Lipocalin 2 contributes to brain iron dysregulation but does not affect cognition, plaque load, and glial activation in the J20 Alzheimer mouse model

Background Lipocalin 2 (Lcn2) is an acute-phase protein implicated in multiple neurodegenerative conditions. Interestingly, both neuroprotective and neurodegenerative effects have been described for Lcn2. Increased Lcn2 levels were found in human post-mortem Alzheimer (AD) brain tissue, and in vitro studies indicated that Lcn2 aggravates amyloid-β-induced toxicity. However, the role of Lcn2 has not been studied in an in vivo AD model. Therefore, in the current study, the effects of Lcn2 were studied in the J20 mouse model of AD. Methods J20 mice and Lcn2-deficient J20 (J20xLcn2 KO) mice were compared at the behavioral and neuropathological level. Results J20xLcn2 KO and J20 mice presented equally strong AD-like behavioral changes, cognitive impairment, plaque load, and glial activation. Interestingly, hippocampal iron accumulation was significantly decreased in J20xLcn2 KO mice as compared to J20 mice. Conclusions Lcn2 contributes to AD-like brain iron dysregulation, and future research should further explore the importance of Lcn2 in AD. Electronic supplementary material The online version of this article (10.1186/s12974-018-1372-5) contains supplementary material, which is available to authorized users.


Mice
Mice had ad libitum access to water and food, and nesting material and a cardboard tube were always present in their cage. Mice were on a 12:12 L/D cycle (lights on at 06:00), in a room with a temperature of 21±2 °C and a humidity of 50%. All mice were housed in the same housing room from 5 months before the start of experiments, and were housed individually starting from approximately 3 months prior to behavioral testing. Behavioral experiments started when mice were 11.5 months of age. All mouse experiments performed in this study were approved by the animal ethics committee of the University of Groningen (DEC6851A). J20 mice were hemizygous for the J20 transgene, established by standardly crossing J20 mice with WT mice, and Lcn2 KO mice were homozygous for the Lcn2 knock-out. Similarly, J20xLcn2 KO mice were hemizygous for the J20 transgene and homozygous for the Lcn2 knock-out. WT mice in this study were the non-transgenic littermates of the hemizygous J20 transgenic mice. Genotypes of the mice were determined by polymerase chain reaction (PCR) on genomic DNA isolated from tail or ear. The used primers are listed in Supplementary   Table 1. Table S1. Primers used for mouse genotyping.

Target
Forward Reverse

Behavioral experiments
To study different behaviors and cognitive functions, several behavioral tests were performed, in the following order: home-cage activity measurement, open field, novel location recognition, Y-maze spontaneous alternation, elevated-plus maze and Morris water maze. All behavioral experiments (except home-cage activity recording) were performed in a separate experimental room, located next to the room in which the mice were housed. All tests (except home-cage activity recording) were filmed by a camera hanging centered above the test arena.
For all behavioral tests, the researcher left the experimental room immediately after the start of a test trial. All tests were conducted by the same experimenter, who was blinded for the mouse genotypes. The arenas used for the behavioral tests (except the Morris water maze) were cleaned with ethanol in between mice. In total, 61 mice were studied, with n=13-17 mice per genotype (WT: n=17, J20: n=13, Lcn2 KO: n=16, J20xLcn2 KO: n=15). Mice were studied in two cohorts of 30-31 mice, balanced for genotype.

Habituation
Before start of further behavioral tests, mice were habituated to handling by the researcher,

Open field
For the open field test, mice were allowed to freely explore a square arena (50x50x36 cm grey Plexiglas floor and walls, no ceiling) for 5 minutes. An overhead camera recorded the mice, and the movements of the mice were tracked with specialized software (EthoVision XT version 11.5, Noldus, The Netherlands). As a measure of locomotor activity, the total distance moved was analyzed. Also, as indications of anxiety-like behavior, the time spent in the center zone (inner 30x30cm) of the arena and the latency to first enter the center zone were measured. Light intensity in the center of the arena was 10-12 lux. The arena was cleaned with 30% ethanol between mice.

Novel location recognition
The day after the open field, the novel location recognition (NLR) test started. The NLR test is a hippocampus-dependent recognition memory task, and is based on rodent's natural exploratory behavior and preference for novelty [1]. The NLR test consisted of a training and test session, which were 24 hours apart. In the NLR training, mice were again placed in the center of the square arena, in which two identical objects were present (metallic soft drink cans, 5.5 cm in diameter and 14 cm high, extended further with a plastic-sheet cone until 24 cm in height to prevent mice from climbing onto the objects). An intra-maze cue (a black and white checkerboard) was added to one of the walls, to allow mice to spatially orient themselves in the arena. Mice were allowed to freely explore the arena and the two objects for 5 10 minutes. In the NLR training session, the two objects were placed in parallel to either the top or the bottom wall of the arena, separated 10 cm from the walls of the arena.
Twenty-four hours after the training, the NLR test was performed. In the test setting, one of the two objects was placed at its original (training) location, while the other object was relocated towards the other side of the arena (again distanced 10 cm from the walls), creating a diagonal. The intra-maze cue, serving as a stable spatial mark, remained at the same position. Mice were placed in the center of the arena, and were again allowed to explore the arena and objects for 10 minutes. The NLR training and test sessions were recorded by camera, and locomotor behavior and exploration of the objects was analyzed (object exploration could be reliably tracked by the nose-tracking function in EthoVision XT (Noldus)). Object exploration was scored when the nose of the mouse was within a 2 cm radius from the object. Mice with intact recognition memory are expected to recognize objects that are at a familiar place and therefore to spend more time exploring an object that has been placed in a novel location, while mice with memory impairments may not be able to distinguish familiar and novel object locations. As a measure of recognition memory, the discrimination index was calculated as: (time spent exploring the (to be) relocated object/total exploration time)*100. A discrimination index of 50% indicated no preference for one of the objects above the other, while a discrimination index of above 50% indicated a preference for the relocated object. Light intensity in the center of the arena was 10-12 lux. The arena was cleaned with 30% ethanol between mice.

Y-maze spontaneous alternation
As an indication of spatial working memory, mice were studied in the Y-maze spontaneous alternation task [2]. Mice were placed in the center of a white Y-shaped maze (with three 6 arms of 40 cm long, 8 cm wide, and walls of 20 cm high), and allowed to freely explore all three arms for 10 minutes. Trials were recorded by camera and then scored for arm entries (by a blinded observer), after which the percentage of spontaneous alternation was calculated by: (total number of triads/total number of arm entries -2)*100. Herein, a triad is scored when in three consecutive arm entries all three different arms are visited. Light intensity in the center of the arena was 10 lux. The arena was cleaned with 30% ethanol between mice.

Elevated-plus maze
To further assess anxiety-like behavior, exploratory behavior was studied in the elevated-plus maze (EPM) [3,4].

Morris water maze (hidden-platform)
The Morris water maze (MWM) was performed to assess hippocampus-dependent spatial learning and memory [5]. The MWM was performed as described by Van Dam et al. [6,7], After eight MWM training days, two probe trials followed, performed 24h and 48h after the last training block. For the probe trial, the platform was removed from the maze. Mice were inserted into the maze in the quadrant opposite to the target quadrant, after which they were allowed to swim freely for 100 s. As a measure of spatial accuracy, the number of crossings through the original platform position was scored, as well as the time spent swimming in the target quadrant and the other quadrants. Light intensity was 30 lux in the center of the maze. 8

Immunohistochemistry for detection of Aβ, GFAP, Iba1 and Lcn2
A week after the last behavioral test was finished, mice were terminally anesthetized by intraperitoneal injection with sodium pentobarbital and transcardially perfused with saline and 4% paraformaldehyde (PFA). Brains were then post-fixed in PFA for an additional 24h, followed by washing in 0.01 M phosphate buffer, and transfer to 30% sucrose for overnight dehydration. Next, dehydrated brains were frozen and then stored at -80 °C until sectioning. Fluorescent stainings were imaged using the Leica SP8 confocal laser scan microscope. These stainings were performed to obtain a qualitative indication of Lcn2 localization, and were not used for further quantitative analyses.

Histochemistry for detection of iron
For detection of non-heme (mostly ferric Fe 3+ ) iron the DAB-enhanced Perls' iron stain was used, as described previously [8][9][10][11]  included, as well as the stratum lacunosum moleculare (analyzed hippocampal sub-regions are also indicated in Supplementary Fig.1). Of note: the (intracellular) iron staining in the CA1 pyramidal and DG granular cell layers was less intense (yet clearly present), as compared to the intense (plaque-associated) iron staining in the other studied hippocampal sub-regions.

Quantification of (immuno)histochemical stainings
Therefore, proper detection of the iron staining in the CA1 pyramidal and DG granular cell layers required an adjusted detection threshold during analysis, which explains the higher relative coverages of iron staining found for these regions (see Figure 4). For all stainings, 3-6 hippocampi were analyzed per mouse.

Figure S1
Figure S1 Illustration of hippocampal sub-regions that were analyzed for the Perls' iron staining.
Analyzed hippocampal sub-regions include the CA1 oriens, CA1 pyramidal layer (CA1 pyr),   Additional measurements of the GFAP staining, including the coverage of the GFAP staining in specific hippocampal regions: CA1, dentate gyrus inner blade (DG ib), dentate gyrus outer blade (DG ob) and the hilus. In addition, the coverage of GFAP staining was analyzed in the corpus callosum. Also, the optical density (OD) of the GFAP staining in the hippocampus (combined CA1, GD and hilus measurements) was analyzed. n= 9-10 mice per group (WT n=10, J20 n=8, Lcn2 KO n=9, J20xLcn2 KO n=9), 3-6 hippocampi were analyzed per mouse.
Tested with one-way ANOVA with Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01 and *** p < 0.0001 compared to WT. No significant differences were present between J20 and J20xLcn2 KO mice. The four bar graphs on the right show the number of platform crossings in the probe trial, 24h