Efficacy of Donepezil for Cognitive Impairments in Familial Hypercholesterolemia: Preclinical Proof of Concept

Conflict of Interest

The authors declare no conflict of interest.

Familial hypercholesterolemia (FH) is caused by genetic abnormalities that directly or indirectly affect low‐density lipoprotein receptor (LDLr) function, implying a hypocatabolism of lipoproteins mainly the LDL particles. Nowadays, with a frequency of about one in 200 individuals, FH is one of the most common inherited monogenic disorder in the general population 1. In addition to the well‐established high association of FH and cardiovascular disease development, Zambón and colleagues recently reported that middle‐aged patients with FH also show a high incidence of mild cognitive impairment (MCI), a prodromal stage of abnormal cognitive performance, which in many cases precedes Alzheimer's disease (AD) dementia 2. It is worth mentioning that the percentage of FH patients exhibiting abnormal cognitive function and meeting criteria for MCI far exceeded the age‐specific prevalence predicted from either epidemiological studies in the general population or the prevalence observed in follow‐up of large cohorts with milder sporadic hypercholesterolemia 2. We experimentally corroborated this observation using LDLr knockout (LDLr^−/−) mice, a widely used rodent model of FH. Specifically, we found that middle‐aged LDLr^−/− mice exhibited working, spatial reference, and procedural memory impairments accompanied by increased acetylcholinesterase (AChE) activity in the prefrontal cortex, brain area associated with learning and memory processes 3. In addition, we also demonstrated that these hypercholesterolemic mice are more susceptible to beta‐amyloid peptide (Aβ)‐induced neurotoxic effects. LDLr^−/− mice treated with Aβ_1–40 via acute intracerebroventricular injection (an experimental tool to study AD), when compared to identically wild‐type C57BL/6 mice, presented more severe memory dysfunction, which was accompanied by increased AChE activity in the hippocampus 4. Accordingly, using an experimental protocol to induce mild hypercholesterolemia in Swiss mice, we demonstrated that increased AChE activity within the hippocampus and prefrontal cortex is an early event associated with hypercholesterolemia‐induced cognitive impairments 5. Noteworthy, Ullrich and colleagues observed memory impairment in rats exposed to hypercholesterolemic diet, which was associated with cholinergic dysfunction characterized by a reduction in the number of cholinergic neurons in the basal nucleus of Meynert and in acetylcholine levels in the cortex 6. Therefore, the cholinergic system seems to be particularly affected by hypercholesterolemia. In an in vitro assay, we exposed SH‐SY5Y cells to human isolated LDL, in order to manipulate cell cholesterol levels. The exposition to LDL increased in a concentration‐dependent manner the activity of AChE in cultured neuronal cells 5. The AChE plays an important role in modulating the acetylcholine activity, a neurotransmitter markedly implied in learning and memory function. The elevation of acetylcholine activity by inhibition of its metabolizing enzyme is an interesting approach to improve cognitive impairments. In fact, AChE inhibitors (AChEIs) have been widely used for treating AD and represent the most realistic approach to the symptomatic treatment of this neurodegenerative disease 7, 8. In this regard, we hypothesized that an AChEI could improve the FH‐related cognitive impairments. To experimentally test this hypothesis, three‐month‐old C57BL/6 wild‐type and LDLr^−/− mice were treated with donepezil (3 mg/kg i.p.) or saline during twenty days 9. After this period, the animals were submitted to memory and learning paradigms.

Confirming our previous studies 3, 4, the present findings demonstrate impaired learning and memory function in hypercholesterolemic LDLr^−/− mice when compared to wild‐type controls. Firstly, we evaluated the recognition memory of mice using the novel object recognition task. Unlike the wild‐type mice, LDLr^−/− mice did not show any preference toward the novel object, presenting the discrimination score similar to the random chance. Notably, donepezil treatment reversed the memory impairment in LDLr^−/− mice. LDLr^−/− mice treated with donepezil explored for a longer time the new object. The two‐way ANOVA revealed that LDLr^−/− mice treated with donepezil presented greater discrimination score than untreated hypercholesterolemic animals (Figure 1A). We also investigated the short‐term spatial memory using the location object task. In contrast to wild‐type mice, LDLr^−/− mice were not able to identify the spatial alteration in the open field arena. Donepezil treatment mitigated the cognitive function in the LDLr^−/− mice. Actually, LDLr^−/− mice treated with donepezil displayed higher location index in comparison with LDLr^−/− mice treated with saline (Figure 1B). Moreover, the T‐maze was used to assess the spontaneous alternation and working memory. Once again, only wild‐type mice showed a percentage of alternations significantly higher than chance performance, while the LDLr^−/− mice displayed a spontaneous alternation index similar to the chance level. Donepezil significantly enhanced the percentage of spontaneous alternations in the hypercholesterolemic mice. A higher percentage of alternations were observed when LDLr^−/− mice were treated with donepezil in comparison with vehicle‐treated LDLr^−/− mice (Figure 1C). As previously described 2, LDLr^−/− mice presented increased locomotor activity. Interestingly, donepezil mitigates the locomotor activity in LDLr^−/− mice, decreasing the total number of crossings of these mice in the open field arena (Figure 1D).

Figure 1.

Effects of donepezil treatment on memory performance in C57BL/6 wild‐type and LDLr^−/− mice. (A) Object recognition, (B) Object location, (C) Spontaneous alternation tasks and (D) Open field. Each value represents the mean ± SEM of ten animals in each group. + P < 0.05 versus chance level (50% of new object or displaced object investigation in test trial and spontaneous alternations, respectively; one sample t‐tests ). *P < 0.05 versus C57BL/6 wild‐type vehicle group and ^# P < 0.05 versus LDLr^−/− vehicle group (two‐way ANOVA following Duncan's post hoc test).

It is assumed that hypercholesterolemia affects the cholinergic system, but the exact mechanisms are still unclear. We can speculate that the activity of the brain AChE, an enzyme located in the neuronal membrane, might be modulated by cholesterol content alterations mainly in the lipid rafts 5. Nowadays, three AChEI are widely prescribed for patients with AD, donepezil, galantamine, and rivastigmine, which exhibit a wide variation in their pharmacological properties and affinities for AChE. Donepezil is a highly selective and reversible derivator of piperidine with AChEI activity. There are multiple clinical trials proving the beneficial effects of donepezil in mild, moderate, and severe AD 8. For instance, in a previous placebo‐controlled study, patients in severe stage of AD (n = 248) treated with donepezil presented significant improvements in cognition and global function 10. In addition, growing evidence suggests that donepezil treatment is also able to delay progression to AD in MCI subjects carrying the apolipoprotein‐E4 genotype, which is the best‐established risk gene for sporadic AD 8.

In summary, our results indicated that donepezil, an AChEI, mitigates the cognitive impairments in LDLr^−/− mice, a mouse model of FH, therefore providing preclinical proof of concept that donepezil can be potentially used to treat cognitive decline and prevent progression toward dementia in FH subjects.