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Overexpression of DYRK1A inhibits choline acetyltransferase induction by oleic acid in cellular models of Down syndrome
Experimental Neurology 239 (2013) 229–234
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Overexpression of DYRK1A inhibits choline acetyltransferase induction by oleic acid in cellular models of Down syndrome Maruan Hijazi a, Cristina Fillat b, José M. Medina a, Ana Velasco a,⁎ a b
Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, (IBSAL), Spain Instituto de Investigaciones Biomédicas August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Raras (CIBERER) Barcelona, Spain
a r t i c l e
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Article history: Received 26 March 2012 Revised 19 October 2012 Accepted 26 October 2012 Available online 1 November 2012 Keywords: Down syndrome DYRK1A Oleic acid Albumin Differentiation
a b s t r a c t Histological brain studies of individuals with DS have revealed an aberrant formation of the cerebral cortex. Previous work from our laboratory has shown that oleic acid acts as a neurotrophic factor and induces neuronal differentiation. In order to characterize the effects of oleic acid in a cellular model of DS, immortalized cell lines derived from the cortex of trisomy Ts16 (CTb) and normal mice (CNh) were incubated in the absence or presence of oleic acid. Oleic acid increased choline acetyltransferase expression (ChAT), a marker of cholinergic differentiation in CNh cells. However, in trisomic cells (CTb line) oleic acid failed to increase ChAT expression. These results suggest that the overdose of specific genes in trisomic lines delays differentiation in the presence of oleic acid by inhibiting acetylcholine production mediated by ChAT. The dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) gene is located on human chromosome 21 and encodes a proline-directed protein kinase. It has been proposed that DYRK1A plays a prominent role in several biological functions, leading to mental retardation in DS patients. Here we explored the potential role of DYRK1A in the modulation of ChAT expression in trisomic cells and in the signaling pathways of oleic acid. Down-regulation of DYRK1A by siRNA in trisomic CTb cells rescued ChAT expression up to levels similar to those of normal cells in the presence of oleic acid. In agreement with these results, oleic acid was unable to increase ChAT expression in neuronal cultures of transgenic mice overexpressing DYRK1A. In summary, our results highlight the role played by DYRK1A in brain development through the control of ChAT expression. In addition, the overexpression of DYRK1A in DS models prevented the neurotrophic effect of oleic acid, a fact that may account for mental retardation in DS patients. © 2012 Elsevier Inc. All rights reserved.
Introduction Down syndrome (DS) is a genetic disease characterized by the presence of an extra copy of the human chromosome 21 (HSA21) (Lejeune et al., 1959). Intellectual disability is a common trait in DS individuals, primarily as a consequence of alterations in neurogenesis, neuronal differentiation, myelination, dendritogenesis and synaptogenesis during embryonic and fetal development (Becker et al., 1991; Coyle et al., 1986). Neurotransmitter alterations and changes in the activity and function of their receptors have been described and they may be responsible for some of the phenotypes described (reviewed in Rueda
Abbreviations: DS, Down syndrome; CNh, cerebral cortex cells of normal mice; CTb, cerebral cortex cells of trisomic 16 mice; DYRK1A, dual-specificity tyrosine-phosphorylated and regulated kinase 1A; ChAT, choline acetyltransferase; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; siRNA, small interfering RNA. ⁎ Corresponding author at: Instituto de Neurociencas de Castilla y León (INCYL), Universidad de Salamanca, C/ Pintor Fernando Gallego 1, 37007 Salamanca, Spain. Fax: + 34 923 29 45 64. E-mail address: [email protected] (A. Velasco). 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2012.10.016
et al. (2012)). The DS neurological alterations affect key brain regions involved in learning and memory processes, and they could be the origin of the observed intellectual disability (see review Rachidi and Lopes (2010)). Trisomy 16 (Ts16) mice were initially generated to model HSA21 trisomy due to the synteny of both chromosomes. Unfortunately the Ts16 condition is unviable and the animals die in utero. However Ts16-derived neuronal cultures are good models to study DS-related cellular phenotypes (Cheng et al., 2004), (for a review, see Antonarakis et al., 2004). Several DS-related genes or their combination and interaction with genes in other chromosomes may underlie the biological mechanisms of the different DS phenotypes. DYRK1A (dual-specificity tyrosine (Y)-regulated kinase) has been proposed as one of the genes on human chromosome 21 with a relevant role in those brain functions altered in DS (reviewed by Dierssen and de Lagran, 2006; Hammerle et al., 2003). DYRK1A is overexpressed in DS brains (Dowjat et al., 2007; Guimera et al., 1999) and is involved in neurogenesis and learning/memory processes (Tejedor et al., 1995). Transgenic mice overexpressing DYRK1A (TgDyrk1A) exhibit DS-like features such as neurodevelopmental delay, motor abnormalities and cognitive deficits (Altafaj et al., 2001).
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Cholinergic deficits in the brain are a hallmark in both human DS and Ts16 mice. In this context, synaptic neurochemical studies have demonstrated that the brains of DS individuals exhibit a significant reduction in choline acetyltransferase (ChAT) activity in the cerebral cortex (Yates et al., 1983), which is consistent with the impaired development of the basal forebrain cholinergic system exhibited by Ts16 mice (Corsi and Coyle, 1991). Previous work carried out at our laboratory has shown that the neurotrophic factor oleic acid is synthesized by astrocytes after exposure to albumin. The oleic acid is then released to the extracellular space, thereby becoming available for neurons (Tabernero et al., 2002). Thus, oleic acid induces the differentiation of neurons in culture, promoting cell clustering, neurite growth, and the expression of GAP-43 and MAP-2, markers of axonal and dendrite growth respectively (Rodriguez-Rodriguez et al., 2004; Tabernero et al., 2001). Additionally, it has been observed that oleic acid is synthesized in the periventricular zone of lateral ventricles promoting axonogenesis in the striatum during brain development (Polo-Hernandez et al., 2010). Indeed, the oleic acid–albumin complex may have pharmacological properties because treatment with the complex causes a significant recovery after medullar section (Avila-Martin et al., 2011). It is noteworthy that albumin is present in high concentrations during brain development (Mollgard et al., 1988; Velasco et al., 2003). Interestingly, it has been reported that individuals with DS have lower albumin concentrations than people without the syndrome (Clarke and Bannon, 2005). In light of this background, we were prompted to study the effects of the neurotrophic factor oleic acid in cellular models of DS. Material and methods Cell lines CNh (cerebral cortex cells derived from wild type mice) and CTb (cerebral cortex cells obtained from mouse fetuses with trisomy 16), were previously described by Cardenas et al. (1999). These cell lines were developed at the laboratory of Dr. Pablo Caviedes (University of Chile) and are marketed through an agreement with the University of South Florida. CNh and CTb cells retain neuronal markers such as neuronal specific enolase—NSE, choline acetyl transferase—ChAT, synaptophysin, microtubule-associated protein 2—MAP2 and a lack of the glial markers (GFAP, galactocerebroside, S-100) (Saud et al., 2006). Cells were plated on Petri dishes in modified DMEM/Ham's F12 medium (6 g/L glucose and supplemented with 1 mM pyruvate and N2 (Invitrogen, Barcelona, Spain) containing 10 mM human transferrin, 10 nM insulin, 20 nM progesterone, 0.1 mM putrescine and 30 nM selenium) for treatment with oleic acid. 0.1% BSA and 33 μM oleic acid (Sigma, St. Louis, USA) were also added in the specified conditions. Culture of primary neurons from transgenic mice overexpressing DYRK1A TgDyrk1A mice were previously generated at the laboratory of Cristina Fillat as described in Altafaj et al. (2001). The animals were housed under standard conditions and experiments were performed according to local and EU Ethical Committee guidelines. Hemizygous TgDyrk1A mice were mated with wild-type females. Conception was confirmed by the presence of a vaginal plug. For preparing neurons in primary culture, fetuses at 15 days of gestation were delivered by rapid hysterectomy after cervical dislocation of the mother. Neuronal cultures were prepared as previously reported (Tabernero et al., 1993). Neurons were grown in DMEM/Ham's F12 medium, supplemented with 5 μg/mL insulin, 10 μg/mL transferrin, 1 mM pyruvate, 50 U/mL penicillin and 37.5 U/mL streptomycin. Neuronal cultures were performed by plating dissociated embryonic brain cells onto poly-L-lysine (10 μg/mL-coated Petri dishes) at a density of 1.0 × 10 6
cells/cm2. After plating, neurons were cultured in defined serum-free medium in the presence of 2% (w/v) BSA or 2%(w/v) BSA plus 50 μM oleic acid. Cells were grown for 3 days and were then fixed or collected for analysis by immunocytochemistry or western blot. Twenty-three fetuses were analyzed (15 wild-type and 8 TgDyrk1A). Genotyping transgenic mice with the polymerase chain reaction (PCR) Genomic DNA was obtained from 25 mg of fetal tissue and purified with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Genotyping of the fetuses was performed by PCR analysis using the Dyrk1A transgene-specific primers 5′-GTCCAAACTCATCAATGTATC and 5′-CTTGAGCACAGCACTGTTG and the 5-GAGCACCCTGTGCTGCTCACCGAGG and 5-GTGGTGGTGAAGCTGT AGCCACGCT primer pair for β-actin amplification. The PCR conditions were as follows: 5 min of denaturation at 94 °C, 32 (Dyrk1A) or 35 (β-actin) cycles of denaturation at 94 °C for 30 s, annealing at 54 °C (Dyrk1A) or at 55 °C (β-actin) for 30 s, and extension at 72 °C for 45 s, followed by 10 min at 72 °C. DNA amplification was visualized on 2% agarose gel. Immunocytochemistry Cells were fixed with 4% (w/v) formaldehyde in PBS for 30 min and permeabilized in 100% methanol for 20 min at −20 °C. Cells were then incubated with rabbit polyclonal antibody against ChAT overnight at 4 °C and then incubated with goat anti-rabbit AlexaFluor488 secondary antibody and TOPRO-3-iodide (used to visualized cell nuclei (Invitrogen)) for 1 h at room temperature. Cells were then mounted using the Slowfade Gold Antifade kit (Invitrogen). A Zeiss LSM510 confocal microscope was used to obtain images. Determination of protein expression by Western blot analysis Cell proteins were extracted using RIPA buffer containing 25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS and cocktail protease inhibitors (Calbiochem, Darmstadt, USA). Lysates were centrifuged at 14.000 ×g for 15 min at 4 °C. 20 μg of protein extract was analyzed in 8% precast commercial gels (NuPAGE Novex 8% Bis-Tris Midi Gel 1.0 mm) or in 10% precast commercial gels (NuPAGE Novex 10% Bis-Tris Midi Gel 1.0 mm). The buffer used for protein electrophoresis was NuPAGE MOPS SDS Running Buffer 20×. NuPAGE Sample Reducing Agent 10 × and NuPAGE LDS Sample Buffer 4 × were used to prepare the samples. Electrophoresis was run at room temperature using a constant voltage. After electrophoresis, gels were washed in transfer buffer (10% Panreac methanol, 0.1% NuPAGE Antioxidant diluted in NuPAGE Transfer Buffer 2 ×) for 10 min. Then, the proteins separated were transferred to a nitrocellulose membrane (iBlot Gel Transfer Stacks Nitrocellulose) for 10 min, applying a constant voltage. All products used for electrophoresis and subsequent electrotransfer were purchased from Invitrogen (Life Technologies S.A. Madrid, Spain). After blocking for non-specific binding, the membranes were incubated for 14 h at 4 °C with rabbit polyclonal antibodies against ChAT (Cat. AB143 Millipore, CA, USA), DYRK1A (Cat. ab69811 Abcam, Cambridge, UK) or GAP-43 (Cat. AB5220 Millipore, CA, USA). Mouse monoclonal antibody against GAPDH (Cat. AM4300 Ambion, Life Technologies S.A., Madrid, Spain) was used to quantify and normalize protein expression. Following this, after several washes membranes were incubated with secondary antibody against mouse or rabbit immunoglobulin conjugated with peroxidase. Finally, the membranes were incubated for 1 min with peroxidase substrates, which afforded a chemiluminescence reaction. The signal collected on the autoradiographic film was proportional to the amount of protein in the membrane. The bands were quantified using an image analysis program.
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Transfection of siRNA CNh and CTb cells were transfected with a validated non-targeting siRNA (NT-siRNA) or with a siRNA specific for Dyrk1A suppression (Dyrk1A-siRNA; 5′-CACUGAAGCUCCUACACAATT-3′). Three different siRNA sequences for this protein were tested and the same phenotype was found. Transfections were performed with Lipofectamine 2000 reagent, according to the manufacturer's instructions. Cell treatments were performed 24 h after transfections. After the siRNA transfections, the reduction in protein expression was assessed by Western blot analysis. Statistical analysis Results are mean± SEM of at least three independent experiments. Statistical analyses were carried out with Student's t-test. Values were considered significant when p b 0.05. Results Oleic acid induces choline acetyltransferase expression in normal cells but not in trisomic cells In order to characterize whether oleic acid modifies ChAT levels, we cultured cerebral cortex cells from normal and trisomic mice (CNh and CTb, respectively) in the absence or presence of oleic acid and we measured ChAT expression by immunocytochemistry and by western blot analysis. As shown in Fig. 1, treatment with oleic acid significantly increased ChAT expression in CNh cells. Quantification of the data revealed a 50% increase when compared to CNh untreated cells. In contrast, ChAT expression was not significantly modified by oleic acid in CTb cells. These results are in agreement with the work of Allen et al. (2000) describing alterations in the cholinergic system and reinforce the presence of an impaired cholinergic function in CTb cells. DYRK1A inhibition increases ChAT expression in trisomic cells To better understand the molecular determinants involved in ChAT regulation in response to oleic acid we studied the effects of oleic acid in DYRK1A knockdown CNh and CTb cells. Cells treated with oleic acid were transfected with siRNA sequences against DYRK1A or with an oligonucleotide validated as a non-targeting siRNA (NT-siRNA). Western blot analysis demonstrated a 1.5-fold overexpression of DYRK1A in CTb cells compared to CNh cells, in keeping with the presence of an extra-copy of the DYRK1A gene in the trisomic cells. siRNA-Dyrk1A knockdown induced a significant reduction in DYRK1A protein expression, estimated at 75% in CNh cells and 70% in CTb cells (Figs. 2A and B). Expression analysis of the ChAT protein revealed reduced enzyme expression in the control and NT-siRNA-treated CTb cells, similar to what was observed previously in non-silenced cells (Fig. 1). However, in CNh and CTb DYRK1A knockdown cells the levels of ChAT expression were similar (Figs. 2A and C). These results suggest that the overexpression of DYRK1A prevents the induction of ChAT promoted by oleic acid in CTb cells.
Fig. 1. Oleic acid increases choline acetyltransferase expression in normal cells but not in trisomic cells. ChAT expression in CNh and CTb cells in the absence or presence of oleic acid was analyzed by immunocytochemistry (A) and Western blot analysis (B). Data were obtained by measuring the fluorescent areas of the previous images using the image-analyzer software (Scion Image). For immunocytochemistry of ChAT, three cultures with three plates per condition were made to take four photomicrographs from each plate. Approximately, 60 cells per plate were analyzed. Results are expressed as percentages of CNh as means±SEM (n=5), **pb 0.01; ***pb 0.001 as compared with CNh cells in the absence of oleic acid.
ChAT expression is inhibited in cultured neurons from transgenic mice overexpressing DYRK1A (TgDyrk1A) To further evaluate the potential involvement of DYRK1A in the regulation of the ChAT expression promoted by oleic acid, we analyzed ChAT levels in primary neuronal cultures from TgDyrk1A (Altafaj et al., 2001). We analyzed 23 independent fetuses: 8 fetuses were positive for transgenic DYRK1A, and 15 were wild-type (Fig. 3A). Each brain was cultured individually in the absence (A) or presence of oleic acid (O). After 3 days, photomicrographs were taken to evaluate
ChAT expression and other possible changes induced by oleic acid. Immunocytochemical analysis against ChAT revealed an increase in ChAT expression in wild-type cells caused by oleic acid (Fig. 3B). These results are in agreement with previous data showing an increase in ChAT expression due to the presence of fatty acids (Hattori et al., 1987; Machova et al., 2006). However, no differences were observed
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Fig. 2. The inhibition of DYRK1A expression in CTb cells rescues the phenotype of normal cells. A) DYRK1A expression was inhibited by RNA interference technique, as described in Material and methods. CNh and CTb cells were incubated with 33 μM oleic acid and transfected with non-targeting siRNA (NT-siRNA) or with siRNA specific for DYRK1A (Dyrk1A-siRNA). Western blot analyses of DYRK1A were carried out. B) Values of DYRK1A expression were quantified and normalized with GAPDH and are means ± SEM of at least three independent experiments **p b 0.01; ***p b 0.001 vs. CNh. C) Analyses of ChAT protein after the inhibition of DYRK1A expression by siRNA. After 3 days of transfection, cells were collected to analyze ChAT expression by Western blot analyses. ChAT values were quantified and normalized with GAPDH and are means ± SEM of at least three independent experiments **p b 0.01 vs. CNh.
in ChAT staining in neuronal cultures from TgDyrk1A mice upon treatment with oleic acid. Furthermore, western blot analysis showed increased expression levels of ChAT in the presence of oleic acid in normal cells but not in TgDyrk1A cells (Fig. 3C). Interestingly, the expression of GAP-43 was also increased in the presence of oleic acid in wild-type neurons, while it was almost unaltered in TgDyrk1A cultures (Fig. 3D). Taken together these results support the hypothesis that the overexpression of Dyrk1A acts as a negative regulator of cholinergic differentiation promoted by oleic acid. Discussion This study presents a previously uncharacterized connection between DYRK1A overexpression and ChAT expression in DS models. Our results support those of Chen et al. (2009), who used magnetic resonance imaging in disomic and trisomic mice and reported that forebrain cholinergic neurons were reduced in a DS model, a fact that may account for the failures in the neurotransmitter system and alterations in the electrical properties of cell membranes observed in DS patients (Caviedes et al., 1991). In fact, these defects are probably due to the overexpression of certain genes, some of which may affect the membrane composition and subsequently the function of some receptors (Caviedes et al., 1990). Using neurons in primary cultures from Ts16 mice, a significant decrease in choline
uptake by the high-affinity choline transporter has been reported, together with a decrease in acetylcholine release following depolarization (Fiedler et al., 1994). Here we demonstrate that oleic acid induces the expression of ChAT in CNh cells whereas it failed to induce the expression of ChAT in CTb cells. Pioneer reports have highlighted a potential role of oleic acid in the cholinergic system (Massarelli et al., 1988). These authors showed that there was a choline acetyltransferaselike activity bound to neuronal plasma membranes that was responsible for the increase in the production of the neurotransmitter acetylcholine. Thus, acetylcholine is synthesized from radioactive acetyl-CoA by isolated rat brain synaptosomes but it was released to the incubation medium only when the synthesis of choline was stimulated by oleic acid through the activation of phospholipase D. In this context, results from our laboratory revealed that oleic acid was incorporated in neurons, preferentially into phosphatidylcholine and phosphatidylethanolamine (Tabernero et al., 2001), probably to increase membrane fluidity. Moreover, the hydrolysis of phosphatidylcholine can provide lipid second messengers, which participate in signal transduction mechanisms. In addition, Granda et al. (2003) showed that oleic acid induced neuronal differentiation through a PKC-mediated mechanism that was not mediated by other neurotrophic factors, but that strongly synergized with neurotrophins NT-3 and NT-4/5. The results presented here suggest that the overexpression of some genes in the trisomic region hinders the effect of oleic acid on the expression of ChAT, which probably affects acetylcholine production to coordinate cell signaling regulatory mechanisms. In our study, we observed that after silencing DYRK1A expression with the RNA interference trisomic cells rescued the phenotype of normal cells. Moreover, measurements of ChAT levels by Western blot revealed that ChAT expression increased in DYRK1A-silenced cells up to the same levels found in non-silenced cells. These results suggest that DYRK1A may inhibit the effect of oleic acid on ChAT expression in CTb cells. Since silencing Dyrk1A did not affect ChAT expression in non-trisomic cells, it is reasonable to suggest that the expression of ChAT was maximal in the presence of oleic acid. In addition to the changes in DYRK1A activity, the occurrence of some additional regulatory mechanisms controlling ChAT expression in non-trisomic cells remains to be elucidated. In keeping with these results, cultured neurons from transgenic mice overexpressing DYRK1A exposed to oleic acid failed to increase ChAT expression as compared to wild-type cells. The reduction in immunolabeled ChAT in neuronal outgrowth is also noteworthy. In support of this, the presence of oleic acid did not promote profound changes in the shape of the neurons from TgDyrk1A mice, unlike the situation in wild-type cells. Thus, whereas wild-type neurons exposed to oleic acid tended to aggregate to form clusters and elongated axons and dendrites, these morphological changes were not observed in TgDyrk1A neurons, again suggesting impairment in neuronal differentiation in TgDyrk1A mice. In agreement with this observation, the expression of GAP-43 in TgDyrk1A cells failed to increase in response to oleic acid. In addition, these data proposes Dyrk1A as a target gene involved in the cholinergic system. In this respect, the role of the cholinergic system in cognition is well established, as supported by cognitive deficits observed in transgenic mice overexpressing DYRK1A (Ahn et al., 2006; Altafaj et al., 2001). Moreover, some cognitive enhancers are based on the increase in the tonic levels of acetylcholine (Demeter and Sarter, 2013). Earlier reports have highlighted the idea that DS as well as Alzheimer's patients have decreased levels of monounsaturated fatty acids in their brain phospholipids (Martin et al., 2010; Shah, 1979). These biochemical alterations may be responsible for the decrease in neuronal interconnections frequently observed in such patients (Elul et al., 1975). It could be speculated that the normalization of DYRK1A activity in DS patients could facilitate the positive effects of oleic acid in neuronal transmission.
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Fig. 3. The increase of ChAT expression by oleic acid is inhibited by DYRK1A overexpression. A) DYRK1A and β-actin polymerase chain reaction (PCR) of ten TgDyrk1A fetuses. PCR was carried out as described in Material and methods. Results were visualized on agarose gel with ethidium bromide in a UV transilluminator. B) Pattern of ChAT immunoreactivity in wild-type (WT) and transgenic DYRK1A (TgDyrk1A) neurons cultured separately for 3 days in defined medium in the absence (A) or presence of 50 μM oleic acid (O). Bar 50 μm. C) and D) Western blot analyses of ChAT protein and GAP-43 protein respectively, in WT and TgDyrk1A mice in the absence or presence of oleic acid. After 3 days, cells were collected and analyzed by Western blot as described in Material and methods. Values for ChAT and GAP-43 were quantified and normalized with GAPDH and are means ± SEM of at least three independent experiments **p b 0.01 vs. WT (A).
Taken together, our results confirm the importance of oleic acid as a neurotrophic factor and highlight the involvement of DYRK1A in the development of the brain.
Acknowledgments This work was supported by the Ramón Areces Foundation (A.V.), Spain and the Jêrome Lejeune Foundation (C.F.), France. We are grateful to S. Capdevila and C. Rodriguez for helping with the animals and for the technical assistance of T. del Rey.
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Overexpression of DYRK1A inhibits choline acetyltransferase induction by oleic acid in cellular models of Down syndrome Maruan Hijazi a, Cristina Fillat b, José M. Medina a, Ana Velasco a,⁎ a b
Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, (IBSAL), Spain Instituto de Investigaciones Biomédicas August Pi i Sunyer (IDIBAPS), CIBER de Enfermedades Raras (CIBERER) Barcelona, Spain
a r t i c l e
i n f o
Article history: Received 26 March 2012 Revised 19 October 2012 Accepted 26 October 2012 Available online 1 November 2012 Keywords: Down syndrome DYRK1A Oleic acid Albumin Differentiation
a b s t r a c t Histological brain studies of individuals with DS have revealed an aberrant formation of the cerebral cortex. Previous work from our laboratory has shown that oleic acid acts as a neurotrophic factor and induces neuronal differentiation. In order to characterize the effects of oleic acid in a cellular model of DS, immortalized cell lines derived from the cortex of trisomy Ts16 (CTb) and normal mice (CNh) were incubated in the absence or presence of oleic acid. Oleic acid increased choline acetyltransferase expression (ChAT), a marker of cholinergic differentiation in CNh cells. However, in trisomic cells (CTb line) oleic acid failed to increase ChAT expression. These results suggest that the overdose of specific genes in trisomic lines delays differentiation in the presence of oleic acid by inhibiting acetylcholine production mediated by ChAT. The dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) gene is located on human chromosome 21 and encodes a proline-directed protein kinase. It has been proposed that DYRK1A plays a prominent role in several biological functions, leading to mental retardation in DS patients. Here we explored the potential role of DYRK1A in the modulation of ChAT expression in trisomic cells and in the signaling pathways of oleic acid. Down-regulation of DYRK1A by siRNA in trisomic CTb cells rescued ChAT expression up to levels similar to those of normal cells in the presence of oleic acid. In agreement with these results, oleic acid was unable to increase ChAT expression in neuronal cultures of transgenic mice overexpressing DYRK1A. In summary, our results highlight the role played by DYRK1A in brain development through the control of ChAT expression. In addition, the overexpression of DYRK1A in DS models prevented the neurotrophic effect of oleic acid, a fact that may account for mental retardation in DS patients. © 2012 Elsevier Inc. All rights reserved.
Introduction Down syndrome (DS) is a genetic disease characterized by the presence of an extra copy of the human chromosome 21 (HSA21) (Lejeune et al., 1959). Intellectual disability is a common trait in DS individuals, primarily as a consequence of alterations in neurogenesis, neuronal differentiation, myelination, dendritogenesis and synaptogenesis during embryonic and fetal development (Becker et al., 1991; Coyle et al., 1986). Neurotransmitter alterations and changes in the activity and function of their receptors have been described and they may be responsible for some of the phenotypes described (reviewed in Rueda
Abbreviations: DS, Down syndrome; CNh, cerebral cortex cells of normal mice; CTb, cerebral cortex cells of trisomic 16 mice; DYRK1A, dual-specificity tyrosine-phosphorylated and regulated kinase 1A; ChAT, choline acetyltransferase; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; siRNA, small interfering RNA. ⁎ Corresponding author at: Instituto de Neurociencas de Castilla y León (INCYL), Universidad de Salamanca, C/ Pintor Fernando Gallego 1, 37007 Salamanca, Spain. Fax: + 34 923 29 45 64. E-mail address: [email protected] (A. Velasco). 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2012.10.016
et al. (2012)). The DS neurological alterations affect key brain regions involved in learning and memory processes, and they could be the origin of the observed intellectual disability (see review Rachidi and Lopes (2010)). Trisomy 16 (Ts16) mice were initially generated to model HSA21 trisomy due to the synteny of both chromosomes. Unfortunately the Ts16 condition is unviable and the animals die in utero. However Ts16-derived neuronal cultures are good models to study DS-related cellular phenotypes (Cheng et al., 2004), (for a review, see Antonarakis et al., 2004). Several DS-related genes or their combination and interaction with genes in other chromosomes may underlie the biological mechanisms of the different DS phenotypes. DYRK1A (dual-specificity tyrosine (Y)-regulated kinase) has been proposed as one of the genes on human chromosome 21 with a relevant role in those brain functions altered in DS (reviewed by Dierssen and de Lagran, 2006; Hammerle et al., 2003). DYRK1A is overexpressed in DS brains (Dowjat et al., 2007; Guimera et al., 1999) and is involved in neurogenesis and learning/memory processes (Tejedor et al., 1995). Transgenic mice overexpressing DYRK1A (TgDyrk1A) exhibit DS-like features such as neurodevelopmental delay, motor abnormalities and cognitive deficits (Altafaj et al., 2001).
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Cholinergic deficits in the brain are a hallmark in both human DS and Ts16 mice. In this context, synaptic neurochemical studies have demonstrated that the brains of DS individuals exhibit a significant reduction in choline acetyltransferase (ChAT) activity in the cerebral cortex (Yates et al., 1983), which is consistent with the impaired development of the basal forebrain cholinergic system exhibited by Ts16 mice (Corsi and Coyle, 1991). Previous work carried out at our laboratory has shown that the neurotrophic factor oleic acid is synthesized by astrocytes after exposure to albumin. The oleic acid is then released to the extracellular space, thereby becoming available for neurons (Tabernero et al., 2002). Thus, oleic acid induces the differentiation of neurons in culture, promoting cell clustering, neurite growth, and the expression of GAP-43 and MAP-2, markers of axonal and dendrite growth respectively (Rodriguez-Rodriguez et al., 2004; Tabernero et al., 2001). Additionally, it has been observed that oleic acid is synthesized in the periventricular zone of lateral ventricles promoting axonogenesis in the striatum during brain development (Polo-Hernandez et al., 2010). Indeed, the oleic acid–albumin complex may have pharmacological properties because treatment with the complex causes a significant recovery after medullar section (Avila-Martin et al., 2011). It is noteworthy that albumin is present in high concentrations during brain development (Mollgard et al., 1988; Velasco et al., 2003). Interestingly, it has been reported that individuals with DS have lower albumin concentrations than people without the syndrome (Clarke and Bannon, 2005). In light of this background, we were prompted to study the effects of the neurotrophic factor oleic acid in cellular models of DS. Material and methods Cell lines CNh (cerebral cortex cells derived from wild type mice) and CTb (cerebral cortex cells obtained from mouse fetuses with trisomy 16), were previously described by Cardenas et al. (1999). These cell lines were developed at the laboratory of Dr. Pablo Caviedes (University of Chile) and are marketed through an agreement with the University of South Florida. CNh and CTb cells retain neuronal markers such as neuronal specific enolase—NSE, choline acetyl transferase—ChAT, synaptophysin, microtubule-associated protein 2—MAP2 and a lack of the glial markers (GFAP, galactocerebroside, S-100) (Saud et al., 2006). Cells were plated on Petri dishes in modified DMEM/Ham's F12 medium (6 g/L glucose and supplemented with 1 mM pyruvate and N2 (Invitrogen, Barcelona, Spain) containing 10 mM human transferrin, 10 nM insulin, 20 nM progesterone, 0.1 mM putrescine and 30 nM selenium) for treatment with oleic acid. 0.1% BSA and 33 μM oleic acid (Sigma, St. Louis, USA) were also added in the specified conditions. Culture of primary neurons from transgenic mice overexpressing DYRK1A TgDyrk1A mice were previously generated at the laboratory of Cristina Fillat as described in Altafaj et al. (2001). The animals were housed under standard conditions and experiments were performed according to local and EU Ethical Committee guidelines. Hemizygous TgDyrk1A mice were mated with wild-type females. Conception was confirmed by the presence of a vaginal plug. For preparing neurons in primary culture, fetuses at 15 days of gestation were delivered by rapid hysterectomy after cervical dislocation of the mother. Neuronal cultures were prepared as previously reported (Tabernero et al., 1993). Neurons were grown in DMEM/Ham's F12 medium, supplemented with 5 μg/mL insulin, 10 μg/mL transferrin, 1 mM pyruvate, 50 U/mL penicillin and 37.5 U/mL streptomycin. Neuronal cultures were performed by plating dissociated embryonic brain cells onto poly-L-lysine (10 μg/mL-coated Petri dishes) at a density of 1.0 × 10 6
cells/cm2. After plating, neurons were cultured in defined serum-free medium in the presence of 2% (w/v) BSA or 2%(w/v) BSA plus 50 μM oleic acid. Cells were grown for 3 days and were then fixed or collected for analysis by immunocytochemistry or western blot. Twenty-three fetuses were analyzed (15 wild-type and 8 TgDyrk1A). Genotyping transgenic mice with the polymerase chain reaction (PCR) Genomic DNA was obtained from 25 mg of fetal tissue and purified with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Genotyping of the fetuses was performed by PCR analysis using the Dyrk1A transgene-specific primers 5′-GTCCAAACTCATCAATGTATC and 5′-CTTGAGCACAGCACTGTTG and the 5-GAGCACCCTGTGCTGCTCACCGAGG and 5-GTGGTGGTGAAGCTGT AGCCACGCT primer pair for β-actin amplification. The PCR conditions were as follows: 5 min of denaturation at 94 °C, 32 (Dyrk1A) or 35 (β-actin) cycles of denaturation at 94 °C for 30 s, annealing at 54 °C (Dyrk1A) or at 55 °C (β-actin) for 30 s, and extension at 72 °C for 45 s, followed by 10 min at 72 °C. DNA amplification was visualized on 2% agarose gel. Immunocytochemistry Cells were fixed with 4% (w/v) formaldehyde in PBS for 30 min and permeabilized in 100% methanol for 20 min at −20 °C. Cells were then incubated with rabbit polyclonal antibody against ChAT overnight at 4 °C and then incubated with goat anti-rabbit AlexaFluor488 secondary antibody and TOPRO-3-iodide (used to visualized cell nuclei (Invitrogen)) for 1 h at room temperature. Cells were then mounted using the Slowfade Gold Antifade kit (Invitrogen). A Zeiss LSM510 confocal microscope was used to obtain images. Determination of protein expression by Western blot analysis Cell proteins were extracted using RIPA buffer containing 25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS and cocktail protease inhibitors (Calbiochem, Darmstadt, USA). Lysates were centrifuged at 14.000 ×g for 15 min at 4 °C. 20 μg of protein extract was analyzed in 8% precast commercial gels (NuPAGE Novex 8% Bis-Tris Midi Gel 1.0 mm) or in 10% precast commercial gels (NuPAGE Novex 10% Bis-Tris Midi Gel 1.0 mm). The buffer used for protein electrophoresis was NuPAGE MOPS SDS Running Buffer 20×. NuPAGE Sample Reducing Agent 10 × and NuPAGE LDS Sample Buffer 4 × were used to prepare the samples. Electrophoresis was run at room temperature using a constant voltage. After electrophoresis, gels were washed in transfer buffer (10% Panreac methanol, 0.1% NuPAGE Antioxidant diluted in NuPAGE Transfer Buffer 2 ×) for 10 min. Then, the proteins separated were transferred to a nitrocellulose membrane (iBlot Gel Transfer Stacks Nitrocellulose) for 10 min, applying a constant voltage. All products used for electrophoresis and subsequent electrotransfer were purchased from Invitrogen (Life Technologies S.A. Madrid, Spain). After blocking for non-specific binding, the membranes were incubated for 14 h at 4 °C with rabbit polyclonal antibodies against ChAT (Cat. AB143 Millipore, CA, USA), DYRK1A (Cat. ab69811 Abcam, Cambridge, UK) or GAP-43 (Cat. AB5220 Millipore, CA, USA). Mouse monoclonal antibody against GAPDH (Cat. AM4300 Ambion, Life Technologies S.A., Madrid, Spain) was used to quantify and normalize protein expression. Following this, after several washes membranes were incubated with secondary antibody against mouse or rabbit immunoglobulin conjugated with peroxidase. Finally, the membranes were incubated for 1 min with peroxidase substrates, which afforded a chemiluminescence reaction. The signal collected on the autoradiographic film was proportional to the amount of protein in the membrane. The bands were quantified using an image analysis program.
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Transfection of siRNA CNh and CTb cells were transfected with a validated non-targeting siRNA (NT-siRNA) or with a siRNA specific for Dyrk1A suppression (Dyrk1A-siRNA; 5′-CACUGAAGCUCCUACACAATT-3′). Three different siRNA sequences for this protein were tested and the same phenotype was found. Transfections were performed with Lipofectamine 2000 reagent, according to the manufacturer's instructions. Cell treatments were performed 24 h after transfections. After the siRNA transfections, the reduction in protein expression was assessed by Western blot analysis. Statistical analysis Results are mean± SEM of at least three independent experiments. Statistical analyses were carried out with Student's t-test. Values were considered significant when p b 0.05. Results Oleic acid induces choline acetyltransferase expression in normal cells but not in trisomic cells In order to characterize whether oleic acid modifies ChAT levels, we cultured cerebral cortex cells from normal and trisomic mice (CNh and CTb, respectively) in the absence or presence of oleic acid and we measured ChAT expression by immunocytochemistry and by western blot analysis. As shown in Fig. 1, treatment with oleic acid significantly increased ChAT expression in CNh cells. Quantification of the data revealed a 50% increase when compared to CNh untreated cells. In contrast, ChAT expression was not significantly modified by oleic acid in CTb cells. These results are in agreement with the work of Allen et al. (2000) describing alterations in the cholinergic system and reinforce the presence of an impaired cholinergic function in CTb cells. DYRK1A inhibition increases ChAT expression in trisomic cells To better understand the molecular determinants involved in ChAT regulation in response to oleic acid we studied the effects of oleic acid in DYRK1A knockdown CNh and CTb cells. Cells treated with oleic acid were transfected with siRNA sequences against DYRK1A or with an oligonucleotide validated as a non-targeting siRNA (NT-siRNA). Western blot analysis demonstrated a 1.5-fold overexpression of DYRK1A in CTb cells compared to CNh cells, in keeping with the presence of an extra-copy of the DYRK1A gene in the trisomic cells. siRNA-Dyrk1A knockdown induced a significant reduction in DYRK1A protein expression, estimated at 75% in CNh cells and 70% in CTb cells (Figs. 2A and B). Expression analysis of the ChAT protein revealed reduced enzyme expression in the control and NT-siRNA-treated CTb cells, similar to what was observed previously in non-silenced cells (Fig. 1). However, in CNh and CTb DYRK1A knockdown cells the levels of ChAT expression were similar (Figs. 2A and C). These results suggest that the overexpression of DYRK1A prevents the induction of ChAT promoted by oleic acid in CTb cells.
Fig. 1. Oleic acid increases choline acetyltransferase expression in normal cells but not in trisomic cells. ChAT expression in CNh and CTb cells in the absence or presence of oleic acid was analyzed by immunocytochemistry (A) and Western blot analysis (B). Data were obtained by measuring the fluorescent areas of the previous images using the image-analyzer software (Scion Image). For immunocytochemistry of ChAT, three cultures with three plates per condition were made to take four photomicrographs from each plate. Approximately, 60 cells per plate were analyzed. Results are expressed as percentages of CNh as means±SEM (n=5), **pb 0.01; ***pb 0.001 as compared with CNh cells in the absence of oleic acid.
ChAT expression is inhibited in cultured neurons from transgenic mice overexpressing DYRK1A (TgDyrk1A) To further evaluate the potential involvement of DYRK1A in the regulation of the ChAT expression promoted by oleic acid, we analyzed ChAT levels in primary neuronal cultures from TgDyrk1A (Altafaj et al., 2001). We analyzed 23 independent fetuses: 8 fetuses were positive for transgenic DYRK1A, and 15 were wild-type (Fig. 3A). Each brain was cultured individually in the absence (A) or presence of oleic acid (O). After 3 days, photomicrographs were taken to evaluate
ChAT expression and other possible changes induced by oleic acid. Immunocytochemical analysis against ChAT revealed an increase in ChAT expression in wild-type cells caused by oleic acid (Fig. 3B). These results are in agreement with previous data showing an increase in ChAT expression due to the presence of fatty acids (Hattori et al., 1987; Machova et al., 2006). However, no differences were observed
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Fig. 2. The inhibition of DYRK1A expression in CTb cells rescues the phenotype of normal cells. A) DYRK1A expression was inhibited by RNA interference technique, as described in Material and methods. CNh and CTb cells were incubated with 33 μM oleic acid and transfected with non-targeting siRNA (NT-siRNA) or with siRNA specific for DYRK1A (Dyrk1A-siRNA). Western blot analyses of DYRK1A were carried out. B) Values of DYRK1A expression were quantified and normalized with GAPDH and are means ± SEM of at least three independent experiments **p b 0.01; ***p b 0.001 vs. CNh. C) Analyses of ChAT protein after the inhibition of DYRK1A expression by siRNA. After 3 days of transfection, cells were collected to analyze ChAT expression by Western blot analyses. ChAT values were quantified and normalized with GAPDH and are means ± SEM of at least three independent experiments **p b 0.01 vs. CNh.
in ChAT staining in neuronal cultures from TgDyrk1A mice upon treatment with oleic acid. Furthermore, western blot analysis showed increased expression levels of ChAT in the presence of oleic acid in normal cells but not in TgDyrk1A cells (Fig. 3C). Interestingly, the expression of GAP-43 was also increased in the presence of oleic acid in wild-type neurons, while it was almost unaltered in TgDyrk1A cultures (Fig. 3D). Taken together these results support the hypothesis that the overexpression of Dyrk1A acts as a negative regulator of cholinergic differentiation promoted by oleic acid. Discussion This study presents a previously uncharacterized connection between DYRK1A overexpression and ChAT expression in DS models. Our results support those of Chen et al. (2009), who used magnetic resonance imaging in disomic and trisomic mice and reported that forebrain cholinergic neurons were reduced in a DS model, a fact that may account for the failures in the neurotransmitter system and alterations in the electrical properties of cell membranes observed in DS patients (Caviedes et al., 1991). In fact, these defects are probably due to the overexpression of certain genes, some of which may affect the membrane composition and subsequently the function of some receptors (Caviedes et al., 1990). Using neurons in primary cultures from Ts16 mice, a significant decrease in choline
uptake by the high-affinity choline transporter has been reported, together with a decrease in acetylcholine release following depolarization (Fiedler et al., 1994). Here we demonstrate that oleic acid induces the expression of ChAT in CNh cells whereas it failed to induce the expression of ChAT in CTb cells. Pioneer reports have highlighted a potential role of oleic acid in the cholinergic system (Massarelli et al., 1988). These authors showed that there was a choline acetyltransferaselike activity bound to neuronal plasma membranes that was responsible for the increase in the production of the neurotransmitter acetylcholine. Thus, acetylcholine is synthesized from radioactive acetyl-CoA by isolated rat brain synaptosomes but it was released to the incubation medium only when the synthesis of choline was stimulated by oleic acid through the activation of phospholipase D. In this context, results from our laboratory revealed that oleic acid was incorporated in neurons, preferentially into phosphatidylcholine and phosphatidylethanolamine (Tabernero et al., 2001), probably to increase membrane fluidity. Moreover, the hydrolysis of phosphatidylcholine can provide lipid second messengers, which participate in signal transduction mechanisms. In addition, Granda et al. (2003) showed that oleic acid induced neuronal differentiation through a PKC-mediated mechanism that was not mediated by other neurotrophic factors, but that strongly synergized with neurotrophins NT-3 and NT-4/5. The results presented here suggest that the overexpression of some genes in the trisomic region hinders the effect of oleic acid on the expression of ChAT, which probably affects acetylcholine production to coordinate cell signaling regulatory mechanisms. In our study, we observed that after silencing DYRK1A expression with the RNA interference trisomic cells rescued the phenotype of normal cells. Moreover, measurements of ChAT levels by Western blot revealed that ChAT expression increased in DYRK1A-silenced cells up to the same levels found in non-silenced cells. These results suggest that DYRK1A may inhibit the effect of oleic acid on ChAT expression in CTb cells. Since silencing Dyrk1A did not affect ChAT expression in non-trisomic cells, it is reasonable to suggest that the expression of ChAT was maximal in the presence of oleic acid. In addition to the changes in DYRK1A activity, the occurrence of some additional regulatory mechanisms controlling ChAT expression in non-trisomic cells remains to be elucidated. In keeping with these results, cultured neurons from transgenic mice overexpressing DYRK1A exposed to oleic acid failed to increase ChAT expression as compared to wild-type cells. The reduction in immunolabeled ChAT in neuronal outgrowth is also noteworthy. In support of this, the presence of oleic acid did not promote profound changes in the shape of the neurons from TgDyrk1A mice, unlike the situation in wild-type cells. Thus, whereas wild-type neurons exposed to oleic acid tended to aggregate to form clusters and elongated axons and dendrites, these morphological changes were not observed in TgDyrk1A neurons, again suggesting impairment in neuronal differentiation in TgDyrk1A mice. In agreement with this observation, the expression of GAP-43 in TgDyrk1A cells failed to increase in response to oleic acid. In addition, these data proposes Dyrk1A as a target gene involved in the cholinergic system. In this respect, the role of the cholinergic system in cognition is well established, as supported by cognitive deficits observed in transgenic mice overexpressing DYRK1A (Ahn et al., 2006; Altafaj et al., 2001). Moreover, some cognitive enhancers are based on the increase in the tonic levels of acetylcholine (Demeter and Sarter, 2013). Earlier reports have highlighted the idea that DS as well as Alzheimer's patients have decreased levels of monounsaturated fatty acids in their brain phospholipids (Martin et al., 2010; Shah, 1979). These biochemical alterations may be responsible for the decrease in neuronal interconnections frequently observed in such patients (Elul et al., 1975). It could be speculated that the normalization of DYRK1A activity in DS patients could facilitate the positive effects of oleic acid in neuronal transmission.
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Fig. 3. The increase of ChAT expression by oleic acid is inhibited by DYRK1A overexpression. A) DYRK1A and β-actin polymerase chain reaction (PCR) of ten TgDyrk1A fetuses. PCR was carried out as described in Material and methods. Results were visualized on agarose gel with ethidium bromide in a UV transilluminator. B) Pattern of ChAT immunoreactivity in wild-type (WT) and transgenic DYRK1A (TgDyrk1A) neurons cultured separately for 3 days in defined medium in the absence (A) or presence of 50 μM oleic acid (O). Bar 50 μm. C) and D) Western blot analyses of ChAT protein and GAP-43 protein respectively, in WT and TgDyrk1A mice in the absence or presence of oleic acid. After 3 days, cells were collected and analyzed by Western blot as described in Material and methods. Values for ChAT and GAP-43 were quantified and normalized with GAPDH and are means ± SEM of at least three independent experiments **p b 0.01 vs. WT (A).
Taken together, our results confirm the importance of oleic acid as a neurotrophic factor and highlight the involvement of DYRK1A in the development of the brain.
Acknowledgments This work was supported by the Ramón Areces Foundation (A.V.), Spain and the Jêrome Lejeune Foundation (C.F.), France. We are grateful to S. Capdevila and C. Rodriguez for helping with the animals and for the technical assistance of T. del Rey.
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