Down syndrome is the most common genetic cause of mental retardation due to triplication of all or part of chromosome 211 and occurs in 1 in 800 live births. In the newborn period, neonates with Down syndrome have hypotonia and delay in achievement of developmental motor2 and sensory milestones as well as in olfactory, auditory, and visual sensitivity.3,4 At the neuropathologic level, neonates with Down syndrome have a smaller brain, delayed myelination of neurons, and glial alterations.5 These early developmental anomalies may predispose to abnormalities in adulthood, including mental retardation and early onset of Alzheimer’s disease.
The Ts65Dn mouse is a well-characterized model for Down syndrome,6 with triplication of a segment of chromosome 16 that includes more than 55% of the genes present on human chromosome 21. In the newborn period, Ts65Dn mice mimic the human condition, presenting developmental delay in motor and sensory milestones.7 Microscopically, Ts65Dn neonates have fewer granule cells in the hippocampus, reduced long-term potentiation, and abnormal synaptic plasticity.8–10 In adulthood, Ts65Dn mice have a deficit in short-term and long-term memory and learning and early onset of the neuropathology of Alzheimer’s disease.7
Vasoactive intestinal peptide (VIP) levels are altered in Down syndrome. Human newborns with Down syndrome have increased VIP blood levels11; Ts65Dn mice have elevated brain VIP mRNA,12 and cortical astrocytes from postnatal day 8 brains show a defect in the signal transduction mechanism of the VIP receptor VPAC-1 with astrocyte dysfunction.13 Furthermore, blockade of VIP during embryogenesis is followed by hypotonia and growth and developmental delays14 in both humans with Down syndrome and Ts65Dn mice.
Vasoactive intestinal peptide stimulation of astrocytes results in the release of numerous neurotrophic factors, including activity-dependent neuroprotective protein and activity-dependent neurotrophic factor, which have demonstrated neuroprotective proprieties.15 Active fragments of activity-dependent neuroprotective protein and activity-dependent neurotrophic factor, NAPVSIPQ and SALLRSIPA, respectively, have shown therapeutic potential for developmental delay and learning deficit. Treatment of Down syndrome cortical neurons with SALLRSIPA or NAPVSIPQ resulted in a twofold increase in neuronal survival as well as a reduction of degenerative morphological changes.16 NAPVSIPQ+SALLRSIPA are neuroprotective in vivo against diverse neuronal insults, including excitotoxicity, closed head injury, ischemic brain injury, apoplipoprotein E deficiency and alcohol-induced microcephaly, fetal death, growth restriction, and learning deficits in fetal alcohol syndrome.15,17,18
To date, there is no therapy for the prevention of developmental delays in Down syndrome (MEDLINE, 1966–June 1, 2008; keywords: Down syndrome, treatment, development, fetus; all languages). Our hypothesis was that prenatal treatment with NAPVSIPQ+SALLRSIPA may prevent the developmental delay in the Ts65Dn mouse model for Down syndrome. In addition, because one quarter of the progeny inherits the trisomy, three quarters of the pups treated with the peptides are controls, thus we also sought to explore the peptides’ effects on achievement of normal development.
MATERIALS AND METHODS
Pregnant Ts65Dn females were randomly assigned to NAPVSIPQ+SALLRSIPA or control and were treated by investigators blinded to treatment and genotype on gestational days 8–12. This time period was chosen based on previous studies that showed that this is a critical time for VIP action during in utero development.19 Offspring were weighed and tested from postnatal day 5 to 21 for motor and sensory milestones with standardized tests7 by operators blinded to the pup’s treatment and genotype. The pup’s genotype was determined after completion of all tests.
Ts65Dn female mice (The Jackson Laboratory, Bar Harbor, ME) were kept in a 12-hour light/12-hour dark regimen; food and water were available at all times with a 6% fat diet. The mice received humane animal care in compliance with the National Institutes of Health guidelines for care and use of experimental animals. The protocol was approved by the Eunice Kennedy Shriver National Institute of Child Health and Human Development Animal Care and Use Committee. Females were mated with B6EiC3SnFi male mice; the presence of vaginal plug was checked twice daily, and the day of its appearance was considered day 0 (E0) of pregnancy.
Ts65Dn pregnant females were treated (intraperitoneal) on days E8, 9, 10, 11, and 12 (a typical mouse gestation is 18–21 days long) with saline (placebo, n=6) or peptides (n=4). The researchers providing the treatments and performing the developmental assessments were blinded to the treatments and to the genotyping. The female breeders were eartagged. Treatments were made by a single investigator (C.Y.S.) and were labeled with the eartag number. Treatments were given by other investigators (L.T. and D.A.). After delivery, the litters were coded and each animal was labeled with standard markings. After completion of the developmental tests, tail tips were collected and sent to Jackson Laboratory for genotyping. Treatment was performed with VIP-derived peptides from activity-dependent neurotrophic protein and activity-dependent neurotrophic factor, whose active fragments are, respectively, NAPVSIPQ (20 micrograms) and SALLRSIPA (20 micrograms) (SynPep, Dublin, CA). NAPVSIPQ was diluted in 50 microliters of dimethyl sulfoxide and in filtered Dulbecco’s phosphate-buffered saline; activity-dependent neurotrophic factor–9 was dissolved and diluted in filtered Dulbecco’s phosphate-buffered saline.
Six trisomic+peptides from four litters, 14 trisomic+ placebo from five litters, 18 control+placebo from six litters, and nine control+peptides pups from three litters were tested.
From neonatal days 5 to 21, newborn pups were weighed and tested daily for developmental milestones as previously published7 by the same operators blinded both to the genotype and the treatment group (Fig. 1). These standardized tests assess the achievement of motor and sensory behavior that would be expected for age. Tests were performed on the days behavior was expected to appear based on developmental curves from normal mice.20 A score was given every day for each behavior as follows: 0, behavior not present; 1, behavior weakly present; 2, first day of behavior presence; 3, second day of behavior presence. Tests included7,20,21 (Fig. 1):
- Weak tactile stimulation, which assesses the head-turning response triggered by the application of tactile stimuli (cotton swab) in the perioral area on both sides of the head; it is a sensory response mediated by the 5th cranial nerve
- Righting reflex, which assesses the ability of the pup to right itself when placed upside down; it is principally a test for labyrinthine and body-righting mechanism
- Vibrissa-placing reflex—when the mouse is suspended by the tail and lowered so that the vibrissae make contact with a solid object, the pup raises its head and forelimbs to grasp the object; this is principally a measure of neck and forelimb tone and strength
- Forelimb grasp, which measures the pup’s ability to flex its forelimbs and grasp a blunt instrument that strikes its forefeet; thus it is a measure of forelimb muscle tone and strength
- Cliff aversion, which is the ability of the mouse to turn and crawl away when placed on the edge of a cliff with its fore paws and face over the edge; this milestone requires intact feet sensitivity and muscle strength to turn the body
- Audio-startle response—a loud, sharp noise causes an immediate startle response; this is a test for auditory sensitivity
- Eye opening—the day on which the pup opens its eyes is recorded
- Forelimb placing—contact of the dorsum of the foot against the edge of an object will cause the foot to be raised and placed on the surface of that object when the animal is suspended and no other foot is in contact with a solid surface; this is a test of placing-reflex development with sensory and motor components
- Ear twitch, which tests the twitching of the pup’s ears at the touch of a cotton swab; this is a sensitivity test
- Screen climbing—pups climb a vertical wire-mesh (5×5 mm) screen using both fore paws and hind paws; this is a motor test
Statistical analysis of behavior included post hoc comparisons using paired t-tests with conservative Bonferroni-Dunn’s correction with significance level 5%.
Mouse genotyping revealed that 14 trisomic pups from five litters, 18 control from six litters, six trisomic+peptides from four litters, and nine control+peptides pups from three litters were included in the neonatal tests.
For genotyping, tail tips (2 mm/sample) from newborn pups from 7 to 21 days of age were collected. A previously published method was used.22 Briefly, tail tips were digested using an alkali method,23 samples were neutralized and diluted with Tris to about 0.1 ng/L based on an expected yield of approximately1 to 2 g DNA from a 1- to 2-mm tail sample. For genotyping, we used real-time quantitative polymerase chain reaction (PCR) to amplify myxovirus (influenza virus) resistance 1 (Mx1) gene, which is close to the distal end of the chromosome 16 segment in the trisomic chromosome. For Mx1 (Gen-Bank accession no. M21117), exon 14 sequence was used; the PCR product size is 74 bp. The sequences for primers and probe are: forward primer 5′-TCTCCGATTAACCAGGCTAGCTAT-3′, reverse primer 5′-GACATAAGGTTAGCAGCTAAAGGATCA-3′, and probe 5′-FAM-TGGCTTTCCTGGTCGCTGTGCA-TAMRA-3′. The apolipoproteinB gene (Apob; GenBank accession no. X15191) was used as an internal control to normalize variations of the amount of input DNA. The product size is 73 bp. The primer and probe sequences are: forward primer 5′-CACGTGGGCTCCAGCATT-3′, reverse primer 5′-TCACCAGTCATTTCTGCCTTTG- 3′, and probe 5′-VIC-CCAATGGTCGGGCACTGCTCAA-TAMRA-3′.The TaqMan probes for the target gene Mx1 and the control gene Apob were labeled with two different fluorescent reporters (FAM and VIC) so that a multiplexed PCR could be used. The PCR was set up as follows: 12.5 microliter of 2×TaqMan universal PCR master mix (Applied Biosystems, Foster City, CA), 0.25 microliter of each primer (40 micromolar), 0.75 microliter of each probe (5 micromolar), and 10 microliters of diluted template DNA (about 0.1 ng/microliter). The reaction was carried out at 50°C for 2 minutes and at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute on an ABI PRISM 7000 sequence detection system (Applied Biosystems) with a 96-well format. For each sample, triplicate PCRs were carried out.
After real-time PCRs, the average change (Δ) in cycle threshold (CT) of target gene Mx1 from that of the internal control Apob was calculated (ΔCT=CT for App or Mx1-CT for Apob) and analyzed. For each PCR experiment, we included a standard curve. These were done by using four 1:2 serial dilutions over a range of eightfold for one sample, including a dilution of about 0.1 ng/microliter DNA. Also, known trisomic and wild type samples were included in each run. Because the Ts65Dn mouse has three copies of the Mx1 gene, the expected difference between the ΔCT for Ts65Dn mice and the ΔCT for diploid control mice should be 0.585 (2–0.585=1.5, where 1.5 is the fold difference between three copies of Mx1 in trisomic mice compared with two copies in wild-type mice). The cycle threshold was adjusted so that wild type samples would show an average ΔCT close to 0 and trisomics would show an average ΔCT close to -0.6. Anything less obvious would suggest a failure, and the whole experiment would need to be repeated. The 95% confidence interval was determined for the trisomic and wild type samples, which then was used as the criterion to genotype whether a given unknown sample fell into the range of the diploid or trisomic samples. All genotyping samples were run after the completion of all testing.
To better understand the NAPVSIPQ+SALL RSIPA mechanism of action, we explored whether NAPVSIPQ+SALLRSIPA prevented the glial deficit in Ts65Dn mice by evaluating expression of VIP, the glial marker glial fibrillary acidic protein (GFAP), and activity-dependent neuroprotective protein. To evaluate long-term alterations in gene expression, adult brains were removed and immediately frozen in dry ice. Four trisomic, four control, and three trisomic+peptides adult brains were collected separately and immediately frozen in dry ice. Each group represented three litters, and mice were 40 weeks old.
For RNA isolation, samples were homogenized by a sonicator (Janke & Kunkel, Wilmington, NC) and processed with SV Total RNA Isolation System (Promega, Madison, WI). A 5-microliter aliquot was taken for spectrophotometeric determination of RNA content. The remaining sample was stored at -80°C. Using 5 micrograms of total RNA, the reverse transcriptase reaction was performed (Applied Biosystems) in a final volume of 150 microliters. Each RNA sample was run in duplicate.
For PCR, glyceraldehyde-3-phosphate dehydrogenase (GAPD) primers were synthesized by IDT (Integrated DNA Technologies, Coralville, IA), GFAP primer pair was synthesized by Superarray (Frederick, MD), activity-dependent neuroprotective protein primer pair was synthesized by Biosynthesis (Louisville, TX), and VIP was synthesized by TIB Molbiol (Adelphia, NJ). The GAPD gene, a housekeeping gene located on mouse chromosome 6, frequently is used as a normalized gene because of its stability in cells. In our experiments and in experiments reported by other groups24 we did not detect a significant difference in GAPD expression between the trisomic and wild type samples. The gene for the GFAP subunit is located on chromosome 11, VIP is located on chromosome 10, and activity-dependent neuroprotective protein on chromosome 2. With the use of the FastStart DNA Master SYBR Green 1 dye-base detection (Roche, Basel, Switzerland), VIP, activity-dependent neuroprotective protein, GFAP, and GAPD 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. Optimization for VIP-, activity-dependent neuroprotective protein–, GFAP-, and GAPD-specific primers was performed with separate runs by varying magnesium chloride and primer concentrations, amount of template, and annealing temperature. The presence and purity of target gene sequence expression in the reverse transcriptase PCR reaction were confirmed by gel electrophoresis. Samples were run in duplicate, and relative quantification was performed by using calibrator-normalized data with efficiency correction. Results are presented as the normalized ratio of VIP, GFAP, and activity-dependent neuroprotective protein–to–GAPD expression.
An analysis of variance test was performed for overall comparisons between the treatment groups, and Fisher’s protected least significance difference test was used for post hoc analysis, with P<.05 considered significant (StatView 5.0.1, SAS Institute Inc., Cary, NC).
Trisomic mice achieved milestones with a significant delay in four of five motor (forelimb grasp, vibrissa placing, righting, and cliff aversion) and four of five sensory (eye open, ear open, weak tactile stimulation, forelimb placing) milestones and showed growth delay compared with the controls. These results confirm a previous study that showed developmental delay in the trisomic mice and is consistent with human findings.7 Trisomic mice that were prenatally exposed to NAPVSIPQ+SALLRSIPA achieved developmental milestones at the same time as the controls in three of four motor (righting, vibrissa placing, and forelimb grasp) and one of four sensory (weak tactile stimulation) milestones (P<.01), and there was a trend toward better performance in the trisomic+peptides compared with the trisomic group in forelimb placing and weight (Fig. 2).
Control pups prenatally treated with NAPVSIPQ+ SALLRSIPA achieved developmental milestones significantly earlier than did the controls treated with placebo in four of five motor (righting, vibrissa placing, cliff aversion, and screen climbing) and two of five sensory (ear twitch and weak tactile stimulation) milestones (all P<.01; Fig. 3).
To better understand the NAPVSIPQ+SALLR SIPA mechanism of action, we explored whether NAPVSIPQ+SALLRSIPA prevented the glial deficit in Ts65Dn mice by evaluating expression of VIP, the glial marker GFAP, and activity-dependent neuroprotective protein. Polymerase chain reaction analysis showed that, although activity-dependent neuroprotective protein was significantly downregulated in the Ts65Dn brains compared with the controls, prenatal treatment with NAPVSIPQ+SALLRSIPA prevented the activity-dependent neuroprotective protein decrease in the Ts65Dn brains and the expression was not different from the controls. The glial marker GFAP demonstrated the known glial deficit in the Ts65Dn mice, and treatment with NAPVSIPQ+SALLRSIPA prevented its downregulation. Lastly, VIP levels were increased in the trisomic brains, whereas treatment with NAPVSIPQ+ SALLRSIPA did not prevent its upregulation (Fig. 4).
We found that prenatal treatment with VIP-regulated peptides prevented developmental delay in the neonatal period and resulted in prevention of the glial deficit in adulthood. In the neonatal period, Ts65Dn pups were delayed in achieving important milestones, as is seen in the human condition. These developmental milestones are ultimately achieved; the delay may represent a nervous system deficit that may result in neurodevelopmental and learning deficits in adulthood. We hypothesize that by preventing the developmental delay with the peptides, we may be able to prevent longer term neurodevelopmental and learning abnormalities in the adult offspring. One of the neuropathologic characteristics of Down syndrome is a glial deficit that likely induced alterations in VIP and its related neuropeptides.
In cortical neurons of the brains of people with Down syndrome, activity-dependent neuroprotective protein and VIP receptor VPAC-1 expression have been shown to be decreased,16 whereas VIP levels were found to be elevated in the blood of neonates with Down syndrome,11 possibly as a feedback mechanism to overcome the glial deficit. In the Ts65Dn mouse also, we and others13 show a decrease in activity-dependent neuroprotective protein and an upregulation of VIP, changes that are present already in postnatal day 8 neonates. Astrocytes in adult Ts65Dn brains are abnormal, as indicated by the downregulation of the glial marker GFAP. Here we show that prenatal treatment with peptides derived from activity-dependent neuroprotective protein and activity-dependent neurotrophic factor resulted in a normalization of activity-dependent neuroprotective protein and of the glial marker GFAP in adult brains; treatment with the peptides may have overcome the glial deficit by restoring the appropriate neuropeptides. Nonetheless, treatment did not prevent VIP upregulation; it is possible that other mechanisms regulate VIP release (more comments on NAPVSIPQ+SALLRSIPA mechanism of action are in the Appendix, available online at www.greenjournal.org/cgi/content/full/112/6/1242/DC1.).These findings may explain, at least in part, NAPVSIPQ+SALLRSIPA prevention of glial deficit, developmental delay in the Ts65Dn pups, and developmental enhancement in the wild type mice.
In this study, we also show that control animals prenatally treated with NAPVSIPQ+SALLRSIPA had accelerated development. Previously, we have shown learning enhancement after treatment with NAPVSIPQ+SALLRSIPA in adulthood,18,25 and these results suggest that the adult enhancement observed might be a consequence of faster developmental-milestone achievement.
Because Down syndrome can be diagnosed prenatally, it is possible that an intervention during pregnancy may improve the outcome. The prevention of developmental delays in patients with Down syndrome may both improve the neonatal period as well as affect learning skills in adulthood. We are currently studying the effects of prenatal treatment with NAPVSIPQ+SALLRSIPA on prevention of learning deficit in adult mice. These findings highlight a possibility for the prevention of developmental sequelae in Down syndrome and other conditions with developmental delay and learning deficit.
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© 2008 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.
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