Gene knockout of 5-lipoxygenase rescues synaptic dysfunction and improves memory in the triple-transgenic model of Alzheimer's disease.

5-Lipoxygenase (5LO) is upregulated in Alzheimer's disease (AD) and in vivo modulates the amyloidotic phenotype of amyloid precursor protein transgenic mice. However, no data are available on the effects that 5LO has on synaptic function, integrity and cognition. To address this issue, we used a genetic and a pharmacological approach by generating 3 × Tg mice deficient for 5LO and administering 3 × Tg mice with a 5LO inhibitor. Compared with controls, we found that even before the development of overt neuropathology, both animals manifested significant memory improvement, rescue of their synaptic dysfunction and amelioration of synaptic integrity. In addition, later in life, these mice had a significant reduction of Aβ and tau pathology. Our findings support a novel functional role for 5LO in regulating synaptic plasticity and memory. They establish this protein as a pleiotropic contributor to the development of the full spectrum of the AD phenotype, making it a valid therapeutic target for the treatment of AD.

Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and leading cause of dementia worldwide for which no effective treatments exist[1,2]. Memory loss is the most prominent clinical aspect of AD, and it typically manifests prior to the development of overt brain pathologies. While there is still debate on the actual contributors to the development of memory impairments, there is a consensus that alteration at the synaptic level, a phenomenon also known as synaptic dysfunction, is probably one of the most significant factors in the initial stages of memory loss[3,4].

In the last decade the development of transgenic mice has represented an invaluable tool for modeling diverse aspects of the AD phenotype. Although no model exactly and fully recapitulates it, the consensus is that the triple-transgenic mice, also known as 3xTg, have the advantage of presenting the most salient features of the human disease including synaptic dysfunction, memory impairments, Aβ and tau pathology [5].

The 5-lipoxygenase (5LO) is a lipid-peroxidizing enzyme which inserts molecular oxygen into fatty acids leading to the biosynthesis of bioactive lipids such as leukotrienes[6]. The protein is widely expressed in the brain where its expression and activity increase in an age-dependent manner [7]. Previous work showed that levels of 5LO are elevated in AD brains [8], and its genetic absence or pharmacological blockade reduced Aβ levels and deposition in a transgenic APP mouse model, Tg2576 [9,10]. More recently, we demonstrated that 5LO neuronal over-expression in the 3xTg exacerbated their neuro-pathologies [11]. However, no data are currently available on the effect that 5LO genetic deficiency has on the AD-like synaptic phenotype, which includes synaptic function, synaptic integrity and cognition.

To address this issue we used a genetic and a pharmacologic approach by generating 3xTg mice genetically deficient for 5LO (3xTg-5LOKO), and administering 3xTg mice which a selective 5LO inhibitor, zileuton [10]. Compared with controls, we found that even before the development of overt neuropathology, 3xTg-5LOKO mice and 3xTg mice receiving zileuton manifested a significant improvement in cognition and memory, which was associated with a rescue of their synaptic dysfunctions and amelioration of synaptic integrity. In addition, later in life these mice had a significant reduction of their Aβ and tau pathology.

Our findings support a novel role for 5LO at the synapse level whereby modulating synaptic plasticity and integrity as well as memory. Taken together, these new data establish the 5-LO as a key player in the development of the full spectrum of the AD-like phenotype and an important therapeutic target with true disease-modifying potential for the treatment of AD.

Methods

All animal procedures were approved by the Institutional Animal Care and Usage Committee, in accordance with the U.S. National Institutes of Health guidelines. The 3xTg mice harboring a mutant amyloid precursor protein (APP; KM670/671NL), a human mutant PS1 (M146V) knockin, and tau (P301L) transgenes; 3xTg wild type (WT), and mice genetically deficient for 5LO (5LOKO) used in the study were reported previously [5,12]. All the animals were backcrossed 10 times on the same genetic background C57BL6/SJL. The 5LOKO mice were crossbred several times with 3xTg mice to obtain founder animals (3xTg/5LOKO), which were then crossed with each other and the animals from these crosses used for the studies. They were kept in a pathogen-free environment, on a 12hour light/dark cycle, and fed a normal chow and water ad libitum. Male and female mice were used throughout the studies. Animals underwent behavioral testing at two different age groups (6–8 months and 12–14 months). A separate group of five months old 3xTg mice were also randomized to receive zileuton (200 mg/L) or vehicle in their drinking water for a month. After this period they underwent to behavioral testing as described below then sacrificed for electrophysiology study. After sacrifice, mice were perfused with ice-cold 0.9% PBS containing EDTA (2mmol/L) pH7.4. Brain was removed, gently rinsed in cold 0.9% PBS and immediately dissected in two halves. One half was immediately stored at −80°C for biochemistry; the other half was fixed in 4% paraformaldehyde in PBS, pH7.4 for immunohistochemistry studies. The cortex and hippocampus were the two brain regions always used for biochemistry and immunohistochemistry studies, as also indicated in figure legends.

Behavioral tests

All the animals were handled for at least 3–4 days prior to testing. They were tested in random order and the experimenter conducting the tests was unaware of the genotype or treatment.

Y-maze

The Y-maze behavioral paradigm, a test widely used to assess working memory in rodents, was carried out as previously described [11]. Briefly, each mouse was placed in the center of the Y-maze and allowed to explore freely through the maze during a 5-min session for assessment of spontaneous alternating behavior. The sequence and total number of arms entered were video recorded. Any entry into an arm was considered valid if all four paws entered the arm. An alternation was defined as three consecutive entries in three different arms (1,2,3 or 2,3,1, etc.). The percentage alternation score was calculated using the following formula: total alternation number/total number of entries-2)*100. Testing was always performed in the same room and at the same time to ensure environmental consistency.

Fear-conditioning

Two weeks before sacrifice, fear conditioning experiments were performed following methods previously described [11, 13,14]. Briefly, on day one animals are placed into the conditioning chamber for 2 min before the onset of a sound, which in its last 2 sec is paired with a foot shock. Mice are removed from the chamber 1 min after the shock, and on day two tested for contextual and cued fear conditioning, which is basically a 24hr memory retention test. Conditioning is measured by recording the freezing behavior as the complete absence of movement both during training as well as testing. The percentage time during which the mouse froze is calculated for analysis of cintexual ans cued fear memory assessments. ests were conducted in a conditioning chamber equipped with black methacrylate walls, a transparent front door, a speaker and grid floor (Start Fear System; Harvard Apparatus).

Immunoblot Analyses

Primary antibodies used in this paper are summarized in the Table. Proteins were extracted in EIA buffer containing 250mM Tris base, 750mM NaCl, 5% NP-40, 25mM EDTA, 2.5% Sodium Deoxycholate, 0.5% SDS and an EDTA-free protease and phosphatase inhibitors cocktail tablet (Roche Applied Science), sonicated, centrifuged at 13,000 rpm for 45 min at 4°C, and supernatants used for immunoblot analysis, as previously described [9,10]. Total protein concentration was determined by using BCA Protein Assay Kit (Pierce, Rockford, IL). Samples were electrophoretically separated using 10% Bis–Tris gels or 3–8% Tris–acetate gel (Bio-Rad, Richmond, CA), according to the molecular weight of the target molecule, and then transferred onto nitrocellulose membranes (Bio-Rad). They were blocked with Odyssey blocking buffer for 1 hr; and then incubated with primary antibodies overnight at 4°C. After three washing cycles with T-TBS, membranes were incubated with IRDye 800CW or IRDye 680CW-labeled secondary antibodies (LI-COR Bioscience, NE) at 22°C for 1 hr. Signals were developed with Odyssey Infrared Imaging Systems (LI-COR Bioscience). Actin was always used as an internal loading control.

Table 1

Antibodies used in this study

Antibody	Immunogen	Host	Application	Source
4G8	aa 18-22 of human beta amyloid (VFFAE)	Mouse	IHC	Covance
APP	aa 66-81 of APP {N-terminus}	Mouse	WB	Millipore
BACE-1	aa human BACE (CLRQQHDDFADDISLLK)	Rabbit	WB	IBL
ADAM10	aa 732-748 of human ADAM 10	Rabbit	WB	Millipore
PS-1	aa arround valine 293 of human presenilin 1	Rabbit	WB	Cell Signaling
Nicastrin	aa carboxy-terminus of human Nicastrin	Rabbit	WB	Cell Signaling
APH-1	Synthetic peptide from hAPH-1a	Rabbit	WB	Millipore
Pen-2	aa N-terminal of human and mouse Pen-2	Rabbit	WB	Invitrogen
sAPPα	Synthetic peptide of the C-terminal part of Human sAPPα (DAEFRHDSGYEVHHQK)	Rabbit	WB	Cell Signaling
sAPPβ	Synthetic peptide of the C-terminal part of human sAPPβ-sw (ISEVNL)	Rabbit	WB	Cell Signaling
CTFs	a synthetic peptide [(C)KMQQNGYENPTYKFFEQMQN] corresponding to amino acids 751-770 of human precursor protein (APP), conjugated to KLH	Rabbit	WB	Santa Cruz
HT-7	aa 159-163 of human tau	Mouse	WB,IHC	Pierce
AT-8	Peptide containing phospho-S202/T205	Mouse	WB,IHC	Pierce
AT-180	Peptide containing phospho-T231/S235	Mouse	WB,IHC	Pierce
AT-270	Peptide containing phospho-T181	Mouse	WB,IHC	Pierce
PHF-13	Peptide containing phospho-Ser396	Mouse	WB,IHC	Cell Signaling
PHF-1	Peptide containing phospho-Ser396/S404	Mouse	WB,IHC	Dr. P. Davies
GFAP	aa spinal chord homogenate of bovine origin	Mouse	WB	Santa Cruz
CD45	Mouse thymus or spleen	Rat	WB	BD Pharmingen
SYP (H-8)	aa 221-313 of SYP of human origin	Mouse	WB,IHC	Santa Cruz
PSD95 (7E3-1B8)	Purified recombinant rat PSD-95	Mouse	WB,IHC	Thermo Scientific
MAP2	Bovine brain microtubule protein	Rabbit	WB,IHC	Millipore
GSK3α/β	aa 1-420 full length GSK-3β of Xenopus origin	Mouse	WB	Millipore
p-GSK3α/β	aa around Ser21 of human GSK-3a.	Rabbit	WB	Cell Signaling
JNK2	aa of human JNK2	Rabbit	WB	Cell Signaling
SAPK/JNK	aa of recombinant human JNK2 fusion protein	Rabbit	WB	Cell Signaling
Phospho-SAPK/JNK	aa Thr183/Tyr185 of human SAPK/JNK	Mouse	WB	Cell Signaling
p38 MAPK	aa sequence of human p38MAPK	Rabbit	WB	Cell Signaling
p-p38 MAPK	Aa resides surrounding Thr180/Tyr182 of human p38 MAPK	Rabbit	WB	Cell Signaling
Cdk5	aa C-terminus of Cdk5 of human origin	Rabbit	WB	Santa Cruz
P35/25	aa C-terminus of p35/25 of human origin	Rabbit	WB	Santa Cruz
Actin	aa C-terminus of Actin of human origin	Goat	WB	Santa Cruz

Sarkosyl insolubility assay

The assay for insoluble tau was performed as previously described [15]. Briefly, ultracentrifugation and sarkosyl extraction (30 min in 1% sarkosyl) was used to obtain soluble and insoluble fractions of tau. Insoluble fractions were washed one time with 1% sarkosyl, then immunoblotted with HT-7 antibody (Table).

Biochemical Analyses

Mouse brain homogenates were sequentially extracted first in RIPA for the Aβ 1-40 and 1-42 soluble fractions, then in formic acid for the Aβ 1-40 and 1-42 insoluble fractions, and then assayed by a sensitive sandwich ELISA kits (WAKO Chem.), as previously described [10,14].

Immunohistochemistry

Primary antibodies used in this study are listed in Table. Immunostaining was performed as reported previously [9,13,14]. Briefly, serial 6μm-thick coronal sections were mounted on 3-aminopropyl triethoxysilane-coated slides. Every eighth section from the habenular to the posterior commissure (8–10 sections per animal) was examined using unbiased stereological principles.

The sections for testing Aβ were deparaffinized, hydrated, and pretreated with formic acid (88%) and subsequently with 3% H2O2 in methanol. The sections for testing total tau (HT7), phospho-tau (PHF-1, PHF-13, AT8, AT180, AT270), Synaptophysin (SYP), Postsynaptic Density Protein 95 (PSD95) and Microtubule associated protein-2 (MAP-2) were deparaffinized, hydrated, subsequently pretreated with 3% H2O2 in methanol, and then treated with citrate (10mM) or IHC-Tek Epitope Retrieval Solution (IHC World, Woodstock, MD) for antigen retrieval. Sections were blocked in 2% fetal bovine serum and then incubated with primary antibody overnight at 4°C. The following day, sections were incubated with biotinylated anti-mouse immunoglobulin G (Vector Laboratories, Burlingame, CA) and then developed by using the avidin-biotin complex method (Vector Laboratories) with 3,3′-diaminobenzidine as a chromogen. Light microscopic images were used to calculate the area occupied by Aβ immunoreactivity, and the cell densities of GFAP- and CD45-immunopositive reactions by using the software Image-Pro Plus for Windows version 5.0 (Media Cybernetics, Bethesda, MD). The threshold optical density that discriminated staining from background was determined and held constant for all quantifications. The area occupied by Aβ immunoreactivity was measured by the software and divided by the total area of interest to obtain the percentage area of immunoreactivity.

Electrophysiology

Six month old mice (n= # slices/# of animals): WT (n=23/8); 3xTg (n=21/7); 3xTg-5LOKO (n=20/6); 3xTg plus zileuton (n= 5/2) were sacrificed by rapid decapitation and brains placed into ice-cold artificial cerebral spinal fluid (ACSF) in which sucrose (248mM) was substituted for NaCl. Transverse hippocampal slices (400 μm thick) were cut using a Vibratome 3000 plus (Vibratome, Bannockburn, IL) and placed in ACSF (124 m MNaCl, 2.5 mM KCl, 2mM NaH2PO4, 2.5 mM CaCl2, 2 mM MgSO4, 10 mM dextrose, and 26 mM NaHCO3) at room temperature to recover for 1 hr bubbled with 95% O2/5% CO2. Slices were transferred to a recording chamber (Warner Instruments, Hamden, CT) and continuously perfused with ACSF at 1.5–2.0 ml/min flow, bubbled with 95% O2/5% CO2, and maintained by an in-line solution heater (TC-324; Warner Instruments) at 32–34 °C. We recorded field excitatory postsynaptic potentials (fEPSPs) from the CA1 stratum radiatum by using an extracellular glass pipette (3–5 MΩ) filled with ACSF. Schaffer collateral/commissural fibers in the stratum radiatum were stimulated with a bipolar tungsten electrode placed 200–300 μm from the recording pipette. Stimulation intensities were chosen to produce a fEPSP that was 1/3 of the maximum amplitude, based on an input/output curve using stimulations of 0–300 μA, in increments of 20μAs. Paired-pulse facilitation experiments were performed using a pair of stimuli of the same intensity delivered 20, 50, 100, 200 and 1000 ms apart. Baseline was recorded for 20 mins prior to tetanization with pulses every 30 seconds. LTP at CA3–CA1 synapses was induced by four trains of 100 Hz stimulation delivered in 20 second intervals. Recordings were made every 30 seconds for 3 hours following tetanization. The fEPSP rise/slope (mV/ms) between 30 and 90% was measured offline using Clampfit 10.3 (Molecular Devices, LLC) and normalized to the mean rise/slope of the baseline. Slices were eliminated if an unstable baseline was produced or if the normalized rise/slope dropped more than 20–50mV/ms in an approximately 10 min period. All the tests were always performed by an experimenter who was unaware of the different genotypes and treatment.

Data Analysis

One-way analysis of variance (ANOVA), unpaired Student’s t-test (two-sided) and Bonferroni multiple comparison tests were performed using Prism 5.0 (GraphPad Software, La Jolla, CA). All data are presented as mean +/− standard error of the mean. Significance was set at p <0.05.

Results

Genetic absence of 5LO ameliorates cognition

To assess the effect of 5LO genetic absence on behavior, mice were initially tested in the Y-maze at two different ages: 6–8 and 12–14 months old. As shown in Figure 1A, initially we did not notice any differences among the four groups of mice considered in regard to their general activity as assessed by the total number of arm entries for each group at both ages (Fig. 1A). When we considered the number of alternations in the same test, we observed that 3xTg mice had a much lower number of alternations resulting in a significant lower percentage when compared with wild type and 5LOKO mice, suggesting that they have impairment in their working memory. However, compared with the 3xTg mice, the 3xTg-5LOKO mice had a greater number of alternations resulting in a significantly higher percentage at both ages, suggesting an improvement of their working memory (Fig. 1B). Next, mice underwent fear conditioning testing, which is a measure of a 24 hr retention memory for both the cued and the contextual form. No differences among the groups of mice were observed during the training session (not shown). When the four groups of mice were subjected to contextual fear conditioning they did not manifest any significant differences (Fig. 1C). In the cued phase of the conditioning we observed that 5LOKO and wild type mice exhibited similar levels of freezing at both ages. On the other hand, 3xTg mice had significant lower freezing percentages which were normalized in the 3xTg5LOKO mice (Fig. 1D).

Figure 1

Genetic absence of 5LO ameliorates behavioral deficits of 3xTg mice. (A) Total number of arm entries for WT, 5LOKO, 3xTg and 3xTg-5LOKO mice at 6–8 and 12–14 months of age. (B) Percentage of alternations between the same groups of mice (*p <0.001). (C) Contextual fear memory response in WT, 5LOKO, 3xTg and 3xTg-5LOKO mice. (D) Cued recall fear memory response in the same groups of mice (6–8 months: n=8 (4M,4F) for WT, n=7 (4M, 3F) for 5LOKO, n=12 (6M, 6F) for 3xTg, n=13(7M, 6F) for 3xTg-5LOKO; 12–14 months: n=7(3M,4F) for WT, n= 7(4M, 3F) for 5LOKO, n=11 (6M,5F) for 3xTg, n=10 (4M, 6F) for 3xTg-5LOKO) (*p<0.001). Values represent mean +/− standard error of the mean.

Genetic absence of 5LO decreases brain Aβ level and deposition

Two weeks after completion of the last behavior tests (14 months of age), mice were sacrificed, brains harvested and assayed for Aβ levels and deposition. In comparing the two groups, we observed that 3xTg mice genetically deficient for 5LO displayed a significant decrease in the amount of RIPA-soluble and formic-acid soluble Aβ 1-40 and 1-42 (Fig. 2A). Similar results were obtained when brain tissues for 8-month old mice were assayed (Supplementary Fig. 1). Confirming the ELISA data, we found that absence of 5LO led also to a significant decrease of their brain Aβ immunoreactive areas (Fig. 2B). To explore for possible mechanisms responsible for this change, we assessed the steady-state levels of APP along with its cleavage products in the same samples. As shown in figure 2C, no differences were detected for total APP, the α-secretase (ADAM-10) and β-secretase (BACE-1) pathways between the two groups. By contrast, compared with controls, 3xTg-5LOKO mice had a significant decrease in the steady-state levels of the 4 components of the γ-secretase complex, PS1, nicastrin, Pen-2 and APH-1, which was associated with lower levels of C-terminal fragments (CTFs) (Fig. 2C, D).

Figure 2

Genetic absence of 5LO reduces Aβ peptide levels of deposition in brains of 3xTg mice. (A) Radioimmunoprecipitation assay (RIPA)-soluble and formic acid (FA)-extractable Aβ1-40 and 1-42 levels in cortex of 3xTg mice and 3xTg-5LOKO mice at 14 months of age [n=9 (5M, 4F)for 3xTg and n=10 (5M,5F) for 3xTg-5LOKO] (*p<0.001). (B) Quantification of the area occupied with Aβ immunoreactivity in the brain cortices of the same group of mice (*p<0.001). (C) Representative western blots of amyloid precursor protein (APP), ADAM-10, BACE-1, sAPPα, sAPPβ, CTFs, PS1, Nicastrin, APH-1 and Pen-2 in brain cortex homogenates from 14 month old 3xTg and 3xTg-5LOKO mice. (D) Densitometric analyses of the immunoreactivities to the antibodies shown in the panel C (*p<0.01) [n=4 (2M, 2F) for 3xTg; n=4 (2M, 2F) for 3xTg-5LOKO]. Values represent mean +/− standard error of the mean.

Genetic knockout of 5LO modulates tau metabolism

We then examined the effect of 5LO knockout on tau metabolism. As shown in Figure 3, while we did not observe any change in levels of total soluble tau between the two groups, compared with the 3xTg, the 3xTg-5LOKO mice had a significant decrease in its phosphorylated forms at epitopes S396, as recognized by the specific antibody PHF-13, at S396/S404 as recognized by the specific antibody PHF-1, and S202/T205, as recognized by the specific antibody AT8 (Fig. 3A, B). By contrast, no changes were detected for other phosphorylation sites as recognized by the antibodies AT180 (T231/S235) and AT270 (T181). Additionally, compared with 3xTg we observed that 3xTg-5LOKO had a significant reduction in the levels of insoluble tau (Fig. 3C,D). In accordance with the western blot results, immunohistochemical staining showed decreased somatodendritic accumulations of the phosphorylated epitopes recognized by the antibodies PHF-1, PHF-13 and AT-8 in the 3xTg-5LOKO mice (ratios: PHF-13/tau=0.57; PHF-1/tau=0.64; AT8/tau=0.52) (Fig. 3E).

Figure 3

5LO modulates tau phosphorylation and metabolism in the brains of 3xTg mice. (A) Representative Western blot analyses for total tau (HT7) and phosphorylated tau at residues S202/T205 (AT8), T231/S235 (AT180), T181(AT270), S396(PHF-13) and S396/S404(PHF-1) in brain cortex homogenates of 3xTg and 3xTg-5LOKO mice at 14 months of age. (B) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A (*p <0.01). (C) Representative Western blot analysis of sarkosyl-soluble tau (HT7) in brain cortex homogenates from the same mice. (D) Densitometric analyses of the immunoreactivities shown in panel D (*p <0.001). (E) Representative immunohistochemical stainings for AT8, PHF-13, PHF-1 positive areas in brain sections of 3xTg and 3xTg-5LOKO mice at 14 months of age. (F) Representative Western blot analyses for Cdk5, p35, p25, GSk3α, GSK3β, p-GSK3α, p-GSK3β, p38,p-p38, SAPK/JNK1, SAPK/JNK2, p-SAPK/JNK1 and p-SAPK/JNK2 protein levels in brain cortex homogenates from 3xTg and 3xTg-5LOKO mice 14 mo (G) Densitometric analyses of the immunoreactivities to the antibodies from the previous panel [3xTg, n=4 (2M,2F); 3xTg-5LOKO n=4 (2M,2F)] (*p<0.001). Values represent mean +/− standard error of the mean.

To explore the molecular mechanism responsible for the hypophosphorylation of tau, next we assayed some of the kinases which are considered major regulators of tau post-translational modification. In comparing the two groups of mice, we did not observe any significant differences in the levels of total and phosphorylated GSK3-α and GSK3-β, JNK2, total and phosphorylated p38 and total and phosphorylated SAPK/JNK (Fig. 3F). However, we found that compared with controls, 3xTg-5LOKO mice had a statistically significant decrease in levels Cdk5 kinase along with its coactivators p35 and p25 (Fig. 3F, G).

Absence of 5LO increases synaptic integrity

Since changes in tau phosphorylation state have been implicated in alterations of synaptic integrity in AD, next we assessed this aspect of the 3xTg mice phenotype. Compared with the control group, 3xTg-5LOKO mice had a significant increase in the steady state levels of two main synaptic proteins: post-synaptic density protein 95 (PSD-95) and synaptophysin (Fig. 4A, B). A similar result was obtained also when the dendritic protein MAP2 was assayed (Fig. 4A, B). These results were further confirmed in brain sections of the same mice when they were assessed by immunohistochemical analyses (Fig. 4C).

Figure 4

Genetic absence of 5LO ameliorates synaptic biomarkers and decreases neuroinflammation in 3xTg mice. (A) Representative Western blot analyses for synaptophysin (SYP), post-synaptic density protein 95 (PSD95) and MAP2 in brain cortex homogenates from wild type (WT), 3xTg and 3xTg-5LOKO mice. (B) Densitometric analyses of the immunoreactivities from panel A (*p<0.001). (C) Representative immunohistochemical staining for SYP, PSD95 and MAP2 positive areas in brain sections of WT, 3xTg and 3xTg-5LOKO mice. (D) Representative Western blot analyses for GFAP and CD45 in brain cortex homogenates from 3xTg and 3xTg-5LOKO mice. (D) Densitometric analyses of the immunoreactivities to the antibodies from the previous panel (3xTg, n=4; 3xTg-5LOKO; n=4), (*p<0.001). Values represent mean +/− standard error of the mean.

Finally, we observed that compared with brain homogenates from 3xTg, the ones from 3xTg-5LOKO mice had a significant decrease in GFAP and CD45 immunoreactivities, markers of astrocytes and microglia cells activation respectively (Fig. 4D, E).

Genetic knockout of 5LO ameliorates synaptic deficits

Since the absence of 5LO in 3xTg-AD mice yielded an improvement in memory at a very early stage of their AD-like phenotype (6 months; prior to plaque and tangle pathology), we then explored its effect on synaptic function at this age. To this end, first we investigated basal synaptic transmission by generating input/output (I/O) curves and measuring field-excitatory postsynaptic potentials (fEPSPs) elicited in CA1 by stimulation of the Schaffer collaterals at increasing strength of stimulations and intensities. As shown in Figure 5A, there were no differences observed in the I/O curves between any of the groups considered (WT, 3xTg, 3xTg-5LOKO).

Figure 5

Genetic absence or pharmacologic inhibition of 5LO rescues synaptic dysfunction in 3xTg mice. (A) Input/output (I/O) curves and representative fEPSPs at increasing stimulus strengths (0–300 μA) are shown for Wildtype, 3xTg, 3xTg/5LOKO and 3xTg+zileuton mice at 6 months of age. (B) Mean fEPSP slopes as a function of interpulse interval between the first and second fEPSPs evoked at CA3-CA1 synapses in slices from the same mice at 20, 50, 100, 200 and 1000 ms in the same animals. (C) fEPSP slopes were recorded for 3 hours and expressed as the percentage of pretetanus baseline in the same mice. (D) LTP magnitudes expressed as the percentages of baseline for 0–10 minutes post-tetanus [274.6% +/− 8.5% for WT (n=23 slices); 159.9% +/− 13.8% (n=19 slices) for 3xTg; 272.7% +/− 9.6% (n=20 slices) for 3xTg/5LOKO; 268.5% +/− 14.3% (n=5 slices) for 3xTg+zileuton)]. (E) For the same groups of mice, LTP magnitudes expressed as the percentages of baseline for 170–180 minutes post-tetanus (202.6% +/− 6.5% for WT; 121.0% +/− 3.4% for 3xTg; 197.1% +/− 11.5% for 3xTg/5LOKO; 156.9% +/− 6.9%; 199.5% +/− 7.4% for 3xTg+zileuton). (*p<0.0001). Values represent mean +/− standard error of the mean.

Next, we measured short-term plasticity by examining paired-pulse facilitation (PPF), which is due to an activity-dependent presynaptic modulation of transmitter release [16]. Similar to the observation in the I/O curves, there were no differences in PPF between any of the groups analyzed (Fig. 5B). Finally, we investigated long-term potentiation (LTP) in the CA1 region of the hippocampus, which is thought to be a measure of neuronal plasticity and a major player in cognition[17]. In this test we found that, compared with WT, 3xTg mice had a significant reduction in LTP responses. However, the genetic absence of 5LO in the 3xTg completely restored the LTP responses to a level comparable to that of the WT mice (Fig. 5C–E).

5LO Pharmacologic blockade improves memory and rescues synaptic deficits

To further confirm the involvement of the 5LO in the memory improvement and rescue of the pathological synaptic phenotype, 3xTg mice were randomized to receive zileuton, a selective and specific 5LO inhibitor in their drinking water for a month at a concentration we previously showed to completely block 5LO activation [10]. At the end of this period, mice underwent memory assessment in the Y-maze as well as the fear conditioning paradigm. As shown in supplemental figure 2, first we observed that zileuton had no effect on the general motor activities of these mice. By contrast, we observed that compared with vehicle group, 3xTg mice receiving zileuton had a significant improvement in the number of alternations, suggesting an improvement of their working memory (Supplemental Fig. 2). In the fear conditioning paradigm, while we did not observe any differences between the two groups in the training phase, compared with their controls 3xTg mice receiving zileuton had a significant increase in the freezing percentage time in the cued phase, but no changes were detected in the contextual phase of the conditioning (Supplemental Fig. 2).

Next, mice were sacrificed and their brains harvested for electrophysiology studies. First, we observed that 3xTg mice treated with zileuton did not differ from their controls in terms of basal synaptic transmission as measured by the field-excitatory postsynaptic potentials or short-term plasticity by examining paired-pulse facilitation (Fig. 5A,B). However, analysis of the LTP responses demonstrated that pharmacological blockade of 5LO was sufficient to restore the impairments noticed in the 3xTg mice back to levels indistinguishable from wild type animals (Fig. 5C–E).

Discussion

The data in the present paper unravel a new aspect of the neurobiology of 5LO by demonstrating its functional role in synaptic function and plasticity as well as memory. Together with the previous knowledge on this protein, they establish 5LO as a key player in the development of the full spectrum of the AD phenotype and an important therapeutic target with true disease-modifying potential for the treatment of AD. In recent years, there has been increasing evidence suggesting that alterations in synaptic integrity and function are the earliest phenotypic manifestations during the evolution of AD pathogenesis [18,19]. Thus, synaptic loss, as reflected by changes in synaptic markers, is a constant feature of early stage AD pathology, and better correlates with clinical cognitive impairments than classical assessment of Aβ or tau brain lesions [20,21]. However, the mechanisms involved in this phenomenon are still under investigation.

Recent developments in gene-targeted and transgenic mice represent an invaluable tool for modeling diverse aspects of the AD phenotype, including synaptic dysfunction and pathology. Notably, the 3xTg mice exhibit deficits in synaptic plasticity, such as LTP which occurs at an early age prior to extracellular Aβ deposition and tau pathology [5]. Previously, we showed that genetic manipulation or pharmacologic inhibition of the 5LO modulates the phenotype of the APP transgenic mice [9,10]. However, this mouse model represents a limited version of AD since it manifests mainly AD-like brain amyloidosis and lacks the tau pathology. The availability of the 3xTg mice offered us the unique opportunity to better explore the biological relevance of this pathway in the context of the AD pathogenesis. In the current paper, first we showed that genetic absence of 5LO per se did not influence any of the memory tests performed in our study. Next, we observed that compared with their controls, the 3xTg-5LOKO mice did not manifest any difference in general motor activities. By contrast, they had a significant improvement in their memory performance already at 6 months of age, as demonstrated in the Y-maze paradigm, which by recording spontaneous alternation behavior, assesses working memory in rodents [11]. Additionally, the same animals had an improvement in their learning memory ability, as assessed by the fear conditioning test. Thus, in this setting we observed that 3xTg mice performed significantly worse than the ones genetically deficient for the 5LO in the cue recall, but not in the contextual paradigm, suggesting a possible amygdala involvement.

Consistent with these results, we observed that compared with 3xTg, the 3xTg-5LOKO mice had a significant increase in 3 distinct protein markers of synaptic integrity (i.e., synaptophysin, PSD95 and MAP2) suggesting an improvement of this important function for memory and learning secondary to the 5LO deficiency.

In association to these changes, we observed a significant reduction in Aβ levels and deposition, which, confirming our previous report was secondary to an effect on the γ-secretase pathway [11]. Besides the effect on the Aβ pathology, we also observed that 5LO genetic absence had an influence on tau metabolism. Thus, we observed that genetic absence of 5LO resulted in a significant decrease in phosphorylated tau epitopes as recognized by the immunoreactivity of the specific antibodies PHF-1, PHF13 and AT8, but not for the other two tested, AT180 and AT270, and confirmed the cdk-5 kinase pathway but not other kinases or phosphatases were involved [11].

To corroborate the involvement of the 5LO in the improvement of the behavioral deficits we detected as early as at 6 months of age, we adopted a pharmacologic approach by administering a selective 5LO inhibitor to five-month old 3xTg mice for a month. The dosage selected was based on our previous published work where we showed that zileuton at this concentration significantly reduced 5LO activation up to 85% [10]. Similar to the genetic study, we observed that 3xTg mice receiving the active drug had a significant amelioration of their working memory and conditioning learning.

Because of the early improvements in memory deficits we noticed in both the 3xTg mice genetically deficient for 5LO and the ones administered with a 5LO inhibitor, next we assessed the effects that both conditions had on synaptic function by implementing an electrophysiological approach.

First, we investigated basal synaptic transmission by measuring field-excitatory postsynaptic potentials elicited in CA1 by stimulation of the Schaffer collaterals. We found that there were no differences in this parameter amongst the four groups of mice, suggesting that synaptic transmission is not altered in any of the conditions implemented. Next, we measured short-term plasticity by examining paired-pulse facilitation, which is secondary to an activity-dependent presynaptic modulation of transmitter release [16]. Similar to the observation for the basal synaptic transmission, there were no significant differences in paired-pulse facilitation among any of the groups investigated. These results suggest that there is not an increase or decrease in the probability of transmitter release in any of these groups of mice [5,22].

Finally, we assessed the LTP response which is a type of plasticity that is thought to play a major role in learning and memory functions. As reported previously [5], there was a significant difference in LTP responses between the wild type and 3xTg mice with the latter showing deficits. However, the genetic absence of 5LO in the 3xTg mice was sufficient to restore their LTP responses to a level comparable to the ones measured in the wild type mice. The biological importance of this finding was further corroborated by the demonstration that 5LO pharmacologic blockade was also sufficient to rescue the abnormal synaptic phenotype of the 3xTg mice. Taken together these data unravel a new aspect of the neurobiology of 5LO pathway by demonstrating its functional role in synaptic function and plasticity as well as memory and learning.

Interestingly, the above described 5LO-dependent beneficial effects on the LTP parameters are in line with previously reported enhancements of the GluR1/AMPA receptor phosphorylation by 5LO genetic absence or pharmacologic inhibition [23,24]. Importantly, despite the fact that some of the 5LO biologic effects are mediated by an involvement of the γ-secretase pathway, no alteration of the Notch signaling has been reported [10]. This observation makes any potential Notch-related side effect secondary to a chronic 5LO inhibition unlikely.

Considering the anti-Aβ and tau effect of 5LO, our findings have important patho-physiological implications for AD since they establish this pathway as a major active contributor to all of the aspects of the disease phenotype. This new information by demonstrating the pleiotropic role of this protein in AD pathogenesis makes it not only a valid pharmacological target, since 5LO inhibitors are already FDA approved, but most importantly represents a unique therapeutic opportunity with a true disease-modifying potential for the treatment of AD.

Supplementary Figure 1

Genetic absence of 5LO affects Aβ peptide levels of deposition in brains of 8month old 3xTg mice. (A) Radioimmunoprecipitation assay (RIPA)-soluble and formic acid (FA)-extractable Aβ1-40 and 1-42 levels in brain cortex of 3xTg mice and 3xTg-5LOKO mice at 8 months of age [n=8 (5M, 3F) for 3xTg and n=9 (4M, 5F) for 3xTg-5LOKO] (*p<0.001). Values represent mean +/− standard error of the mean.

Supplementary Figure 2

Pharmacological blockade of 5LO improves memory in 3xTg mice. (A) Total number of arm entries for 3xTg and 3xTg+zileuton mice at 6 months of age (B) Percentage of alternations between the same groups of mice (*p <0.001). (C) Contextual fear memory response in 3xTg and 3xTg+zileuton mice. (D) Cued recall fear memory response in the same groups of mice [n=4 (2M, 2F) for 3xTg, n=4 (2M, 2F) for 3xTg+zileuton (*p<0.001). Values represent mean +/− standard error of the mean.