Neuroprotective effect of PPAR alpha and gamma agonists in a mouse model of amyloidogenesis through modulation of the Wnt/beta catenin pathway via targeting alpha- and beta-secretases
Abstract
This study evaluated the efficacy of fenofibrate and pioglitazone in a mouse model of amyloidogenesis induced by amyloid beta (βA) peptide. Mice received a single intracerebroventricular injection of βA1-40 (400 pmol/mouse), followed by daily treatment with fenofibrate (300 mg/kg), pioglitazone (30 mg/kg), or their combination for 21 days. Memory impairment and cognitive function were assessed using the Morris water maze, Y-maze, and object recognition tests. On day 22, mice were sacrificed, and hippocampal tissues were analyzed for levels of α- and β-secretase, peroxisome proliferator-activated receptors (PPARα and PPARβ), Wnt, and β-catenin. The amyloid-induced model displayed significant memory deficits and cognitive dysfunction, accompanied by increased α- and β-secretase levels and decreased Wnt, β-catenin, and PPARα and β expression. Histopathological examination confirmed neuronal damage. Treatment with fenofibrate, pioglitazone, and their combination significantly improved behavioral outcomes and neurochemical alterations induced by βA injection. Combined administration was more effective than either monotherapy in mitigating behavioral, neurochemical, and histopathological changes, suggesting a promising therapeutic approach for Alzheimer’s disease complicated by diabetes and hypercholesterolemia.
Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder affecting elderly populations worldwide. It is characterized by cognitive decline and impaired learning and memory, associated with hippocampal and cortical senile plaques and neurofibrillary tangles. The accumulation of soluble amyloid beta (βA) peptides and extracellular fibrillar βA deposits are key pathological features of AD. Failure to clear these fibrillar deposits can exacerbate AD pathology and neuronal death through chronic production of cytotoxic factors. βA peptides are produced through sequential cleavage of amyloid precursor protein (APP) by β- and α-secretase enzymes, leading to oligomer formation and eventual deposition of senile plaques.
Wnt ligands interact with cell membrane receptors and coreceptors to activate the intracellular Wnt signaling pathway. Dysfunctional Wnt signaling is implicated in various diseases including metabolic disorders, cancer, epilepsy, schizophrenia, osteoporosis, mood disorders, and AD. During embryonic development, Wnt pathways regulate neuronal activities such as synaptic differentiation, function, circuit operation, and plasticity. Wnt proteins are involved in remodeling pre- and postsynaptic regions and are continuously released to maintain basal neuronal activity. Dysfunction of Wnt signaling has been linked to βA neurotoxicity in AD, leading to neuronal damage and synaptic dysfunction. β-catenin is a central mediator in canonical Wnt signaling, with its intracellular levels and phosphorylation status influencing downstream signaling cascades. Reduced nuclear translocation of β-catenin, indicating impaired Wnt signaling, occurs in transgenic mouse models of familial AD mutations.
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate cellular metabolism by binding to specific DNA sequences. The three isoforms—PPARα, PPARβ/δ, and PPARγ—act as lipid sensors and regulate cholesterol and fatty acid metabolism. PPARs also modulate inflammatory responses in microglia and macrophages by inhibiting cytokine production, enhancing phagocytosis, and promoting tissue repair. Their roles in neurodegenerative diseases such as AD, multiple sclerosis, amyotrophic lateral sclerosis, and Parkinson’s disease have been established. PPARα agonists suppress proinflammatory responses in various cell types, including microglia and astrocytes, and provide neuroprotection in models of stroke, AD, Parkinson’s disease, traumatic brain injury, diabetic peripheral neuropathy, and retinopathy. These effects are attributed to antioxidant, anti-inflammatory properties and improved glucose and lipid metabolism.
Fenofibrate is a fibric acid derivative used to treat mixed dyslipidemia, primary hypertriglyceridemia, and hypercholesterolemia. It has demonstrated pharmacological effects on the central nervous system, including maintaining hippocampal neurogenesis and preventing memory disturbances after cerebral ischemia in rats, as well as neuroprotective effects against Parkinson’s disease. Fenofibrate activates PPARα, reducing inflammatory cytokine and protein formation, while stimulating antioxidant enzyme expression. PPARα agonists also exhibit anti-inflammatory effects in several CNS diseases.
Pioglitazone is an insulin sensitizer belonging to the thiazolidinedione class of nuclear PPARγ agonists. It binds PPARγ, forming a heterodimer with retinoid X receptor (RXR), which regulates gene transcription involved in glucose and lipid metabolism and inflammation reduction. PPARγ has become a target for AD drug development due to the overlap between metabolic diseases and AD risk factors, particularly insulin metabolism. Pioglitazone has been shown to improve motor coordination and synaptic plasticity in transgenic mouse models of AD before βA accumulation.
The present study aims to evaluate the role of PPAR receptor agonists in a mouse model of amyloidogenesis induced by intracerebroventricular injection of amyloidβ 1-40 peptide, with a focus on possible modulation of the Wnt/β-catenin signaling pathway.
Materials and Methods
Animals
Adult male Swiss albino mice weighing between 25 and 30 grams were utilized in this study. These animals were sourced from the National Research Centre’s animal house in Giza, Egypt, and allowed to acclimate to laboratory conditions for one week before the start of experimentation. The mice were housed in polycarbonate cages, with ten mice per cage, under controlled temperature and a 12-hour light/dark cycle. They had unrestricted access to standard rodent chow and water throughout the study. All experimental protocols were approved by the Ethics Committee of the Faculty of Pharmacy at Cairo University and were conducted in accordance with NIH guidelines for the care and use of laboratory animals.
Materials
The amyloid beta 1-40 peptide was acquired from Sigma-Aldrich, Germany. Fenofibrate was provided by Egyphar Pharmaceutical Company in Egypt, while pioglitazone was obtained from Chemipharm Pharmaceutical Company, Egypt. The amyloid beta 1-40 peptide was dissolved in 0.9% saline solution and administered intracerebroventricularly at a dose of 410 pmol per mouse. Fenofibrate was dissolved in saline and given intraperitoneally at a dose of 300 mg/kg daily for 21 consecutive days. Pioglitazone was similarly dissolved in saline and administered intraperitoneally at 30 mg/kg daily for 21 days.
Induction of Amyloidogenesis
Amyloid beta 1-40 peptide was injected into the lateral ventricle of each mouse using a Hamilton syringe via freehand intracerebroventricular injection. The volume injected was 100 microliters, delivering a dose of 410 pmol per mouse. The procedure followed was adapted from previously reported methods and involved fixing the mouse’s head with gentle downward pressure above the ears. The injection needle was carefully positioned into the lateral ventricle using the bregma as a reference point, which is identified by forming an equilateral triangle between the eyes and the center of the skull. The needle was inserted approximately one millimeter lateral to this reference. Normal behavior was observed within one minute following the injection. Control mice received an intracerebroventricular injection of saline solution at a volume of 100 microliters per mouse.
Experimental Design
The study included seven groups of mice, each consisting of ten animals. The first group served as the control and received a single intracerebroventricular injection of saline, followed by daily saline treatment for 21 days. The second group was the amyloidogenesis model, injected intracerebroventricularly with amyloid beta 1-40 on day one and treated daily with saline for 21 days. Groups three and four received daily treatments with fenofibrate at 300 mg/kg and pioglitazone at 30 mg/kg, respectively, for 21 days. Groups five and six consisted of amyloid beta 1-40-injected mice treated daily with fenofibrate or pioglitazone at the same doses for 21 days. The seventh group comprised amyloid beta 1-40-injected mice treated daily with a combination of fenofibrate and pioglitazone for 21 days. On the 22nd day, all mice were sacrificed, and the hippocampus was carefully dissected, weighed, and stored at −80 °C until further neurochemical analyses were conducted.
Behavioral Studies
Morris Water Maze Test
The Morris water maze test was employed to evaluate the learning ability and visuospatial memory of the mice. The test used a circular pool with a diameter of 150 centimeters and a height of 60 centimeters, filled halfway with water maintained at room temperature. The pool was divided into four quadrants by two perpendicular lines crossing at the center. A black platform measuring 10 centimeters in width and 28 centimeters in height was placed in a fixed target quadrant, submerged 2 centimeters below the water surface. The water was colored with a non-toxic dye to conceal the platform. Normal mice were trained to swim to the platform in the shortest time possible. Testing was conducted over four consecutive days starting on the 18th day of the experiment. Each mouse underwent two trials per day, separated by a 15-minute rest period. Mice were allowed up to 120 seconds to locate the platform and remained on it for 20 seconds before removal. If a mouse failed to find the platform, it was gently guided to it. On the 21st day, the platform was removed, and each mouse was placed facing the fourth quadrant. The time spent in this quadrant over one minute was recorded, with longer durations indicating better spatial memory.
Y-Maze Test
The Y-maze test assessed short-term memory and was performed on the last two days of the experiment. The apparatus consisted of three arms arranged in a Y shape, each arm measuring 35 centimeters in length, 25 centimeters in height, and 10 centimeters in width, separated by 120 degrees from a central platform. The test is based on the natural tendency of mice to explore a new arm rather than one they have already visited. On the first day, mice were habituated by allowing free exploration of the maze for eight minutes. On the following day, the sequence of arm entries was recorded over an eight-minute period. The maze was cleaned with 70% ethanol between trials to eliminate olfactory cues. The spontaneous alternation percentage was calculated by dividing the number of actual alternations by the total number of possible alternations and multiplying by 100. This measure is considered a direct indicator of spatial memory performance.
Novel Object Recognition Test
The novel object recognition test was used to assess long-term memory and cognitive function by leveraging the natural tendency of mice to explore new objects more than familiar ones. The test was conducted over three consecutive days in three phases. The first phase, habituation, involved placing each mouse in an empty wooden box for ten minutes to allow acclimatization. The second phase was the training phase, during which mice explored two identical wooden objects positioned in opposite corners of the box for ten minutes. These objects were uniform in size, shape, and color, and made from non-toxic materials. On the third day, the test phase, one of the familiar objects was replaced with a novel object that differed in size, shape, and color. Mice were then allowed to explore both objects for five minutes. To avoid odor interference, the arena and objects were cleaned with 70% ethanol after each trial. The recognition index was calculated as the percentage of time spent exploring the novel object relative to the total exploration time for both objects. The discrimination index, ranging from +1 to −1, was calculated by subtracting the time spent exploring the familiar object from the time spent on the novel object, divided by the total exploration time. A positive score indicated preference for the novel object, a negative score indicated preference for the familiar object, and a zero score indicated no preference.
Quantitative RT-PCR for PPARα, PPARγ, α-Secretase, and β-Secretase Gene Expression
Total RNA was extracted from tissue using a Qiagen extraction kit according to the manufacturer’s instructions. Between 0.5 and 2 micrograms of RNA were reverse transcribed into cDNA using a high-capacity reverse transcription kit. PCR primers were designed with Gene Runner software based on RNA sequences from GenBank. The annealing temperature for all primer sets was 60 °C. Quantitative RT-PCR reactions were performed in a 25-microliter volume containing SYBR Green PCR Master Mix, primers at 900 nM concentration, and 2 microliters of cDNA. The amplification protocol consisted of initial incubation at 50 °C for 2 minutes, denaturation at 95 °C for 10 minutes, followed by 40 cycles of denaturation at 95 °C for 15 seconds and annealing/extension at 60 °C for 10 minutes. Data from the real-time PCR assays were analyzed with Applied Biosystems software using the comparative threshold cycle method to calculate relative gene expression, which was normalized to beta actin gene expression.
Detection of Beta-Catenin and Wnt Protein by Western Blot
Proteins were extracted from brain tissue homogenates using ice-cold RIPA buffer supplemented with phosphatase and protease inhibitors including sodium vanadate, phenylmethylsulphonyl fluoride, aprotinin, and leupeptin. After extraction, samples were centrifuged at 12,000 rpm for 20 minutes. Protein concentrations were measured using the Bradford method. Twenty to thirty micrograms of total protein were separated by SDS-polyacrylamide gel electrophoresis on a 10% acrylamide gel. Proteins were then transferred to polyvinylidene difluoride membranes. The membranes were washed with PBS and blocked with 5% skimmed milk in PBS for one hour at room temperature. Primary antibody incubation was performed overnight at 4 °C with antibodies targeting β-catenin, Wnt, and β-actin. After washing, membranes were incubated with peroxidase-labelled secondary antibodies at 37 °C for one hour. Band intensities were analyzed using a ChemiDoc imaging system and Image Lab software. Protein expression levels were normalized to β-actin and expressed in arbitrary units.
Histopathological Examination
Brains were excised from mice and fixed in 10% formalin for 24 hours. Each brain was then processed individually for histopathological evaluation. The tissues were cleared in xylene, embedded in paraffin at 56 °C in a hot air oven for 24 hours, and sectioned at 4 micrometers thickness. Sections were processed through an alcohol-xylene series and stained with alum hematoxylin and eosin for microscopic examination.
Statistical Analysis
Data were expressed as mean ± SEM. Mean escape latency in the Morris water maze was analyzed by repeated measures analysis of variance (ANOVA). Other data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. Statistical analyses were performed using GraphPad Prism software version 6.01. The significance threshold was set at p < 0.05. Results Behavioral Results Effect of Fenofibrate, Pioglitazone, or Their Combination on Mean Escape Latency and Time Spent in the Target Quadrant in the Morris Water Maze On the first training day, there were no significant differences in mean escape latency among groups. From the second day onward, mice treated with beta-amyloid showed significantly longer escape latencies compared to controls and groups treated with fenofibrate, pioglitazone, or their combination. Treatment with fenofibrate, pioglitazone, or their combination reduced escape latency times in the Alzheimer’s disease model mice to levels comparable to controls. Treatment of normal mice with fenofibrate or pioglitazone did not significantly affect escape latency. On the fifth day, during the probe test, Alzheimer’s model mice spent significantly less time in the target quadrant compared to other groups. Effect of Fenofibrate, Pioglitazone, or Their Combination on Spontaneous Alternation Behavior in the Y-Maze Test in Alzheimer’s Disease Model Mice The Y-maze test, which evaluates short-term spatial memory, showed a significant reduction of 31.01% in spontaneous alternation percentage in Alzheimer’s disease model mice compared to controls. This deficit was reversed to control-like levels by daily treatment with fenofibrate and/or pioglitazone. Normal mice treated with these drugs exhibited no significant changes compared to controls. Effect of Fenofibrate, Pioglitazone, or Their Combination on Recognition Index and Discrimination Ratio in the Novel Object Recognition Test in Alzheimer’s Disease Model Mice The recognition index, which reflects the percentage of time spent exploring the novel object out of the total exploration time, was significantly decreased by 22.59% in Alzheimer’s disease model mice compared to controls. Treatment with fenofibrate, pioglitazone, or their combination restored the recognition index to levels comparable to controls and significantly improved it relative to untreated Alzheimer’s model mice. Normal mice treated with fenofibrate or pioglitazone showed no significant changes in recognition index compared to controls but had a significant increase compared to Alzheimer’s disease model mice. The discrimination ratio, calculated as the difference in exploration time between novel and familiar objects divided by the total exploration time, was +0.126 in controls but dropped to −0.005 in Alzheimer’s disease model mice. Treatment with fenofibrate, pioglitazone, and their combination increased the discrimination ratio in Alzheimer’s mice to +0.054, +0.255, and +0.114, respectively. Normal mice treated with fenofibrate and pioglitazone had discrimination ratios of +0.027 and +0.225, respectively. Biochemical Results Secretase Levels The study showed a significant increase in hippocampal α-secretase by 82.5% and β-secretase by 403.7% in Alzheimer’s disease model mice compared to controls, with the increase being more pronounced for β-secretase. Daily treatment of Alzheimer’s model mice with fenofibrate, pioglitazone, or their combination for 21 days significantly decreased α-secretase levels by 41.6%, 33.4%, and 42.7%, respectively, compared to untreated Alzheimer’s mice. The elevated β-secretase content induced by beta-amyloid was significantly reduced by 52.4% with pioglitazone treatment and by 72.2% with the combination treatment. Fenofibrate alone caused a non-significant decrease in β-secretase compared to Alzheimer’s mice. Normal mice treated with either fenofibrate or pioglitazone showed no changes in hippocampal α- or β-secretase levels. Wnt and β-Catenin Levels Hippocampal levels of Wnt and β-catenin were significantly decreased by 83.5% and 73.7%, respectively, in Alzheimer’s disease model mice compared to controls. Fenofibrate or pioglitazone treatment restored hippocampal Wnt levels to values not significantly different from controls or Alzheimer’s model mice, while the combination of both drugs fully restored Wnt to control-like levels. Treatment with fenofibrate or pioglitazone alone did not restore β-catenin levels, which remained decreased by 33.25% and 50.8%, respectively, compared to controls. However, combined treatment returned β-catenin levels to control-like values with a significant increase compared to Alzheimer’s model mice. Neither fenofibrate nor pioglitazone affected Wnt or β-catenin levels in normal mice. PPARα and PPARβ Levels Intracerebroventricular injection of beta-amyloid resulted in a significant reduction in hippocampal PPARα by 46.66% and PPARβ by 76.73% compared to controls. Treatment with fenofibrate or pioglitazone alone failed to restore the decrease in PPARα levels. However, combined treatment elevated PPARα to levels not significantly different from controls and significantly higher than in Alzheimer’s mice. The reduction in PPARβ was elevated by fenofibrate or pioglitazone treatment to levels not significantly different from controls or Alzheimer’s model mice. Combined treatment restored PPARβ to near control values and significantly increased it above Alzheimer’s mice. Normal mice treated with fenofibrate or pioglitazone showed no changes in PPARα or PPARβ levels. Histopathological Results No histopathological changes were observed in the hippocampi of control mice treated with fenofibrate or pioglitazone. However, Alzheimer’s disease model mice showed extensive neuronal nuclear pyknosis and degeneration in the hippocampus. Daily treatment with fenofibrate or pioglitazone for 21 days reduced these pathological alterations. Combined treatment with both drugs completely prevented histopathological changes in Alzheimer’s model mice. Discussion This study aimed to evaluate the effects of fenofibrate and/or pioglitazone on cognitive and memory impairments induced in amyloidogenesis model mice. The data demonstrated a decline in both long-term and short-term memory and cognition after 21 days following intracerebral injection of β-amyloid. This was evident by the increased mean latency escape time in spatial memory tasks, where mice required more time to locate the platform, indicating spatial memory deterioration. Additionally, a reduction in spontaneous alternation percentage in the Y-maze test indicated short-term memory impairment. The cognitive decline was further confirmed by a decrease in the recognition index in Alzheimer’s model mice. The Morris Water Maze test is widely recognized as a reliable assessment of hippocampal synaptic plasticity and NMDA receptor function, relying on hippocampal place cells, which makes it suitable for evaluating spatial learning. The Y-maze test measures short-term memory, locomotor activity, and stereotypic behavior and is used to assess working memory and anxiety in rodents. The spontaneous alternation task in the Y-maze correlates strongly with hippocampal function, supporting its use to evaluate pharmacological effects on memory acquisition and consolidation. The Novel Object Recognition test is based on the natural tendency of mice to explore new objects, serving as a pure working memory assessment independent of reference memory. Both the hippocampus and the perirhinal cortex are crucial in recognition memory processes in humans and animals. The observed behavioral changes confirm amyloidogenesis-induced cognitive dysfunction and were accompanied by neurochemical alterations in the hippocampus. These included a significant increase in the transcription levels of α- and β-secretases and a marked decrease in Wnt, β-catenin, PPARα, and PPARβ levels. Secretases play vital roles in Alzheimer’s disease due to their involvement in β-amyloid formation. β-Amyloid is generated through the activities of α- and β-secretases. The enzymatic activity of these secretases leads to β-amyloid accumulation in senile plaques, a hallmark of Alzheimer’s disease. Processing of amyloid precursor protein (APP) by α-secretase is considered protective as it cleaves within the β-amyloid sequence, preventing its formation. In contrast, cleavage by β-secretase promotes amyloid aggregation. The current study found that β-secretase levels increased more than α-secretase levels following β-amyloid injection, potentially favoring β-amyloid production by disrupting the balance between these enzymes. The amyloidogenic or non-amyloidogenic processing of APP depends on the relative levels of α- and β-secretases. Previous research has shown that beta-site APP cleaving enzyme 1 (BACE1), the main β-secretase, is negatively regulated by canonical Wnt signaling, while ADAM10, the principal α-secretase, is positively regulated by this pathway. Activation of Wnt signaling reduces β-amyloid peptide concentration by repressing BACE1 transcription via β-catenin binding to TCF4. Conversely, loss of Wnt function increases BACE1 expression. Impairment of Wnt signaling by β-amyloid is associated with neuronal degeneration and synaptic dysfunction in Alzheimer’s disease. Therefore, the increase in β-secretase observed may result from decreased Wnt levels, which play a key regulatory role. The reduction in Wnt induced by β-amyloid could also explain the decrease in β-catenin levels. Normally, Wnt binding to Frizzled receptors inhibits β-catenin phosphorylation by glycogen synthase kinase 3 beta (GSK3β), preventing β-catenin degradation. When Wnt signaling is inactivated, β-catenin is phosphorylated and degraded via the proteasome. β-Catenin is essential for memory formation through its role in neuroplasticity, so its reduction may mediate memory impairments caused by β-amyloid. Furthermore, decreased Wnt signaling may underlie the reduced expression of PPARα and PPARβ observed. Activation of Wnt/β-catenin signaling promotes nuclear translocation of β-catenin, which, combined with LEF/TCF transcription factors, induces transcription of Wnt-responsive genes including PPARβ/δ. Wnt signaling also influences β-amyloid formation; its inhibition increases β-amyloid 42 levels and the β-amyloid 42/40 ratio, facilitating amyloid formation. Impaired Wnt signaling also promotes tau phosphorylation via GSK3β, contributing to Alzheimer’s pathology. Active Wnt signaling maintains tau in a dephosphorylated state and prevents β-amyloid 1-42 accumulation, while dysfunctional Wnt signaling results in tau phosphorylation, β-amyloid accumulation, and disease progression. This study highlights the critical role of Wnt in Alzheimer’s disease initiation. Low Wnt levels trigger a cascade including β-catenin degradation, increased β-secretase, and decreased PPARα and PPARβ, leading to β-amyloid accumulation and tau pathology. The neurochemical alterations induced by β-amyloid likely mediate the neuronal damage observed histopathologically in the hippocampus. These behavioral, neurochemical, and histopathological changes validate the use of this mouse model for screening PPARα and PPARγ agonists as potential anti-Alzheimer’s agents. Activation of PPARα in the hippocampus induces ADAM10 expression and α-secretase activity, promoting non-amyloidogenic APP proteolysis. Thus, PPAR agonists may have therapeutic potential in managing β-amyloid and Alzheimer’s disease. Fenofibrate, a PPARα agonist, modulates neurorepair mechanisms and inhibits amyloid formation, providing neuroprotection in chronic neurodegenerative diseases and brain injury models. It also preserves adult hippocampal neurogenesis and prevents memory impairment in rodents. However, its limited brain availability reduces these effects, as it crosses the blood-brain barrier slowly. The current results show that fenofibrate ameliorated β-amyloid-induced short-term and long-term memory deficits, as evidenced by improved performance in spatial memory and recognition tests. Fenofibrate also reduced β-secretase levels, although not to normal, which may contribute to decreased β-amyloid accumulation. Additionally, fenofibrate increased Wnt and PPARβ levels but did not fully restore PPARα or β-catenin levels. Its partial effectiveness may relate to its low brain penetration. Pioglitazone, a PPARγ agonist, reduces glial inflammation and β-amyloid levels in transgenic models. It enhances microglial β-amyloid uptake by upregulating the scavenger receptor CD36. However, pioglitazone also exhibits low brain penetration, limiting its bioavailability. Similar to fenofibrate, pioglitazone increased PPARβ and Wnt levels without fully normalizing them, but still improved memory and cognition. Combined treatment with fenofibrate and pioglitazone restored levels of α- and β-secretases, β-catenin, Wnt, PPARβ, and PPARα to values comparable to controls. These neurochemical restorations correlated with improved behavioral performance, suggesting a synergistic effect of both agents. Although fenofibrate and pioglitazone act through different mechanisms, their combined use effectively restored Wnt agonist 1 signaling and its downstream targets.
The improvements in neurochemical markers induced by fenofibrate or pioglitazone were accompanied by reduced neuronal damage. This protective effect was most prominent in mice receiving combined treatment, which showed no histopathological alterations in the hippocampus.
Conclusion
The co-administration of fenofibrate and pioglitazone was more effective than either treatment alone in reversing Alzheimer’s disease-related changes in this mouse model. These findings support further preclinical investigation of these agents as potential therapies to alleviate Alzheimer’s disease.