Down syndrome is the most common genetic etiology of mental retardation and affects 1 out of 800 newborn infants.1 Down syndrome is associated with characteristic facial features, structural anomalies, abnormal central nervous system function typified by developmental delay, hypotonia in the newborn period, mental retardation, and early onset of Alzheimer disease.2,3 Although there are various antenatal screening and diagnostic tests for Down syndrome, there is no known cure or intervention that improves outcome. Treatment focuses on early intervention programs, such as physical, occupational, and speech therapy.
Vasoactive intestinal peptide and other neuropeptides are altered in Down syndrome. Vasoactive intestinal peptide levels are increased in the blood of children with Down syndrome, and vasoactive intestinal peptide mRNA expression is elevated in the brain of a mouse model of Down syndrome (Ts65Dn).4,5 Vasoactive intestinal peptide regulates neuropeptide release from glial cells, including activity-dependent neuroprotective protein (ADNP) and activity-dependent neurotrophic factor (ADNF).6–9 These two proteins have active peptide fragments, NAPVSIPQ (NAP) and SALLRSIPA (SAL), that mimic the activity of their parent proteins and have been shown to prevent deficits in Down syndrome.10,11 Treatment of Down syndrome cortical neurons with NAPVSIPQ and SALLRSIPA resulted in a significant increase in neuronal survival as well as a reduction of degenerative morphological changes.10 Prenatal treatment with NAPVSIPQ and SALLRSIPA in the Ts65Dn model prevented the delay of neurodevelopmental milestones in Down syndrome offspring as well as the alterations of ADNP and vasoactive intestinal peptide.11
Impairment of cognitive function is one of the most important alterations in Down syndrome, with characteristic and specific deficits in learning and memory.12 The learning deficit includes impaired hippocampal function with dysfunctional pathways.13,14 Understanding the mechanism of impairment includes evaluation of long-term potentiation, the electrophysiological model of learning. An essential first step in long-term potentiation is the activation of the N-methyl-D-aspartate (NMDA) receptors, which are composed of different subunits that determine their functionality. NMDA receptors function in a multisynaptic circuit and are regulated through the inhibitory tone of the γ-aminobutyric acid type A (GABAA) receptors in the hippocampus.15,16 Alterations in NR2B, NR2A, and GABAAα5 have been found in Ts65Dn mice.11
Our objective was to evaluate whether adult administration of these neuropeptides D-NAP+D-SAL could reverse the learning deficit in the Ts65Dn model of Down syndrome and evaluate if the reversal of learning deficits is mediated through alterations of neuropeptides (ADNP, vasoactive intestinal peptide), glial fibrillary acidic protein (GFAP), an astrocyte marker as well as through long-term potentiation by evaluating subunits of the NMDA and GABA receptors.
MATERIALS AND METHODS
We used the Ts65Dn mouse, a well-characterized model which closely mimics the genetic abnormality of Down syndrome.17 The Ts65Dn model is trisomic for a segment of mouse chromosome 16, which is orthologous to the “critical region” of human chromosome 21. The mice exhibit features characteristic of Down syndrome, including developmental delay, impairment of hippocampal function as evidenced by impaired spatial memory and deficits in learning, and early onset of the neuropathology of Alzheimer disease.18–23
The protocol was approved by the Eunice Kennedy Shriver National Institute of Child Health and Development (NICHD) Animal Care and Use Committee and the mice received humane care in compliance with the National Institutes of Health Guidelines for Care and Use of Experimental Animals.
Ts65Dn female mice were mated with wild-type (normal) C57B16/J male mice (The Jackson Laboratory) for 24 hours and were kept in a 12-hour light/dark cycle. Presence of vaginal plug was considered day 0 of pregnancy. The litters were allowed to deliver and the pups were weaned on day 21. The pups were ear-tagged and genotyped using a small segment of their tail tip as previously described.11 The study investigators were blinded to genotype during all experiments. At approximately 10 months of age, the mice were treated once a day for 9 days via oral gavage with either 40 micrograms of D-NAP and 40 micrograms of D-SAL or placebo 0.2-mL dose. Each treatment was blinded by labeling each solution with the animal's eartag. D-NAP was diluted in 50 microliters of dimethyl sulfoxide and diluted in 2.5 mL of filtered Dulbecco phosphate-buffered saline solution; D-SAL was dissolved and diluted in filtered Dulbecco solution. For placebo solution we added 50 microliters of DMSO at 2.5 mL of Dulbecco phosphate-buffered solution. Animals were fasted 1 hour before treatment to avoid interference with drug absorption. Food and water were replaced immediately following treatment. The all-D amino acid configurations of the peptides were used that allow for absorption after oral administration.24 During days 4–9 of treatment, the mice were assessed for learning and memory in the Morris watermaze. Testing began 3 hours after treatment.
Two independent trials were conducted. In the first 4 Ts65Dn, 15 wild type (control) and 7 Ts65Dn+ (D-NAP+D-SAL) were tested. In the second 4 Ts65Dn, 4 wild type (control) and 5 Ts65Dn+(D-NAP+D-SAL) were tested.
The Morris watermaze is a well-established test that evaluates spatial learning, a measure of cognitive function.25 The apparatus consists of a circular pool with a water level of approximately 30 cm and maintained at 24–26°C. Nontoxic tempura paint is added to make the water opaque and blend with the color of the pool wall. External cues (arrow, star, circle, and rectangle) are placed around the pool as a reference for the mice. A transparent platform is placed in the pool and kept in a fixed location throughout the testing period. The platform surface is hidden, submerged under the water surface. Each day, the mouse was positioned on the platform for 15 seconds before being placed into the pool. The mouse was placed into the water at the same location for each trial and allowed to swim freely to find the hidden platform using the external visual cues. On the first day of testing, animals were allowed to remain on the platform for 60 seconds. Each animal underwent four consecutive trials daily, with the average time or latency required to find the platform recorded over 5 consecutive days. Each trial was tracked using an overhead camera interfaced with a computer that recorded the time and path traveled. On treatment day 9, the mice underwent the probe test, a measure of memory retention. In this test, the hidden platform was removed and the time spent in each quadrant of the pool was recorded. Then after 10 days of no treatment or testing, the mice were evaluated by the probe test again to measure memory retention without peptide treatment. The probe test evaluates how much time the mice spent in the correct quadrant, which was the quadrant where the hidden platform had been during the active 5 days of testing. All examiners were blinded to the treatment groups and genotype.
Before treatment and then on day 9 of treatment, a set of animals was tested in the open field for 30 minutes. The open field measures motor activity and anxiety. Individual animals were transferred from the home cage to an open field arena made of Plexiglas (17×17 in2). The Auto-Track System senses motion with a grid of infrared photocells surrounding the arena. Vertical motion is detected by a second array of photocells placed above the animal. Horizontal activity, vertical activity, total distance traveled, overall movement number, movement time, and rest time were recorded by the system connected to the arena. Immediately after each test the apparatus was thoroughly cleaned.
Upon completion of all testing, the animals were sacrificed by CO2 asphyxiation and in the first set of animals the brain was collected and immediately frozen on liquid N2. RNA extraction was performed by homogenizing each brain sample using a sonicator (Janke & Kunkel) and the samples were processed with SV Total RNA Isolation System (Promega). A 1-microliter aliquot was used for spectrophotometric determination of RNA content. The remaining sample was stored at −80°C. Using 5 micrograms of total RNA, the reverse transcriptase reaction was performed to synthesize cDNA (Applied Biosystems) with a final volume of 150 microliters. Each sample was run in duplicate. Negative controls included reverse transcriptase reactions omitting RNA or reverse transcriptase. In the second set of animals, the hippocampus was dissected separately to evaluate expression of gene products specifically in the hippocampus, due to its importance in learning and memory. In addition, to elucidate the changes while treatment was ongoing, the second group of animals was sacrificed immediately after the first probe test, on the last day of treatment.
Calibrator normalized real-time polymerase chain reaction (PCR) was performed using at least four samples from each group from at least three different litters per treatment. The primers used for real-time PCR included the primer pairs for ADNP, vasoactive intestinal peptide, GFAP, NR2A, NR2B, and GABAAα5. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) was used as an internal standard. Primer sequences and producer companies are available upon request. With the use of the FastStart DNA Master SYBR Green 1 dye-base detection (Roche), vasoactive intestinal peptide, ADNP, GFAP, NR2A, NR2B, and GABAAα5 expression were measured by real-time PCR using the LightCycler with relative quantification software (Roche) and melting point analysis to assess the specificity of the amplified genes. To eliminate the risk of cross-contamination, LightCycler Uracil-DNA Glycosylase (Roche) was added to the master mix. Each sample was run in duplicate and relative quantification was performed using calibrator-normalized data with efficiency correction. Results are presented as a normalized ratio of ADNP, vasoactive intestinal peptide, GFAP, NR2A, NR2B, or GABAAα5-to-GAPD.
Statistical analysis included ANOVA and the Fisher protected least significant difference (PLSD) and Kruskal-Wallis for nonparametric evaluation of the gene expression studies with P<.05 considered significant (Statview 5.0.1).
Adult treatment with D-NAP+D-SAL prevented the Ts65Dn learning deficits in the Morris watermaze. Ts65Dn animals treated with placebo did not learn over the 5-day period as compared to the wild-type animals (P<.001). Ts65Dn animals treated daily with D-NAP+D-SAL learned significantly better than the Ts65Dn+placebo mice (P<.05) and similar to the wild-type animals (Fig. 1A). On treatment day 9, Ts65Dn+(D-NAP+D-SAL) retained learning during the probe test, with results similar to the wild-type animals (P=.48). Ten days after stopping treatment, the Ts65Dn+(D-NAP+D-SAL) animals did not maintain learning in the probe test with results similar to the Ts65Dn+placebo (P=.93, Fig. 1B).
The animals that were assessed in the open field were tested initially before treatment and then on day 9 after treatment with placebo or peptides. Nine Ts65Dn animals and four control (wild-type) animals were tested before treatment. After treatment there were three groups, Ts65Dn+placebo (n=4), Ts65Dn+(D-NAP+ D-SAL) (n=5) and wild type+placebo (n=4). Comparing Ts65Dn with wild type, there were significance differences in the total distance traveled (P=.003) and movement time (P=.007), no differences were seen for horizontal and vertical activity or movement number (all P>.05). Rest time correlated with movement time and is not reported separately. Treatment with placebo did not change the parameters for Ts65Dn or wild-type animals (Fig. 2A,B). Treatment with peptides brought the total distance traveled (P=.042, Fig. 2A) and movement time (P=.002, Fig. 2B) significantly less than Ts65Dn+placebo and similar to the wild-type animals (all P>.05).
In Ts65Dn brain after the second probe test, we found decreased GFAP and ADNP expression and increased vasoactive intestinal peptide compared with wild-type animals (P=.01, P=.04; P=.002, respectively). Treatment of adult Ts65Dn mice with D-NAP+D-SAL prevented the alterations in ADNP and vasoactive intestinal peptide expression, with levels similar to wild-type animals (P=.001; P=.036, respectively). However, the peptides did not prevent GFAP down-regulation (P=.90, Fig. 3A). NR2B, NR2A, and GABAAα5 are decreased in adult Down syndrome mice (P=.010, P=.001, P=.008, respectively). Postnatal administration of D-NAP+D-SAL increased NR2B expression to control levels; Ts65Dn+(D-NAP+D-SAL) compared with wild type, P=.63, but did not affect NR2A or GABAAα5 expression; Ts65Dn+(D-NAP+D-SAL) compared with Ts65Dn+placebo, P=.89, P=.43 (Fig. 3B).
In the second set of animals, the hippocampus was evaluated separately for expression of gene products due to its importance in learning and memory. In addition, to elucidate the changes while treatment was ongoing, the second group of animals was sacrificed immediately after the first probe test, on the last day of treatment.
In the hippocampus, vasoactive intestinal peptide expression was increased in the Ts65Dn, while NR2B and GABAAα5 were decreased (P=.002, .02, and .03, respectively) compared with the controls. Treatment with D-NAP+D-SAL prevented the vasoactive intestinal peptide up-regulation (P=.04) and the NR2B down-regulation (P=.03) in the Ts65Dn, but it did not prevented the GABAAα5 dysregulation (P=.2). There were no differences in ADNP (P=.13), GFAP (P=.8), and NR2A (P=.9) in the hippocampus of the Ts65Dn mice compared with wild-type animals (Fig. 4).
In the Down syndrome model, postnatal treatment with the neuroprotective peptides D-NAP+D-SAL reversed the learning deficits with learning similar to controls. This learning was retained while on daily treatment. However, once daily treatment ceased, memory retention returned to Down syndrome levels. We also found that treatment of Down syndrome with D-NAP+D-SAL resulted in behavior and motor activity at level similar to controls in the open field test. We found that these effects are mediated, at least in part, through the reversal of the alterations in ADNP and vasoactive intestinal peptide levels and by increasing the expression of the more plastic NMDA subunit, NR2B, which facilitates learning.
Our findings extend the benefit shown with prenatal treatment (treatment during pregnancy) in the Ts65Dn model where NAP and SAL prevented developmental delay in the Down syndrome offspring.11 In addition, the findings are consistent with studies of learning enhancement with NAP and SAL in a model of fetal alcohol syndrome26 and in normal animals.27 The increased activity in the Down syndrome animals confirms the hyperactivity of the Ts65Dn mouse model.22
Based on our results and the literature,28 we hypothesize that the glial cell deficit in Down syndrome results in a decrease of ADNP and ADNF and a subsequent up-regulation of vasoactive intestinal peptide via a feedback mechanism. Treatment with D-NAP+D-SAL to the Down syndrome animals reversed the vasoactive intestinal peptide and ADNP expression to control levels. Since GFAP was not altered, the prevention of learning deficits likely includes a mechanism that does not increase glial cell numbers in the less plastic adult brain.
Learning is in part mediated by long-term potentiation, which is initiated by NMDA receptors, and influenced by the inhibitory tone of the GABA receptors. The NMDA receptors are composed of the ubiquitous NR1 subunit and a ratio of NR2A and NR2B subunits. The NR2B subunit is the most critical for long-term potentiation induction. It is prevalent during development and associated with greater plasticity of brain circuits and learning. The NR2A subunit is predominantly found in the adult brain, it is less plastic and thus less capable of triggering long-term potentiation. The NMDA receptors are regulated through the inhibitory tone of GABAA receptors in the hippocampus. The GABAAα5 subunit is involved in spatial learning. GABAAα5 knockout mice have shown increased long-term potentiation and learning.29,30 Previous work has found increase in the GABA inhibitory tone in Down syndrome, which may underlie the NMDA-induced long-term potentiation learning dysfunction.14 Our work is consistent with these findings; we found decreased levels of NR2A, NR2B, and GABAAα5 in adult Down syndrome brain. Adult administration of D-NAP+D-SAL did not affect NR2A or GABAAα5 levels; however, it did increase NR2B to levels similar to controls. Paralleling the findings in the whole brain, we found decreased expression of NR2B in the hippocampus in Down syndrome; treatment with D-NAP+D-SAL restored the level to control. The reversal of learning deficits may be due to the increase in the more plastic NR2B receptor subunit instead of decreasing the expression of the less plastic NR2A subunit or the inhibitory tone of GABAAα5. Our findings of NAP and SAL affecting the NMDA and GABA receptor subunits are consistent with work in a model of fetal alcohol syndrome.31
One limitation of the study is that the PCR for gene expression in the whole brain was conducted after the second probe test, in which the mice had not had D-NAP+D-SAL for 10 days and thus did not show memory retention. A hypothesis could be made that changes in the NR2A and GABAAα5 levels in the whole brain would be detected if PCR was performed immediately after the first probe test when memory was retained. Another limitation of our study is that the results on the hippocampus gene expression are available only on the animals sacrificed immediately after treatment and not after the second probe test.
The peptides NAP and SAL are 8 and 9 amino acids, respectively, with actions noted in femtomolar concentrations.8,32 We used the all-D amino acid configuration of these peptides, thus all amino acids were D-amino acids (not the typical L-amino acids). This allows for oral absorption of the peptides without being rapidly degraded. The potency of the all-D amino acid form of these peptides has been demonstrated in culture24 as well as in vivo.24,27
The adult treatment with D-NAP+D-SAL reversed the learning deficits, memory retention, and hyperactivity, but it could not maintain the memory retention after cessation of treatment, suggesting the need for a continuous treatment to obtain long-term effect. As the most common genetic etiology of mental retardation, Down syndrome remains one of the most common and challenging genetic disorders. To date, the treatment for children with Down syndrome focuses on early childhood intervention, screening for common problems, medical treatment where indicated, a conducive family environment, and vocational training, which can improve overall development.33 Our findings are novel in that we propose the possibility of an intervention that may enhance learning via reverse the learning deficits and potentially improve the quality of life. Another group, Fernandez et al, has found that GABAA antagonists may be useful therapeutic agents for the intellectual disabilities associated with Down syndrome.34
Our findings are supported by alterations in neuropeptides and mediators of long-term potentiation. These mechanisms, which include the neuroprotective actions of vasoactive intestinal peptide and ADNP as well as alterations in the NMDA receptor subunit, NR2B, help to understand the action of the peptides NAP and SAL. Further studies are encouraged to delineate possible pathways underlying the mechanism behind learning enhancement in the Ts65Dn mouse model of Down syndrome.
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