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Impaired brain development and reduced cognitive function in phospholipase D-deficient mice
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Impaired brain development and reduced cognitive function in phospholipase D-deficient mice
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Ute Burkhardt a , David Stegner c , Elke Hattingen b , Sandra Beyer a , Bernhard Nieswandt c , Jochen Klein a,∗ a
Department of Pharmacology, Goethe University College of Pharmacy, Frankfurt, Germany Institute of Neuroradiology, Goethe University Medical Center, Frankfurt, Germany Q2 c Vascular Medicine, University Hospital Würzburg & Rudolf Virchow Center, University of Würzburg, Germany b
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h i g h l i g h t s • • • •
Genetic deletion of phospholipase D 1 or 2 delays brain development in mice. Cognitive function is reduced in adult PLD-deficient mice. PLD deficiency impairs release of acetylcholine after behavioral stimulation. Disruption of PLD signaling may contribute to fetal alcohol syndrome and Alzheimer’ disease.
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Article history: Received 28 February 2014 Received in revised form 29 April 2014 Accepted 30 April 2014 Available online xxx
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Keywords: Acetylcholine Alzheimer’ disease Brain growth spurt Fetal alcohol syndrome Microdialysis Phospholipase D
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1. Introduction
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The phospholipases D (PLD1 and 2) are signaling enzymes that catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid, a lipid second messenger involved in cell proliferation, and choline, a precursor of acetylcholine (ACh). In the present study, we investigated development and cognitive function in mice that were deficient for PLD1, or PLD2, or both. We found that PLD-deficient mice had reduced brain growth at 14–27 days post partum when compared to wild-type mice. In adult PLD-deficient mice, cognitive function was impaired in social and object recognition tasks. Using brain microdialysis, we found that wild-type mice responded with a 4-fold increase of hippocampal ACh release upon behavioral stimulation in the open field, while PLD-deficient mice released significantly less ACh. These results may be relevant for cognitive dysfunctions observed in fetal alcohol syndrome and in Alzheimer’ disease. © 2014 Published by Elsevier Ireland Ltd.
The phospholipases D (PLD) are ubiquitous enzymes which catalyze the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. Phosphatidic acid is a lipid second messenger which regulates cytoskeletal organization and vesicular trafficking and is involved in cell proliferation. Choline is a precursor of acetylcholine (ACh) synthesis and of choline-containing phospholipids
Abbreviations: ACh, acetylcholine; ACSF, artificial cerebrospinal fluid; MRI, magnetic resonance imaging; ORT, object recognition task; PA, phosphatidic acid; PC, phosphatidylcholine; PLD, phospholipase; DSRT, social recognition task. ∗ Corresponding author at: Goethe University College of Pharmacy, Max-vonLaue-Str. 9, 60438 Frankfurt, Germany. Tel.: +49 69 798 29366; fax: +49 69 798 29277. E-mail address: [email protected] (J. Klein).
such as PC and sphingomyelin. The two major mammalian isoforms of PLD are PLD1 and PLD2. PLD1 is located in the perinuclear region, is activated by small GTPases such as ARF and Rho and participates in budding and fusion of secretory vesicles and in stress fiber formation. In contrast, PLD2 is located at the cellular membrane, shows high basal activity, is regulated by tyrosine kinases and protein kinase C and participates in receptor endocytosis (reviewed in [20]). PLD1 and 2 are capable of transphosphatidylation, a reaction in which PC is transformed into phosphatidylalcohols (e.g., phosphatidylethanol) when alcohols (e.g. ethanol) are present; thus, the PLD signaling pathway is suppressed in the presence of alcohols because PA formation is reduced [1,14]. Numerous studies have implicated PLD activation in the control of cell proliferation [9]. Importantly, several lines of evidence support a role of PLDs in cognitive function or dysfunction. Previous work of our group and others demonstrated a role for PLD in early
http://dx.doi.org/10.1016/j.neulet.2014.04.052 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: U. Burkhardt, et al., Impaired brain development and reduced cognitive function in phospholipase D-deficient mice, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.052
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development of the brain. In fact, growth factors stimulate PLD activity in neurons and astrocytes which results in cell proliferation [19,26]. Astrocytes interact closely with neurons during the brain growth spurt enabling neuritogenesis and formation of synapses, two processes in which PLDs are involved [13]. Accordingly, interruption of PLD signaling, e.g. by alcohols, may inhibit normal brain development [14] by inhibiting the formation of the lipid messenger, phosphatidic acid; this mechanism may be partly responsible for ethanol toxicity to the fetus (“fetal alcohol syndrome”) [1]. There is also evidence that links PLD to neurotransmitter release, especially in cholinergic neurons. In whole cells, PLD activity can be activated by muscarinic receptors [14]. In synaptosomes, PLD is activated by depolarization and by calcium influx [22,28]. Both PLD isoforms may be involved in neurotransmitter release. In one study, catalytically inactive PLD1 mutants (K898R) reduced ACh release in Aplysia neurons by reducing the number of active presynaptic releasing sites [12]. In another study, PLD2 was responsible for the generation of choline for ACh synthesis, and downregulation of PLD2 led to reduce ACh release in cholinergic neurons [29]. Another line of investigation links PLD activities to Alzheimer’ disease (AD), which is also characterized by cognitive deficits and by widespread cholinergic dysfunction. PLD activity was reduced in cells with AD-like mutations, and overexpression of PLD1 in these cells corrected trafficking alterations of the amyloid precursor protein [2]. In contrast, amyloid  toxicity in cell cultures was promoted by PLD2 activity [18]. In addition, a third PLD form (“PLD3”) was associated with Alzheimer’ disease in a recent genome-wide association study [5], but further characterization of this new isoform is required to address its potential role in lipid signaling. In the present study, we made use of recently developed mouse models that are deficient in PLD1 or PLD2. In light of the previous reports summarized above, we investigated brain growth in juvenile PLD-deficient mice and tested cognitive function and cholinergic activity in adult mice in vivo. We found that characteristic impairments can be seen in both PLD-deficient strains, with stronger effects in PLD2-deficient mice.
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2. Materials and methods
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2.1. Materials
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Chemicals for artificial cerebrospinal fluid (aCSF; 147 mM NaCl, 4 mM KCl, 1.2 mM CaCl2 , and 1.2 mM MgCl2 ) were obtained from Sigma–Aldrich (Seelze, Germany). Neostigmine bromide was obtained from Acros Organics (Thermo Fisher Scientific, Schwerte, Germany) and scopolamine bromide from TCI Europe (Eschborn, Germany). Ketamine/xylazine was prepared with Rompun® 2% (Bayer Co.) and Ketavet® 100 mg/ml (Pfizer Co.).
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2.2. Animals
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Gender-mixed Pld1−/− , Pld2−/− mice (generated on a B6 background) and wild-type controls [7,27] were housed in a facility with controlled temperature and humidity and a day/night cycle of 12/12 h. They had free access to food and water. All animal experiments were performed in agreement with EU directive 2010/63 and were registered with the local animal committee (Regierungspräsidium Darmstadt, Germany).
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2.3. Magnetic resonance imaging (MRI)
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The MRI study was performed in a 3 T Magnetom® Trio (Siemens, Erlangen, Germany) with circular polarized wrist coil. The mice were assessed once a week over a time period of 5 weeks beginning at the age of P7. The litter size of all groups was 6–8 pups and the mothers had free access to food and water. Prior to
MRI, pups were anesthetized with ketamine/xylazine (70/7 mg/kg body weight i.p.) and kept warm throughout the entire measurement. The brain size of the pups was measured by MRI in 19–20 transversal T2-weighted spin echo sequences with a slice thickness of 0.9 mm without gap and an in-plane resolution of 0.2 × 0.2 mm. The measurements lasted around 9 min. Resulting DICOMs were analyzed by marking the brain area in each slice using free MRIcro software. The brain volume was calculated by addition of brain areas (cortices) in all slices. 2.4. Behavioral testing 3–4 months old mice were placed in the behavioral testing room at least 1 week before the experiments took place. Behavioral testing was performed during their active time (night cycle) using red light enabling observation. All mice moved freely and showed no sign of fear or stress during the experiments. Mice which did not interact with objects (object recognition test) and mice which fell off the platform (passive avoidance) several times were excluded (one each in control and Pld1−/− group and three in the Pld2−/− group). After each trial, objects and environment were cleaned to eliminate odor cues. The social recognition task [10] tests short-term memory [24]. In an open field of 75 × 43 × 20 cm, each mouse was presented for 5 min with an empty cage and a second cage containing an unknown mouse of the same gender. Contacts with the mouse and the empty cage were scored. After a break of 15 min, the mouse was confronted for 5 min with the familiar mouse of the first session in one cage and a novel mouse (same gender) in the other cage and the contacts with both mice were counted. Learning and memory were scored using the level of discrimination (LD). It was calculated by the formula (b − a)/(b + a), where a and b are the contacts with the familiar and novel mouse, respectively. The object recognition task (ORT; [8]) is a test for long-term memory. In a grey box of 45 × 30 × 15 cm, each mouse was presented for 5 min with two identical objects (cylinders) and contacts with both objects were scored. On the next day, mice were confronted with two familiar and an additional novel object (pyramid) and contacts with each object were counted again. The LD was calculated as (b − a)/(b + a), in which “a” is the mean of contacts with the familiar objects and “b” the contacts with the novel object. The passive avoidance task [4] was used to assess learning based on aversive stimuli. Briefly, mice were put on a small, 20 cm high platform in front of an opening to a dark box of 20 × 20 × 20 cm. The platform was lighted to further facilitate entering into the box. A grid at the bottom of the box transmitted a mild foot shock (0.5 mA, 1 s), delivered immediately when the mouse entered the box. The time till entering the box was measured (s). On day 2, the mouse was placed on the platform again and the time till entering the box was measured again (s). The experiment was stopped when mice stayed on the platform longer than 3 min. 2.5. Microdialysis Mice were anesthetized with isoflurane (induction dose, 4%; maintenance dose, 1–1.5% v/v) in synthetic air and placed in a stereotaxic frame. Self-made, I-shaped, concentric dialysis probes with an exchange length of 2 mm were implanted in the right ventral hippocampus using the following coordinates (from bregma): anterior–posterior, −2.7 mm; lateral, −3.0 mm; dorsoventral, −3.8 mm [19]. Mice were allowed to recover overnight, and experiments were carried out on the following day in awake, freely moving animals [17]. The microdialysis probes were perfused with artificial cerebrospinal fluid (aCSF) supplemented with 0.1 M neostigmine bromide. The perfusion rate was 1 l/min, and efflux from the microdialysis probe was collected in intervals of 15 min.
Please cite this article in press as: U. Burkhardt, et al., Impaired brain development and reduced cognitive function in phospholipase D-deficient mice, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.052
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Fig. 1. Brain development of wild-type (“wt”), PLD1−/−, PLD2−/− and doubleknockout (“DKO”) juvenile mice. Brain size was obtained by MRI scan (see Section 2). Data is presented as means ± SD (n = 4). Statistics: *, p < 0.05 **, p < 0.01; ***, p < 0.001 vs. the same developmental age in wild-type mice (one-way ANOVA plus Dunnett’s post-test for unpaired data, GraphPad Prism 5.0).
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First, samples were collected for 60 min to establish baseline values of acetylcholine (ACh); choline, glucose, lactate and glutamate were also measured. Subsequently, animals were placed into a novel environment (“open field”), consisting of a grey, rectangular plastic box (45 × 30 × 15 cm). After 90 min in the open field, mice were returned to their home cages, and dialysis was continued for 2 h. On the next day, mice were sacrificed, and probe location in the hippocampus was verified. ACh and choline in dialysates were determined by microbore HPLC-ECD using the Eicom HTEC-500 system (Kyoto, Japan) as described before [17]. Glucose, lactate and glutamate concentrations in the microdialysis samples were determined by a CMA/Microdialysis (Solna, Sweden) 600 microanalyzer [11].
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2.6. Statistics
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Statistical comparisons were done by one-way ANOVA plus Dunnett’s post-test for unpaired data (Fig. 1) and by two-way ANOVA for repeated measurements plus Bonferroni’s post-test for time courses (Fig. 3) using the GraphPad® Prism 5.0 program. The behavioral data (Fig. 2) were analyzed by one-way ANOVA plus Kruskal–Wallis test.
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3. Results
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3.1. Delayed brain development in PLD-deficient mice
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Since PLDs play a crucial role in glial proliferation in vitro [1], it was of interest to study the development of the brain in PLD-deficient mice in vivo. Magnetic resonance imaging (MRI) offers the opportunity to observe brain growth in a non-invasive manner. Using high-resolution MRI at high field strengths, we recorded brain architectures of mice at the age of P7 through P33. In wild-type mice, the brain grew rapidly between P7 and P21 when it approached its final size (Fig. 1). In Pld1−/− mice, the development of total brain volumes was delayed at days 21 and 27 (Fig. 1), and an even stronger delay of brain growth was observed with Pld2−/− mice and Pld1−/− /PLD2−/− double knockouts (Fig. 1). PLD-deficient mice caught up with wild-type mice at P33, and in adult mice, no difference in brain weights could be observed between wild-type mice (128 ± 20 mg per hemisphere),
Fig. 2. Learning and memory of wild-type and PLD-deficient mice as tested by (A) social recognition task, (B) object recognition task and (C) passive avoidance task. Learning and memory were expressed as “level of discrimination” as described in Section 2. Data is presented as means ± min/max (n = 12–13). Statistics: *, p < 0.05; **, p < 0.01 vs. wild-type mice; n.s., not significant (by one-way ANOVA plus Kruskal–Wallis test).
Pld1−/− mice (123 ± 46 mg) and Pld2−/− mice (134 ± 19 mg) (means ± SD; n = 8) (not illustrated). 3.2. Impaired cognitive function in PLD-deficient mice Since brain development is delayed in PLD-deficient mice, we studied cognitive function in adult, 3–4 months mice. Levels of discrimination (LD) were calculated for group comparisons (Fig. 2); positive values indicate learning while zero values represent impaired learning and memory. In the social recognition
Please cite this article in press as: U. Burkhardt, et al., Impaired brain development and reduced cognitive function in phospholipase D-deficient mice, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.052
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not illustrated). Moreover, for unknown reasons, wild-type mice continued to release large amounts of ACh when back in the home cage while ACh levels decreased rapidly in PLD-deficient mice. In separate measurements, choline levels in wild-type mice were 1.58 ± 0.39 M, and were unchanged in PLD-deficient mice. Moreover, glucose levels were 0.54 ± 0.1 mM and lactate levels were 0.19 ± 0.1 mM (n = 8), and extracellular concentrations of glutamate were estimated at 6.4 ± 3.5 M, in wild-type mice. These values are in agreement with previous measurements (Hartmann et al. [11]). Of note, none of these values were significantly changed in PLD-deficient mice (data not shown).
4. Discussion
Fig. 3. Stimulated release of ACh in mouse hippocampi of wild-type, Pld1−/− and Pld2−/− mice measured by microdialysis. After a 1-h collection of basal acetylcholine, changes of ACh upon behavioral activation (exposure of mice to a novel environment) were observed from 0 to 90 min; then, mice were returned to the home cage. Data is represented as mean ± SEM (n = 7). Statistical evaluation: F2,19 = 4.85; p = 0.02 (two-way ANOVA for repeated measurements plus Bonferroni’s post-test using the GraphPad® Prism 5.0 program); individual time points significant as indicated.
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task (SRT), wild-type mice had 1.6-fold more contacts with the unknown mouse than with the familiar mouse, resulting in a LD value of 0.24. In contrast, Pld1−/− and Pld2−/− mice, as well as double knockouts, failed to remember the familiar mouse (Fig. 2A). In the curiosity-driven object recognition task (ORT), wild-type mice, on average, had 11 ± 6 contacts with the unfamiliar object vs. 6 ± 3 contacts with the familiar object, indicating successful recognition of the new object. PLD-deficient mice performed worse; the LD values were significantly reduced in the case of Pld2−/− and double knockout mice (Fig. 2B). Fear-driven memory, however, is evidently intact in PLD-deficient mice. In the passive avoidance task, all mice entered the dark box within 20–40 s on day one, but on the second day, all mice stayed on the platform for an average of ∼120 s (Fig. 2C).
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3.3. Cholinergic function is impaired in PLD-deficient mice
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The hippocampus plays a crucial role in short-term and longterm memory and in spatial navigation [4,24], and failure of hippocampal function likely contributed to the poor outcome of recognition tests in PLD-deficient mice (Fig. 2). Moreover, blockade of cholinergic function is known to disrupt social and object recognition [6,21]. Therefore, we chose the hippocampal area for an investigation of cholinergic function in PLD-deficient mice. In the presence of 0.1 M neostigmine, basal levels of ACh were 18.6 ± 5.2 nM in wild-type mice, and similar values were obtained in PLD-deficient mice (data not shown). Importantly, when the mice were exposed to a novel environment (open field) to stimulate explorative behavior, wild-type mice responded with a 4-fold increase of cholinergic activity as reflected by increased extracellular ACh levels (86.0 ± 20.2 nM after 90 min; Fig. 3). In comparison, Pld1−/− mice only had a 2.5-fold increase (51.3 ± 11.9 nM after 90 min), and Pld2−/− mice produced even less neurotransmitter (2fold increase at 34.2 ± 7.3 nM after 90 min; Fig. 3). The differences between the time courses were significant as tested by two-way ANOVA (see legend of Fig. 3). Similarly, the calculation of the “area under the curve” (AUC) values showed that Pld1−/− mice released 57% less ACh (p < 0.05), and Pld2−/− mice 65% less ACh (p < 0.01), than wild-type mice during 90 min of behavioral stimulation (data
As described in the introduction, PLD may have a role in early brain development, and disruption of PLD signaling pathways in the presence of ethanol may be involved in the development of fetal alcohol syndrome which is characterized by delayed brain growth and life-long cognitive dysfunction [3,16,25]. The present finding of delayed brain growth in PLD-deficient mice supports a role for PLD in brain development and corroborates our previous hypothesis about the importance of PLD signaling in astroglial proliferation [1,23]. As recent publications also linked PLD activity to cognitive function in old age, e.g. in Alzheimer’ disease [2,5,18], we decided to carry out a short, preliminary investigation into cognitive and cholinergic function in PLD-deficient mice. Because of the link of PLD to Alzheimer’ disease, we chose to perform cognitive tests that are based on hippocampal function, and in parallel investigated cholinergic activity in the hippocampus which is known to be involved in memory processes required for social and object recognition. For this purpose, we used brain microdialysis, a minimal-invasive method to monitor neurotransmitters and metabolites in awake, freely moving animals. Our results demonstrate that both PLD1 and PLD2 are required for normal brain development, cognitive function and cholinergic neurotransmission. In the case of PLD1, genetic ablation caused a minor delay in brain development, and a more limited impairment of cognitive function. ACh release was clearly reduced, however, upon behavioral stimulation. These findings are in agreement with previous reports on the role of PLD1 in vesicular transport and endo- and exocytosis of neurotransmitters [12,22]. The case of impaired brain function is even stronger for PLD2. Compared to wild-type mice, brain development of Pld2−/− mice was significantly delayed throughout early life (P14–P27) (Fig. 1). In adults, PLD2 deficiency caused severe impairments of brain function: social recognition was basically absent, and object recognition significantly impaired (Fig. 2). Only fear-related memory was retained, probably because the severe stimulus in the passive avoidance task (foot shock) overrode more subtle changes of recognition memory. Concomitantly, hippocampal ACh release during behavioral stimulation was rather limited, particularly when compared with the strong increase in wild-type animals (Fig. 3). In a smaller series of experiments, we could also test mice that were deficient for both, PLD1 and PLD2; these double-knockouts showed basically the same delay of brain growth and similar impairments in cognitive testing, as PLD2-deficient mice. Unfortunately, the limited supply of mice prevented extensive studies with microdialysis. Summarizing, Pld2−/− mice and, to a smaller extent, Pld1−/− mice have cognitive dysfunction and reduced ACh release upon physiological stimulation. Our results suggest that disruption of the PLD signaling pathway early in life leads to long-lasting dysfunctions of brain growth and brain function. It seems likely that a delay in early brain development causes long-lasting changes of the corticohippocampal neuronal architecture which limits the proper activity of
Please cite this article in press as: U. Burkhardt, et al., Impaired brain development and reduced cognitive function in phospholipase D-deficient mice, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.052
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the septohippocampal pathway, and probably many more pathways, for adequate cognitive function. The hypothesis that PLD deficiency causes persistent changes in central cholinergic systems should be fruitful for further investigations.
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[15]. Acknowledgments
The authors thank M. Hardt and S. Pelikan for assistance with 328 the MRI study; F. Mohr for implanting microdialysis probes; and 329 Q4 H. Lau for HPLC analysis. Funding was provided by a stipend from 330 Q5 the FIRST Graduate School to U.B. and by start-up funds of Goethe 331 University to J.K. 327
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Impaired brain development and reduced cognitive function in phospholipase D-deficient mice
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Ute Burkhardt a , David Stegner c , Elke Hattingen b , Sandra Beyer a , Bernhard Nieswandt c , Jochen Klein a,∗ a
Department of Pharmacology, Goethe University College of Pharmacy, Frankfurt, Germany Institute of Neuroradiology, Goethe University Medical Center, Frankfurt, Germany Q2 c Vascular Medicine, University Hospital Würzburg & Rudolf Virchow Center, University of Würzburg, Germany b
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h i g h l i g h t s • • • •
Genetic deletion of phospholipase D 1 or 2 delays brain development in mice. Cognitive function is reduced in adult PLD-deficient mice. PLD deficiency impairs release of acetylcholine after behavioral stimulation. Disruption of PLD signaling may contribute to fetal alcohol syndrome and Alzheimer’ disease.
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Article history: Received 28 February 2014 Received in revised form 29 April 2014 Accepted 30 April 2014 Available online xxx
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Keywords: Acetylcholine Alzheimer’ disease Brain growth spurt Fetal alcohol syndrome Microdialysis Phospholipase D
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1. Introduction
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The phospholipases D (PLD1 and 2) are signaling enzymes that catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid, a lipid second messenger involved in cell proliferation, and choline, a precursor of acetylcholine (ACh). In the present study, we investigated development and cognitive function in mice that were deficient for PLD1, or PLD2, or both. We found that PLD-deficient mice had reduced brain growth at 14–27 days post partum when compared to wild-type mice. In adult PLD-deficient mice, cognitive function was impaired in social and object recognition tasks. Using brain microdialysis, we found that wild-type mice responded with a 4-fold increase of hippocampal ACh release upon behavioral stimulation in the open field, while PLD-deficient mice released significantly less ACh. These results may be relevant for cognitive dysfunctions observed in fetal alcohol syndrome and in Alzheimer’ disease. © 2014 Published by Elsevier Ireland Ltd.
The phospholipases D (PLD) are ubiquitous enzymes which catalyze the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. Phosphatidic acid is a lipid second messenger which regulates cytoskeletal organization and vesicular trafficking and is involved in cell proliferation. Choline is a precursor of acetylcholine (ACh) synthesis and of choline-containing phospholipids
Abbreviations: ACh, acetylcholine; ACSF, artificial cerebrospinal fluid; MRI, magnetic resonance imaging; ORT, object recognition task; PA, phosphatidic acid; PC, phosphatidylcholine; PLD, phospholipase; DSRT, social recognition task. ∗ Corresponding author at: Goethe University College of Pharmacy, Max-vonLaue-Str. 9, 60438 Frankfurt, Germany. Tel.: +49 69 798 29366; fax: +49 69 798 29277. E-mail address: [email protected] (J. Klein).
such as PC and sphingomyelin. The two major mammalian isoforms of PLD are PLD1 and PLD2. PLD1 is located in the perinuclear region, is activated by small GTPases such as ARF and Rho and participates in budding and fusion of secretory vesicles and in stress fiber formation. In contrast, PLD2 is located at the cellular membrane, shows high basal activity, is regulated by tyrosine kinases and protein kinase C and participates in receptor endocytosis (reviewed in [20]). PLD1 and 2 are capable of transphosphatidylation, a reaction in which PC is transformed into phosphatidylalcohols (e.g., phosphatidylethanol) when alcohols (e.g. ethanol) are present; thus, the PLD signaling pathway is suppressed in the presence of alcohols because PA formation is reduced [1,14]. Numerous studies have implicated PLD activation in the control of cell proliferation [9]. Importantly, several lines of evidence support a role of PLDs in cognitive function or dysfunction. Previous work of our group and others demonstrated a role for PLD in early
http://dx.doi.org/10.1016/j.neulet.2014.04.052 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: U. Burkhardt, et al., Impaired brain development and reduced cognitive function in phospholipase D-deficient mice, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.052
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development of the brain. In fact, growth factors stimulate PLD activity in neurons and astrocytes which results in cell proliferation [19,26]. Astrocytes interact closely with neurons during the brain growth spurt enabling neuritogenesis and formation of synapses, two processes in which PLDs are involved [13]. Accordingly, interruption of PLD signaling, e.g. by alcohols, may inhibit normal brain development [14] by inhibiting the formation of the lipid messenger, phosphatidic acid; this mechanism may be partly responsible for ethanol toxicity to the fetus (“fetal alcohol syndrome”) [1]. There is also evidence that links PLD to neurotransmitter release, especially in cholinergic neurons. In whole cells, PLD activity can be activated by muscarinic receptors [14]. In synaptosomes, PLD is activated by depolarization and by calcium influx [22,28]. Both PLD isoforms may be involved in neurotransmitter release. In one study, catalytically inactive PLD1 mutants (K898R) reduced ACh release in Aplysia neurons by reducing the number of active presynaptic releasing sites [12]. In another study, PLD2 was responsible for the generation of choline for ACh synthesis, and downregulation of PLD2 led to reduce ACh release in cholinergic neurons [29]. Another line of investigation links PLD activities to Alzheimer’ disease (AD), which is also characterized by cognitive deficits and by widespread cholinergic dysfunction. PLD activity was reduced in cells with AD-like mutations, and overexpression of PLD1 in these cells corrected trafficking alterations of the amyloid precursor protein [2]. In contrast, amyloid  toxicity in cell cultures was promoted by PLD2 activity [18]. In addition, a third PLD form (“PLD3”) was associated with Alzheimer’ disease in a recent genome-wide association study [5], but further characterization of this new isoform is required to address its potential role in lipid signaling. In the present study, we made use of recently developed mouse models that are deficient in PLD1 or PLD2. In light of the previous reports summarized above, we investigated brain growth in juvenile PLD-deficient mice and tested cognitive function and cholinergic activity in adult mice in vivo. We found that characteristic impairments can be seen in both PLD-deficient strains, with stronger effects in PLD2-deficient mice.
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2. Materials and methods
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2.1. Materials
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Chemicals for artificial cerebrospinal fluid (aCSF; 147 mM NaCl, 4 mM KCl, 1.2 mM CaCl2 , and 1.2 mM MgCl2 ) were obtained from Sigma–Aldrich (Seelze, Germany). Neostigmine bromide was obtained from Acros Organics (Thermo Fisher Scientific, Schwerte, Germany) and scopolamine bromide from TCI Europe (Eschborn, Germany). Ketamine/xylazine was prepared with Rompun® 2% (Bayer Co.) and Ketavet® 100 mg/ml (Pfizer Co.).
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2.2. Animals
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Gender-mixed Pld1−/− , Pld2−/− mice (generated on a B6 background) and wild-type controls [7,27] were housed in a facility with controlled temperature and humidity and a day/night cycle of 12/12 h. They had free access to food and water. All animal experiments were performed in agreement with EU directive 2010/63 and were registered with the local animal committee (Regierungspräsidium Darmstadt, Germany).
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2.3. Magnetic resonance imaging (MRI)
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The MRI study was performed in a 3 T Magnetom® Trio (Siemens, Erlangen, Germany) with circular polarized wrist coil. The mice were assessed once a week over a time period of 5 weeks beginning at the age of P7. The litter size of all groups was 6–8 pups and the mothers had free access to food and water. Prior to
MRI, pups were anesthetized with ketamine/xylazine (70/7 mg/kg body weight i.p.) and kept warm throughout the entire measurement. The brain size of the pups was measured by MRI in 19–20 transversal T2-weighted spin echo sequences with a slice thickness of 0.9 mm without gap and an in-plane resolution of 0.2 × 0.2 mm. The measurements lasted around 9 min. Resulting DICOMs were analyzed by marking the brain area in each slice using free MRIcro software. The brain volume was calculated by addition of brain areas (cortices) in all slices. 2.4. Behavioral testing 3–4 months old mice were placed in the behavioral testing room at least 1 week before the experiments took place. Behavioral testing was performed during their active time (night cycle) using red light enabling observation. All mice moved freely and showed no sign of fear or stress during the experiments. Mice which did not interact with objects (object recognition test) and mice which fell off the platform (passive avoidance) several times were excluded (one each in control and Pld1−/− group and three in the Pld2−/− group). After each trial, objects and environment were cleaned to eliminate odor cues. The social recognition task [10] tests short-term memory [24]. In an open field of 75 × 43 × 20 cm, each mouse was presented for 5 min with an empty cage and a second cage containing an unknown mouse of the same gender. Contacts with the mouse and the empty cage were scored. After a break of 15 min, the mouse was confronted for 5 min with the familiar mouse of the first session in one cage and a novel mouse (same gender) in the other cage and the contacts with both mice were counted. Learning and memory were scored using the level of discrimination (LD). It was calculated by the formula (b − a)/(b + a), where a and b are the contacts with the familiar and novel mouse, respectively. The object recognition task (ORT; [8]) is a test for long-term memory. In a grey box of 45 × 30 × 15 cm, each mouse was presented for 5 min with two identical objects (cylinders) and contacts with both objects were scored. On the next day, mice were confronted with two familiar and an additional novel object (pyramid) and contacts with each object were counted again. The LD was calculated as (b − a)/(b + a), in which “a” is the mean of contacts with the familiar objects and “b” the contacts with the novel object. The passive avoidance task [4] was used to assess learning based on aversive stimuli. Briefly, mice were put on a small, 20 cm high platform in front of an opening to a dark box of 20 × 20 × 20 cm. The platform was lighted to further facilitate entering into the box. A grid at the bottom of the box transmitted a mild foot shock (0.5 mA, 1 s), delivered immediately when the mouse entered the box. The time till entering the box was measured (s). On day 2, the mouse was placed on the platform again and the time till entering the box was measured again (s). The experiment was stopped when mice stayed on the platform longer than 3 min. 2.5. Microdialysis Mice were anesthetized with isoflurane (induction dose, 4%; maintenance dose, 1–1.5% v/v) in synthetic air and placed in a stereotaxic frame. Self-made, I-shaped, concentric dialysis probes with an exchange length of 2 mm were implanted in the right ventral hippocampus using the following coordinates (from bregma): anterior–posterior, −2.7 mm; lateral, −3.0 mm; dorsoventral, −3.8 mm [19]. Mice were allowed to recover overnight, and experiments were carried out on the following day in awake, freely moving animals [17]. The microdialysis probes were perfused with artificial cerebrospinal fluid (aCSF) supplemented with 0.1 M neostigmine bromide. The perfusion rate was 1 l/min, and efflux from the microdialysis probe was collected in intervals of 15 min.
Please cite this article in press as: U. Burkhardt, et al., Impaired brain development and reduced cognitive function in phospholipase D-deficient mice, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.052
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Fig. 1. Brain development of wild-type (“wt”), PLD1−/−, PLD2−/− and doubleknockout (“DKO”) juvenile mice. Brain size was obtained by MRI scan (see Section 2). Data is presented as means ± SD (n = 4). Statistics: *, p < 0.05 **, p < 0.01; ***, p < 0.001 vs. the same developmental age in wild-type mice (one-way ANOVA plus Dunnett’s post-test for unpaired data, GraphPad Prism 5.0).
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First, samples were collected for 60 min to establish baseline values of acetylcholine (ACh); choline, glucose, lactate and glutamate were also measured. Subsequently, animals were placed into a novel environment (“open field”), consisting of a grey, rectangular plastic box (45 × 30 × 15 cm). After 90 min in the open field, mice were returned to their home cages, and dialysis was continued for 2 h. On the next day, mice were sacrificed, and probe location in the hippocampus was verified. ACh and choline in dialysates were determined by microbore HPLC-ECD using the Eicom HTEC-500 system (Kyoto, Japan) as described before [17]. Glucose, lactate and glutamate concentrations in the microdialysis samples were determined by a CMA/Microdialysis (Solna, Sweden) 600 microanalyzer [11].
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2.6. Statistics
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Statistical comparisons were done by one-way ANOVA plus Dunnett’s post-test for unpaired data (Fig. 1) and by two-way ANOVA for repeated measurements plus Bonferroni’s post-test for time courses (Fig. 3) using the GraphPad® Prism 5.0 program. The behavioral data (Fig. 2) were analyzed by one-way ANOVA plus Kruskal–Wallis test.
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3. Results
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3.1. Delayed brain development in PLD-deficient mice
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Since PLDs play a crucial role in glial proliferation in vitro [1], it was of interest to study the development of the brain in PLD-deficient mice in vivo. Magnetic resonance imaging (MRI) offers the opportunity to observe brain growth in a non-invasive manner. Using high-resolution MRI at high field strengths, we recorded brain architectures of mice at the age of P7 through P33. In wild-type mice, the brain grew rapidly between P7 and P21 when it approached its final size (Fig. 1). In Pld1−/− mice, the development of total brain volumes was delayed at days 21 and 27 (Fig. 1), and an even stronger delay of brain growth was observed with Pld2−/− mice and Pld1−/− /PLD2−/− double knockouts (Fig. 1). PLD-deficient mice caught up with wild-type mice at P33, and in adult mice, no difference in brain weights could be observed between wild-type mice (128 ± 20 mg per hemisphere),
Fig. 2. Learning and memory of wild-type and PLD-deficient mice as tested by (A) social recognition task, (B) object recognition task and (C) passive avoidance task. Learning and memory were expressed as “level of discrimination” as described in Section 2. Data is presented as means ± min/max (n = 12–13). Statistics: *, p < 0.05; **, p < 0.01 vs. wild-type mice; n.s., not significant (by one-way ANOVA plus Kruskal–Wallis test).
Pld1−/− mice (123 ± 46 mg) and Pld2−/− mice (134 ± 19 mg) (means ± SD; n = 8) (not illustrated). 3.2. Impaired cognitive function in PLD-deficient mice Since brain development is delayed in PLD-deficient mice, we studied cognitive function in adult, 3–4 months mice. Levels of discrimination (LD) were calculated for group comparisons (Fig. 2); positive values indicate learning while zero values represent impaired learning and memory. In the social recognition
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not illustrated). Moreover, for unknown reasons, wild-type mice continued to release large amounts of ACh when back in the home cage while ACh levels decreased rapidly in PLD-deficient mice. In separate measurements, choline levels in wild-type mice were 1.58 ± 0.39 M, and were unchanged in PLD-deficient mice. Moreover, glucose levels were 0.54 ± 0.1 mM and lactate levels were 0.19 ± 0.1 mM (n = 8), and extracellular concentrations of glutamate were estimated at 6.4 ± 3.5 M, in wild-type mice. These values are in agreement with previous measurements (Hartmann et al. [11]). Of note, none of these values were significantly changed in PLD-deficient mice (data not shown).
4. Discussion
Fig. 3. Stimulated release of ACh in mouse hippocampi of wild-type, Pld1−/− and Pld2−/− mice measured by microdialysis. After a 1-h collection of basal acetylcholine, changes of ACh upon behavioral activation (exposure of mice to a novel environment) were observed from 0 to 90 min; then, mice were returned to the home cage. Data is represented as mean ± SEM (n = 7). Statistical evaluation: F2,19 = 4.85; p = 0.02 (two-way ANOVA for repeated measurements plus Bonferroni’s post-test using the GraphPad® Prism 5.0 program); individual time points significant as indicated.
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task (SRT), wild-type mice had 1.6-fold more contacts with the unknown mouse than with the familiar mouse, resulting in a LD value of 0.24. In contrast, Pld1−/− and Pld2−/− mice, as well as double knockouts, failed to remember the familiar mouse (Fig. 2A). In the curiosity-driven object recognition task (ORT), wild-type mice, on average, had 11 ± 6 contacts with the unfamiliar object vs. 6 ± 3 contacts with the familiar object, indicating successful recognition of the new object. PLD-deficient mice performed worse; the LD values were significantly reduced in the case of Pld2−/− and double knockout mice (Fig. 2B). Fear-driven memory, however, is evidently intact in PLD-deficient mice. In the passive avoidance task, all mice entered the dark box within 20–40 s on day one, but on the second day, all mice stayed on the platform for an average of ∼120 s (Fig. 2C).
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3.3. Cholinergic function is impaired in PLD-deficient mice
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The hippocampus plays a crucial role in short-term and longterm memory and in spatial navigation [4,24], and failure of hippocampal function likely contributed to the poor outcome of recognition tests in PLD-deficient mice (Fig. 2). Moreover, blockade of cholinergic function is known to disrupt social and object recognition [6,21]. Therefore, we chose the hippocampal area for an investigation of cholinergic function in PLD-deficient mice. In the presence of 0.1 M neostigmine, basal levels of ACh were 18.6 ± 5.2 nM in wild-type mice, and similar values were obtained in PLD-deficient mice (data not shown). Importantly, when the mice were exposed to a novel environment (open field) to stimulate explorative behavior, wild-type mice responded with a 4-fold increase of cholinergic activity as reflected by increased extracellular ACh levels (86.0 ± 20.2 nM after 90 min; Fig. 3). In comparison, Pld1−/− mice only had a 2.5-fold increase (51.3 ± 11.9 nM after 90 min), and Pld2−/− mice produced even less neurotransmitter (2fold increase at 34.2 ± 7.3 nM after 90 min; Fig. 3). The differences between the time courses were significant as tested by two-way ANOVA (see legend of Fig. 3). Similarly, the calculation of the “area under the curve” (AUC) values showed that Pld1−/− mice released 57% less ACh (p < 0.05), and Pld2−/− mice 65% less ACh (p < 0.01), than wild-type mice during 90 min of behavioral stimulation (data
As described in the introduction, PLD may have a role in early brain development, and disruption of PLD signaling pathways in the presence of ethanol may be involved in the development of fetal alcohol syndrome which is characterized by delayed brain growth and life-long cognitive dysfunction [3,16,25]. The present finding of delayed brain growth in PLD-deficient mice supports a role for PLD in brain development and corroborates our previous hypothesis about the importance of PLD signaling in astroglial proliferation [1,23]. As recent publications also linked PLD activity to cognitive function in old age, e.g. in Alzheimer’ disease [2,5,18], we decided to carry out a short, preliminary investigation into cognitive and cholinergic function in PLD-deficient mice. Because of the link of PLD to Alzheimer’ disease, we chose to perform cognitive tests that are based on hippocampal function, and in parallel investigated cholinergic activity in the hippocampus which is known to be involved in memory processes required for social and object recognition. For this purpose, we used brain microdialysis, a minimal-invasive method to monitor neurotransmitters and metabolites in awake, freely moving animals. Our results demonstrate that both PLD1 and PLD2 are required for normal brain development, cognitive function and cholinergic neurotransmission. In the case of PLD1, genetic ablation caused a minor delay in brain development, and a more limited impairment of cognitive function. ACh release was clearly reduced, however, upon behavioral stimulation. These findings are in agreement with previous reports on the role of PLD1 in vesicular transport and endo- and exocytosis of neurotransmitters [12,22]. The case of impaired brain function is even stronger for PLD2. Compared to wild-type mice, brain development of Pld2−/− mice was significantly delayed throughout early life (P14–P27) (Fig. 1). In adults, PLD2 deficiency caused severe impairments of brain function: social recognition was basically absent, and object recognition significantly impaired (Fig. 2). Only fear-related memory was retained, probably because the severe stimulus in the passive avoidance task (foot shock) overrode more subtle changes of recognition memory. Concomitantly, hippocampal ACh release during behavioral stimulation was rather limited, particularly when compared with the strong increase in wild-type animals (Fig. 3). In a smaller series of experiments, we could also test mice that were deficient for both, PLD1 and PLD2; these double-knockouts showed basically the same delay of brain growth and similar impairments in cognitive testing, as PLD2-deficient mice. Unfortunately, the limited supply of mice prevented extensive studies with microdialysis. Summarizing, Pld2−/− mice and, to a smaller extent, Pld1−/− mice have cognitive dysfunction and reduced ACh release upon physiological stimulation. Our results suggest that disruption of the PLD signaling pathway early in life leads to long-lasting dysfunctions of brain growth and brain function. It seems likely that a delay in early brain development causes long-lasting changes of the corticohippocampal neuronal architecture which limits the proper activity of
Please cite this article in press as: U. Burkhardt, et al., Impaired brain development and reduced cognitive function in phospholipase D-deficient mice, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.04.052
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the septohippocampal pathway, and probably many more pathways, for adequate cognitive function. The hypothesis that PLD deficiency causes persistent changes in central cholinergic systems should be fruitful for further investigations.
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[15]. Acknowledgments
The authors thank M. Hardt and S. Pelikan for assistance with 328 the MRI study; F. Mohr for implanting microdialysis probes; and 329 Q4 H. Lau for HPLC analysis. Funding was provided by a stipend from 330 Q5 the FIRST Graduate School to U.B. and by start-up funds of Goethe 331 University to J.K. 327
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