Role of aspartyl-(asparaginyl)-β-hydroxylase mediated notch signaling in cerebellar development and function
© Silbermann et al; licensee BioMed Central Ltd. 2010
Received: 11 August 2010
Accepted: 4 November 2010
Published: 4 November 2010
Aspartyl-(Asparaginyl)-β-Hydroxylase (AAH) is a hydroxylating enzyme that promotes cell motility by enhancing Notch-Jagged-HES-1 signaling. Ethanol impaired cerebellar neuron migration during development is associated with reduced expression of AAH.
To further characterize the role of AAH in relation to cerebellar development, structure, and function, we utilized an in vivo model of early postnatal (P2) intracerebro-ventricular gene delivery to silence AAH with small interfering RNA (siAAH), or over-express it with recombinant plasmid DNA (pAAH). On P20, we assessed cerebellar motor function by rotarod testing. Cerebella harvested on P21 were used to measure AAH, genes/proteins that mediate AAH's downstream signaling, i.e. Notch-1, Jagged-1, and HES-1, and immunoreactivity corresponding to neuronal and glial elements.
The findings demonstrated that: 1) siAAH transfection impaired motor performance and blunted cerebellar foliation, and decreased expression of neuronal and glial specific genes; 2) pAAH transfection enhanced motor performance and increased expression of neuronal and glial cytoskeletal proteins; and 3) alterations in AAH expression produced similar shifts in Notch-1, Jagged-1, and HES-1 protein or gene expression.
The results support our hypothesis that AAH is an important mediator of cerebellar development and function, and link AAH expression to Notch signaling pathways in the developing brain.
Aspartyl-(asparaginyl)-β-hydroxylase (AAH) is an ~86 kD Type 2 transmembrane protein and member of the α-ketoglutarate-dependent dioxygenase family that includes prolyl-3, prolyl-4, and lysyl hydroxylases [1–3]. AAH's carboxyl region can be proteolytically cleaved to generate ~52 kD or ~56 kD catalytically active fragments [1, 3, 4]. Site-directed mutagenesis studies demonstrated that the 675His residue present in the C-terminal fragment is essential for catalytic activity [1, 5]. AAH catalyzes post-translational hydroxylation of β carbons of specific aspartate and asparagine residues in epidermal growth factor (EGF)-like domains  of proteins such as Notch and Jagged [5, 7], which have known roles in cell growth, differentiation, and neuronal migration during development [8, 9], and in extracellular matrix molecules, such as tenascin , which mediate adhesion, motility, and cell process extension [10–12]. Correspondingly, previous studies showed that Jagged, the ligand for Notch [13, 14], is indeed a substrate for AAH hydroxylation , and that AAH is capable of physically interacting with both Notch and Jagged . Moreover, over-expression of AAH results in increased nuclear translocation and accumulation of Notch, and activation of Notch's downstream target genes, including Hairy and Enhancer of Split 1 (HES-1) .
A direct role for AAH in cell motility and invasion was demonstrated by the findings that: 1) over-expression of AAH by transfection with recombinant plasmid DNA increases cell motility; 2) inhibition of AAH via gene silencing with small interfering (si) RNA duplexes reduces cell motility; and 3) inhibition of signaling pathways required for AAH expression and function impairs cell motility [15–21]. The AAH gene is regulated by insulin and insulin-like growth factor (IGF) signaling through insulin receptor substrate (IRS)-dependent pathways that activate Erk MAPK and phosphatidylinositol-3-kinase (PI3 kinase)-Akt [15, 17, 19]. However, AAH is also regulated by post-translational mechanisms, since chemical inhibition of glycogen synthase kinase 3β (GSK-3β) by LiCl or transfection with si-GSK-3β [16, 19] increased AAH protein without altering its mRNA levels, and over-expression of catalytically active GSK-3β increased AAH phosphorylation and reduced AAH protein expression .
Previous studies demonstrated that ethanol inhibits insulin and IGF signaling in immature neuronal cells [22–26], and that chronic in utero exposure to ethanol causes fetal alcohol spectrum disorders (FASD). FASD is associated with impaired cerebellar development including hypoplasia, disordered neuronal migration, insulin and IGF resistance, and reduced AAH expression [18, 24–27]. Ethanol's inhibitory effects on AAH are mediated at transcription and post-translation levels . Since insulin and IGF signaling pathways mediate cerebellar growth and development , and AAH is a downstream target of insulin and IGF stimulation [15, 19], we hypothesize that in FASD, ethanol impaired cerebellar development is mediated, in part, by inhibition of AAH expression and/or function. Herein, we used in vivo models to determine if inhibition of AAH is sufficient to cause some of the functional and neuro-developmental abnormalities observed in FASD. The strategy used was to transfect immature brains with siRNA targeting AAH, or recombinant plasmid carrying the full length AAH cDNA, and examine the long-term consequences in terms of function, structure, and gene expression in the brain. We focused our investigations on the cerebellum because this structure: 1) develops mainly in the early postnatal period; 2) is a primary target of ethanol-mediated neurotoxicity; and 3) exhibits impaired AAH expression in experimental models of FASD .
Gene delivery model
Two-day-old (P2) Long Evans rat pups were given a single intracerebroventricular injection of small interfering RNA duplexes (siRNA) that targeted AAH (siAAH) [ASPH NM_001009716] or no specific sequences (scrambled; siScr) [NM D-00121001-20], or recombinant plasmid DNA containing the complete coding sequence of human AAH (pAAH), or Green fluorescent protein (pGFP). The cDNAs were ligated into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA) in which gene expression was under the control of a CMV promoter. Supercoiled plasmid DNA was purified using endotoxin-free columns (Qiagen Inc., Valencia, CA). For each animal, 10 μg of recombinant plasmid DNA or 0.4 nmol siRNA were complexed with 10 μl of Dharmafect reagent (Dharmacon, Inc., Chicago, IL), and injected into the right lateral ventricle using a Hamilton syringe with a 26-gauge needle as previously described [29, 30]. All animals survived the procedure, and there were no consequential aberrant behaviors or adverse effects such as failure to thrive, poor grooming, reduced physical activity, or weight loss. The rats were subjected to rotarod testing on P20, and sacrificed on P21 (N = 8 per group). However, several rats were sacrificed on P35 for longer observation (N = 6 per group). Cerebella were divided in the mid-sagittal plane. One half was fixed in Histochoice (Amresco, Solon, OH) and embedded in paraffin. Histological sections were stained with Luxol fast blue, hematoxylin and eosin (LHE) to detect morphological abnormalities. The other half was snap-frozen in a dry ice/methanol bath and stored at -80°C for later mRNA and protein studies. Our experimental protocol was approved by the Institutional Animal Care and Use Committee at Lifespan-Rhode Island Hospital, and it conforms to the guidelines set by the National Institutes of Health.
We used rotarod testing to assess long-term effects on motor function  resulting from the siAAH or pAAH treatments. On P19, rats were trained to remain balanced on the rotating Rotamex-5 apparatus (Columbus Instruments) at 1-5 rpm. On P20, rats (N = 8-10 per group) were administered 10 trials at incremental speeds up to 10 rpm, with 10 minutes rest between each trial. The latency to fall was automatically detected and recorded with photocells placed over the rod. However, trials were stopped after 30 seconds to avoid exercise fatigue. Data from trials 1-3 (2-5 rpm), 4-7 (5-7 rpm), and 8-10 (8-10 rpm) were culled and analyzed using the Mann-Whitney test.
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis
Primer Pairs Used for Quantitative RT-PCR Analysis
Amplicon Size (bp)
CAC TGT GTG AGG GTC CAT CTT CTG
TCA AGC CAT TCC ACT CCA TCT G
AAC CTT CTG TAT CAG TGC TCC TCG
CAG TCA ACC AAG TCT CTT CCG TG
TGG TAA AGA CGG TGG AGA TGC G
GGC ACT AAA ACA GAA GCA AGG GG
CGC CAG GAG TTT GAC ACA ATG
CCT TCT TGG TCT TGG AGC ATA GTG
TGC CTG CTC GTC TTG TTT GTC
ATC CGT TCT GTA ACC CGT TGG
AGC GCT ACC GAT CAC AAA GT
TCA GCT GGC ATT TTC CTT TT
CTG AGG ACT ACG AGG GCA AG
ACA GGT GAA TTT GCC TCC TG
GGT GGA CAT TGA CGA GTG TG
CCC TTG AGG CAT AAG CAG AG
GGA CAC GGA CAG GAT TGA CA
ACC CAC GGA ATC GAG AAA GA
Enzyme linked immunosorbent assay (ELISA)
Cerebellar homogenates were prepared in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors [30, 33]. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). We performed direct binding ELISAs to measure immunoreactivity. Samples containing 50 ng protein diluted in Tris buffered saline, pH 7.4 (TBS) were adsorbed to the bottom flat surfaces of 96-well polystyrene plates (Nunc, Rochester, NY) overnight at 4°C . Non-specific binding sites were blocked by a 3-hour room temperature incubation with 300 μl/well of TBS + 0.05% Tween 20 + 3% BSA. Samples were then incubated with 0.1-0.5 μg/ml primary antibody for 1 h at 37°C. Immunoreactivity was detected with horseradish peroxidase (HRP)-conjugated secondary antibody and Amplex Red soluble fluorophore (Molecular Probes, Eugene, OR) [18, 33]. Fluorescence was measured (Ex 530/Em 590) in a SpectraMax M5 microplate reader (Molecular Devices Corp., Sunnyvale, CA). Parallel negative control assays had primary, secondary, or both antibodies omitted. Between steps, reactions were rinsed 3 times with TBS + 0.05% Tween 20 using a Nunc ELISA plate washer.
Sources of reagents
QuantiTect SYBR Green PCR Mix was obtained from (Qiagen Inc, Valencia, CA). Monoclonal antibodies to Notch-1, Jagged-1, β-Actin and were purchased from Abcam Inc. (Cambridge, MA). Antibodies to Hu, glial fibrillary acidic protein (GFAP), myelin-associated glycoprotein 1 (MAG-1), synaptophysin, SNAP-25, and GAP-43 were purchased from Molecular Probes (Eugene, OR), Santa Cruz Biotechnology Inc. (Santa Cruz, CA), or Chemicon International (Tecumsula, CA). The 85G6 AAH mAb was generated to human recombinant protein and purified over Protein G columns (Healthcare, Piscataway, NJ) .
Data depicted in the graphs represent the means ± S.E.M.'s for each group. Inter-group comparisons were made using Student t-tests since the siAAH and siScr groups, and the pAAH and pGFP groups were studied in separate experiments. Statistical analyses were performed using the GraphPad Prism 5 software (San Diego, CA) and significant P-values (<0.05) are indicated over the graphs.
Effects of siAAH on growth and brain weight
Rotarod test performance
Cerebellar hypofoliation in siAAH-treated rats
Long-term effects of siAAH and pAAH on cellular gene expression in cerebella
Effects of siAAH and pAAH on downstream notch signaling mechanisms
This study investigated the role of AAH in cerebellar development and function. The goal was to determine the degree to which ethanol's inhibition of AAH expression contributes to FASD-associated structural and functional abnormalities in the cerebellum. We demonstrated that intracerebroventricular transfection with siAAH significantly impairs motor function, while over-expression of AAH in the cerebellum enhances motor performance as demonstrated by rotarod testing. Importantly, although the siRNA and recombinant plasmid DNA transfections were performed on P2, the CNS effects persisted for several weeks, corresponding with results in a previous report utilizing this same experimental approach .
The siAAH-induced impairments in motor function were associated with conspicuous structural abnormalities in the cerebellum, including reduced foliation and decreased expression of genes that mark neurons (Hu), astrocytes (GFAP), and oligodendroglia (MAG-1). In addition, siAAH brain transfections reduced tau and GFAP expression. Together, these findings suggest that siRNA-mediated inhibition of AAH expression during postnatal cerebellar development results in net losses of neurons, oligodendroglia, and astrocytes. Since tau and GFAP represent major cytoskeletal proteins expressed in neurons and astrocytes, respectively, inhibition of AAH could promote cytoskeletal collapse and reduced inter-cellular connectivity and signaling, irrespective of relatively preserved or marginally reduced expression of synaptic plasticity proteins, including SNAP-25 and synaptophysin [37–39]. On the other hand, the significantly improved motor function and increased expression of tau associated with pAAH transfection indicate that robust AAH expression in the cerebellum during the early postnatal period could have a positive impact on subsequent cerebellar development and motor function. Therefore, ethanol's inhibition of AAH in the developing cerebellum most likely contributes to the cerebellar motor deficits in FASD.
The adverse effects of siAAH on cerebellar structure and function are highly reminiscent of previous findings in experimental models of FASD [18, 25, 27]. In particular, chronic gestational exposure to ethanol results in reduced cerebellar foliation with loss of neurons and oligodendrogial cells, and reduced expression of neuronal cytoskeletal proteins [18, 25, 27]. Moreover, ethanol exposure during development impairs motor performance due to cerebellar hypoplasia. Since AAH has a demonstrated role in mediating cell migration, which is needed for proper cerebellar foliation, reduced AAH expression in brains of siAAH-transfected rats could account for the associated perturbations in cerebellar architecture. Therefore, ethanol's inhibition of insulin/IGF stimulation of target genes, e.g. AAH, that mediate neuronal motility, contributes to some of the major CNS teratogenic effects of ethanol, including reduced cerebellar foliation and function. The adverse effects of siAAH were much less severe than those caused by early exposure to ethanol during development [18, 25, 27], perhaps because ethanol inhibits expression and function of many genes regulated by insulin/IGF signaling pathways, whereas siAAH targets just one of those genes and its downstream signaling through Notch .
Previous studies demonstrated that AAH mediates its effects on cell motility by interacting with, and hydroxylating Notch and Jagged , and that a downstream target of Notch signaling is the effector gene, HES-1 [40, 41]. Since Notch-1 stimulates HES-1 transcription , the reductions in HES-1 mRNA caused by siAAH support the notion that Notch signaling is regulated by AAH. As demonstrated herein, and in previous reports, over-expression of AAH increases Notch-1 protein levels and HES-1 gene expression . Previously, we showed that AAH over-expression stimulates Notch's translocation to the nucleus where it regulates gene expression . Once in the nucleus, Notch-1 serves as a transcription factor for other genes involved in various functions, including motility.
Since siAAH and pAAH transfections had no significant effects on Notch's mRNA levels, AAH's regulation of Notch is likely mediated by post-translational mechanisms. For example, AAH hydroxylation of Notch leading to its translocation to the nucleus reflects post-translational regulation of Notch protein. Jagged is a ligand for Notch, and its binding to Notch is needed for Notch cleavage and release from the membrane for translocation to the nucleus [13, 43, 44]. The finding that pAAH increased Jagged-1 protein expression suggests an additional mechanism by which AAH regulates Notch signaling. The relevance of this observation is that Jagged and Notch are known to play critical roles in neuronogenesis and gliogenesis, and in maintaining the specialized functions of oligodendrocytes and radial glia [45–50]. Since oligodendrocytes produce central nervous system myelin and radial glia are needed for proper neuronal migration and organization of the cerebellar cortex [51, 52], the impaired cerebellar foliation coupled with significantly reduced expression of Hu, MAG-1 and GFAP in siAAH-transfected brains correlate with the associated inhibition of Notch and HES-1 expression/signaling. Furthermore, siAAH may also have mediated its adverse effects on cerebellar structure by interfering with Notch signaling through sonic hedgehog , as sonic hedgehog mediates cerebellar foliation . While all of the effects of siAAH or pAAH cannot be explained readily, conceivably some of the responses were either compensatory or regulated by yet unknown mechanisms involving pathways affected by AAH but not investigated herein.
Together, these studies demonstrate a pivotal role for AAH in cerebellar development, structure, and function, and confirm that AAH expression is integrally tied to Notch-Jagged-HES-1 signaling, which regulates target genes that mediate neuronal migration and cerebellar cortical foliation in the brain. Moreover, the findings herein support the concept that ethanol inhibition of AAH expression during development mechanistically contributes to the cerebellar dysgenesis, and attendant impairments in motor function.
E. Silbermann, P. Moskal, and N. Bowling are all young investigators who worked diligently to complete their first research project as pre-medical and medical students.
List of abbreviations
epidermal growth factor
fetal alcohol spectrum disorders
glial fibrillary acidic protein
glycogen synthase kinase 3β
Hairy and Enhancer of Split 1
insulin like growth factor
insulin receptor substrate
myelin-associated glycoprotein 1
recombinant plasmid DNA expressing AAH mRNA
- PI3 kinase:
quantitative Reverse Transcriptase Polymerase Chain Reaction
siRNA targeting AAH
small interfering RNA
synaptosome-associated protein of 25 kD
Tris buffered saline, pH 7.4.
Supported by AA-11431, AA-12908, and AA-16126 from the National Institutes of Health
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