AβPP/PS1 Transgenic Mice Show Sex Differences in the Cerebellum Associated with Aging

Abstract

Cerebellar pathology has been related to presenilin 1 mutations in certain pedigrees of familial Alzheimer’s disease. However, cerebellum tissue has not been intensively analyzed in transgenic models of mutant presenilins. Furthermore, the effect of the sex of the mice was not systematically analyzed, despite the fact that important gender differences in the evolution of the disease in the human population have been described. We analyzed whether the progression of amyloidosis in a double transgenic mouse, AβPP/PS1, is susceptible to aging and differentially affects males and females. The accumulation of amyloid in the cerebellum differentially affects males and females of the AβPP/PS1 transgenic line, which was found to be ten-fold higher in 15-month-old females. Amyloid-β accumulation was more evident in the molecular layer of the cerebellum, but glia reaction was only observed in the granular layer of the older mice. The sex divergence was also observed in other neuronal, survival, and autophagic markers. The cerebellum plays an important role in the evolution of the pathology in this transgenic mouse model. Sex differences could be crucial for a complete understanding of this disease. We propose that the human population could be studied in this way. Sex-specific treatment strategies in human populations could show a differential response to the therapeutic approach.

Keywords

AβPP/PS1 AD mouse models aging Alzheimer’s disease amyloid-β autophagy cerebellum glial sex differences transgenic mice

INTRODUCTION

Alzheimer’s disease (AD) is the most prevalent type of dementia. Neurodegeneration, neurofibrillary tangles, and amyloid-β (Aβ) are the hallmark pathologic characteristics of this disease [1, 2]. Most AD cases are sporadic (unknown cause); however, a minority of these cases is genetic in origin. This is known as familial Alzheimer’s disease (FAD) and is related to some mutations in the amyloid-β protein precursor (AβPP) [3], presenilin 1 [4], or presenilin 2 [5]. In studies of AD whose aim is to elucidate therapies for this disease, different transgenic mouse models expressing AβPP and/or PS1/2 human mutations have been reported. These transgenic mice accumulate Aβ as diffuse neuritic plaques [6].

Non-familial cases of AD are referred to as “sporadic” AD, where initial mechanisms remain unclear; however, some risk factors have been reported with age as a strong one (see, for example, [7, 8]). Epidemiological studies show an increased risk of AD with the age-related loss of sex steroid hormones. In medical research, the term ‘sex’ refers to biological differences, whereas ‘gender’ refers to psychosocial and cultural issues. Some authors have pointed to gender as one of the causes of the differences between men and women and their susceptibility to AD [9]; almost two-thirds of the individuals diagnosed are women [10]. In addition, sex-related differences in the rate of progression after diagnosis of AD, and in the response to treatment have also been reported. These findings prompted us to carry out research into the sex bias of AD [11].

During the past century, cerebellar function has been concerned with motor function. However, recent anatomical studies have demonstrated that the output of the cerebellum targets multiple nonmotor areas in the cortex. Neuroimaging and psychological data associate the cerebellum with executive control, language, working memory, learning, pain. or emotion (for review, see [12]). The presence of Aβ plaques in the cerebellum of AD patients has been described [13, 14], and other research has shown significant atrophy and the reduction of Purkinje and granular cells [15, 16]. Cerebellar pathology has been related to PS1 mutations [17, 18] that are the most common cause of FAD [19]. PS1 mutations can increase the ratio of longer Aβ peptides that are more prone to aggregation and show higher neurotoxicity [20]. The AβPP/PS1 transgenic mouse line is one of the most suitable models because of the early deposition at the beginning, and an exponential increase with age [21 –23]. This mouse model has already been used to describe cerebellar pathology caused by Aβ deposition [24, 25]. However, whether these alterations are developed differentially in males or females, and/or are age-dependent has not beentested.

The aim of the present study is to evaluate whether a double-transgenic (AβPP/PS1) mouse model presents differences associated with aging and/or sex in the cerebellar tissue. We have analyzed some biochemical markers, and evaluated males and females separately at ages 6 and 15 months, to assess the evolution of the amyloidosis with age. Transgenic and wild type individuals were also tested to investigate whether any potential modifications were related to the pathology, or due to aging.

MATERIALS AND METHODS

Experimental animals

The double transgenic mice used in the present study incorporate a human AβPP construct bearing the Swedish double mutation and the exon-9-deleted PSEN1 mutation: B6.Cg-Tg (AβPPSwe, PSEN1dE9) (Jackson Laboratory, Bar Harbor: stock no. 005864). The genotype of the mice was confirmed by polymerase chain reaction (PCR) of DNA isolated from tail biopsies [26]. We have used 6- and 15-month-old, male and female, transgenic and wild type mice. Each group consisted of 4 to 6 mice. All animal care and handling strictly followed the current Spanish legislation and guidelines, and those of the European Commission (directive 2010/63/EU). The use of wild type and transgenic animals was an absolute requirement for this project; however, experiments were designed to minimize the use of animals.

Mouse sacrifice and tissue sampling

Mice were sacrificed by CO₂ inhalation, and brain was removed and split into two hemibrains. One hemibrain was dissected and each zone (cortex and cerebellum) was snap frozen separately on dry ice for subsequent homogenization, while the other was fixed for 24 h in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS, pH 7.4) and cryoprotected in graded concentrations of sucrose (15–30%) in PBS. The fixed tissue was then set in O.C.T.^TM compound (Tissue-Tek^® Sakura) and frozen at –80°C.

Tissue processing

Previously frozen brain tissue was homogenized in 3 volumes of ice-cold lysis buffer (20 mM Hepes, 100 mM NaCl, 100 mM NaF, 1 mM NaVO₄, 5 mM EDTA, 1% Triton X100) with protease inhibitor cocktail (Roche Diagnostic) and 1 μM okadaic acid (Calbiochem) as phosphatase inhibitor. The homogenate was then centrifuged at 4°C for 20 min at 16000×g and the supernatant stored at –80°C. The protein concentration was measured using the BioRad DC Protein Assay (BioRad) following the manufacturer’s protocol. Prior to resolving the proteins by SDS/PAGE (PolyAcrylamide Gel Electrophoresis), loading buffer (10% SDS, 0.5 mM DTT, 325 mM TrisHCl [pH 6.8], 87% glycerol and bromophenol blue) was added to thesupernatants.

Tissue samples used for ELISA tests were homogenized in 8 volumes of ice-cold guanidine buffer (5 M guanidine HCl/50 mM Tris HCl, pH 8). The homogenates were mixed for 3 h at room temperature and stored at –20°C.

ELISA quantification of Aβ₄₀ and Aβ₄₂

Brain homogenates were diluted 1 : 50 in PBS-Tween-BSA buffer (0.03% Tween-20, 5% Bovine Serum Albumin in PBS) before centrifugation (16000×g for 20 min at 4°C) and the supernatant was analyzed immediately. Brain amyloid burden was measured using the Aβ₄₀ and Aβ₄₂ Human ELISA kit (Invitrogen) following the manufacturer’s protocol. Plate absorbance was measured at 450 nm using an Opsys MR Microplate reader (Dynex Technologies).

Gel electrophoresis and western blots

Cerebellum extracts were resolved by SDS/PAGE (10 or 20 μg protein/lane) and transferred onto nitrocellulose (Whatman) or PVDF (Millipore) membranes. To detect Aβ peptide, Tris-Tricine gels were performed [27] and transferred to nitrocellulose membranes. After blocking in 10% solution of non-fat milk, membranes were incubated overnight at 4°C with the primary antibody (listed in Supplementary Table 1). After washing in 0.1% Tween-PBS, membranes were incubated with the secondary horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology). The antibody binding was detected with Western lighting^TM Plus ECL (Perkin-Elmer), using β-actin as internal control. The intensity of each band was determined using GS-800™ Calibrated Densitometer (BioRad) and Quantity One^® program (Bio-Rad).

Immunofluorescence studies

Coronal sections (30 μm) of fixed hemibrains were obtained with freezing cryostat (Thermo Scientific) and stick on Superfrost Ultra Plus cryoslices (Thermo Scientific). Sections were pre-treated with 70% formic acid and incubated with Glycine 0.1 M in PBS to eliminate auto-fluorescence caused by PFA. After blocking (1% BSA, 1% serum, 1% Triton in PBS) for 1 h, slices were incubated with anti-GFAP (1 : 1000) and 6E10 anti-Aβ antibody (1 : 1000) at 4°C for overnight. After washing, the sections were incubated with anti-rabbit Alexa 488 and anti-mouse Alexa 555 conjugated secondary antibodies (Invitrogen) and DAPI (Invitrogen) was used to label the cell nuclei. The sections were mounted with Fluoromount G (Southern Biotech) and were visualized by fluorescence microscopy (Axiovert200, Zeiss). All images were captured using a SPOT RT Slider camera (Diagnostic) and the analysis and quantification of amyloid plaque load was performed using ImageJ^TM software (NIH–National Institutes of Health) and the area occupied by amyloid plaques represented as a percentage of the total area analyzed.

Statistical analyses

Two-way ANOVA (genotype and sex variables) with Holm-Sidak post hoc analysis for multiple comparisons was employed for data analysis. In all cases, differences were considered statistically significant when ^*p≤0.05, ^**p≤0.01, or ^***p≤0.001.

RESULTS

We had previously characterized the amyloid deposition in this lineage of AβPP/PS1 double transgenic mice and our data indicated that 6-month-old mice already showed an important Aβ burden in the cortex that appeared as diffuse plaques [21]. We therefore decided to analyze the period from 6 to 15 months of age, as a reflection of an advanced status of the disease with plaques extensively distributed in the cortex and hippocampus. We therefore analyzed the cortex and cerebellum Aβ levels in adult (6 months) and old (15 months) mice. The ELISA analysis showed that the Aβ₄₀ and Aβ₄₂ levels in cortex and cerebellum from both males and females increased from 6 to 15 months (Fig. 1A, C). However, there was an important difference between the evolutions in the cerebellum, which occurred in the different sexes. The females reached a higher burden at 15 months, which was 5-fold and 10-fold more than the males in the case of Aβ₄₂ and Aβ₄₀, respectively. Surprisingly, these differences were not found in the cortex analysis in which males and females showed a parallel increase in the Aβ burden with age (Fig. 1B, D).

Next, we performed an immunofluorescence analysis to check the deposition of Aβ in the cerebellum, considering both the area and density of the plaques. The data confirmed the evolution in the development of the plaques in the cerebellum with a significant increase from 6 to 15 months of age (Fig. 2A). The quantitative data of the images showed an important augment in the area and density of the plaques per section in the older mice (p < 0.001). There were no differences between males and females at 6 months, while older females, as was already revealed by the ELISA assays, showed a greater increase than males at 15 months (p≤0.05). Interestingly, the plaques in the molecular layer (ML) were more abundant (Fig. 2B) than in the granular layer (GL). As hypothesized, the wild type mice did not show any Aβ deposition at any time (data notshown).

Glial fibrillary acidic protein (GFAP) is localized in the reactive glial cells and is another marker of the pathology that usually appears surrounding Aβ plaques in the cortex and hippocampus. Immunofluorescence analysis performed in the cerebellum samples showed no evident GFAP staining in the 6-month-old mice. Meanwhile, the 15-month-old mice showed that the GFAP-positive cells were only present in the GL (Fig. 2B). We have observed reactive glia cells near Purkinje cells, but Aβ plaques that were localized far from GL were not surrounded by reactive glia, independently of the sex. Western blot analysis showed no significant differences between groups, even sex or genotype dependent (Fig. 2C). Next we analyzed some of the proteins involved in the formation of Aβ (AβPP and BACE, β-secretase enzyme) and some pathological markers that accompany the amyloidosis, such as phospho-epitopes of the tau protein, were measured (PHF1 and Tau1). As hypothesized, the data showed important differences in AβPP between the transgenic and the wild type mice, but no differences between the males and the females. Oligomer and monomer forms of the Aβ peptide can be detected by the 6E10 antibody, and these showed higher levels in the 15-month-old females, although this was not statistically significant, maybe because of the reduced number of samples analyzed by western blot. BACE, the enzyme that processes AβPP to form the Aβ peptide, showed no differences between either genotypes or sex (Fig. 3). After that, we checked two phospho-tau antibodies, Tau1 and PHF1, and found a lower level of expression of Tau 1 in the 6-month-old transgenic males when compared with their wild type littermates. Surprisingly, PHF1 antibodies were significantly reduced in the 6-month-old transgenic mice (Fig. 3).

AD has been considered a ‘synaptophathy’, and we therefore decided to analyze several synaptic markers such as synapsin, p120-catenin, synaptophysin, PSD95 and α-N-catenin, in all the transgenic and non-transgenic groups (Fig. 4). Our quantitative data showed that phospho-synapsin, N-catenin, and p120 in 6-month-old female transgenic mice were lower than in wild type; but the levels in the male mice were not modified by the transgenic genotype/phenotype. An analysis at 15 months showed increased levels in postsynaptic PSD95 protein in the transgenic mice, both males and females. The presynaptic p120 protein showed a statistically significant increase only in the case of the transgenic males compared with the wild type at 15 months of age.

Several studies have shown aberrant activation of the PI3K/AKT signaling pathway in AD [28]. Therefore we analyzed PI3K, AKT, and GSK3 levels in all the groups (Fig. 5). The analysis of phosphorylated forms of AKT and GSK3 revealed that there were no statistically significant differences between the groups; they were therefore sex and genotype-independent. The same result was found in the analysis of the total proteins, PI3K, AKT, and GSK3.

Another important marker that is usually modified in patients with AD is the AMPK (AMP-activated protein kinase), a regulator of the cellular energy homeostasis, whose activity decreases in AD brain. AMPK in combination with mTORC1 (mammalian target of rapamycin complex 1) are key regulators of protein synthesis versus autophagy (for review, see [29, 30]). Our quantitative data did not show any statistically significant differences in young mice; however, the older transgenic mice showed reduced levels in the activated form of the AMPK, pAMPK, when compared with wild type (Fig. 6). The reduction was more important in the case of females, which might be linked to the differences in the Aβ burden observed in the cerebellum. We therefore decided to analyze some of the proteins that have been used as autophagy markers such as LC3I/II, p62, or NBR1, in combination with some mTORC1 reporters such as pS6K or pS6. Our data showed that the 6-month-old mice presented significant differences in both NBR1 and p62 (Fig. 6). NBR1 was decreased in the transgenic mice when compared with the wild type, independent of the sex. The results of p62 showed an important increase in transgenic females when compared with the males. The analysis of the older mice showed that both pS6K and NBR1 were different in the transgenic versus wild type, although they were only statistically significant in the case of NBR1. The older transgenic females showed increased levels of pS6 that were not modified in the case of transgenic males. An analysis of p62 showed an important reduction in the levels of this protein in the transgenic female group when compared with the wild type at the same age. The levels of LC3II in the 6-month-old transgenic mice were lower than in the wild type, although this was not statistically significant. In the 15-month-old mice, these differences disappeared between both sex and genotype(Fig. 6).

DISCUSSION

Our data show that sex plays an important role in the evolution of the pathology in the cerebellum of transgenic models of FAD. In recent studies we did not find any significant differences in the Aβ burden in the cortex between the males and females aged from 3 to 12 months, although sex differences were more prominent in Aβ levels in the blood of older mice [21]. However, other authors have described sex differences in amyloid levels in the hippocampus [31]. Besides, the accumulation of senile plaques in the cerebellum is a common feature of FAD [32, 33] and other authors have described that levels of Aβ in the cortex and cerebellum may be similar to this AD model [25]. In addition, the distribution of Aβ in several regions of the brain of the AβPP/PS1 transgenic mouse model has been previously described [34]. These authors have measured Aβ levels by ELISA and have obtained twice the quantity of amyloid in the cerebellum when compared with the cortex at 19 months, even though they confirmed that the most affected region in the brain was the cortex, followed by the hippocampus, striatum, and cerebellum, which was in agreement with the results obtained by PET. However, these results were obtained using male mice only, and no correlation with females or different ages were reported. Another study performed on 12-month-old females revealed that amyloid levels were lower in cerebellum than in the frontal cortex [35], however no correlation with males was reported. So we can confirm that this is the first study that confirms the differences between males and females in the accumulation of Aβ in the cerebellum of AβPP/PS1 mice. Moreover, our immunofluorescence results showed the evolution in the Aβ plaques density. Even other authors have not described any Aβ plaques in the cerebellum of 6-month-old mice [25].

The difference in the distribution of Aβ plaques in the cerebellum between sporadic AD (SAD) and FAD cases has already been reported [18]. SAD patients showed diffuse plaques mainly in the Purkinje and granular layers. However, patients that carried PS1 mutations showed abundant deposits of Aβ mainly localized in the Purkinje and molecular layers. The presence of Aβ plaques in the molecular layer of the cerebellum in transgenic mice has been already described [25, 36], confirming our results and those obtained in patients.

The gliotic reaction has been associated with senile plaques in FAD models [37]. In the AβPP/PS1 mice model, GFAP-positive cells appear usually surrounding Aβ plaques, in the cortex and hippocampus [21]. Cerebellum Aβ plaques have been described as being surrounded by microglia, astrocytes, and dystrophic neurites [36]. These authors described the presence of GFAP reaction surrounded Aβ deposits in the ML. However, other authors found no GFAP in the ML [25] that they related with the presence of Bergmann glia that may substitute the gliosis reaction. We have obtained similar results with presence of GFAP in the Purkinje and the granular layers surrounding Aβ plaques and the absence of GFAP in the ML even near Aβ plaques. These data agree with the observation in the cerebellum of AD patients [38] that were compared with healthy and young individuals.

The AβPP/PS1 transgenic model was used to study α and β secretases which showed higher levels in the cortex and hippocampus of the transgenic mice, when compared with the wild type, although the cerebellum analysis was similar. Surprisingly, no differences in the γ-secretase activity between the wild type and transgenic mice in the cortex and hippocampus have been reported, whereas there were statistically significant differences in the cerebellum, with higher levels in the wild type mice than in the transgenic mice [35]. These authors suggested that the PS1dE9 transgene does not lead to a higher γ-secretase activity, but is rather more efficient cleaving AβPP into Aβ peptides [35]. All these data led us to formulate a tantalizing hypothesis that the increase in amyloid in the female cerebellum, or the delay in its accumulation in the male cerebellum, is not the result of a direct modification of γ- or β-secretase activity per se, but could be explained by hormonal differences as has been deeply hypothesized. In fact, some authors related sex differences with brain testosterone levels that increase in triple transgenic males with age while there is a reduction in females. Surprisingly, estrogen levels were not modified in either males and females [39].

According to the amyloid cascade hypothesis, as a consequence of the presence of oligomeric amyloid and/or accumulation of Aβ there is usually an increase of some kinases that affect phospho-tau levels in the cortex [2]. Our results are in agreement with previous data that have indicated that the levels of tau and mature AβPP are reduced in the cerebellum when compared with the frontal cortex or the hippocampus [40]. This fact could be related to the reduced levels of PHF1 in our young transgenic mice samples. Besides, phospho-tau levels in the cerebellum of FAD patients were described to be increased compared to SAD [41].

The amyloid cascade hypothesis proposes that toxic oligomers may affect different aspects of the normal neuronal physiology, such as axonal transport and neurotransmission, and in parallel survival signaling. For instance, reduced levels of phosphorylated synapsin in AD postmortem tissues has been described [42]. The important of this feature is focused on the significant contribution of synapsin to the formation and maintenance of the presynaptic structure and neurotransmission [43]. Besides, double-labeling immunocytochemistry at the ultrastructural level in the adult cerebellum locates presenilin and AβPP in single postsynaptic Purkinje cell spines and in single axonal boutons of presynaptic afferences as well [44]. AβPP/PS1 transgenic mice have already been described to have synaptic changes [45] and the accumulation of Aβ in the cerebellum as disturbing synaptic activity [24]. Effectively, transgenic mice have modified levels of several synaptic markers that could be affected by the presence of Aβ and its toxic effect on the synaptic activity. However, we could not find sex differences that relate Aβ levels and synaptic markers proportional and directly.

With respect to signaling pathways, the PI3K-Akt pathway has been proposal to be essential in neuronal survival. In fact, some differences in the levels of p-AKT and p-GSK3 between transgenic and wild type mice have been described in the cortex [21], however, in our analysis there were no differences in the cerebellum between the sexes or the genotypes. It is possible that the cerebellum tissue is more resistant to the Aβ toxicity and the transgenic phenotype/genotype does not affect this metabolic pathway as occurs in the cortex or hippocampus. However, we cannot discard other interpretations. For instance, if we consider that oligomeric amyloid is lower in the cerebellum than in the hippocampus, the survival signaling would obviously be less affected in the cerebellum; or perhaps it is a factor of time, and we may be able to observe a greater effect after a longer period of aging.

We should therefore conclude that the presence of Aβ in the cerebellum tissue does not affect any PI3K/AKT pathway but alters the activation of AMPK, which usually occurs in AD patients (for review, see [29]). In fact, the autophagy pathway has been recently described to be altered in AD; this complex pathway may be regulated by mTORC1, as well as AMPK [29]. In AD models, the inhibition of mTORC1 has been correlated with a reduction of Aβ_40/42 [46]. The autophagy pathway has been described as being altered in AD patients and in transgenic mouse models (for review, see [47]). Increased autophagy levels have been described in the cerebellum prior to neuronal death [48] as an increment in the LC3II levels and a reduction in p62 in Purkinje cells. Although we did not find statistically significant differences in LC3, we can confirm a reduction in both p62 and NBR1 in older transgenic females. Other studies have described a reduction in both activated forms of mTOR and S6K in the cortex of AβPP/PS1 mice while both proteins were increased in the cerebellum, although this was not statistically significant in the case of S6K when compared with wild type [49]. In conclusion, we would infer that the reduction in p62 and NBR1 is due to an increase in autophagic activity. However, these data are still in conflict with the fact that activated autophagy, through the inhibition of mTORC1, showed a reduction of amyloid [30] in contrast with other authors [50] that support our present data in in vitro and in vivo experiments (unpublished data).

Taking into account all our data, it is tantalizing to propose that the factor that may correlate with the differences observed between older males/females would be related with the deregulation of autophagy markers, p62, NBR1, and AMPK activity. Obviously more work has to be done to understand the autophagy process in normal versus AD brains.

Cerebellar dysfunction has already been described at the pre-Aβ accumulation stage in AβPP/PS1 mice [25, 51] so this could explain the modifications observed in younger mice. Even some of the markers were evidently modified by the transgenic genotype/ phenotype; most of them were only altered in the transgenic females group. This could be a consequence of the increased levels of Aβ in older female’s cerebellum.

There is an important deposition of Aβ in the cerebellum similar to those observed and previously described in the cortex and hippocampus of the AβPP/PS1 transgenic mice. This accumulation was greater in females than in males, and it occurred differentially in the granular and molecular layers. It would appear that the Aβ deposition in the cerebellum starts at the molecular layer and in time spreads to the granular layer as was observed in the 15-month-old mice. GFAP-positive cells were detected in GL at 15 months while they were starkly reduced in the ML at the age analyzed.

We can conclude that the effect of amyloidosis in the cerebellum differs from the cortex and that this permits further investigation. Our data indicated different accumulation rates of amyloid and subsequently different signaling effects males versus females. When we contemplate all data from cortex, hippocampus, and cerebellum, and the differences in the evolution of the disease between males and females, we support the hypothesis that specific sex-designed therapies to treat FAD should be considered.

Footnotes

ACKNOWLEDGMENTS

We thank the animal and microscopy facility for technical support and Dr. JJ Lucas for the use of his cryostat. This work was supported by grant from the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED; an initiative of the ISCIII). In addition, work in FW’s lab was supported by grants from the “Plan Nacional”, “Dirección General de Ciencia y Tecnología -DGCYT” SAF2012-39148-C03-01; CAM S2010/BMD-231-(2010-14) and EU-FP7-2009-CP-IP 212043-2 NAD, and an Institutional grant from the “Fundación Areces”.

Authors’ disclosures available online ().

Appendix

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AβPP/PS1 Transgenic Mice Show Sex Differences in the Cerebellum Associated with Aging

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Experimental animals

Mouse sacrifice and tissue sampling

Tissue processing

ELISA quantification of Aβ40 and Aβ42

Gel electrophoresis and western blots

Immunofluorescence studies

Statistical analyses

RESULTS

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

Appendix

References

ELISA quantification of Aβ₄₀ and Aβ₄₂