Background
Besides aggregation of amyloid-β (Aβ) and tau, it has become clear that Alzheimer’s disease (AD) pathology is also characterized by neuroinflammation and iron dysregulation.
Neuroinflammation has been recognized to play an important role in AD, and involves chronic activation of microglia and astrocytes in brain regions affected by AD pathology [
1‐
3]. While microglia- and astrocyte-mediated immune responses may initially be neuroprotective, chronically activated brain immune cells may lose certain protective functions and acquire toxic properties [
2,
4,
5]. For example, chronic production of reactive oxygen species and pro-inflammatory cytokines by activated glia contributes significantly to AD pathology, by activating pro-apoptotic signaling pathways and promoting further aggregation of Aβ [
6‐
10].
Iron dysregulation is also known to play an important part in AD pathology [
11]. Iron accumulation occurs in brain regions affected by AD pathology, particularly in plaques and cell types including neurons and microglia [
12‐
15]. Iron accumulation may contribute significantly to AD pathology by promoting Aβ aggregation, enhancing further pro-inflammatory processes, disturbing mitochondrial respiration, stimulating oxidative stress, and inducing ferroptosis [
11,
16].
Interestingly, findings suggest that the protein Lipocalin 2 (Lcn2) may be involved in both neuroinflammation and iron regulation, and might contribute to AD pathology. Lcn2, also known as siderocalin, 24p3, uterocalin, and neutrophil gelatinase-associated lipocalin (NGAL), is an acute-phase protein that is rapidly produced and secreted in response to a wide range of inflammatory and pathological stimuli [
17,
18]. Lcn2 plays a role in various processes, including the defense against certain bacterial infections by sequestering iron-loaded bacterial siderophores, mammalian iron metabolism, anti- and pro-inflammatory responses, anti- and pro-apoptotic signaling, and cell migration and differentiation [
17,
19‐
21]. Studies showed that Lcn2 protein levels in blood increase with age and mild cognitive impairment, and are increased in human post-mortem brain tissues in different diseases of the central nervous system (CNS), including AD, Parkinson’s disease, and multiple sclerosis [
22‐
27]. Studies in mice and cell culture showed that Lcn2 contributes significantly to neurodegenerative processes, for example by aggravating pro-inflammatory responses, silencing neuroprotective signaling pathways, and sensitizing brain cells to cell death [
25,
27‐
34]. In the context of AD pathology, in vitro experiments showed that astrocytes produce high levels of Lcn2 in response to Aβ, and that Lcn2 renders brain cells more vulnerable to Aβ-induced cell death [
27,
31]. Contrastingly, a few studies concerning sepsis, stroke, and multiple sclerosis reported that Lcn2 exerts significant neuroprotective functions, by promoting anti-inflammatory responses and glial pro-recovery phenotypes [
35‐
37].
Whether Lcn2 exerts significant—either neuroprotective or neurodegenerative—effects on AD pathology has not yet been studied in vivo. Therefore, the aim of the present study was to determine the effect of Lcn2 on AD-like pathology in a mouse model of AD. To this end, we compared behavior, memory functioning, and Aβ-associated neuropathology in J20 mice (a transgenic amyloid precursor protein (APP) overexpressing AD mouse model) and Lcn2-deficient J20 (J20xLcn2 KO) mice. Neuropathological investigation included analysis of hippocampal Aβ plaque load, activation of microglia and astrocytes, and iron load.
Discussion
We report that J20 and Lcn2-deficient J20 (J20xLcn2 KO) mice at 12 months of age show equally severe AD-like behavioral changes, cognitive impairment, plaque formation, and glial activation. Intriguingly, J20xLcn2 KO mice presented significantly decreased AD-like iron accumulation in the hippocampus, as compared to J20 mice.
The reduced hippocampal iron load in J20xLcn2 KO versus J20 mice indicates that Lcn2 contributes to AD-like iron accumulation in the brain. This result corresponds with the finding of Lcn2-mediated brain iron accumulation in an animal model of hemorrhagic stroke [
34]. Also, Lcn2 was reported to stimulate ferritin mRNA expression in Aβ-treated cultured astrocytes, indicating a Lcn2-mediated increase in iron storage upon Aβ-challenge [
31].
Lcn2 is known to be involved in iron metabolism by binding to bacterial and mammalian siderophores (small iron-binding molecules) present in the body [
49‐
51]. Lcn2 is able to mediate both import and export of iron into/from cells [
52,
53]. Under inflammatory conditions, Lcn2 contributes to an iron-retentive response known as the anemia of inflammation, by promoting iron retention in macrophages [
54,
55]. Possibly a similar iron-retentive response occurs in the inflamed brain. Indeed, astrocytes, neurons, and especially microglia (the macrophages of the brain) were found to accumulate high levels of non-transferrin bound iron upon inflammatory stimulation [
15,
56‐
61], which might possibly in part be regulated by Lcn2.
This possibility may explain the observed Lcn2-mediated iron retention in J20 versus J20xLcn2 KO brains, and warrants further investigation of involved cell types and siderophores. Moreover, the pathophysiological relevance of Lcn2-mediated brain iron accumulation in AD requires further research. Namely, despite the well-known important role of iron dysregulation in different AD-related pathophysiological processes, the alleviated iron accumulation in J20xLcn2 KO mice as compared to J20 mice is not accompanied by significant changes in other AD characteristics, such as behavior, cognition, Aβ aggregation, and glial activation. It may be possible that the hippocampal iron accumulation in 12-month-old J20 mice is not severe enough (yet) to overwhelm the stable iron storage facilities in the brain, and/or that other compensatory mechanisms induced upon iron dysregulation are (still) sufficient to protect the brain from iron toxicity. This scenario would explain why the difference in iron load between J20 and J20xLcn2 KO mice does not result in significant differences in other AD characteristics. It would be of great interest to study this hypothesis, including the possibility that (Lcn2-mediated) iron dysregulation may become more severe in more advanced AD mice. In these future studies, it would be interesting to for example assess potential differences in levels of labile reactive iron, oxidative stress, and ferroptosis.
Interestingly, the results suggests that Lcn2 may also affect iron homeostasis under healthy unchallenged conditions, as indicated by the increased hippocampal iron levels in Lcn2 KO mice as compared to WT mice. It should be noted that significance between WT and Lcn2 KO mice was often not reached when all four genotypes were compared, while significant differences were found when only WT and Lcn2 KO mice were compared. This finding corresponds with previous reports, in which increased iron levels were found in macrophages and neural stem cells in healthy Lcn2 KO mice [
62‐
64].
Taken together, basal physiological levels of Lcn2 under healthy conditions may be required to maintain iron homeostasis (with absence of Lcn2 leading to iron accumulation in certain cell types, possibly by reduced iron export), while increased Lcn2 levels under pathological conditions may contribute to iron dysregulation and accumulation as well (with increased Lcn2 levels leading to iron accumulation in plaques and certain cell types, possibly by increased iron import). Importantly, although iron homeostasis is tightly regulated, it thus appears that Lcn2 has an important function in iron regulation and distribution in the brain.
No differences were detected in behavior, cognition, plaque load, and glial activation between J20 and J20xLcn2 KO mice. The absence of significant detrimental or beneficial effects of Lcn2 on these AD-like characteristics is notable, taking into account the strong neurodegenerative [
22,
25,
27‐
29,
31‐
34,
65‐
69] and neuroprotective [
35‐
37] effects that were reported for Lcn2 in various in vitro and animal models of brain injury and disease. It is possible that Lcn2 indeed does not affect cognition and glial activation in AD, which would provide interesting contrasting information on the role of Lcn2 in neurodegeneration. Alternatively, it is possible that Lcn2 might affect AD pathology in other models of AD. For example, it would be important to validate if Lcn2 might affect cognitive function and pathology in AD models that present more severe neuronal loss (for example APP/PS1KI mice [
70]), or in mice at other ages. Furthermore, although clear Lcn2-positive astrocytes were detected, it should be determined whether Lcn2 in the J20 mouse brain reaches concentrations that are comparable to the Lcn2 levels in the human AD brain. Related to this, it would be of interest to investigate AD models which overexpress Lcn2 (besides Lcn2-deficient AD models). Of note, most studies regarding the role of Lcn2 in CNS disease/injury until now were performed in acute models of CNS injury (which may cause stronger, short-term, induction of Lcn2), in contrast to the chronic transgenic AD model studied here (in which—in comparison to models of acute injury—Lcn2 expression may be less strong, which may also be the case for the expression of other inflammatory mediators such as interleukin 1 beta (IL-1β, also see, Additional file
1: Figure S9)). Finally, it should be taken into account that J20 mice overexpress human Aβ while expressing murine Lcn2 (62% identical to human Lcn2 on the amino acid level [
71]), which might impact their potential direct or indirect interactions and the translational value to the human disease.
Taken together, more insight into the protective and toxic effects of Lcn2 is required to understand its importance in AD, and its potential as a therapeutic target. Of note, while Lcn2 KO mice are of great value in the study of Lcn2, it should be considered that potential mild developmental disturbances may be present in these mice. As shown in the current and previous studies [
62,
63], healthy unchallenged Lcn2 KO mice may display disturbed iron regulation in the periphery and the brain, which might affect important physiological processes such as synaptic plasticity and neurogenesis [
62,
72,
73]. Possibly related to this, Lcn2 KO mice appear to present a mild cognitive impairment, as seen from the MWM learning curves in Fig.
3e, f (Additional file
1: Figure S10) and previous reports [
62,
72]. Interestingly, this mild cognitive impairment might in part be overcome with age (Additional file
1: Figure S10). To circumvent potential mild developmental disturbances in Lcn2 KO mice, it would be of interest to investigate conditional Lcn2 KO (or conditional Lcn2-overexpressing) mice.
Acknowledgments
We thank Wendy Kaspers, Roelie Veenstra-Wiegman, Benjamin Otten, and Diane ten Have for their fantastic work in mouse breeding and animal caretaking, and Wanda Douwenga and Kunja Slopsema for excellent technical assistance. We also thank Dr. Debby Van Dam and Elly Geerts from the University of Antwerp for very helpful advice on Morris water maze set-up, and Klaas Sjollema for excellent technical assistance with imaging of immunofluorescent stainings at the UMCG Microscopy and Imaging Center (UMIC).