Age-dependent roles of peroxisomes in the hippocampus of a transgenic mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of dementia, characterized by progressive neurodegeneration, particularly affecting the hippocampal formation. Impairment of cognitive and memory functions is associated with amyloid β-peptide (Aβ) accumulation, increased oxidative stress, lipid metabolism alteration, and inflammation [1‐5].

The central role of peroxisomes in reactive oxygen species (ROS) metabolism has emerged since their discovery [6]. In fact, peroxisomes participate in both ROS generation and removal, under physiological or pathological conditions. Moreover, peroxisomes are involved in a wide range of catabolic and anabolic functions, including β-oxidation of very long chain fatty acids (VLCFAs) [7, 8], biosynthesis of polyunsaturated fatty acids and plasmalogens [9, 10], and calcium homeostasis [11, 12]. Emergent studies have revealed that peroxisomes can also function as intracellular signaling compartments and organizing platforms that orchestrate important developmental decisions from inside the cell [13]. These dynamic and versatile organelles respond to physiological and pathological changes in cellular environment by adapting their morphology, number and enzyme content accordingly [14]. Peroxisomal dysfunction has been shown to be associated with cellular aging as well as with age-related degenerative diseases [13]. The potential role of peroxisomes in human AD, especially in relationship with lipid metabolism, has recently been suggested [15]. Indeed, a decrease in plasmalogens and, conversely, an increase in VLCFAs, were described in cases with advanced Braak stages [16]. Moreover, in an in vitro model of advanced AD a decrease of peroxisomes in hippocampal neurons was reported, while induction of peroxisomal proliferation attenuated Aβ-dependent toxicity [17]. We previously demonstrated that peroxisomes are involved in early stages of AD, as studied either in vivo, in a transgenic mouse model [18], or in vitro, on Aβ treated cortical neurons [19].

The aim of the present work was to investigate the role of peroxisomes during the progression of AD and in normal aging. To this purpose, we utilized the Tg2576 (Tg) mouse model, compared to its wild-type (WT) counterpart. Differently from other mouse models, this strain displays a slowly progressive AD pathology, offering the opportunity to study even subtle age-dependent alterations [20‐23]. By combined molecular and morphological approaches, we examined the expression of peroxisome-related proteins in the hippocampal formation at early and advanced AD stages (3, 6, 9, 12, and 18 months). We focused on the CA1 hippocampal region, as pyramidal cells in this field are the most profoundly affected cell type in human AD [24]. Specifically, the expression and immunolocalization patterns of peroxisomal membrane protein of 70 kDa (PMP70), peroxin 14p (Pex14p), catalase (CAT), acyl-CoA oxidase 1 (AOX), and 3-ketoacyl-CoA thiolase (THL) were studied, to get an insight into the biogenesis and functioning of peroxisomes in the diseased brain. Given the involvement of peroxisomes in the maintenance of the redox status, antioxidant enzymes other than CAT, namely selenium-dependent glutathione peroxidase (GPX1), Cu,Zn-superoxide dismutase (SOD1), and Mn-superoxide dismutase (SOD2) were also investigated in Tg animals and in their WT littermates. It is worth recalling that these proteins, though mainly localized to different cell compartments, were also found in peroxisomes [25]. At selected stages, markers for oxidative damage to lipids and nucleic acids - acrolein and 8-hydroxy(deoxy)guanosine (8-OH(d)G), respectively - were also studied.

Notably, the size and functions of peroxisomal population are regulated by a class of ligand-activated transcription factors (peroxisome proliferator-activated receptors, PPARs) [26]. Among these, we studied PPARα which is directly involved in peroxisomal induction [27], plays a neuroprotective role in age-related inflammation [28] and enhances memory consolidation [29]. It is worth-mentioning in this context that PPARα specific agonists have been demonstrated to exert a neuroprotective action against Aβ-mediated toxicity in vitro [17]. Finally, we examined the expression of PPARγ coactivator-1γ (PGC-1α), in view of its synergism with PPARα and its implication in peroxisomal remodeling and biogenesis [30, 31].

The distribution of peroxisomes in Tg and WT hippocampal formation from 3-, 6-, 9-, 12-, and 18-month-old mice was investigated by analyzing the expression of peroxisomal membrane (PMP70, Pex14p) and matrix (CAT, AOX, THL) proteins. Markers of oxidative damage to nucleic acids (8-OH(d)G) and to lipids (acrolein), as well as antioxidant enzymes (SOD1, SOD2, GPX1), were also studied. Further, to get an insight into the transcriptional regulation of these genes, PPARα and PGC-1α expression was examined. At selected stages, the presence of PMP70 and PPARα in neuronal and/or astroglial cells was studied by immunofluorescence and their ultrastructural localization was investigated by immunoelectron microscopy.

The aim of the present study was to investigate the role of peroxisomes in AD onset and progression. In a previous work [18], peroxisomal involvement in early AD was demonstrated in the Tg2576 mouse model, also used in this work. Differently from that study, in which male animals were analyzed, we here utilized females, since epidemiological and histopathological data indicate this as the most AD-affected gender in mice and humans [41, 42]. We focused on the hippocampal formation, i.e., the primarily affected area of the diseased brain [43]. The expression of peroxisome-related proteins was studied by combined molecular and morphological approaches, at the onset (3 months) and during AD progression (6, 9, 12, 18 months) in Tg animals, compared to their WT counterparts.

Our results demonstrate that significant peroxisomal alterations occur in Tg mice, as early as 3 months. These early changes are consistent with behavioral, electrophysiological, ultrastructural and molecular modifications reported in this model at the same age [22]. Indeed, PMP70, long been considered marker of the numerical size of peroxisomal population [36] is upregulated, as assessed by WB. Consistently, PMP70 immunohistochemistry reveals especially intense immunostaining in the pyramidal layer of Tg hippocampal CA1 subdivision, and immunoelectron microscopy shows numerous positive peroxisomes in the somata of these neurons.

Differently from PMP70, the biogenesis marker Pex14p, also used to evaluate peroxisome number [35], is unchanged in 3-month-old Tg animals. The absence of correlation between PMP70 and Pex14p levels in early AD could either indicate specific concentration of PMP70 in a numerically unchanged peroxisomal population, or impoverishment of Pex14p in proliferated peroxisomes. In either case, the altered content in these proteins likely results in profound modifications of peroxisomal functions. Interestingly, Pex14p has recently been involved in microtubule-based peroxisome motility [44], suggesting that at this early AD stage, peroxisomes, endowed with a relatively low Pex14p/PMP70 ratio are altered in their transport along neurites. To this respect, it is worth mentioning that a recent work from Berger’s group demonstrates an increased peroxisomal volume density in neuronal cell bodies of human AD brain, associated with impaired peroxisome trafficking [16].

The specific increase in early AD of PMP70, presumably involved in fatty acyl-CoA import across peroxisomal membranes [37], could reflect the need for a more efficient acyl-CoA β-oxidation. In agreement with this hypothesis, AOX, rate-limiting enzyme of peroxisomal β-oxidation pathway, is upregulated in 3-month-old Tg hippocampus. This increase could thus represent a compensatory response to early Aβ-mediated mitochondrial insult, to cope with the impaired energy metabolism, occurring in AD [45, 46]. Indeed, abnormalities in glucose metabolism/mitochondrial function are invariant features of early AD [46‐49]. Consistently, positron emission tomography studies in AD patients report abnormal cerebral glucose utilization, particularly in the hippocampus, decades prior to the onset of histopathological and clinical features [50]. A consequent shift in brain metabolism, from a primarily aerobic glycolysis pathway to a ketogenic/fatty acid β-oxidation pathway is thought to occur [51]. In this context, we suggest that peroxisomal β-oxidation contributes to such metabolic change, providing mitochondria with acetyl-CoA and fatty acyl substrates. Induction of peroxisomal β-oxidation pathway may also be neuroprotective, by enhancing the clearance of potentially toxic VLCFAs and by promoting the biosynthesis of the neuroprotective docosahexaenoic acid (DHA) [15]. Relevantly, elevated levels of VLCFA have been found in human AD brain lesions [16] and the ability of these fatty acids to trigger oxidative stress and mitochondrial dysfunction on neural cells has recently been demonstrated [52, 53].

A biogenetic/metabolic relationship between peroxisomes and mitochondria has long been established and recently emphasized [13, 30, 31]. In yeast, experimentally induced or age-associated mitochondrial dysfunction activates the so-called retrograde (RTG) signaling pathway, leading to the transcription of RTG-target genes, including those required for peroxisome biogenesis and function [13]. Our results showing increased PPARα nuclear immunoreactivity allow us to speculate that this gene be included among the above mentioned RTG-target genes. The increased PPARα expression could in turn be responsible for the induction of its target genes PMP70 and AOX [54, 55]. Even though the molecular triggers of PPARα activation remain to be determined in our model, endogenous production of PPARα ligands, e.g., oxidized lipid molecules, is likely to occur due to Aβ-mediated ROS generation.

While, in principle, AOX induction in 3-month-old Tg hippocampus may represent a positive response to enhanced need for lipid substrates to be processed in mitochondria, this induction may also result in increased H₂O₂ production by the oxidase. Moreover, mitochondrial respiration supported by fatty acids generates substantial rates of ROS production [56], thus contributing to cellular redox status alteration. In this context, even the observed early increase of SOD2 expression, in agreement with reports demonstrating induction of mitochondrial genes in young Tg mice [57], may contribute itself to redox imbalance, through enhanced conversion of superoxide anion to H₂O₂.

Notably, GPX1, the major cytosolic H₂O₂–scavenging enzyme, is significantly down-regulated in our early AD samples, further exacerbating imbalance between generation and removal of hydrogen peroxide. GPX1 seems especially critical in AD, since its deletion increases susceptibility of neurons to Aβ-mediated damage, while its overexpression protects against neurodegeneration [58, 59]. The postulated pro-oxidant environment likely results in post-translational modifications of GPX1 itself, leading to its irreversible inactivation [60]. Even the low levels of SOD1 detected in the young Tg hippocampus may be explained taking into account that this cytosolic protein is selectively damaged by H₂O₂ [61]. Importantly, SOD1 deficiency in Tg2576 mice has been associated with oxidative damage, accelerated Aβ oligomerization and memory impairment [62].

Therefore, our data strongly support the idea that oxidative stress is the primary culprit in AD pathogenesis [63]. Consistently, 8-OH(d)G and acrolein, markers of oxidative modification to biomolecules [64, 65], show DNA/RNA oxidation and lipoperoxidation in the hippocampus of young Tg mice. Interestingly, 8-OH(d)G immunostaining is predominantly localized in the cytoplasm, suggesting that mitochondrial nucleic acids are primarily affected. To our knowledge, this is the first report of such a modification occurring at 3 months in this mouse strain. In this context, it is worth mentioning that acrolein has been suggested not only as a marker of lipoperoxidation, but also as initiator of oxidative stress [65].

Between 3 and 6 months of age, most peroxisomal markers decrease in WT hippocampal formation, indicating a numerical reduction and/or a metabolic change of the organelles in the physiological course of maturity. By contrast, in Tg mice of the same age, when several hallmarks of AD pathology make their appearance [21], marker-specific patterns are observed. In particular, while high levels of CAT and Pex14p are maintained, PMP70 and AOX are significantly decreased, suggesting metabolic, rather than numerical, modifications of peroxisomes. These changes, indicating decreased efficiency of peroxisomal β-oxidation, presumably lead to VLCFA accumulation, consistent with what reported in human AD [16]. Interestingly, pharmacological inhibition of peroxisomal β-oxidation, resulting in VLCFA accumulation, is positively correlated with Aβ40 levels in rat cerebral cortex [66].

The relatively high CAT levels observed in 6-month-old Tg hippocampus are conceivably sustained by transcription factors activated in response to oxidative stress. To this respect, the cellular redox state sensor PGC-1α may play a role in regulating CAT expression [67]. Our immunohistochemical data support this relationship, in that at 6 months intense PGC-1α nuclear staining, suggestive of its induction and activity, is observed in CA1 pyramidal neurons, exclusively in Tg. The decrease in PGC-1α nuclear immunostaining seen at later AD stages, also parallels the behavior of CAT.

The same transcriptional co-activator, which has been involved in aging and age-associated diseases [68], may be responsible for the pattern of other antioxidant enzymes. Indeed, the increased PGC-1α nuclear immunoreactivity observed around 9–12 months in Wt neurons is paralleled by the raise in SOD1 and SOD2 levels, occurring in 12-month-old normal hippocampus. Conversely, the dramatically low expression of PGC-1α at 12 months in AD neurons, when we first observe hippocampal amyloid deposits (not shown), is concomitant with the lower levels of SOD1, SOD2 and, particularly, GPX1, compared to WT. Accordingly, altered glutathione redox status has been associated to the onset of amyloid plaques in a mouse model of AD [69] and decreased activities of SOD1 and GPX1 have been reported in symptomatic human AD [62, 70]. By contrast, SOD2 overexpression in Tg mice results in diminished plaque formation [71].

In 18-month-old mouse hippocampus, genotype-dependent variations are observed for most peroxisomal and antioxidant proteins. At this stage, gliosis, known to occur during brain aging and exacerbated in AD [72‐74], should be taken into account, when interpreting WB quantitative data. In fact, astrogliosis may be at least in part responsible for the peaks of PMP70 and Pex14p observed in old Tg mice. An important contribution to the overall brain peroxisomal population by astrocytes, highlighted by our GFAP/PMP70 double immunofluorescence images, is consistent with the notion that peroxisomes are abundant in this cell type [75‐77]. Nevertheless, our immunohistochemical data on 18-month-old hippocampus clearly show enhanced positivity to PMP70 and Pex14p also in CA1 pyramidal neurons, markedly in the Tg, suggesting a global increase of peroxisome number in the hippocampal tissue, contributed by both neuronal and glial cells. Differently from the above membrane proteins, peroxisomal matrix enzymes, namely CAT, AOX and THL are not increased or even decreased, in 18-month-old hippocampus, possibly owing to age-related low efficiency of peroxisomal protein import [13] and in keeping with the impairment of brain peroxisomal acyl-CoA β-oxidation in physiological and pathological human aging [15, 16].

Oxidative stress is known to occur in age-related neurodegeneration [78, 79]. Our results on 18-month-old WT hippocampus show significantly lower levels of SOD1 and SOD2 proteins, while GPX1 is not decreased, with respect to previous ages. Thus, it seems that oxidative damage in the aging hippocampus may result from defective scavenging of superoxide anion, rather than hydrogen peroxide. In 18-month-old Tg hippocampus, GPX1 levels are significantly lower than in the corresponding WT, indicating impaired removal of H₂O₂, thus exacerbating the physiological age-related alteration of the cellular redox status. Concerning SODs, while SOD1 follows the decreasing trend of the age-matched WT hippocampus, SOD2 is not decreased in 18-month-old Tg, being significantly higher than in its WT counterpart. This finding can be explained by immunohistochemical data, showing particularly high concentration of SOD2 in glial cells surrounding amyloid plaques. Consistently, SOD2 - but not SOD1, CAT, or GPX1 - is induced in astroglia by activated microglia in an in vitro model of neuroinflammation [80].

A pro-oxidant cellular environment in the aged hippocampus is further demonstrated by the presence of oxidative damage markers (acrolein and 8-OH(d)G), particularly concentrated in Tg neurons. However, while in WT hippocampus both markers clearly show age-dependent increase, the same is not true for the Tg, where 8-OH(d)G immunoreactivity appears reduced in 18-month-old hippocampus, compared to the young. This observation is in agreement with data from other Authors indicating a negative correlation between 8-OH(d)G levels and histological amyloid burden [81, 82].

Overall, our results confirm and extend our previous observations on the early involvement of peroxisomes in AD pathogenesis [18]. In particular, we strongly suggest that these organelles constitute a first line of defense in support of mitochondria, primary targets of intracellular Aβ/ROS-mediated damage. This protective role specifically involves increased peroxisomal fatty acyl β-oxidation, likely providing mitochondria with acetyl-CoA and shortened acyl-CoAs. This conclusion may shed new light into the physiological significance of peroxisome metabolism in mature neurons, so far mainly related to the synthesis of plasma membrane components and to neurotransmission [7‐10, 83, 84].

In brain aging, when oxidative stress sensu strictu begins, peroxisomes become target of ROS, their functions irreversibly decline, thus contributing themselves to the “pro-oxidant loop” [13]. Deepening knowledge of these processes, which are associated to aging and accelerated/exacerbated in AD, will add significantly to the dissection of pathogenic mechanisms of the disease. At the same time, the early response shown by peroxisomes supports the potential use of some of their proteins as biomarkers, to recognize the first signs of presymptomatic AD and to follow the effectiveness of novel therapeutic approaches, possibly involving peroxisomal induction. Indeed, preventing or treating the disease earlier than in its mild to moderate stages presently appears a major task, as stressed by the latest commentaries on the topic [85, 86].