabolites; among the metabolites found were amino acids and lipids, specifically PUFAs, ceramides and sphingolipids related to energy deficiencies and oxidative stress [37,38]. Furthermore, metabolomics studies in an AD model in rodents administered with Aβ has been carried out, identifying the up- and downregulation of important metabolites such as aminoacids, phytosphingosine, LPC (14:0), (18:2), (20:4), palmytoylcarnitine, hexadecenoic and docosahexaenoic acids, etc. [38].

Therefore, the aim of this study was to analyze the effect of the chronic administration of scopolamine in Wistar rats on Aβ levels, as well as BACE1, P-BACE1-T252 and PKA expression by Western blot and BACE1 and PKA activity in the hippocampus and prefrontal cortex, and determine if some metabolites that are related with other AD models in rodents are present in the scopolamine model, employing the UHPLC-MS/MS. The establishment of the AD model with repeated doses of scopolamine could be useful in designing and assaying drugs directed against the biological targets BACE1, P-BACE1 and PKA involved in AD.

2. Results

2.1. Aβ_1-42 Levels Increased During Scopolamine Administration

The Aβ concentrations in the hippocampus (Figure 1A) and prefrontal cortex (Figure 1B) samples were quantified by ELISAs. For the hippocampus samples, the mean concentration of Aβ_1-42/μg protein in the control group was 0.012, whereas in the Tx group, it was 0.026 μg of Aβ_1-42/μg protein; thus, in the samples from the Tx group, the Aβ_1-42 concentration increased 2.1-fold relative to that in the control group, indicating a significant difference (p < 0.0001). However, in the prefrontal cortex samples, no significant difference in Aβ concentration was detected between the control and Tx groups (p < 0.05).

Figure 1.

Quantification of the Aβ peptide concentration in the hippocampus and prefrontal cortex. Aβ levels in the hippocampus; n = 11, **** p < 0.0001 (A). Aβ_1-42 levels in the prefrontal cortex; n = 12, ns = not significant (B).

2.2. BACE1 and P-BACE1-T252 Levels Increased in the Hippocampus of the Animals in the Tx Group

Figure 2 shows the bands from Western blot of BACE1 and β-actin (Figure 2A), and P-BACE1-T252 and β-actin (Figure 2B), in the samples from the hippocampus of the control and Tx groups. The band detection approximately at 45 kDa is in accordance with bands around 50 and 45 kDa corresponding to BACE1 proforms or less glycosylated variants in human brain tissue [39]. Also, a band close to 45 kDa has been associated with immature BACE1 in cortical extracts from animal models and in human brains with amyloid pathology [40,41].

Figure 2.

Expression of BACE1 and P-BACE1-T252 in the hippocampus. BACE1 and β-actin bands obtained by Western blotting in the control and Tx groups (A); P-BACE1-T252 and β-actin bands obtained by Western blotting in the control and Tx groups (B); quantitation of the BACE1 bands shown in Figure 2A (C); quantitation of the P-BACE1-T252 bands shown in Figure 2B (D); levels of P-BACE1-T252 obtained by ELISA (E). * p < 0.05; ** p < 0.01; *** p < 0.001. n = 8 (control group); n = 9 (Tx group).

The intensity of each BACE1 band was normalized to that of its corresponding β-actin band and plotted. These graphs show a significant increase in BACE1 (85%, Figure 2C) and P-BACE1-T252 (100%, Figure 2D) in the Tx group, indicating that the expression of these proteins was increased 2-fold compared with that in the control group. The data were analyzed via Student’s t test for unpaired data. Additionally, P-BACE1-T252 was measured by ELISAs, as shown in Figure 2E, and a 40% increase in P-BACE1-T252 levels was detected in the Tx group compared with the control group.

2.3. P-BACE1-T252 Increased in the Prefrontal Cortices of the Animals in the Tx Group

Figure 3 shows BACE1 and P-BACE1-T252 expression in the prefrontal cortex. The bands corresponding to BACE1/β-actin and P-BACE1-T252/β-actin in the control and Tx groups are displayed in Figure 3A and 3B, respectively. Each data point was normalized to its corresponding β-actin band. Figure 3C shows that there was a nonsignificant increase in BACE1 levels in the Tx group. In contrast, P-BACE1-T252 levels increased 53% (3D). The analysis was performed via an unpaired Student’s t test. An ELISA for P-BACE1-T252 was also performed; as shown in Figure 3E, there was a 46% increase in P-BACE1-T252 levels in the Tx group.

Figure 3.

Expression of BACE1 and P-BACE1-T252 in the prefrontal cortex. BACE1 and β-actin bands obtained by Western blotting in the control and Tx groups (A); P-BACE1-T252 and β-actin bands obtained by Western blotting in the control and Tx groups (B); quantitation of the BACE1 bands shown in Figure 3A (C); quantitation of the P-BACE1-T252 bands shown in Figure 3B (D); levels of P-BACE1-T252 obtained by ELISA (E). * p < 0.05; *** p < 0.001; ns = not significant. n = 8 (control group); n = 9 (Tx group).

2.4. BACE1 Activity in Rats Treated with Scopolamine

Figure 4 shows BACE1 activity in the hippocampus expressed in relative units of fluorescence (UF). Compared with the control group, the group treated with scopolamine presented increased BACE1 activity. This finding indicates that BACE1 activity increases with scopolamine treatment, and this increase is correlated with the increased Aβ concentration in hippocampal tissue, as shown in Figure 1.

Figure 4.

BACE1 activity. BACE1 activity in the hippocampus of rats treated with scopolamine (Tx group) was significantly greater than that in the control group. ** p < 0.05; n = 8.

2.5. PKA Is Modified in the Hippocampus but Not in the Prefrontal Cortex of Animals in the Tx Group

Figure 5A shows the PKA bands in the hippocampus, and Figure 5B displays the bands in the prefrontal cortex. The PKA/β-actin intensity for each band was obtained; the results are presented in Figure 5C,D, which compare the control group bands with the Tx group bands. Figure 5C reveals a significant decrease in PKA/β-actin levels in the hippocampus of the Tx group, suggesting an impact of the treatment. In contrast, Figure 5D shows that the changes in PKA/β-actin expression in the prefrontal cortex in the Tx group were not statistically significant.

Figure 5.

Expression of PKA in the hippocampus and the prefrontal cortex. PKA and β-actin bands obtained by Western blotting of hippocampal tissue in the control and Tx groups (A); PKA and β-actin bands obtained by Western blotting of prefrontal cortex samples in the control and Tx groups (B); quantitation of the PKA bands shown in Figure 5A (C); quantitation of the PKA bands shown in Figure 5B (D). * p < 0.05; ns = nonsignificant. n = 5 (hippocampus); n = 8 (prefrontal cortex).

2.6. PKA Activity Decreased in the Tx Group

Figure 6 shows the PKA activity in the hippocampus and prefrontal cortex of the control and Tx groups. Figure 6A shows the PKA activity in the hippocampus; a statistically significant difference (p < 0.05) was observed. Figure 6B shows the PKA activity in the same groups but in the cortex, where no statistically significant difference was found.

Figure 6.

PKA activity in the hippocampus and prefrontal cortex. PKA activity in the hippocampus (A); PKA activity in the prefrontal cortex (B). * p < 0.05, ns = not significant. n = 8.

In the hippocampus, Spearman correlation analyses revealed that pBACE1 expression showed a positive correlation with Aβ (r = 0.490), while PKA levels were inversely correlated with both Aβ (r = −0.587) and pBACE1 (r = −0.588), indicating that increased BACE1 phosphorylation and Aβ production are associated with reduced PKA signaling. Finally, Aβ levels showed a strong negative correlation with PKA activity (r = −0.891). In the prefrontal cortex, a similar trend was observed: pBACE1 positively correlated with Aβ (r = 0.447), and β-amyloid levels negatively correlated with both total PKA (r = −0.217) and its activity (r = −0.857), while PKA and pBACE1 exhibited a weak negative correlation (r = −0.077).

2.7. Identification of Metabolites in Plasma Samples from Rats Treated with Scopolamine

The identification of 19 metabolites in a murine Alzheimer’s disease model in which Aβ_1-42 was administered intracerebroventricularly were previously reported [38]. Additionally, if scopolamine produces changes in Aβ production, similar metabolites could be observed in animals that are administered with repeated doses of scopolamine. The metabolites that were identified in the plasma from rats administered scopolamine for 6 weeks are proline, valine, tryptophan, acetylcarnitine, phytosphingosine, hexadecenoic acid, docosahexaenoic acid, MG (18:3), docosapentaenoic acid, octadecenoic acid, and LPC (17:0); several of these compounds were found in both the control and Tx groups, as shown in Figure 7A.

Figure 7.

Metabolites identified by UHPLC-QTOF MS/MS. Retention times (RTs) and m/z ratios of the metabolites detected in the plasma samples (A); average intensities of each metabolite identified in the samples obtained from the control group and the Tx group (scopolamine) (B); mass spectra of valine (C) and phytosphingosine (D), the two metabolites that showed significant differences between the two groups n = 8. * p < 0.05 compared with the control group via an unpaired Student’s t test.

After statistical processing and analysis were performed using MassHunter LC/MS Qualitative Analysis version B.07.00 software, the parameters used in the analysis (range from 100 to 1000 m/z, peak height, and mass tolerance at 0.01 Da) were adjusted. The Tx group treated with scopolamine presented greater intensities of some metabolites, including phytosphingosine, MG 18:3, docosapentaenoic acid and octadecaenoic acid (Figure 7B). However, some metabolites, such as LPC 14:6, LPC 14:0, LPC 18:2, LPC 20:4, LPC 15:1, LPC 15:0, and palmitoylcarnitine, were not identified in relation to the model administrated with Aβ_1-42. Figure 7C and 7D show the MS/MS spectra obtained from valine and phytosphingosine, respectively; these are significantly different between the groups.

3. Discussion

AD is a neurodegenerative disease with high incidence worldwide, which is partly a consequence of increased life expectancy due to improved medical therapy and lifestyle. However, there are no specific experimental models that simulate all the pathogenesis mechanisms and yield complete biomarkers for AD. Thus, rat models are of great interest due to them allowing us to study the possible biochemical alterations that occur during AD and can be replicated. In this work, the relationship between chronic scopolamine administration and the expression and activity of proteins such as BACE1, Aβ and PKA, as well as the deregulated metabolites related to AD, were analyzed.

It has been widely reported that the repeated doses of scopolamine induce cognitive deficits, loss of memory, and increases in Aβ production, which have occurred in AD patients [41,42]. An increase in Aβ_1-42 levels was observed in rats administered scopolamine for 18 days until 6 weeks. Therefore, the result obtained from brain samples of Wistar rats administered scopolamine by 6 weeks showed a 2.1-fold increase in the concentration of Aβ_1-42 in the hippocampus compared with that in the control group. This finding is consistent with several studies in which scopolamine administration resulted in a 2.4-fold increase in Aβ_1-42 in comparison with the control group [26,43]. Furthermore, an increase in Aβ_1-42 levels in mice after scopolamine administration confirmed the efficacy of scopolamine administration in these species [44]. It is important to note that in the results from scopolamine administration for 6 weeks, significant changes in Aβ levels are observed in the hippocampus but not in the prefrontal cortex, as has been reported in AD patients who do not show significantly increased levels of Aβ in the prefrontal cortex in relation to healthy individuals [45].

BACE1 is the key enzyme in Aβ_1-42 production; it is expressed primarily in the brain, particularly in neurons, oligodendrocytes, and astrocytes. After administration of scopolamine for 6 weeks, BACE1 in the hippocampus was significantly increased by 0.85-fold compared with the control group, which correlates with observations in AD patients [8,46,47]. In addition, P-BACE1-T252 expression increases significantly in the hippocampus and prefrontal cortex of the Tx group; this is consistent with the findings of Woo-Joo Song (2015), who demonstrated BACE1 phosphorylation at T252 in the superior frontal cortex of humans with AD [8]. Therefore, Aβ_1-42 production in the prefrontal cortex could be catalyzed by P-BACE1-T252 as well.

However, scopolamine not only influences Aβ_1-42 production and BACE1 expression but also the kinase’s activity involved in long-term memory, such as protein kinase A [26,48,49]. PKA activity is crucial for memory, and it was decreased in the scopolamine-treated groups for 6 weeks, especially in the hippocampus; this change may be linked to increases in Aβ_1-42, whereas in the prefrontal cortex, neither the level nor the activity of PKA differed significantly between the control group and the Tx group due to PKA also regulating APP cleavage, decreasing Aβ_1-42 production and its accumulation in the brain [50,51]. The decrease in PKA activity reflects how Aβ affects the PKA pathway, especially given that Aβ_1-42 deposits are prominent in the hippocampus and neocortex [11]. In this work, a strong negative correlation was found between Aβ levels and PKA activity (r = −0.891) and expression (r = −0.587). Additionally, in animal models of AD where PKA is diminished, its activation with a drug can improve memory and decrease neuropathological changes [52].

The changes in the BACE1 and PKA pathways observed in the model used in this study agree with other published results showing that BACE1 regulates the cAMP/PKA/CREB pathway in a transgenic mouse model, in which opposite effects on BACE1 and PKA were found; as BACE1 expression increased, PKA activity decreased, and when BACE1 expression decreased, PKA activity increased [11]. Together, these findings support a model in which increased BACE1 phosphorylation is associated with enhanced amyloid accumulation, which in turn may contribute to the suppression of PKA signaling. In this study, a negative correlation was noted between pBACE1 and PKA (r = −0.588).

UHPLC-QTOF MS/MS studies revealed that 11 of the 19 metabolites reported to be associated with AD were identified in plasma samples from rats treated with scopolamine. Among the metabolites found were amino acids and lipids, specifically PUFAs, ceramides and sphingolipids [53,54]. The plasma analysis revealed that this group of metabolites increased in the group treated with scopolamine compared with the control group, particularly phytosphingosine, which is associated with apoptotic events that are activated through different mechanisms. Phytosphingosine induces chromatin DNA fragmentation, a hallmark of apoptosis, through the activity of caspases 3 and 9. Other studies have shown that phytosphingosine can activate two distinct MAPK signaling pathways that amplify the apoptotic cascade [55,56]. It has been reported that scopolamine induces oxidative stress and apoptosis, leading to the death of hippocampal neurons [57,58]; based on our findings, this may possibly be mediated through an increase in phytosphingosine. Importantly, phytosphingosine is formed in mammalian tissues from the hydrolysis of phytoceramides by ceramidases, indicating that the production of phytosphingosine in mammalian tissues is controlled only by ceramidases [59]. Ceramides are lipid molecules that determine cell integrity and intercellular signaling related to behavioral plasticity. High ceramidases activity is also associated with poor long-term object memory; if ceramidases are active, high amounts of sphingosine or phytosphingosine can be produced, resulting in apoptosis. This interesting metabolite was identified in animals that were subjected to chronic administration of scopolamine and has been identified in AD patients [38]. The results suggest that the ceramide/sphingomyelin pathway could represent an effective therapeutic target for AD in patients [60].

Sphingolipids are abundant within cell membranes, and they play a role in neuronal homeostasis and in the immune system [61,62]. In a plasma study of patients with AD and controls, several potential biomarkers, including LPC, sphingosine and tryptophan, were found, and all these metabolites were present at lower levels in AD patients than in the controls. In this study, we observed an increase in the metabolites phytosphingosine and tryptophan in an animal model of AD induced with scopolamine compared with the group treated with saline; the results were different from those reported in the cited study. This could be due to the damage produced because the AD samples do not specify the disease’s evolution, and in the scopolamine model it was administrated for six weeks; so, phytosphingosine could be related to the apoptotic process as was mentioned before. Another metabolite under study was acetylcarnitine (Figure 5). Previous studies have shown that acetylcarnitine levels decrease significantly and progressively in patients with memory loss (mild cognitive impairment and already-established AD) compared with healthy individuals [63]. In this study, a similar phenomenon was observed; the group treated with scopolamine presented the lowest levels of this metabolite, although no statistically significant difference was found between the study groups.

The scopolamine model represents a simpler, more economical, and faster alternative to genetically modified organisms, as it reproduces key features of Alzheimer’s disease, including the overexpression of Aβ, BACE1, and P-BACE1-T252, as well as reduced expression and activity of PKA. In the hippocampus, an increase in BACE1 activity and a significant decrease in PKA activity are observed, whereas in the prefrontal cortex the main alteration corresponds to the overexpression of P-BACE1-T252 (Figure 8). These molecular changes are integrated into a model in which a scopolamine-induced blockade of muscarinic M1 receptors promotes amyloidogenic APP processing, increased β-amyloid production, and mitochondrial dysfunction, leading to metabolomic alterations characterized by increased phytosphingosine and decreased acetylcarnitine, as well as suppression of the cAMP/PKA/CREB pathway, resulting in reduced BDNF expression. These modifications may be further modulated by oxidative stress induced after chronic scopolamine administration [31], in agreement with previous reports in this model describing increased MDA levels and elevated free radical production determined by EPR [30].

Figure 8.

Modifications in protein expression, activity and metabolite levels in the scopolamine model. Scopolamine administration blocks M1 receptors, promotes amyloidogenic APP processing through increased BACE1/P-BACE1 and β-amyloid production, induces mitochondrial dysfunction with metabolomic alterations (↑ phytosphingosine, ↓ acetylcarnitine), and suppresses the cAMP/PKA/CREB pathway, leading to reduced BDNF expression and neuroprotection.

Additionally, the scopolamine model could have some advantages over transgenic models due to their limitations, as they are based on familial mutations where only 5–10% of AD cases are found, while most are sporadic, with no clear mutations [64,65]. In addition, transgenic animals tend to overexpress genes at non-physiological levels, such as APP in PDAPP mice, which increases up to 10 times, altering brain development and not reproducing age-dependent progression [66]. The difference between species for the understanding of AD is part of a work that we are also doing as part of a more robust study; one of the first findings regarding the difference between BACE1 in rats and humans was published [9]. Nevertheless, despite the marked molecular alterations observed within the amyloidogenic pathway, it is important to acknowledge that the scopolamine-induced model represents a pharmacological model that mimics certain aspects of Alzheimer’s disease but does not fully reproduce the disease itself.

4. Materials and Methods

Twenty-seven male Wistar rats (10 weeks old) obtained from the Centro de Investigación y Estudios Avanzados (CINVESTAV) Norte, CDMX and the Universidad Autónoma de México (UNAM) were used. All animals were accompanied by a health certificate issued by the supplier, confirming that they were free of known infectious or transmissible diseases. Before the experiments began, the animals were acclimatized for 3 weeks in the Laboratorio de Biofisica y biocatalisis in the Escuela Superior de Medicina del IPN. The animals were kept in individual cages with a 12 h light/dark cycle and had access to water and rat chow (Purina) ad libitum throughout both the acclimatization period and the duration of the experiment [24]. The experimental protocol was performed according to the guidelines provided by the Norma Oficial Mexicana (NOM-062-ZOO-1999 [67]), which specifies the technical requirements for the production, care, and use of laboratory animals. The protocol was approved by the Committee for the Care and Use of Laboratory Animals: Comité de Investigación para el Cuidado y uso de Animales de Laboratorio (CICUAL) of the Escuela Superior de Medicina of IPN (Approval number: ESM-CICUAL-02/22-05-2019).

No inclusion or exclusion criteria were applied during the experiment or data analysis, and all animals initially allocated to the study were included in the final analysis; these criteria were established a priori. The number of animals was determined based on the amount of the sample (µg) required for the assays and the size of the hippocampal tissue.

4.1. Scopolamine Treatment and Quantification of Protein in Samples Obtained from the Hippocampus and Prefrontal Cortex

The rats were randomly allocated to control or treatment groups using a randomization function in Microsoft Excel and divided into two groups: the control group (control, n = 13) and the treated group (Tx, n = 14). To reduce potential confounding factors associated with animal identification and cage allocation, rats were housed two per cage. Cages were appropriately labeled according to experimental group (control or treatment) and cage number. Individual animals within each cage were distinguished by tail marking (one marked, one unmarked), ensuring consistent identification throughout the experiment. The authors were always aware of the group allocation of each rat. The treatments were administered via i.p. injection; the control group received 1 mL/kg/kg/day isotonic saline solution (0.9% NaCl, PISA), and the Tx group received 2 mg/kg/day scopolamine daily for 6 weeks [24]. Throughout the study, no signs of distress were observed, including hair loss, seizures, tremors, hunched posture indicative of pain, or significant changes in body weight. Blinding was not implemented during the experimental procedures; however, all outcome measures were obtained using objective and quantitative techniques (ELISA, Western blot, enzymatic activity assays, and UHPLC-MS/MS), minimizing potential observer bias.

One day after the treatment was finished, the animals were anesthetized with sodium pentobarbital at a dose of 60 mg/kg. Deep anesthesia was verified based on the plantar reflex, and cardiac puncture was performed to obtain whole blood samples; it is important to mention that this was a humane endpoint for all the animals in the experiment. Five milliliters of blood was obtained by transcardially collecting the sample in heparin tubes and centrifuging at 3000 rpm and 4 °C for 5 min to obtain plasma samples that were then frozen until use at −80 °C. Each animal was then perfused with 100 mL of cold isotonic saline at a constant flow rate of 1.5 mL/min through the carotid artery.

Tissue samples (20 mg) were dissected from the hippocampus and the prefrontal cortex and placed in 100 µL of CelLytic MT lysis reagent (Sigma-Aldrich, St. Louis, MO, USA, C3228) containing cOmplete Mini EDTA-free protease inhibitor cocktail ( Roche, Mannheim, Germany, 11836170001, 0.1%) and the phosphatase inhibitor PhosSTOP (ROCHE 41116000, 1%). The samples were homogenized in an ultrasonic homogenizer for 15 s and then centrifuged at 9000 rpm for 10 min at 4 °C in a UNIVERSAL 320 R centrifuge.

The protein concentration in the supernatant was quantified using a standard curve consisting of serial dilutions of bovine serum albumin (BSA) (100 µL of dilution per well in Milli-Q H₂O) in a 96-well plate. BSA concentrations of 32.0, 24.0, 18.0, 13.5, 10.1, 7.5, 5.6 and 0 µg/mL were used. For the quantification of protein in the samples, 100 μL of a 1:100 dilution of each sample (prepared by adding 10 μL of the supernatant to 990 μL of Milli-Q H₂O) was tested in triplicate. Then, 100 μL of Bradford reagent was added to each well, and the plate was incubated for 5 min at room temperature. Finally, the absorbance at 595 nm was read on a Multiskan Ex plate reader (Thermo Scientific^®, Shanghai, China).

The number of samples used in each assay varied depending on tissue availability and the specific requirements and sensitivity of each experimental technique.

4.2. Quantification of Aβ_1-42 in Hippocampal and Prefrontal Cortex Samples via ELISA

An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of Aβ_1-42 in supernatants obtained from the hippocampus and prefrontal cortex of the animals in the control and Tx groups. A total of 150 μg of protein was taken in triplicate and resuspended in 0.1 M carbonate buffer (pH 9.6) until a final volume of 100 μL per well was reached. For the Aβ_1-42 standard curve, increasing concentrations (0.0039, 0.0078, 0.0156, 0.0312, and 0.0625 μg/100 μL) of Aβ_1-42 peptide (Calbiochem/Merck, Darmstadt, Germany, PP69-0.25 mg) were prepared in carbonate buffer, and 100 μL was added per well. The dilutions were incubated overnight at 4 °C in 96-well Corning high protein-binding plates. Then, three washes (2 min each one) with 0.05% (0.1 M pH 7.4) of PBSTween20 (PBST) were performed, followed by three washes in the same manner as PBS (0.1 M pH 7.4); the wells were then blocked with a milk solution (Svelty Fig 0%, Nestlé, Vevey, Switzerland; 0.6% w/v in PBST) for 2 h at 37 °C without agitation. Three additional washes with PBST and PBS were then performed as described above, 75 μL per well of a 1:500 dilution of an antibody against Aβ (Amyloid Antibody MOAB-2 NBP2-13075, Novus Biological, CO, USA) was applied, and the plate was incubated at for 2 h at 37 °C in the dark. Finally, the wells were washed three times with PBST followed by PBS and developed using 50 μL per well of a solution containing 2.7 mM OPD dissolved in 33% stable hydrogen peroxide solution (Stable Peroxide Substrate Buffer 1X, Thermo Scientific #1855910) in PBS. After the addition of 30 μL 2.5 N sulfuric acid to each well, the plate was read at 490 nm to obtain sample absorbances using Multiskan Ex equipment (Thermo Scientific^®, Shanghai, China).

4.3. Quantification of BACE1, P-BACE1-T252 and PKA in the Hippocampus and Prefrontal Cortex Samples by Western Blot

Prior to electrophoresis of the samples on gels, each sample was heated to boiling point in a water bath in a mixture containing 5 μg of protein, 5 μL of loading Laemmli buffer, and 5 μL of Milli-Q H₂O. The samples were then electrophoresed on a 10% acrylamide gel at 120 V for 1.5 h in a Mini-PROTEAN^® electrophoresis chamber (Bio-Rad Laboratories, Hercules, CA, USA). One lane of the gel contained a BlueRAY Prestained Protein Ladder molecular weight marker (Bio-Helix, New Taipei City, Taiwan; PM006-0500). The transfer was performed in a Bio-Rad Mini Trans-Blot^® Cell chamber for 1.5 h at 4 °C under constant stirring using an Immobilon-P 0.45 µm PVDF membrane (Merck Millipore, Burlington, MA, USA; IPVH00010)that had been previously activated with HPLC-grade methanol (Sigma-Aldrich, St. Louis, MO, USA). Following the transfer, the membrane was blocked for 1 h under constant agitation at room temperature using a 5% Svelty-Nestlé^® solution in 0.1% PBST, followed by three washes in PBST (10 min each). The membrane was then incubated with the primary antibody Immunotag™ BACE monoclonal antibody (G-Biosciences, St. Louis, MO, USA) coupled to HRP (ITM1015, 1:1000 dilution in PBST) at 4 °C for 12 h with constant stirring. Finally, the membrane was developed via the SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Scientific 34095) kit on a Fusion SL imaging system (Vilber, Paris, France) with an exposure time of 1.2 s.

For quantification of the immunoreactive bands, the public domain software ImageJ 1.53k version was used to measure the pixels within each band [68].

To evaluate whether T252 BACE1 was phosphorylated, a Western blot analysis was performed. Electrophoresis, transfer, and blocking were performed as described above; the membrane was then incubated with a primary antibody against phospho-BACE1 (T252) (Affinity Biosciences, Cincinnati, OH, USA, AF3650, 1:1000 dilution in blocking solution) with constant agitation for 12 h. After five washes with PBST (10 min each), the membrane was incubated with an HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (Novus Biologicals HRP NB730-H, 1:1000 dilution in PBST) with constant agitation at room temperature. Then, five 10 min washes were performed, and the blot was developed as described for BACE1.

To quantify PKA, electrophoresis, transfer and blocking were performed as described above. A PKA antibody (Bioss Antibodies, Beijing, China, bs-0520R, ratio of 1:1000) dissolved in TBS-Tween 20 (TBST) was added, and the mixture was gently shaken (5 rpm) for 12 h. At the end of the 12 h period, four 7 min washes in TBST were performed. The membranes were subsequently developed and analyzed as described above.

All assays were normalized using β-actin as constitutive protein. For the Western blots, the antibody used was Bioss Antibodies bs-0061R-HRP, following the conditions specified in the datasheet.

The molecular weight of each protein was estimated using the Thermo Scientific™ Spectra™ Multicolor Broad Range Protein Ladder (catalog no. 26634). Based on this reference, the bands corresponding to BACE1 (Figure S1) and P-BACE1 (Figure S2) were identified at approximately 45 kDa, PKA at 40 kDa (Figure S3), and β-actin at 42 kDa.

4.4. Measurement of P-BACE1-T252 by ELISA in Hippocampal and Prefrontal Cortex Samples

Supernatants (80 μg of protein) from the hippocampus or prefrontal cortex samples were incubated in high-affinity 96-well plates overnight. To avoid nonspecific binding of the antibody, the wells were blocked with 5% (w/v) bovine serum albumin (BSA) at 37 °C for 2 h with constant stirring. After the blocking solution was washed off, the primary antibody (phospho-BACE1-T252 AF3650 Affinity Biosciences) was added (100 µL per well of a 1:10,000 dilution in PBS-Tween), and the plate was incubated overnight at 4 °C in the dark. Finally, the secondary antibody (Novus Biologicals HRP NB730-H, diluted 1:10,000 in PBS-Tween) was applied for 2 h at room temperature. The plate was developed with TMB, and the reaction was stopped after 5 min with stop solution. The absorbance was read at 450 nm using Multiskan Ex equipment (Thermo Scientific^®, Shanghai, China). For the case of P-BACE1-T252, obtaining the same trend using two different immunoassays, such as ELISA and Western blot, strengthens data reliability, confirming protein presence, expression changes, and reducing method-specific biases.

4.5. Measurement of BACE1 Activity in Hippocampal Samples

BACE1 activity in rat hippocampal tissue was measured using a fluorometric assay kit((BioVision, Inc., Milpitas, CA, USA; catalog no. K360-100).”), which uses a specific peptide labeled with EDANS and DABCYL. Hydrolysis of the peptide by BACE1 separates EDANS and BABCYL, resulting in fluorescence. Two hundred micrograms of protein were used in the assay; to this, 50 μL of 2X BACE1 reaction buffer was added, and the mixture was incubated at 37 °C for 20 min with gentle stirring, followed by the addition of 2 μL of BACE1 substrate. After mixing and covering the plate, the plate was incubated for 1 h at 37 °C. Fluorescence was quantified using a fluorometer (Model L355, PerkinElmer, Seer Green, Buckinghamshire, UK) at Ex/Em wavelengths of 345/500 nm, and the data were analyzed with FL WinLab software (Version 4.00.03, PerkinElmer^® Inc.).

4.6. Measurement of PKA Activity in Hippocampal and Prefrontal Cortex Samples

A PKA kinase activity kit (Enzo Life Sciences, Farmingdale, NY, USA; #ADI-EKS-390) was used. Fifty microliters of kinase assay dilution buffer were added to the wells of a multi-well plate 10 min after the buffer was removed; then, 30 μL of the control or Tx hippocampus and prefrontal cortex samples and 10 μL of ATP were added to each well to initiate the reaction, and the plate was incubated for 90 min at 30 °C. The wells were emptied, 40 μL of specific antibody was added to the phosphorylated substrate, and the plate was incubated at room temperature for 60 min. The wells were washed four times with 100 μL of 1X wash buffer; then, 40 μL of rabbit anti-IgG conjugate was added to each well, and the plate was incubated at room temperature for 30 min. After washing, 60 μL of TMB was added to each well, and the mixture was incubated for 30 min at room temperature. Finally, 20 μL of stop solution was added, and the absorbance was measured at 450 nm to obtain values that were proportional to the amount of substrate phosphorylated by PKA.

4.7. Metabolomic Studies by LC–MS

The plasma samples were thawed at room temperature prior to analysis. Two hundred microliters of each plasma aliquot was mixed with 400 μL of acetonitrile in an Eppendorf tube. The mixture was then vortexed for 3 min to allow protein precipitation and centrifuged at 13,000 rpm and 4 °C for 10 min. The supernatant was separated and evaporated to dryness under a moderate nitrogen stream in a water bath at 30 °C. The dry residue was reconstituted with 200 μL of acetonitrile in water (5:95, v/v), mixed for 3 min, sonicated for 3 min, and centrifuged at 13,000 rpm for 5 min. An aliquot of the supernatant was stored at −20 °C for UHPLC-MS/MS analysis. All reagents used in the experimental assays were HPLC analytical grade. Analysis of specific metabolites (proline, valine, tryptophan, lysophosphatidylcholine (LPC 14:0, 18:2, 20:4, 15:1, and 17:0), acetylcarnitine, palmitoylcarnitine, phytosphingosine, hexadecenoic acid, docosahexaenoic acid, docosapentaenoic acid, octadecenoic acid, n-eicosanolethanolamine, MG (18:3), and vaccenyl carnitine) was performed on a UHPLC 1290 Infinity machine coupled to a mass Q-TOF mass spectrometer 6545 (Agilent Technologies, Palo Alto, CA, USA).

All operations, data acquisition and analysis were performed using Mass Hunter software B.07.00 version. Chromatographic separation was performed on a C-15 column (Agilent Technologies Model G7130A). The column was kept at a constant temperature of 35 °C, and an injection rate of 0.25 mL/min was used. Five microliters of each sample was analyzed, and chromatographic analysis was completed with an elution gradient consisting of 0.1% formic acid dissolved in water and 0.1% formic acid in acetonitrile. The mass spectrometry analysis was carried out by analysis with a double-source AJS; the ESI was in positive mode as previously reported with the following parameters: drying gas (N2), flow rate 9 L/min, gas temperature 350 °C, nebulizer gas pressure 40 psig, VCap 3500, fragmenter velocity 100 V, and skimmer velocity 65 V [69]. The MS/MS analysis was performed in energy collision mode. The data were acquired in 42 Centroid storage mode using a 100–1000 m/z scan interval at a rate of 6 spectra per second throughout the analysis. The program is shown in Figure S1.

4.8. Statistical Analysis

The results of each assay are presented as the means ± SEs. Each in vitro assay was performed in triplicate, and the data were analyzed using Student’s t test in GraphPad Prism 7.0 software. Differences were considered statistically significant when p < 0.05.

Correlation analyses were performed to explore potential associations among the molecular markers involved in amyloidogenic processing. The variables included in the analysis were BACE1, P-BACE1, and Aβ levels. A nonparametric Spearman correlation was computed using GraphPad Prism software, and the correlation coefficients were calculated for every pair of data sets; the statistical significance was assessed using two-tailed p-values with a 95% confidence interval. The results were interpreted to identify possible molecular relationships that may underline treatment-induced changes in amyloidogenic signaling pathways.

5. Conclusions

In this study, the effects of chronic administration of scopolamine to Wistar rats were investigated, with a focus on key proteins and metabolites associated with AD. Scopolamine is a muscarinic antagonist that is capable of inducing AD-like cognitive dysfunction in animal models of AD. The results revealed several significant alterations in protein and metabolite levels in the hippocampus and prefrontal cortex.

The fact that an increase in BACE1 phosphorylation in the hippocampus was observed suggests that increased activity in the amyloidogenic pathway may contribute to the accumulation of Aβ in the brain. This finding was corroborated by the results of BACE1 activity assays, which revealed that there was an increase in the proteolytic activity of BACE1 in the Tx group, leading to a greater increase in Aβ levels. Similarly, alterations in the activity and expression of PKA in the hippocampus were detected, suggesting that PKA plays a neuroprotective role that is affected by the administration of scopolamine.

This study revealed that chronic administration of scopolamine induces a series of biochemical and molecular changes that mimic key aspects of AD; these changes include increased β-amyloid production, decreased PKA activity, and alterations in lipid metabolism. The significant deregulation of metabolites, such as increased levels of phytosphingosine and reduced levels of acetylcarnitine, suggests a link between lipid metabolism and neurodegenerative processes. These findings underscore the usefulness of the scopolamine model in the study of AD and highlight the potential of targeting lipid metabolism as a therapeutic strategy. This study provides a comprehensive view of how scopolamine induces AD-like pathological changes and emphasizes the importance of investigating both the underlying mechanisms of its effects and potential therapeutic interventions to counteract these effects and improve cognitive function in neurodegenerative contexts.

Abbreviations

The following abbreviations are used in this manuscript:

AD	Alzheimer’s disease
Aβ	Amyloid beta
Aβ1-42	Amyloid beta 1-42 peptide
ACh	Acetylcholine
AChE	Acetylcholinesterase
APP	Amyloid Precursor Protein
ATP	Adenosine Triphosphate
BACE1	β-site amyloid precursor protein-cleaving enzyme 1
BSA	Bovine Serum Albumin
CDK5	Cyclin-dependent kinase 5
CREB	cAMP response element-binding protein
ELISA	Enzyme-linked immunosorbent assay
GSK3β	Glycogen synthase kinase 3 beta
HRP	Horseradish peroxidase
i.p.	Intraperitoneal
LC-MS/MS	Liquid Chromatography-tandem mass spectrometry
LPC	Lysophosphatidylcholine
MG	Monoacylglucerol
m/z	Mass-to-charge ratio
OPD	O-Phenylenediamine
PBS	Phosphate-buffered saline
PBST	Phosphate-buffered saline with Tween20
PKA	Protein Kinase A
PTM	Post-translational modifications
PUFAs	Polyunsaturated fatty acids
PVDF	Polyvinylidene difluoride
SE	Standard error
Tx	Treated group
UHPLC-MS/MS	Ultra-high-performance liquid chromatography–tandem mass spectrometry
β-actin	Beta-actin

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27052241/s1.

Author Contributions

M.C.R.H. and J.C.B. directed the research and contributed to the experimental design. J.V.T., I.G.G. and J.P.-Y. conducted experiments and experimental design. All authors participated in the data analysis and writing of the paper. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The experimental protocol was performed according to the guidelines of the Norma Oficial Mexicana (NOM-062-ZOO-1999), which specifies the technical requirements for the production, care, and use of laboratory animals. The protocol was approved by the Comité de Investigación para el Cuidado y uso de Animales de Laboratorio (CICUAL) of the Escuela Superior de Medicina of IPN (Approval number: ESM-CICUAL-02/22-05-2019, approved on 22 May 2019).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was supported by a project grant from CONACYT Ciencia Básica y/o de Frontera: Paradigmas y controversias de la ciencia 2022; 319355, SIP-IPN multidisciplinario 202400059. The project Desarrollo Tecnológico o Innovación para alumnos del IPN—2024: 2024-A187 and to the Apoyo para actividades de investigación 2024-SIP-IPN.