LDL Receptor Deficiency Does not Alter Brain Amyloid-β Levels but Causes an Exacerbation of Apoptosis

Abstract

Familial hypercholesterolemia (FH) is a genetic disorder caused by dysfunction of low density lipoprotein receptors (LDLr), resulting in elevated plasma cholesterol levels. FH patients frequently exhibit cognitive impairment, a finding recapitulated in LDLr deficient mice (LDLr^-/-), an animal model of FH. In addition, LDLr^-/- mice are more vulnerable to the deleterious memory impact of amyloid-β (Aβ), a peptide linked to Alzheimer’s disease. Here, we investigated whether the expression of proteins involved in Aβ metabolism are altered in the brains of adult or middle-aged LDLr^-/- mice. After spatial memory assessment, Aβ levels and gene expression of LDLr related-protein 1, proteins involved in Aβ synthesis, and apoptosis-related proteins were evaluated in prefrontal cortex and hippocampus. Moreover, the location and cell-specificity of apoptosis signals were evaluated. LDLr^-/- mice presented memory impairment, which was more severe in middle-aged animals. Memory deficit in LDLr^-/- mice was not associated with altered expression of proteins involved in Aβ processing or changes in Aβ levels in either hippocampus or prefrontal cortex. We further found that the expression of Bcl-2 was reduced while the expression of Bax was increased in both prefrontal cortex and hippocampus in 3- and 14-month-old LDLr^-/-mice Finally, LDLr^-/- mice presented increased immunoreactivity for activated caspase-3 in the prefrontal cortex and hippocampus. The activation of caspase 3 was predominantly associated with neurons in LDLr^-/- mice. Cognitive impairment in LDLr^-/- mice is thus accompanied by an exacerbation of neuronal apoptosis in brain regions related to memory formation, but not by changes in Aβ processing or levels.

Keywords

Familial hypercholesterolemia memory impairment amyloid-β apoptosis

INTRODUCTION

Familial hypercholesterolemia (FH) is an autosomal co-dominant disease caused by impaired function of low-density lipoprotein (LDL) receptor, resulting in high levels of plasma LDL-cholesterol. With an incidence of about 1 in 200 individuals in the general population, FH is one of the most frequent inherited monogenic disorders [1 –3]. In FH subjects, lifelong exposure to high LDL cholesterol levels since birth is associated with greater risk of atherosclerotic cardiovascular disease, the most important clinical consequence of this disorder [4].

In 2010, Zambón and colleagues (2010) [5] first reported that middle-aged FH patients frequently present with mild cognitive impairment (MCI). MCI is an intermediate phase between normal cognition and dementia, and is frequently considered a prodromal stage to Alzheimer’s disease (AD). That study showed that the percentage of FH patients meeting criteria for MCI far exceeded both the age-specific prevalence predicted from epidemiological studies in the general population and the prevalence observed in follow-up studies of large cohorts affected by milder sporadic hypercholesterolemia [5]. More recently, Ariza et al. (2016) [6] reported that young FH patients also exhibit neuropsychological deficits. Those studies suggested that both hypercholesterolemia, i.e., high levels of plasma LDL cholesterol, and lack of functional brain LDL receptors are involved in cognitive impairment in FH [5, 6].

Studies employing animal models of FH, namely LDL receptor knockout (LDLr^-/-) mice and ApoB100/LDLr^-/- mice, recapitulate findings from clinical studies with FH patients, including cognitive deficits [7 –10]. Learning and memory impairments in hippocampus-dependent tasks are already evident in LDLr^-/- mice at the early stages of life (3 months of age), and become more severe with aging [8, 11]. Hippocampal astrocytosis and dysfunction, increased blood-brain barrier (BBB) permeability, neuronal damage, and impaired neurogenesis accompany cognitive impairments in hypercholesterolemic mice [9, 12].

In addition to the role of LDL receptor dysfunction and/or hypercholesterolemia in cognitive impairment, previous reports have shown an interplay between LDL receptor and amyloid-β peptide (Aβ) metabolism [7 , 13–15]. Ramírez and colleagues (2011) [7] described cognitive impairment and vascular deposition of the Aβ (vascular amyloidosis) in young ApoB100/LDLr^-/- mice, which worsened with increasing age of animals. Lack of LDL receptors has been shown to potentiate amyloid deposition and spatial learning deficits in Tg2576 mice, a transgenic mouse model of AD [13], whereas overexpression of LDLr in the brain markedly reduced amyloid plaque formation in APPswe/PSEN1ΔE9 transgenic mice [14]. Consistent with the notion that LDL receptors in the brain are implicated in brain-to-blood Aβ elimination, we have reported that LDLr^-/- mice are more vulnerable to Aβ-induced neurotoxicity than wild-type (WT) mice [9].

The mechanisms connecting cognitive decline and hippocampal dysfunction in LDLr^-/-mice are not yet fully understood. Although the impact of LDL receptor dysfunction has been explored in transgenic models of AD, which are characterized by excessive brain accumulation of Aβ, whether alterations in Aβ metabolism and load are relevant to trigger cognitive deficits in LDLr^-/- mice is not known. This prompted us to investigate brain Aβ content and expression of proteins related to AβPP processing in LDLr^-/- mice, with the goal to determine whether Aβ pathology is involved in cognitive deficits in this experimental model of FH.

METHODS AND MATERIALS

Animals

WT C57BL/6 and LDL receptor knockout (LDLr^–/–) mice founders were purchased from Jackson Laboratory (Bar Harbor, ME) and were bred at the Federal University of Santa Catarina (UFSC, Florianópolis, Brazil). Mice were maintained in groups of 4 to 5 animals per cage, under controlled temperature (22±1°C) and 12-h light cycle (lights on at 7 : 00 AM), with free access to food and water. All efforts were made to minimize the number of animals used and their suffering. The procedures used in the present study complied with the guidelines for animal care of the UFSC Ethics Committee on the Use of Animals (protocol # PP00948).

Experimental design

Young adult (3 month-old) and middle-aged (14 month-old) male C57BL/6 WT and age-matched LDLr^-/- mice fed a standard commercial chow (Nuvilab CR-1; Nuvital Nutrientes S/A; Paranaá, Brazil) were tested on a spatial memory version of the water maze (n = 7–8 mice per experimental group) as described below. After the memory task, animals were subjected to overnight food deprivation and blood samples were collected by cardiac puncture for determination of total cholesterol levels. Prefrontal cortex and hippocampus were then dissected and processed (see below) for determination of mRNA levels for lipoprotein receptor-related protein 1 (LRP-1), Bcl-2, Bax, amyloid-β precursor protein (AβPP), β-secretase (BACE-1), and presenilin 1 (PS-1), as well as determination of Aβ₄₂ levels by ELISA.

Water maze task

The water maze test was performed to assess spatial learning and memory. The water maze apparatus was made of fiberglass (97×60×60 cm³), filled with water maintained at 23±2°C. A target platform (10×10 cm²) of transparent Plexiglas was submerged about 1.0 cm beneath the water surface and two distant visual cues (55×55 cm²) were placed on the walls of the room. Starting points were marked outside of the pool as north, south, east, and west. All experiments were monitored through a video-recording system. Latency times to reach the platform and swimming speed were measured using ANY-maze™ video tracking system (Stoelting Co.; Wood Dale, IL, USA). Animals were assessed as previously described [16]. Briefly, the training session consisted of 10 consecutive trials (on the same day) during which the animals were left in the tank and allowed to swim freely to the escape platform, remaining on it for 10 s. The platform remained in a fixed location throughout the training session. If the animal did not find the platform during a period of 60 s, it was gently guided to it. For the retention phase of testing, memory for platform location was assessed 24 h after training and consisted of a single probe trial, during which the platform was removed from the pool. Each mouse was allowed to swim for 60 s in the water-maze. The time to first arrive on the platform zone as well as time spent in the platform quadrant (i.e., where the platform was located during the training session) were expressed as percentage of total time in the probe test.

Determination of total cholesterol (TC) levels

TC levels were measured in plasma using an enzymatic kit according to manufacturer’s instructions (Gold Analisa Diagnóstica Ltda.; Minas Gerais, Brazil).

Isolation of mRNA from brain tissue and cDNA synthesis for quantitative real time-PCR

Total RNA was extracted using the SV Total RNA Isolation Kit (Promega) and 0.4 μg of each sample was reverse transcribed to cDNA using TaqMan RT reagents. The reaction mixture was incubated at 25°C for 10 min, 48°C for 1 h, and 95°C for 5 min. Quantitative real-time PCR, using the ABI 7900HT cycler and SYBR Green Master Mix reagent, was performed according to manufacturer’s protocol (Applied Biosystems). Two μL of cDNA were amplified in a total volume of 10 μL. PCR reactions were conducted using primers designed with Primer Express version 3.0 software (Applied Biosystems, USA). The primers were as follows: for APP, forward 5′-CAGAATGGAAAGTGGGAGTCAG-3′, reverse 5’- CCCTCCTTGGTGCCAATG- 3’; PS-1, forward 5’-GCCAGCCCTCCCCATCT, reverse 5’- AAGGTAATCCGTGGCGAAGTAG-3’; BACE-1, forward 5’-CTCTCTTGCCCTCTCCAATG- 3’, reverse 5’-AAAGGCTGCTCTGTCAGGAA-3’; LRP-1, forward 5’-TGAAGAAGATTGCAGCATCG-3’, reverse 5’-TGCAGAGCTGAGAGCAGGTA- 3’; Bcl-2, forward 5’-AAGGGCTTCACACCCAAATCT-3’, reverse 5’- TTCTACGTCTGCTTGGCTTTGA- 3’; Bax, forward 5’- AGGATGCGTCCACCAAGAAG- 3’, reverse 5’-CCATATTGCTGTCCAGTTCATCTC-3’; β-actin, forward 5’-AAATCGTGCGTGACATCAAAGA-3’, reverse 5’-GCCATCTCCTGCTCGAAGTC-3’. Primers were used at a final concentration of 0.3 μM. Reaction conditions were 50°C for 2 min, 90°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Data were analyzed using Sequence Detection Systems (SDS) version 2.4 software (Applied Biosystems). A dissociation step was added for SYBR Green runs. For each sample, gene expression was quantified using a standard curve and normalized against the expression of the β-actin gene. Finally, to assess constitutive differences in LRP1 expression between the two brain structures, the raw data from mRNA levels, which have not been normalized by control group expression, were compared.

Enzyme-linked immunosorbent assay (ELISA) for total Aβ₄₂

Brain tissue was homogenized in buffer containing 10 mM Tris, 0.32 M sucrose, 1 mM EDTA, 0.1% Triton X-100, pH 7.4, plus a cocktail of protease/phosphatase inhibitors. The homogenate was centrifuged at 14,000 rpm for 10 min at 4°C and the supernatant was removed and assayed for total protein concentration using the BCA assay (Pierce). Murine Aβ₄₂ levels in the hippocampus and prefrontal cortex were determined using a sandwich ELISA kit (Invitrogen), according to the manufacturer’s protocol.

Immunofluorescence microscopy

Animals were anesthetized with ketamine and xylazine misture (75 and 10 mg/kg, respectively, intraperitoneally), and then perfused through the left cardiac ventricle with 0.9% saline solution, followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. The brains were removed, post-fixed in the same fixative solution for 24 h at 4°C, and cryoprotected in a 30% sucrose solution in PBS at 4°C. The samples were then frozen by immersion in cooled isopentane and serial coronal sections (30 μm) of prefrontal cortex and hippocampi were obtained with a cryostat (Leica) at –20°C. The free-floating sections were first blocked using 3% albumin in PBS containing 0.2% Triton×100 (PBS-Tx) for 1 h at room temperature. Next the sections were incubated 72 h at 4°C with mouse anti-GFAP (Sigma, 1 : 500) and anti-caspase 3 (cleaved and activated form) (Merck Millipore, 1 : 300) from rabbit, or mouse anti-NeuN (Merck Millipore, 1 : 400) and anti-caspase 3 in PBS-Tx containing 3% albumin. After three washes in PBS-Tx, tissue sections were incubated with anti-mouse Alexa 488 (Invitrogen, 1 : 500) and anti-rabbit Alexa 555 (Invitrogen, 1 : 500) in PBS-Tx for 2 h at room temperature. After incubation in secondary antibodies, the sections were washed three times in PBS-Tx. Thereafter, the sections were incubated with DAPI (Sigma, 1 : 1000) for 5 min, then washed several times in PBS-tx, mounted on slides with CC/Mount (Sigma), and covered with coverslips. Finally, images from mouse prefrontal cortex and hippocampi were obtained with a Microscopy EVOS^® FL Auto Imaging System (AMAFD1000 – Thermo Fisher Scientific; MA, USA) [9, 17].

Statistical analysis

All data are expressed as means±SD. Preliminary, normality was assessed using Shapiro-Wilk test. Data from the training period of the water maze task were analyzed using One-way repeated-measures analysis of variance (ANOVA). The comparison of LRP-1 gene expression between the two brain structures was carried out using Student’s t-test. For other parameters, statistical evaluation was carried out using two-way ANOVA with genotype and age as independent variables. Following statistically significant results in ANOVA, multiple comparisons were performed using Duncan’s post hoc test. The accepted level of significance was p < 0.05. All tests were performed using the Statistic software package (Stat-Soft Inc., Tulsa, OK, USA) or Graphpad Prism software(version 7.4).

RESULTS

Hypercholesterolemia and spatial memory deficit in LDLr^-/- mice

Cholesterol levels were measured in the plasma of young adult (3 month-old) or middle-aged (14 month-old) C57BL/6 WT or LDLr^-/- mice. Two-way ANOVA indicated a significant effect for genotype by aging interaction on plasma cholesterol levels [F(1,16)=5.75, p < 0.05]. Subsequent post-hoc comparisons revealed that 3-month-old LDLr^-/- mice presented higher levels of total cholesterol than age-matched WT mice, and that aging led to an additional increase in plasma cholesterol levels in LDLr^-/- mice (Fig. 1A).

Fig.1

Effect of hypercholesterolemia and aging on plasma total cholesterol levels and spatial memory. Data are expressed as mean±SD (n = 5–8 animals per group). A) Plasma cholesterol levels, B) learning curve, C) latency to first arrive on the platform’s zone, D) percentage of time spent on the platform’s quadrant, and E) occupation plot (red area represents maximum and blue the minimum occupancy pattern) of experimental groups. *p<0.05 compared to 3-month-old C57BL/6 mice, ^#p<0.05 compared to 14-month-old C57BL/6 mice, and ^&p<0.05 compared to 3-month-old LDLr^-/- mice (two-way ANOVA followed by Duncan or Newman-Keuls post-hoc test).

Spatial reference memory of mice was evaluated using the water maze task. Following one day of training, escape latency time to find the platform zone and time spent in the platform quadrant in the probe test without the platform (carried out on the 2th day) were used to assess memory. The analysis revealed no significant learning differences between experimental groups, as indicated by similar escape latencies to find the platform during the training sessions (Fig. 1B). With respect to the test session, two-way ANOVA indicated a significant effect for genotype [F(1, 27)=4.7565, p < 0.05] and aging [F(1, 27)=12.783, p < 0.05] on the escape latency to find the platform zone. Middle-aged LDLr^-/- mice displayed a significant higher escape latency to find the platform zone in the test session than both middle-aged WT and young adult LDLr^-/- mice (Fig. 1 C). In addition, two-way ANOVA indicated a significant effect for genotype [F(1, 24)=8.0543, p < 0.05] on the amount of time spent in the platform quadrant in the test session. Subsequent post-hoc tests revealed that LDLr^-/- mice, regardless of age, spent significantly less time in the correct quadrant (Fig. 1D). These data can be also visualized in occupation plots, which show a decreased spatial location of LDLr^-/- mice (regardless of age) in the target quadrant and platform region (Fig. 1E, dashed line).

Brain Aβ levels and expression of proteins involved in AβPP processing are not altered in LDLr^-/- mice

Expression of AβPP, BACE-1, and PS-1 were measured by qPCR, and total Aβ₄₂ levels were measured by ELISA in the hippocampus and prefrontal cortex (PFC) of 3- and 14-month-old WT and LDLr^-/- mice.

Two-way ANOVA indicated a significant effect for genotype versus aging interaction on AβPP expression [F(1, 26)=5.8301, p < 0.05] in the hippocampi of mice. Subsequent post-hoc comparisons revealed that aging was related to an increase in AβPP expression in WT mice, but not in LDLr^-/- mice. On the other hand, LDL receptor deficiency did not affect AβPP expression in the hippocampus (Fig. 2A). Two-way ANOVA indicated no significant changes in BACE-1 gene expression with either genotype or aging, but revealed a significant effect for aging on PS1 mRNA levels [F(1, 26)=10.105, p < 0.05] in the hippocampi of mice. As illustrated in Figure 2A, aging increased PS-1 expression regardless of genotype.

Fig.2

Effects of hypercholesterolemia and aging in gene expression of Aβ metabolism related proteins in mice’s hippocampus and prefrontal cortex A) Aβ processing pathway in hippocampus, B) Aβ processing pathway in prefrontal cortex, C) Aβ clearance receptor, and constitutive differences in LRP1 expression between the hippocampus and prefrontal cortex of C57BL/6 WT and LDLr^-/- mice, represented by absolute LRP1 expression by the housekeeping gene beta-actin. Data are expressed as mean±SD (n = 6–7 animals per group). *p<0.05 compared to 3-month-old C57BL/6 mice or hippocampus, ^#p<0.05 compared to 14-month-old C57BL/6 mice, and ^&p<0.05 compared to 3-month-old LDLr^-/- mice (two-way ANOVA followed by Duncan post-hoc test and Student’s t-test).

In the PFC, two-way ANOVA revealed a significant effect for aging on AβPP expression [F(1, 28)=4.1533, p < 0.05]. However, subsequent post hoc test indicated that there were no significant differences in AβPP expression in the PFC between experimental groups (Fig. 2B). Two-way ANOVA indicated significant effect for the interaction factory between genotype and aging on BACE-1 gene expression [F(1, 28)=10.441, p < 0.05]. Duncan’s test indicated that aging decreased the expression of BACE-1 in the PFC of LDLr^-/- mice (Fig. 2B). In contrast, increased expression of BACE-1 was verified in the PFC of young LDLr^-/- mice. Moreover, two-way ANOVA indicated significant effect for genotype on PS-1 expression [F(1, 26)=5.4319, p < 0.05]. Middle-aged LDLr^-/- mice exhibited higher expression of PS-1 in the PFC when compared to age-matched WT mice (Fig. 2B).

Finally, two-way ANOVA indicated no differences in total Aβ₄₂ levels in either hippocampus or PFC of young adult or middle-aged WT or LDLr^-/- mice (Fig. 2A, B). Results further showed no difference in hippocampal and prefrontal cortical levels of Aβ between LDLr^-/- mice and matched WT mice, regardless of age (Fig. 2A, B).

LRP-1 expression is differentially modulated in hippocampus and in prefrontal cortex of LDLr^-/- mice

We next evaluated the expression of LRP-1, the main ApoE lipoprotein metabolic receptor in the brain, in the hippocampus and prefrontal cortex of 3- and 14-month-old WT and LDLr^-/- mice. Two-way ANOVA indicated a significant effect for the interaction between genotype and aging on LRP-1 gene expression [F(1, 26)=37.829, p < 0.05] in the hippocampus. Aging did not affect LRP-1 expression in the hippocampus of LDLr^-/- mice. In contrast, LRP-1 expression was significantly higher in middle-aged WT mice in comparison with 3-month-old WT mice (Fig. 2 C). Intriguingly, LRP-1 expression was increased in hippocampi from 3-month-old LDLr^-/- mice in comparison to age-matched WT mice, but was decreased in 14-month-old LDLr^-/- mice compared to WT.

Two-way ANOVA also revealed a significant effect for the interaction between genotype and aging on LRP-1 expression in the PFC [F(1, 28)=9.6750, p < 0.05]. Duncan’s post hoc test indicated that LDLr deletion and aging modulated LRP-1 expression in the mouse PFC in different manners. LRP-1 expression was significantly decreased in the PFC of 3-month-old LDLr^-/- mice and 14-month-old WT mice. On the other hand, aging caused an increase in LRP-1 expression in the PFC of hypercholesterolemic mice (Fig. 2 C).

To assess constitutive differences in LRP1 expression between the two brain structures, the raw data from mRNA levels, which have not been normalized by control group expression, were compared and are shown in Figure 2 C. Results show LRP1 expression normalized by the housekeeping gene beta-actin. Expression of LRP-1 was significantly higher in the prefrontal cortex than in the hippocampus of 3-month-old WT and 14-month-old LDLr^-/- mice.

Exacerbation of apoptosis in hippocampus and PFC of LDLr^-/- mice

We further examined the expression of proteins involved in apoptosis, Bcl-2 (an anti-apoptotic protein), and Bax (a pro-apoptotic protein) in the hippocampus (Fig. 3A) and PFC (Fig. 3B) of young and middle-aged WT and LDLr^-/- mice. Two-way ANOVA indicated a significant effect for genotype on the Bcl-2/Bax ratio [F(1, 26)=7.9067, p < 0.05] in the hippocampus, and a significant effect for the interaction between genotype and aging on Bcl-2/Bax ratio in the PFC [F(1, 28)=5.0091, p < 0.05]. Subsequent post-hoc analysis demonstrated significant decreases in Bcl-2 expression and significant increases in Bax expression, resulting in decreased Bcl-2/Bax ratios, in both hippocampus (Fig. 3A) and PFC (Fig. 3B) of LDLr^-/- mice. In addition, middle-aged WT mice presented a decreased Bcl-2/Bax ratio in the PFC compared to young WT mice (Fig. 3B).

Fig.3

Effects of hypercholesterolemia and aging in gene expression of apoptosis related proteins in mice’s hippocampus and prefrontal cortex A) Apoptotic proteins in hippocampus and B) Apoptotic proteins in prefrontal cortex. Data are expressed as mean±SD (n = 6–7 animals per group). *p<0.05 compared to 3-month-old C57BL/6 mice, #p<0.05 compared to 14-month-old C57BL/6 mice, and ^&p<0.05 compared to 3-month-old LDLr^-/- mice (two-way ANOVA followed by Duncan post-hoc test).

In order to determine the location of apoptosis activation, we performed two double immunofluorescence stainings for visualizing the signals of apoptotic cells and astrocytes or neurons. In the hippocampus, caspase-3 positive cells were analysed in the CA3 region, which presented a more prominent presence of the activated form of caspase 3 (Fig. 4A). LDLr^-/- mice (independent of age), as well as middle-aged WT mice presented high levels of activated caspase-3 in both hippocampus (Fig. 4B, C) and PFC (Fig. 4D, E). In these animals, activated caspase 3 and NeuN (neuronal marker) signals were co-localized, suggesting increased neuronal death (Fig. 4 C, E). On the other hand, cleaved caspase 3 and GFAP (astrocyte marker) signals were not co-localized (Fig. 4B, D).

Fig.4

Effects of hypercholesterolemia and aging in activated caspase 3 protein in mice’s hippocampus and prefrontal cortex. The images of immunofluorescence staining were obtained with a Microscopy EVOS^® FL Auto Imaging System. In order to analysis the location of apoptosis in hippocampus and prefrontal cortex of LDLr^-/- mice, brain sections were co-stained with cleaved (activated) caspase 3 (red) and glial fibrillary acidic protein (GFAP; green) or neuronal nuclear antigen (NeuN; green). DAPI was used to label nuclear DNA of cells. A) The localization in the hippocampus of the areas chosen for the representative images. Scale bars, 1000 μm. B) Representative images of hippocampi of young and middle-aged WT C57BL/6 and LDLr^-/- mice co-stained with activated caspase-3 and GFAP (astrocytes marker). C) Representative images of hippocampi of young and middle-aged WT C57BL/6 and LDLr^-/- mice co-stained with activated caspase-3 and NeuN (neurons marker). D) Representative images of prefronal cortex of young and middle-aged WT C57BL/6 and LDLr^-/- mice co-stained with activated caspase-3 and GFAP. E) Representative images of prefronal cortex of young and middle-aged WT C57BL/6 and LDLr^-/- mice co-stained with activated caspase-3 and NeuN. B-E) Scale bars, 200 μm.

DISCUSSION

Clinical studies [5, 6] have demonstrated that FH individuals display cognitive impairments, and an association between FH and MCI was verified in FH patients over 50 years of age [5]. More recently, Ariza and collaborators (2016) [6] reported that FH subjects aged between 18 and 40 years made more errors than control individuals in verbal memory and executive performance tests. Both studies have suggested that early exposure to elevated cholesterol and/or LDL receptor dysfunction might be triggering events for MCI and, consequently, for AD development later in life [5, 6].

Accumulating evidence indicates that LDLr^-/- mice, a widely used mouse model of FH, present learning and memory impairments. The pioneering study of Mulder and collaborators (2004) [12] demonstrated that 6-month-old LDLr^-/- mice exhibited spatial memory impairment in the water maze task, and impaired working memory performance. Impaired working memory assessed in the radial-arm water maze was also reported in LDLr^-/- mice at the same age [18]. We previously reported impaired spatial and working memory in young adult (3 month-old) LDLr^-/- mice [8 –11]. Herein, we investigated spatial reference memory impairment in young LDLr^-/- mice in the water maze task. In the test phase of the task, 3-month-old LDLr^-/- mice spent significantly less time in the platform quadrant than WT animals, indicative of memory impairment in LDLr^-/- mice even at young age.

We further evaluated age-related alterations in cognitive performance in LDLr^-/- mice. Middle-aged LDLr^-/- mice performed significantly worse than young adult LDLr^-/- mice in the water maze as indicated by longer escape latencies to find the platform zone during the test session. Of note, in a previous study we showed that LDLr^-/- mice were more susceptible to age-related alterations in working memory and in short- and long-term memory retention in a step-down inhibitory avoidance task [8]. Despite the intensification of cognitive deficits in older mice, a point worth mentioning is the fact that cognitive impairment in LDLr^-/- mice occurs regardless of age. Indeed, we and others have observed learning and memory decline in early life in these animals [9 , 18].

A number of studies have attempted to identify the mechanisms underlying the relationship between cholesterol metabolism and memory impairment [9 , 18–21]. Some studies have reported that cholesterol (i.e., hypercholesterolemia) modulates cerebral Aβ metabolism [18 , 20–23], while other works concluded that the LDL receptor, a receptor implicated in peripheral and brain cholesterol metabolism, also participated in the clearance of brain Aβ [13 –15].

Herein, cerebral Aβ metabolism was investigated in LDLr^-/- mice. Results indicate that memory deficits in young or middle-age LDLr^-/- mice were not associated to altered cerebral Aβ metabolism or total Aβ concentration in the parenchyma. No changes were detected in Aβ levels in the hippocampus or cerebral cortex of young or middle-age LDLr^-/- mice. Results also showed no changes in AβPP expression in the hippocampus and PFC of LDLr^-/- mice regardless of age, in line with a previous study that did not find any alteration in AβPP expression in the cerebral cortex of young LDLr^-/- mice [18]. We observed an increase in prefrontal cortex BACE-1 expression in 3-month-old LDLr^-/- mice, but this enhancement was not observed in middle-age mice. On the other hand, PS-1 expression increased in 14-month-old LDLr^-/- mice in both PFC and hippocampus. A possible consequence of higher expression of BACE-1 and/or PS-1 would be an increase in amyloidogenic AβPP processing [18]. However, the isolated alterations in expression of BACE-1 and PS-1 here observed did not lead to changes in hippocampal or cortical Aβ. It thus appears likely that absence of the LDL receptor may contribute to Aβ deposition and plaque formation in conditions favoring amyloidosis, such as in transgenic mouse models of AD, but not in hypercholesterolemic mice alone. These findings suggest that other pathophysiological processes, besides Aβ accumulation and brain aging, are involved in the cognitive deficit associated to FH.

In this context, a previous study by Ettcheto and collaborators (2015) [25] reported that memory deficits are caused by distinct molecular mechanisms in LDLr^-/- mice and in the APPswe/PS1dE9 (APP/PS1) transgenic mouse model of familial AD. Hippocampi of LDLr^-/- mice presented a wider range of gene and protein expression alterations than APPswe/PS1dE9 mice. LDLr^-/- mice present cognitive decline early in life, which involves more complex genetic and neurochemical modulations than APPswe/PS1dE9 mice. Genes involved in the inflammatory process (such as cytokine signaling), lipid metabolism, memory, and others were reported to be altered in the hippocampi of LDLr^-/- mice. In the hippocampi of APP/PS1 mice, only genes related to mitochondrial function were found to be dysregulated. Interestingly, the authors suggested that memory decline in LDLr^-/- mice is not related to hippocampal mitochondrial OXPHOS impairments [25]. However, more accurate functional tests are required to evaluate mitochondrial metabolism in LDLr^-/- mice brains, as those animals coud present post-translational mitochondrial changes. Of note, we previously [11] demonstrated that young LDLr^-/- mice fed a hypercholesterolemic diet displayed decreased Complex I and II activities in cerebral cortex, which was not observed when mice were fed a standard rodent diet.

Aβ levels in the brain are partly regulated by clearance via transport across the BBB. LRP-1, a member of the LDL receptor family, acts as a multi-functional scavenger and signaling receptor, a transporter, and a metabolizer of cholesterol and ApoE-containing lipoproteins [26]. LRP-1 was also identified as an important receptor in the clearance of Aβ [14, 27]. We analyzed the expression of LRP-1 in the brains of mice lacking LDL receptor. Lack of LDLr was accompanied by an increase in expression of LRP-1 in the hippocampi of young mice, but not in LDLr^-/- middle-aged mice. Conversely, we found diminished expression of LRP-1 in the prefrontal cortex of young LDLr^-/- mice, but not in middle-aged animals. AD has been associated with altered distribution of LRP-1 in the BBB in the hippocampus [28, 29], and levels of LRP-1 in the cerebral microvasculature are decreased in severe AD [28]. Moreover, BBB dysfunction and leakage have been reported in MCI and AD before dementia and neurodegeneration. In this regard, Ramírez and collaborators (2011) [7] demonstrated that 18-month-old LDLr^-/-/ApoB100 mice (an experimental model of FH) present cerebral vascular amyloidosis. We further reported that young LDLr^-/- mice present enhanced permeability of the BBB and astrogliosis in the hippocampus [9].

BBB leakage is associated with secondary neuronal injury and neurodegeneration [30]. Interestingly, we found that absence of LDL receptor in mice (regardless of age) was related to a decrease in Bcl-2/Bax expression ratio in both PFC and hippocampus. A similar decrease in Bcl-2/Bax ratio was verified in the PFC of middle-aged WT mice. Members of the Bcl-1 family play key roles in the mitochondrial pathway leading to apoptosis. Bax is a pro-apoptotic factor that initiates the mitochondrial pore opening, which allows cytochrome c release to the cytoplasm and, consequently, apoptosis [31, 32]. Conversely, bcl-2 is an anti-apoptotic protein that stabilizes mitochondrial membrane integrity, thus preventing cytochrome c release and apoptosis [33]. In addition, we observed increased levels of activated caspase-3 protein in neurons of middle-aged WT mice and LDLr^-/- mice already at three months of age. Importantly, caspase 3 is a major mediator of neuronal death. One of the mechanisms known to activate caspase-3 involves pro-apoptotic members of Bcl-2 family (intrinsic apoptotic pathway). Of note, initiation of the intrinsic apoptotic cascade usually is associated to gene modulation, and has been implicated as a key pathway leading to neuronal apoptosis [34]. Results thus indicate activation of pro-apoptotic mechanisms in the brains of LDLr^-/- mice as early as three months of age. These findings are consistent with our previous report that showed increased cell membrane permeability (propidium iodide uptake) in the hippocampi of 3-month-old LDLr^-/- mice [9]. Also in line with our findings, Wang et al. (2014) [35] associated the spatial cognitive impairment induced by hypercholesterolemia and LDL receptor dysfunction to negative effects on hippocampal vulnerability to apoptosis. Those authors demonstrated that 12-month-old LDLr^-/- mice presented higher mean ratios of Bax/Bcl-2 protein and mRNA than WT mice. The induction of neuronal apoptosis related to hypercholesterolemia, here suggested to take place even in young LDLr^-/- mice, likely occurs due to BBB breakdown and consequent neuroinflammation. More recently, we observed that young LDLr^-/- mice also display hippocampal cell proliferation and adult neurogenesis impairment [36]. The alterations in hippocampus adult neural progenitors probably contribute to the development of cognitive dysfunction early in life in this mouse model of familial hypercholesterolemia.

Taken together, our results show that memory impairment in LDLr^-/- mice appears to be associated with increased apoptotic mechanisms in brain regions related to memory formation, but not with cerebral Aβ accumulation, since the lack of LDL receptor did not cause relevant modifications in hippocampal or cortical Aβ content or expression of proteins involved in amyloidogenic AβPP processing (Fig. 5).

Fig.5

The Illustration summarizes the proposed mechanisms underlying memory impairments in LDLr^-/- mice. The spatial memory impairments in 3- and 14-month-old LDLr^-/- mice, hypercholesterolemic and lacking LDL receptor, were associated with an exacerbation of neuronal apoptosis, but no changes in amyloid-β peptide (Aβ) levels, in prefrontal cortex and hippocampus.

Footnotes

ACKNOWLEDGMENTS

This research was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) and Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC).

Authors’ disclosures available online ().

References

Santos

, Maranhao

(2014) What is new in familial hypercholesterolemia? Curr Opin Lipidol 25, 183–188.

Santos

, Gidding

, Hegele

, Cuchel

, Barter

, Watts

, Baum

, Catapano

, Chapman

, Defesche

, Folco

, Freiberger

, Genest

, Hovingh

, Harada-Shiba

, Humphries

, Jackson

, Mata

, Moriarty

, Raal

, Al-Rasadi

, Ray

, Reiner

, Sijbrands

, Yamashita

; International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel (2016) Defining severe familial hypercholesterolaemia and the implications for clinical management: A consensus statement from the International Atherosclerosis Society Severe Familial Hypercholesterolemia Panel. Lancet Diabetes Endocrinol 4, 850–861.

Akioyamen

, Genest

, Shan

, Reel

, Albaum

, Chu

, Tu

(2017) Estimating the prevalence of heterozygous familial hypercholesterolaemia: A systematic review and meta-analysis. BMJ Open 7, e016461.

Santos

, Raal

, Donovan

, Cromwell

(2015) Mipomersen preferentially reduces small low-density lipoprotein particle number in patients with hypercholesterolemia. J Clin Lipidol 9, 201–209.

Zambón

, Quintana

, Mata

, Alonso

, Benavent

, Cruz-Sánchez

, Gich

, Pocoví

, Civeira

, Capurro

, Bachman

, Sambamurti

, Nicholas

, Pappolla

(2010) Higher incidence of mild cognitive impairment in familial hypercholesterolemia. Am J Med 123, 267–274.

Ariza

, Cuenca

, Mauri

, Jurado

, Garolera

(2016) Neuropsychological performance of young familial hypercholesterolemia patients. Eur J Intern Med 34, e29–e31.

Ramírez

, Sierra

, Tercero

, Vázquez

, Pineda

, Manrique

, Burgos

(2011) ApoB100/LDLR-/- hypercholesterolaemic mice as a model for mild cognitive impairment and neuronal damage. PLoS One 6, e22712.

Moreira

, de Oliveira

, Nunes

, Santos

, Nunes

, Vieira

, Ribeiro-do-Valle

, Pamplona

, de Bem

, Farina

, Walz

, Prediger

(2012) Age-related cognitive decline in hypercholesterolemic LDL receptor knockout mice (LDLr-/-): Evidence of antioxidante imbalance and increased acetylcholinesterase activity in the prefrontal cortex. J Alzheimers Dis 32, 495–511.

de Oliveira

, Moreira

, dos Santos

, Piermartiri

, Dutra

, Pinton

, Tasca

, Farina

, Prediger

, de Bem

(2014) Increased susceptibility to amyloid-β-induced neurotoxicity in mice lacking the low-density lipoprotein receptor. J Alzheimers Dis 41, 43–60.

10.

Lopes

, de Oliveira

, Engel

, de Paula

, Moreira

, de Bem

(2015) Efficacy of donepezil for cognitive impairments in familial hypercholesterolemia: Preclinical proof of concept. CNS Neurosci Ther 21, 964–966.

11.

de Oliveira

, Hort

, Moreira

, Glaser

, Ribeiro-do-Valle

, Prediger

, Farina

, Latini

, de Bem

(2011) Positive correlation between elevated plasma cholesterol levels and cognitive impairments in LDL receptor knockout mice: Relevance of cortico-cerebral mitochondrial dysfunction and oxidative stress. Neuroscience 197, 99–106.

12.

Mulder

, Jansen

, Janssen

, van de Berg

, van der Boom

, Havekes

, de Kloet

, Ramaekers

, Blokland

(2004) Low-density lipoprotein receptor-knockout mice display impaired spatial memory associated with a decreased synaptic density in the hippocampus. Neurobiol Dis 16, 212–219.

13.

Cao

, Fukuchi

, Wan

, Kim

, Li

(2006) Lack of LDL receptor aggravates learning deficits and amyloid deposits in Alzheimer transgenic mice. Neurobiol Aging 27, 1632–1643.

14.

Kim

, Castellano

, Jiang

, Basak

, Parsadanian

, Pham

, Mason

, Paul

, Holtzman

(2009) Overexpression of low-density lipoprotein receptor in the brain markedly inhibits amyloid deposition and increases extracellular Abeta clearance. Neuron 64, 632–644.

15.

Castellano

, Deane

, Gottesdiener

, Verghese

, Stewart

, West

, Paoletti

, Kasper

, DeMattos

, Zlokovic

, Holtzman

(2012) Low-density lipoprotein receptor overexpression enhances the rate of brain-to-blood Aβ clearance in a mouse model of β-amyloidosis. Proc Natl Acad Sci U S A 109, 15502–15507.

16.

Prediger

, Franco

, Pandolfo

, Medeiros

, Duarte

, Di Giunta

, Figueiredo

, Farina

, Calixto

, Takahashi

, Dafre

(2007) Differential susceptibility following beta-amyloid peptide-(1-40) administration in C57BL/6 and Swiss albino mice: Evidence for a dissociation between cognitive deficits and the glutathione system response. Behav Brain Res 177, 205–213.

17.

Ribeiro

, Gasparotto

, Petiz

, Brum

, Peixoto

, Kunzler

, da Rosa Silva

, Bortolin

, Almeida

, Quintans-Junior

, Araújo

, Moreira

JCF

, Gelain

(2019) Oral administration of carvacrol/β-cyclodextrin complex protects against 6-hydroxydopamine-induced dopaminergic denervation. Neurochem Int 126, 27–35.

18.

Thirumangalakudi

, Prakasam

, Zhang

, Bimonte-Nelson

, Sambamurti

, Kindy

, Bhat

(2008) High cholesterol-induced neuroinflammation and amyloid precursor protein processing correlate with loss of working memory in mice. J Neurochem 106, 475–485.

19.

Sparks

, Scheff

, Hunsaker III

, Liu

, Landers

, Gross

(1994) Induction of Alzheimer-like beta-amyloid immunoreactivity in the brains of rabbits with dietary cholesterol. Exp Neurol 126, 88–94.

20.

Refolo

, Malester

, LaFrancois

, Bryant-Thomas

, Wang

, Tint

, Sambamurti

, Duff

, Pappolla

(2000) Hypercholesterolemia accelerates the Alzheimer’s amyloid pathology in a transgenic mouse model. Neurobiol Dis 7, 321–331.

21.

Ullrich

, Pirchl

, Humpel

(2010) Hypercholesterolemia in rats impairs the cholinergic system and leads to memory deficits. Mol Cell Neurosci 45, 408–417.

22.

Ghribi

, Larsen

, Schrag

, Herman

(2006) High cholesterol content in neurons increases BACE, beta-amyloid, and phosphorylated tau levels in rabbit hippocampus. Exp Neurol 200, 460–467.

23.

Sharma

, Prasanthi

RPJ

, Schommer

, Feist

, Ghribi

(2008) Hypercholesterolemia-induced Abeta accumulation in rabbit brain is associated with alteration in IGF-1 signaling. Neurobiol Dis 32, 426–432.

24.

Jaya Prasanthi

, Schommer

, Thomasson

, Thompson

, Feist

, Ghribi

(2008) Regulation of beta-amyloid levels in the brain of cholesterol-fed rabbit, a model system for sporadic Alzheimer’s disease. Mech Ageing Dev 129, 649–655.

25.

Ettcheto

, Petrov

, Pedrós

, de Lemos

, Pallàs

, Alegret

, Laguna

, Folch

, Camins

(2015) Hypercholesterolemia and neurodegeneration. Comparison of hippocampal phenotypes in LDLr knockout and APPswe/PS1dE9 mice. Exp Gerontol 65, 69–78.

26.

Herz

, Marschang

(2003) Coaxing the LDL receptor family into the fold. Cell 112,, 289–292.

27.

Cam

, Bu

(2006) Modulation of beta-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family. Mol Neurodegener 1, 8.

28.

Donahue

, Flaherty

, Johanson

, Duncan

3rd , Silverberg

, Miller

, Tavares

, Yang

, Wu

, Sabo

, Hovanesian

, Stopa

(2006) RAGE, LRP-1, and amyloid-beta protein in Alzheimer’s disease. Acta Neuropathol 112, 405–415.

29.

Jeynes

, Provias

(2008) Evidence for altered LRP/RAGE expression in Alzheimer lesion pathogenesis. Curr Alzheimer Res 5, 432–437.

30.

Zhao

, Nelson

, Betsholtz

, Zlokovic

(2015) Establishment and dysfunction of the blood-brain barrier. Cell 163, 1064–1078.

31.

Shimizu

, Narita

, Tsujimoto

(1999) Bcl-2 family proteins regulate the release of apopto-genic cytochrome c by the mitochondrial channel VDAC. Nature 399, 483–487.

32.

Tsujimoto

, Shimizu

(2000) Bcl-2: Life-or-death switch. FEBS Lett 466, 6–10.

33.

Opferman

, Kothari

(2018) Anti-apoptotic BCL-2 family members in development. Cell Death Differ 25, 37–45.

34.

D’Amelio

, Cavallucci

, Cecconi

(2010) Neuronal caspase-3 signaling: Not only cell death. Cell Death Differ 17, 1104–1114.

35.

Wang

, Huang

, Yuan

, Xia

, Wang

, Huang

(2014) LDL receptor knock-out mice show impaired spatial cognition with hippocampal vulnerability to apoptosis and deficits in synapses. Lipids Health Dis 13, 175.

36.

Engel

, Grzyb

, de Oliveira

, Pötzsch

, Walker

, Brocardo

, Kempermann

, de Bem

(2019) Impaired adult hippocampal neurogenesis in a mouse model of familial hypercholesterolemia: A role for the LDL receptor and cholesterol metabolism in adult neural precursor cells. Mol Metab 30, 1–15.

LDL Receptor Deficiency Does not Alter Brain Amyloid-β Levels but Causes an Exacerbation of Apoptosis

Abstract

Keywords

INTRODUCTION

METHODS AND MATERIALS

Animals

Experimental design

Water maze task

Determination of total cholesterol (TC) levels

Isolation of mRNA from brain tissue and cDNA synthesis for quantitative real time-PCR

Enzyme-linked immunosorbent assay (ELISA) for total Aβ42

Immunofluorescence microscopy

Statistical analysis

RESULTS

Hypercholesterolemia and spatial memory deficit in LDLr-/- mice

Brain Aβ levels and expression of proteins involved in AβPP processing are not altered in LDLr-/- mice

LRP-1 expression is differentially modulated in hippocampus and in prefrontal cortex of LDLr-/- mice

Exacerbation of apoptosis in hippocampus and PFC of LDLr-/- mice

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Enzyme-linked immunosorbent assay (ELISA) for total Aβ₄₂

Hypercholesterolemia and spatial memory deficit in LDLr^-/- mice

Brain Aβ levels and expression of proteins involved in AβPP processing are not altered in LDLr^-/- mice

LRP-1 expression is differentially modulated in hippocampus and in prefrontal cortex of LDLr^-/- mice

Exacerbation of apoptosis in hippocampus and PFC of LDLr^-/- mice