Address correspondence and reprint requests to Dr Luca Ferraro, Department of Life Sciences and Biotechnology (SVEB), University of Ferrara, Via Fossato di Mortara 17-19, Ferrara, Italy. E-mail: firstname.lastname@example.org
CHF5074, a new microglial modulator, attenuates memory deficit in Alzheimer's disease transgenic mice. In this study, the effect of an acute or subacute CHF5074 treatment on in vivo novel object recognition test and on [3H]Acetylcholine (ACh) and GABA release in pre-plaque (7-month-old) Tg2576 mice have been compared with those induced by the γ-secretase inhibitor LY450139 (semagacestat). Vehicle-treated Tg2576 mice displayed an impairment of recognition memory compared with wild-type animals. This impairment was recovered in transgenic animals acutely treated with CHF5074 (30 mg/kg), while LY450139 (1, 3, 10 mg/kg) was ineffective. In frontal cortex synaptosomes from vehicle-treated Tg2576 mice, K+-evoked [3H]ACh release was lower than that measured in wild-type mice. This reduction was absent in transgenic animals subacutely treated with CHF5074 (30 mg/kg daily for 8 days), while it was slightly, not significantly, amplified by LY450139 (3 mg/kg daily for 8 days). There were no differences between the groups on spontaneous [3H]ACh release as well as spontaneous and K+-evoked GABA release. These results suggest that CHF5074 has beneficial effects on visual memory and cortical cholinergic dysfunctions in pre-plaque Tg2576 mice. Together with previous findings, these data suggest that CHF5074 could be a possible candidate for early Alzheimer's disease therapeutic regimens.
Alzheimer's disease (AD) is a progressive, neurodegenerative disease characterized by memory and cognitive loss, the formation of senile plaques containing amyloid-beta (Aβ) peptide, degeneration of cholinergic neurons, the development of neurofibrillary tangles, and neuroinflammation (Arendt 2009). The so-called amyloid cascade (Hardy and Allsop 1991; Hardy and Higgins 1992) has long been linked to the pathogenesis of AD. According to this hypothesis, based on studies relying on familial AD patients with mutations in the Aβ (amyloid) precursor protein (APP) or the catalytic subunits of the γ-secretase complex, responsible for the second (Aβ-generating) step of APP processing, the abnormal processing of APP leads to the formation of toxic and self-aggregating Aβ oligomers (Selkoe 2008; Shankar et al. 2008). However, this etiopathogenetic hypothesis has been recently challenged by the failure of several clinical trials testing different drugs targeting Aβ accumulation in the brain (Mangialasche et al. 2010). Moreover, the severity of cognitive impairment is poorly correlated with plaque deposition either in AD patients (Blennow et al. 1996; Berg et al. 1998; Giannakopoulos et al. 2003, 2009) or in animal models of AD (Puoliväli et al. 2002; Christensen et al. 2008; Watanabe et al. 2009; Zhang et al. 2012). On the contrary, soluble Aβ level has been found to correlate with disease progression or decline in synaptic density in AD patients (Lue et al. 1999; McLean et al. 1999). This has led to redirect the research toward new mechanisms possibly underlying cognitive impairment in AD.
Synaptic dysfunction by Aβ oligomers could be involved in memory impairment in the early phase of AD (Arendt 2009). Increased oxidative stress and cholinergic dysfunction might occur well before amyloid plaque formation in transgenic animal models of AD (Knobloch et al. 2007; Christensen et al. 2008; Jazi et al. 2009; Watanabe et al. 2009; Zhang et al. 2012). Taken together, these findings indicate that cholinergic dysfunction possibly induced by soluble forms of Aβ could play a role in early AD cognitive impairment. This is also in line with the findings that, in early AD, memory impairment could be because of cholinergic dysfunctions rather than cholinergic cell loss (for a review, see Schliebs and Arendt 2011). Thus, drugs able to correct the malfunction of the cholinergic system may offer new hopes of successfully treating cognitive decline in early AD.
CHF5074 [1-(3′,4′-dichloro-2-fluoro[1,1′-biphenyl]-4-yl)-cyclopropanecarboxylic acid] is a compound devoid of cyclooxygenase inhibitory activity, but still able to reduce microglia activation in vivo (Peretto et al. 2005; Imbimbo et al. 2009). In transgenic mouse models of AD, CHF5074 has been shown to prevent brain plaque deposition without affecting Notch processing (Imbimbo et al. 2007; Wolfe 2012). In young, plaque-free transgenic mice, chronic oral administration of CHF5074 reversed different types of memory deficits (Imbimbo et al. 2007, 2009, 2010). In 5-month-old Tg2576 mice, acute subcutaneous administration of CHF5074 (30 mg/kg) significantly attenuated contextual memory impairment (Balducci et al. 2011). The beneficial effects of a subchronic CHF5074 treatment in young transgenic mice occurred at a stage that precedes plaque formation and were associated with a reduction in intraneuronal APP/Aβ and hyperphosphorylated tau (Balducci et al. 2011). Although these data suggest that CHF5074 reduces early cognitive deficits in transgenic mouse models of AD, there is no evidence as to its efficacy on cholinergic neuron dysfunction, which can take place along with intraneuronal Aβ accumulation, well before plaque deposition (Watanabe et al. 2009). Thus, in this study, we have investigated the effects of an acute or subacute CHF5074 treatment on in vivo cognitive performance and on [3H]acetylcholine (ACh) release from isolated nerve terminals (synaptosomes) obtained from 7-month-old transgenic Tg2576 mice expressing the Swedish mutations of human APP. This animal age corresponds to the presence of cognitive defects and a rapid rise in soluble forms of Aβ, while plaque deposition is not yet evident (Kawarabayashi et al. 2001; Westerman et al. 2002). We also measured the effects of CHF5074 on GABA and glutamate release from mouse frontal cortex synaptosomes to evaluate whether the drug possesses a widespread action on cortical neurotransmission or preferentially influences cholinergic signaling. Finally, the behavioral and neurochemical effects of CHF5074 have been compared with those induced, under the same experimental conditions, by the γ-secretase inhibitor LY450139 (semagacestat) (Henley et al. 2009).
Materials and methods
Animals and treatments
Tg2576 transgenic mice carry a transgene coding for the 695-amino acid isoform of human APP derived from a large Swedish family with early-onset AD (Hsiao et al. 1996). These mice express high concentrations of the mutant Aβ, develop significant number of amyloid plaques, and display memory deficits. Tg2576 mice and their non-transgenic (i.e., wild-type) littermates (001349-W), which served as controls, were purchased from Taconic Europe (Lille Skensved, Denmark).
Seven-month-old transgenic females and aged-matched non-transgenic littermates were used. For acute behavioral experiments, animals were injected with CHF5074 (30 mg/kg) or LY450139 (1, 3, 10 mg/kg), 24 and 4 h before the habituation trial. Thus, for subacute neurochemistry experiments, the animals were injected daily with CHF5074 (30 mg/kg, s.c.), LY450139 (3 mg/kg, s.c.), or their respective vehicles (Methocel 0.5% and Cromophor 10%) for 8 days. The CHF5074 doses were effective in memory performance, tested with novel object recognition and other tests, in subchronic and chronic treatments (Imbimbo et al. 2010; Balducci et al. 2011). The LY450139 doses have been used in other cognitive tests (Mitani et al. 2012). The number of animals included in each groups in each experiment is indicated in the legend to the respective figure.
Animal care and treatments were in accordance with the EU Directive 2010/63/EU for animal experiments and in conformity with protocols approved by the Ethical Committee of Animal Experimentation, University of Bologna.
Long-term memory under spontaneous behavioral conditions was evaluated using novel object recognition (NOR). For this test, 12 animals were included in each group. Before experiments, all animals were tested for the papillary reflex and only positive animals were included in the study. Mice were tested in an open-square gray arena (46 × 46 cm), 30-cm high (Ugo Basile, Comerio, Italy). The task started with a habituation trial in which the animals were placed into the empty arena for 10 min. The next day, mice were placed into the same arena containing two identical objects (familiarization phase). To evidence side preferences, exploring times spent on left and right familiar objects were recorded separately. The exploratory behavior was analyzed by calculating the investigation time on both objects. Sniffing and touching the object at a distance not greater than 2 cm were scored as object investigation. Four hours later (test trial), mice were placed in the arena containing one object identical to the one presented during the familiarization phase (familiar object), and a new one (novel object); the time spent exploring the two objects was recorded for 10 min. The videotracking software AnyMaze (Stoelting, Wood Dale, IL, USA) was used for analysis. Memory was expressed as discrimination index: (seconds on novel – seconds on familiar)/(total time on objects). Animals with no memory impairment spent longer time investigating the novel object, giving a higher discrimination index.
Frontal cortex synaptosome preparation
Synaptosomes were prepared from frontal cortices of wild-type and Tg2576 transgenic mice. Briefly, the animals were killed, the brains were removed, and frontal cortices were rapidly dissected out. Thereafter, a crude synaptosomal (P2) fraction was prepared as follows: the tissue was suspended in ice-cold buffered sucrose solution (0.32 M, pH 7.4) and homogenized. The homogenate was centrifuged (10 min; 2500 g, 4°C) to remove nuclei and debris, and then synaptosomes were isolated from the supernatant by centrifugation (20 min; 9500 g, 4°C). The P2 pellet was then resuspended in 7 mL of Kreb's solution (mM: NaCl 118.5, KCl 4.7, CaCl2 1.2, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 10; gassed with 95% O2, and 5% CO2).
Spontaneous and K+-evoked [3H]-acetylcholine, GABA, and glutamate release
After the preparation, synaptosomes were incubated (37°C, 20 min) in the presence of [methyl-3H] choline chloride (50 nM; 10 Ci/mmol−1). At the end of this period, identical aliquots of synaptosomal suspension were distributed on microporous filters (0.5 mL/filter), placed at the bottom of a set of parallel superfusion chambers maintained at 37°C, and perfused with aerated (95% O2/5% CO2) Kreb's solution (0.3 mL/min) containing hemicolinium-3 (10 μM) to prevent choline uptake (Moreno et al. 2011). After 30 min of superfusion, to equilibrate the system, 5-min fractions were collected from the 30th to the 75th minute (nine samples). After the collection of three basal samples (used to assess spontaneous neurotransmitter release), synaptosomes were depolarized with 15 mM K+ (substituting for an equimolar concentration of NaCl) for 90 s (Ferraro et al. 2010). This depolarizing stimulus was chosen because it had been previously reported that neurotransmitter release evoked by depolarization of synaptosomes with 12–15 mM KCl occurs almost completely by Ca2+-dependent exocytosis (Raiteri et al. 2009). At the end of the experiment, the radioactivity of the samples and filters was determined by liquid scintillation spectrometry. Aliquots of 100 µL were taken from each sample to measure endogenous GABA levels. In view of the results obtained, in a final set of experiments, the effects of CHF5074 on endogenous glutamate release have also been evaluated.
[3H]-acetylcholine release: data evaluation
The amount of radioactivity released into each fraction was expressed as a percentage of the total synaptosomal tritium present at the start of the respective collection period (fractional release). The K+-evoked release was calculated as net extra-release by subtracting the basal [3H] efflux (determined by interpolation of the outflow measured 5 min before and 10 min after the onset of the stimulation) from the total tritium released during the 90-s stimulation and subsequent 8.5-min washout (i.e., two samples). This difference was calculated as a percentage of the total tissue tritium content at the onset of stimulation (fractional rate net extra-outflow; Ferraro et al. 2010). As the radioactivity released by high-K stimulation from synaptosomes pre-labeled with [3H]Choline consists largely of unmetabolized [3H]ACh (see 'Animals and treatments'), no attempt was made to separate labeled choline from ACh.
Determination of endogenous GABA and glutamate levels and data evaluation
Endogenous GABA levels were measured by HPLC coupled to fluorometric detection. Briefly, 25-μL samples were pipetted into glass microvials and placed in a thermostated (4°C) Triathlon autosampler (Spark Holland, Emmen, The Netherlands). Thirty microliters of o-phthaldialdehyde/mercaptoethanol reagent was added to each sample, and 30 μL of the mixture injected onto a Chromsep analytical column (3-mm inner diameter, 10-cm length; Chrompack, Middelburg, The Netherlands). The column was eluted at a flow rate of 0.48 mL/min (Beckman125 pump; Beckman Instruments, Fullerton, CA, USA) with a mobile phase containing 0.1 M sodium acetate, 10% methanol, and 2.2% tetrahydrofuran (pH 6.5). GABA was detected using a Jasco fluorescence spectrophotometer FP-2020 Plus (Jasco, Tokyo, Japan). GABA retention time was ~ 15.0 min.
Endogenous glutamate levels were quantified using a HPLC/fluorimetric detection system, including pre-column derivatization with o-phthaldialdehyde reagent and a Chromsep 5 (C18) column. The mobile phase consisted of 0.1 M sodium acetate, methanol (100 ml/L), and tetrahydrofurane (22 ml/L), pH 6.5. The limit of detection for glutamate was 30 fmol/sample.
Spontaneous GABA levels in each sample were expressed in nmol/min/g of protein (Chiodi et al. 2012). Protein was determined according to Bradford (1976). The effects of K+ stimulation on endogenous extracellular GABA and glutamate levels during the third fraction were reported and expressed as percentage changes of basal values, as calculated by the means of the two fractions collected prior to treatment.
Statistical analysis was performed using anova followed by Dunnet's test or Newman–Keuls test for multiple comparisons. Statistical significance was assumed for p <0.05.
Novel object recognition memory in 7-month-old wild-type and Tg2576 mice acutely treated with CHF5074 or LY450139
Object recognition memory, measured by the NOR task after two administrations (24 and 4 h before the training session) of CHF5074 (30 mg/kg s.c.) or LY450139 (1, 3, 10 mg/kg s.c.), was evaluated as an indicator of efficacy. As shown in Fig. 1, vehicle-treated Tg2576 mice displayed a significant impairment of recognition memory compared with wild-type animals (Student's t test: p =0.039). This impairment was fully recovered in transgenic animals treated with CHF5074, while it was not significantly modified by LY450139 at all doses tested (one-way anova and post hoc Dunnet's test: p <0.05). Both drugs did not affect recognition memory in wild-type mice and training-phase performances.
[3H]ACh and GABA release from frontal cortex synaptosomes from 7-month-old wild-type and Tg2576 mice subacutely treated with CHF5074 or LY450139
In a further series of experiments, the effects of a subacute treatment with CHF5074 (30 mg/kg s.c. daily; 8 days) or LY450139 (3 mg/kg s.c. daily; 8 days) on spontaneous and K+-evoked [3H]ACh and endogenous GABA release from synaptosomes obtained from wild-type and Tg2576 mice have been evaluated. Control groups were subacutely treated with the drug vehicles (Methocel or Cromophor, respectively).
As reported in Table 1, under basal conditions, there were no significant differences on spontaneous [3H]ACh and GABA release between synaptosomes obtained from the investigated groups.
Table 1. Spontaneous [3H]Acetylcholine (ACh) and GABA release from frontal cortex synaptosomes prepared from wild-type (WT) and Tg2576 subacutely treated with CHF5074 (30 mg/kg s.c.; 8 days) or LY450139 (3 mg/kg s.c.; 8 days). Control mice were treated with the respective drug vehicle (Methocel 0.5% or Cromophor 10%)
Spontaneous neurotransmitter release
The data presented are means ± SEM of eight to nine animals.
[3H]ACh (Fractional rate%)
4.35 ± 0.54
4.41 ± 0.53
3.98 ± 0.48
4.46 ± 0.34
4.38 ± 0.57
4.35 ± 0.44
4.11 ± 0.56
4.29 ± 0.41
GABA (nmol/min/mg protein)
1.98 ± 0.21
2.02 ± 0.0.20
1.78 ± 0.25
1.81 ± 0.27
1.94 ± 0.26
1.98 ± 0.24
1.90 ± 0.22
1.78 ± 0.28
High K+ stimulation increased [3H]ACh release from cortical synaptosomes prepared from all experimental groups under investigation. However, in cortical synaptosomes from vehicle-treated Tg2576 mice, the K+-evoked [3H]ACh release was lower compared with that obtained from wild-type mice (Fig. 2a and b). This reduction was fully recovered in transgenic animals subacutely treated with CHF5074 (Fig. 2a), while it was slightly, not significantly, amplified by LY450139 treatment (Fig. 2b). Both drugs did not affect K+-evoked [3H]ACh release in synaptosomes prepared from the frontal cortex of wild-type mice. HPLC analysis by using the method described by Karanth et al. (2006) demonstrated that the radioactivity released by high K+ stimulation from synaptosomes pre-labelled with [3H]Choline was shown to consist largely of unmetabolized [3H]ACh (data not shown).
No differences between the groups were found on K+-evoked GABA release from frontal cortex synaptosomes (Fig. 3). In a final set of experiments, the effects of CHF5074 on endogenous glutamate release have also been evaluated. There were no differences in endogenous glutamate release between Tg2576 and wild-type mice (data not shown). Furthermore, the subacute treatment with CHF5074 failed to affect K+-evoked glutamate release in synaptosomes from wild-type and Tg2576 mice (wild-type: vehicle = 160 ± 7%, CHF5074 = 152 ± 8%; Tg2576: vehicle = 148 ± 7%, CHF5074 = 147 ± 9% of basal values).
Several studies indicate that synaptic dysfunction is an early event in AD (Schliebs and Arendt 2011; Ardiles et al. 2012; Zhang et al. 2012) and occurs before the formation of amyloid plaques and neurofibrillary tangles (Arendt 2009; Penzes and Vanleeuwen 2011; Schliebs and Arendt 2011). In particular, the appearance of cholinergic neuritic dystrophy, that is, aberrant fibers and fiber swelling are widely common in AD (Schliebs and Arendt 2011). It has been recently suggested that memory impairment in plaque-free Tg2576 mice may be because of cholinergic synapse dysfunction rather than amyloid plaque deposition (Watanabe et al. 2009). Thus, we used Tg2576 mice to compare the effects of CHF5074 and LY450139 (semagacestat) on cognition and pre-synaptic cortical ACh release. We firstly demonstrated that high K+-evoked [3H]ACh release from frontal cortex isolated nerve terminals is reduced in 7-month-old Tg2576 mice as compared with their non-transgenic littermates. In contrast, spontaneous [3H]ACh release was unaltered in Tg2576 mouse frontal cortex synaptosomes, thus suggesting that cholinergic synapses are not damaged. A previous study demonstrated a reduction in high-affinity choline uptake sites in anterior cortical brain regions of 5-month-old Tg2676 mice as compared with wild-type animals (Klingner et al. 2003). However, that study was performed using a single ligand concentration, so the observed changes in binding sites may, therefore, be because of alterations in both choline uptake site number and/or affinity. The possibility that a reduced [3H]ACh uptake is responsible for the present results seems unlikely as: (i) we failed to observe a significant difference in spontaneous [3H]ACh release between synaptosomes from Tg2576 and wild-type mice, (ii) the release experiments have been performed in the presence of hemicolinium-3 to prevent choline uptake (Moreno et al. 2011). Taken together, the present data suggest that in pre-plaque Tg2576 mice, there are cortical cholinergic dysfunctions mainly characterized by the inability of cholinergic nerve endings to provide adequate ACh release in response to incoming depolarizing stimuli. These findings appear to be confirmed by the report that 5-month-old Tg2676 mice present deficit in cholinergic markers, but no signs of cortical degeneration of cholinergic fibers (Klingner et al. 2003). In line with the present results, Watanabe et al. (2009) found a reduction of high K+-evoked, but not in basal, hippocampal ACh release in 9- to 11-month-old Tg2576 mice.
The present results also suggest that the cortical synaptic dysfunction in 7-month-old Tg2576 mice could particularly affect cholinergic neurons, as no alterations in basal and K+-evoked GABA and glutamate release were found. The reduction in frontal cortex K+-evoked [3H]ACh release correlates with a significant impairment of recognition memory in Tg2576 mice, as measured by the NOR test. This test is widely used for memory evaluation in mouse models of AD (Mouri et al. 2007; Scholtzova et al. 2008; Balducci et al. 2011). Several previous studies also reported cognitive dysfunctions in pre-plaque Tg2576 mice (Westerman et al. 2002; Klingner et al. 2003; Comery et al. 2005; Dong et al. 2005; Watanabe et al. 2009; Balducci et al. 2011; Heredia et al. 2012; Mitani et al. 2012).
The main finding of this study is that the cognitive impairment and the reduction of K+-evoked [3H]ACh release from the frontal cortex of Tg2576 mice were completely reversed by subacute treatment with CHF5074. A previous study indicated that CHF5074 treatment may attenuate or reverse contextual memory deficit when given acutely to 5-month-old Tg2576 mice at the same dose (30 mg/kg, s.c.) used in the present experiments (Balducci et al. 2011). Our study extends these data demonstrating that the compound improves, besides contextual memory, also visual memory performance in pre-plaque Tg2576 mice. A CHF5074-induced reduction of spatial memory deficit was similarly observed following chronic treatment of a different mouse model of AD (TADS-41; Imbimbo et al. 2009). Finally, a reversal of object recognition memory as well as hippocampal plasticity deficits were also observed on CHF5074 subchronic treatment of 6-month-old Tg2576 mice (Balducci et al. 2011).
The present data are the first to demonstrate a positive effect of CHF5074 on cortical cholinergic dysfunction in an animal model of AD and raise the exciting hypothesis that a functional recovery of cortical cholinergic synapses could underlie the beneficial effects of the drug on cognition.
Contrarily to CHF5074, LY450139 treatment was ineffective in improving object recognition memory performance in Tg2576 mice. Furthermore, a trend to aggravate the cortical pre-synaptic cholinergic dysfunction in transgenic animals was observed after the subacute LY450139 treatment. Although not significant, this latter result correlates with the worsening of cognition observed in AD patients treated with this γ-secretase inhibitor (Schor 2011). Consistent with this, it has been very recently demonstrated that subchronic treatment with LY450139 impaired normal cognition in young Tg2576 mice (Mitani et al. 2012).
Previous studies (Imbimbo et al. 2010) indicated that chronic treatment with CHF5074 caused a twofold increase in synaptophysin levels in Tg2576 mouse cerebral cortex. As synaptophysin is a protein isolated from pre-synaptic vesicles of neurons and correlates with synaptic density, it cannot be ruled out that this neurotrophic effect could be involved in CHF5074-induced recovery of ACh release from cortical nerve terminals. Finally, mounting evidence suggests that reduced cholinergic activity/signaling in AD can promote the β-amyloidogenic pathway of APP processing, leading to increased Aβ levels and thus creating a sort of a positive feedback or a vicious cycle to accelerate AD pathogenesis (Cheng et al. 2010). In this context, the effects of CHF5074 on cortical cholinergic transmission could also be involved in the ability of the compound to attenuate brain β-amyloid pathology and ameliorate memory impairments (Imbimbo et al. 2009). However, several molecular mechanisms of Aβ synaptotoxicity have been proposed in the literature and there is no agreement about what mechanism, if any, is most relevant to AD (Benilova et al. 2012). Thus, at present, the precise mechanism of action of CHF5074 remains unclear as is any putative relationship with human AD.
In conclusion, this study indicates that the positive effect of CHF5074 on learning and memory in pre-plaque Tg2576 mice is associated with the restore of K+-evoked ACh release from cortical nerve terminals, which is decreased in vehicle-treated Tg2576 compared with wild-type animals. On the contrary, the γ-secretase inhibitor LY450139 has no effect on behavior and ACh release. Taken together, these findings suggest CHF5074 as a possible candidate for early AD therapeutic regimens.
The research has been supported by a grant from Emilia-Romagna Region (‘From industrial districts to technological districts’, DGR 163/2009 – project title: innovative technologies for the causal treatment of neurodegenerative diseases).
Conflict of interest
B.P. Imbimbo is an employee of Chiesi Farmaceutici.