Brain Advance Access originally published online on August 22, 2003
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Brain, Vol. 126, No. 12, 2667-2681,
December 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg277
Gene expression profile of neurodegeneration induced by 
1B-adrenergic receptor overactivity: NMDA/GABAA dysregulation and apoptosis
1 Department of Molecular Cardiology, Lerner Research Institute and 2 Department of Neurology, The Cleveland Clinic Foundation, Cleveland, OH, USA
Correspondence to: Dianne M. Perez, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA E-mail: perezd{at}ccf.org
Received April 21, 2003. Revised May 30, 2003. Accepted June 24, 2003.
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Summary |
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The
1-adrenergic receptors (1ARs) play an important role in mediating sympathetic neurotransmission in peripheral organ systems; however, central 1ARs are not well characterized. Additionally, due to the lack of sufficiently subtype-selective drugs or high avidity antibodies, the contribution of each 1AR subtype to various central functions is currently unclear. Transcription regulation through 1AR subtypes in the CNS is also unknown. Of interest, transgenic mice that systemically overexpress the 1BAR show central symptoms that include age-progressive impaired mobility, neurodegeneration and susceptibility to epileptic seizure. To investigate the molecular basis of this phenotype, oligonucleotide microarray studies of whole brains of various ages were carried out to compare gene expression profiles between transgenic and normal brains. The results indicated changes in expression of apoptotic, calcium regulatory, neurodegenerative and genes involved in neurotransmission. Defects in regulation of intracellular calcium are known to play a role in cell death; thus, these genes may provide clues as to the molecular basis of 1BAR-induced neurodegeneration. Epilepsy is a disorder that can be caused by an imbalance between excitatory (e.g. glutamate) and inhibitory (e.g. GABA) signals. Microarray analysis of transgenic brains showed increased N-methyl-D-aspartate (NMDA) receptors and decreased GABAA, which were confirmed with immunohistochemistry, western blot and radioligand binding studies. The 1BAR also co-localized with the glutamatergic distribution, suggesting a glutamate imbalance as a molecular rationale for the epileptic seizures. Keywords: adrenergic; microarray; NMDA; GABA; transgenic
Abbreviations: 1AR= 1-adrenergic receptor; EGFP = enhanced green fluorescent protein; EST = expressed sequence tag; Ig = immunoglobulin; IHC = immunohistochemistry; MM = mismatched; NMDA = N-methyl-D-aspartate;NT = non-transgenic; PBS = phosphate-buffered saline; PM = perfectly matched; T = transgenic; TBS = Tris-buffered saline; TGF-ß = transforming growth factor-ß; WT = wild-type
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Introduction |
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The
1-adrenergic receptors (1ARs) are members of the superfamily of G protein-coupled receptors that mediate the effects of the endogenous catecholamines adrenaline and noradrenaline. The 1ARs are classified into three subtypes (1A, 1B and 1D) based on molecular cloning and differing affinities for agonists and antagonists (Cotecchia et al., 1988
; Lomasney et al., 1991; Perez et al., 1991, 1994; Graham et al., 1996). All three 1AR subtypes transduce their signals intracellularly primarily via coupling of the receptor to the Gq pathway (Guarino et al., 1996), leading to the activation of downstream signalling molecules such as protein kinase C and the mobilization of calcium. 1ARs participate in a number of physiological processes that are essential in the maintenance of homeostasis, including the regulation of blood pressure and cardiac contractility in the cardiovascular system, as well as sodium reabsorption and glucose metabolism in the kidney and liver, respectively (García-Sáinz et al., 1999).
In the CNS, although 1AR density in the brain is equal to or exceeds that of other adrenergic receptors, the central functions of these receptors remain unclear. Previous studies, however, indicate that 1ARs are postsynaptic and are involved in the regulation of several central functions including synaptic transmission, plasticity, motor activity, as well as attention and memory. For example, 1AR activation is thought to attenuate the excitability of pyramidal neurons in the hippocampus (Mynlieff and Dunwiddie, 1988), whereas in areas of the cerebral cortex, 1AR activation potentiates excitation in response to glutamate or acetylcholine (Mouradian et al., 1991). 1AR activation is primarily excitatory in regions such as the reticular thalamic nuclei, dorsal raphe and spiny motor neurons, largely due to decreased potassium conductance (McCormick et al., 1991). Open field activity assessments also showed that activation of central 1ARs enhances locomotor activity, whereas 1AR-selective blockers depressed motor activity (Stone et al., 1999). 1ARs may also regulate brain functions via glial cells since activation of these receptors has been shown to induce calcium transients in astrocytes (Lerea and McCarthy, 1989). Recent studies from the 1BAR knockout mouse suggest that this subtype may modulate dopamine release that affects the locomotor response to amphetamines (Drouin et al., 2002). While these limited functional responses to 1 AR stimulation have begun to make inroads into 1AR regulation of CNS function, there are no reports of any transcriptional regulation by 1ARs in brain tissue whereby we can begin to analyse the mechanistic reasons for their activity.
Discerning the specific functional contribution of each 1AR subtype in physiological systems, however, has been more challenging. Studies have been hindered primarily by the lack of sufficiently subtype-selective ligands, especially for the 1BAR. To alleviate this constraint, we have examined the function of the 1BAR in transgenic (T) mouse models that overexpress either wild-type (WT) or constitutively active forms of the 1BAR in those tissues that endogenously express the receptor (Zuscik et al., 2000). These T mice exhibit a neurodegenerative phenotype accompanied by motor abnormalities that resemble the Parkinsonian disorder, multiple system atrophy. Additionally, the T brain also possesses increased levels of abnormally nitrated -synuclein inclusion bodies that co-localize with oligodendrocytes and neurons, which is consistent with multiple system atrophy and other synucleinopathies (Papay et al., 2002). T mice also have epileptic seizures that can be induced starting at 
7 months of age and increasing in frequency and occurring spontaneously as the mice grow older (Zuscik et al., 2000). Furthermore, EEG studies indicate that these mice show abnormal ictal and interictal activity similar to human forms of epilepsy, indicating a potential role for 1BARs in the development of epileptogenicity (Kunieda et al., 2002). To identify the molecular mechanisms underlying these central effects of 1BAR activation, we have carried out oligonucleotide microarray analyses on whole brains from T and age-matched normal non-transgenic (NT) control mice. Gene expression profiles were compared at three different ages to correlate gene expression changes with the onset or progression of symptoms. We have identified a number of disease-associated genes, including those involved in apoptosis and calcium/glutamate regulation, providing clues to the molecular bases underlying the central phenotypes observed in the T mice. In particular, we confirm a dysregulation in N-methyl-D-aspartate (NMDA) and GABA signals and apoptosis at the protein level, which may be responsible for the epileptic seizures and/or the neurodegeneration in the brains of the T mice.
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Mice
The T mice (B6CBA) systemically overexpressing the
1BAR have been described previously (Zuscik et al., 2000). Mice used in this study carried a transgene containing a cDNA coding for a constitutively active 1BAR that carries three different mutations: C128F, A204V and A293E (Hwa et al., 1997). Systemic expression was achieved by using the murine 1BAR gene promoter (Zuscik et al., 1999). Founder mice were identified and subsequent generations were genotyped by Southern analysis of genomic DNA isolated from tail biopsies. To show localization of the 1BAR protein, we also constructed an enhanced green fluorescent protein (EGFP)-tagged WT 1BAR in the same manner as above. The EGFP was added to the C-terminal tail of the WT 1BAR. This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and was approved by the Animal Research Committee of The Cleveland Clinic Foundation.
Preparation of cRNA and hybridization to oligonucleotide arrays
Poly(A)+ RNA was isolated from whole brains of five or more T or NT mice each at three different ages, 2–4 months, 8–10 months and 12–18 months, generating six experimental groups: NT(2–4), T(2–4), NT(8–10), T(8–10), NT(12–18) and T(12–18). No difference in phenotype has been observed between male and female T mice, thus each T and NT group at each age contained the same number of male and female mice. RNA was isolated using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA). The RNAs isolated from individual mice were pooled within each group and double-stranded cDNA was synthesized from 1 µg of poly(A)+ RNA using the Superscript Choice double-stranded cDNA synthesis kit (Gibco-BRL, Rockville, MD). Biotin-labelled cRNA was synthesized from the cDNA in an in vitro transcription reaction using the BioArray high yield RNA transcript labelling kit (Enzo Diagnostics, Farmingdale, NY). cRNAs were purified using the RNeasy Mini kit (Qiagen, Valencia, CA). Fragmentation of biotin-labelled cRNAs and subsequent hybridization to the oligonucleotide arrays were carried out by the Gene Expression Core Service at the Cleveland Clinic Foundation. A 50 µg aliquot of biotin-labelled cRNA was then hybridized to the MGU74Av2 oligonucleotide array (Affymetrix, Santa Clara, CA), which represents 12 656 known murine genes and expressed sequence tags (ESTs) from Build 74 of the UniGene database. Each cRNA sample was hybridized to two microarrays and two analyses were carried out for each experimental group. Detection of gene expression changes was determined using Affymetrix software. Briefly, each gene or EST was represented on the chip by 16 pairs of perfectly matched (PM) and mismatched (MM) oligonucleotides. PM–MM pairs differed by only a single base pair change in the MM oligo, and spanned distinct regions of the specified gene/EST represented by the probe set. The MM probes served as specificity controls to allow the direct subtraction of both background and cross-hybridization signals. The number of instances where the PM signal was greater than the MM signal was determined and the average of the logarithm of PM : MM was calculated. A matrix-based algorithm was then used to determine the presence or absence of each cRNA molecule. The abundance of each RNA transcript was then calculated by averaging the differences of PM–MM signals for each probe set after discarding the maximum, minimum and any outliers >3 SDs. Comparisons were then carried out between age-matched NT and T groups resulting in four possible combinations [i.e. NT(chip1) versus T(chip 1), NT(chip 1) versus T(chip2), NT(chip2) versus T(chip1) and NT(chip2) versus T(chip2)] at each age studied. Those transcripts that were found to be significantly increased or decreased in three of the four comparisons and exhibited an average fold change of 1.6 or greater were then included in data tables. This fold cut-off was determined by previous reports that revealed correlations between microarrays and Taqman PCR data at the 1.6- to1.8-fold threshold (Tan et al., 2002).
Northern analysis
Several of the genes that were identified as having increased or decreased transcript levels via microarray studies were examined by northern blot analyses. cDNA probes were isolated from IMAGE Consortium (Lawrence Livermore National Laboratory LLNL) ESTs obtained from the American Type Culture Collection: interferon-
receptor 2 (cloneID: 4164791), insulin receptor subsrate 2 (cloneID: 3164252) and P-type ATPase (cloneID: 3385756). Probes were radiolabelled with [32P]dCTP (6000 Ci/mmol; NEN, Boston, MA) using the random primed DNA labelling kit (Roche, Indianapolis, IN). Equal amounts of poly(A)+ RNA (5 µg) used in the microarray experiments were loaded onto 0.8 % agarose–formaldehyde gels, transferred to nitrocellulose and hybridized to the appropriate probes. Band density was determined by scanning densitometry using NIH image software.
Western blot analyses
Individual whole brains were homogenized in 4 ml of M-PerTM mammalian protein extraction reagent (Pierce, Rockford, IL), supplemented with 0.2 TIU/ml aprotinin, 1 mM EDTA, 2 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 60 µM benzamidine. Homogenates were incubated on ice for 20 min then centrifuged for 10 min at maximum speed at 4°C. The supernatant was collected as total cell lysate and used for subsequent western blots. Protein concentrations were measured with the Bio-Rad (Hercules, CA) protein assay kit, using bovine serum albumin (BSA) as a standard. Equal amounts of protein were solubilized in sample buffer [65.5 mM Tris–HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.001% bromophenol blue, 5% ß-mercaptoethanol], electrophoresed on a 4–15% gradient Tris–HCl SDS–polyacrylamide gel (Bio-Rad) and transferred to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature with 5% non-fat dry milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T), and incubated with a rabbit polyclonal antibody directed against NR1 (0.2 µg/ml; Chemicon, Temecula, CA) or NR2A/B (0.2 µg/ml; Chemicon) for 1 h at room temperature. Blots were washed and subsequently incubated with secondary goat anti-rabbit immunoglobulin (Ig)G conjugated to horseradish peroxidase (1 : 5000; Jackson Immunologicals, West Chester, PA). Proteins were visualized using enhanced chemiluminescence (Pierce).
Immunohistochemistry
For NR1 and activated caspase 3 immunohistochemistry (IHC) experiments, mice were deeply anaesthetized with pentobarbital (100 mg/kg, intraperitonaeally), then transcardially perfused with 20 ml of ice-cold heparin in phosphate-buffered saline (PBS; 10 U/ml), followed by 100 ml of 4% paraformaldehyde in 0.1 M Na2HPO4 buffer pH 7.0. Brains were removed and post-fixed in the same fixative for 3 h. After fixation, brains were cryoprotected in 20% sucrose overnight and sectioned on a cryostat (16 µm in thickness) the following day. Sections were transferred onto electrostatic slides, dried at room temperature then stored at –20°C until use. For experiments, sections were thawed, re-hydrated with PBS, then antigen retrieval was carried out in 10 mM citric acid pH 6.0 for 10 min in a microwave oven. Sections were then incubated successively with 10% normal goat serum for 1 h at room temperature, anti-NR1 (0.7 µg/ml; Chemicon) or anti-activated caspase 3 (1 : 100; Cell Signaling) in PBS containing 1% normal goat serum and 0.03% Triton X-100 overnight at 4°C, biotinylated goat anti-rabbit for 1 h at room temperature, 3% H2O2 in PBS for 20 min at room temperature, and avidin-biotinylated horseradish peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) for 30 min at room temperature. Immunoreactions were visualized with 3,3'-diaminobenzidine (DAB; Vector Laboratories).
For NR2A/B IHC experiments, serial 30 µm thick coronal sections were collected and processed for immunostaining with antibody for NR2A/B (rabbit IgG, 0.5 µg/ml; Chemicon AB 1548, Temecula, CA). The sections were placed in individual 3 ml tissue culture wells containing 0.05 M TBS pH 7.6. TBS was used as the rinsing buffer between each step throughout the IHC staining procedure: (i) 5 min in 3% H2O2/10% methanol in TBS; (ii) 60 min in a blocking solution of 1.5% normal goat serum in TBS; (iii) overnight at room temperature in primary antibody diluted in TBS; (iv) 35 min in diluted biotinylated goat anti-rabbit IgG; and (v) 60 min in a solution of excess avidin and biotinylated horseradish peroxidase (ABC kit, Vector Laboratories). In order to visualize the immunoreactive complex, the sections were reacted for 8 min in 0.05% DAB and 0.01% H2O2 in TBS. Control experiments for IHC were performed by omission of primary antibody using the same staining protocol as mentioned above; no specific IHC staining was seen in the absence of specific primary antibody.
For localization of the 1BAR protein, sections were washed in PBS and blocked by incubation in PBS containing 0.3% Triton X-100 and 2% BSA. The 1BAR tagged receptor was identified with a rabbit anti-GFP (Chemicon) at 1 : 2500. Bound primary antibodies were detected by incubation with fluorescein isothiocyanate (FITC)-coupled anti-rabbit IgG at 1 : 2500. Slides were then treated for 1 h in 10 mM CuSO4 in 50 mM ammonium acetate pH 5.0 to reduce autofluorescence due to lipofuscin. Slides were mounted in Vectashield mounting medium (Vector Laboratories). Sections were analysed on a confocal laser-scanning microscope (Model Aristoplan; Leica, Inc., Deerfield, IL). Confocal images represent optical sections of 1 µm axial resolution. Single images represented an average of 16 line scans of the chosen field.
Radioligand binding studies
GABAA
Membranes were prepared as described (Enna and McCarson, 1998). Briefly, brains were homogenized with a polytron in ice-cold 50 mM Tris-citrate buffer pH 7.1 then centrifuged for 10 min at 50 000 g. The pellet was resuspended in Tris-citrate buffer, and re-centrifuged as described. This wash was repeated five more times to remove endogenous GABA, and the final pellet was resuspended in 10–15 ml of Tris-citrate buffer. For saturation binding studies, membranes (400 µg) were incubated with increasing concentrations of [3H]muscimol (20–300 nM; NEN Life Sciences, Boston, MA), and non-specific binding was measured in the presence of 0.1 mM bicuculline (Sigma, St Louis, MO). Reactions were carried out for 30 min at 4°C, then terminated by addition of 3 ml of Tris-citrate buffer.
GABAB
Membranes were prepared essentially as described (Enna and McCarson, 1998). Briefly, whole brains were homogenized in ice-cold buffer (50 mM Tris–HCl, 2.5 mM CaCl2 pH 7.4). The homogenate was then centrifuged at 20 000 g for 20 min. The pellet was resuspended in homogenization buffer and centrifuged as above; this process was repeated three more times. The final membrane pellet was resuspended in 10–15 ml of the same buffer. For saturation binding studies, membranes (400 µg) were incubated with increasing concentrations of [3H]baclofen (10–100 nM, NEN Life Sciences), and non-specific binding was measured in the presence of 1 mM saclofen (Tocris, Ellisville, MO). Reactions were incubated for 10 min at 25°C, then terminated by addition of ice-cold Tris-CaCl2 buffer.
NMDA
Brain membranes were prepared as described (Ferkany, 1999). Briefly, brains were homogenized in ice-cold 0.32 M sucrose and the sample centrifuged for 10 min at 2000 g. The supernatant was collected, fresh sucrose was added to the sample, and the sample was centrifuged for 20 min at 12 500 g. The pellet was resuspended in deionized water and homogenized. The resulting homogenate was then centrifuged for 20 min at 8000 g. The buffy coat and supernatant were resuspended by inversion, and the sample was centrifuged for 10 min at 40 000 g. The pellet was resuspended in 2% Tris–HCl pH 7.4, and centrifuged as in the previous step. The pellet was washed in the same manner four more times and stored at –70°C until the day of the assay. Membranes were resuspended in 50 mM Tris–HCl, 100 mM potassium thiocyanate buffer and homogenized. The homogenate was centrifuged for 10 min at 40 000 g and resuspended in Tris- potassium thiocyanate buffer. This step was repeated five more times and the final pellet was resuspended in 10–15 ml of the Tris-potassium thiocyanate buffer. The binding experiments were carried out with increasing concentrations of [3H]CGP39653 (5–40 nM; Perkin Elmer Life Sciences) and 400 µg of membrane protein. Non-specific binding was measured in the presence of 0.01 mM (±)CPP (Sigma). The assay was terminated with Tris-potassium thiocyanate buffer.
For competition experiments, membranes were labelled with 70 pM [125I]MK801 (Perkin Elmer Life Sciences) and incubated with increasing concentrations of the NR2B-selective antagonist, ifenprodil (0.01 nM–1 mM; Sigma). Binding was carried out for 60 min at 4°C. The final volume for all binding reactions was 500 µl, and all binding reactions were terminated by addition of 3 ml of the appropriate buffer. Reactions were filtered through GF/B filters with a Brandel cell harvester. Filters were washed three times with the appropriate buffer, placed in 20 ml of scintillation cocktail, and counted on a scintillation counter. Bmax and Kd values were obtained with non-linear regression analyses using GraphPad Prism (San Diego, CA). For competition studies, curves were fitted to a two-site model and the percentage of high and low affinity sites was calculated using GraphPad Prism.
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Gene expression profile
To identify genes that may contribute to the neurological phenotype observed in T mice, gene expression profiles of whole brains from T mice were compared with expression profiles of NT control brains at three different ages: 2–4 months (before disease manifestation), 8–10 months (symptoms begin and worsen) and 12–18 months (diseased state). Age-matched controls negated gene expression changes due to ageing of the mice. Based on our criteria and after ESTs that did not encode a protein were removed, overexpression of
1BARs resulted in the expression changes of 88 genes in 2- to 4-month-old mice (0.6%), 62 genes in 8- to 10-month-old mice (0.5%), and 122 genes in 12- to 18-month-old mice (1.0%). The remaining genes were grouped into functional classifications: glutamate/calcium regulation, apoptotic and neurodegenerative proteins (Table 1); synaptic and structural proteins (Table 2); proliferative/growth and immune proteins (Table 3); and proteins involved in signalling and modifying or transcription and translation (Table 4). Gene expression changes in each table are ranked from highest expression levels to lowest, starting with 2- to 4-month-old mice, then 8- to 10-month-old and, finally, 12- to 18-month-old mice. Of particular interest, expression of genes coding for protein products that are involved in regulation of excitatory or inhibitory signals, e.g. glutamate, GABA or calcium regulation, were found to be primarily changed in 8 month or older T brains as compared with younger T brains (Table 1). In general, genes associated with apoptosis or neurodegeneration (Table 1) were differentially expressed in younger T, whereas genes involved in growth or gene regulation were found to be changing in 8 month or older T brains (Tables 3 and 4).
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Confirmation of microarray data
To confirm the gene expression changes observed in the microarray experiments, northern blotting analysis was carried out with several genes whose expression was found to be increased (P-type ATPase and insulin receptor substrate 2) or decreased (interferon-
receptor 2) at the indicated ages (Fig. 1). Generally, qualitative changes (positive or negative expression) were conserved between the microarray and northern experiments; however, quantitative fold changes were greater in the microarray data compared with the northern blots. This is consistent with previous reports (Yun et al., 2003) in which microarray analysis may be more sensitive than northern analysis.
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Protein levels of NR1 and NR2A/B
One of the more interesting subsets of gene expression changes in T brains, especially in the 8 month or older mice, were those involved in regulating intracellular calcium or glutamate levels. Expression of several glutamate receptor genes such as the major NMDA receptor subunit NR1 were increased in T brains compared with NT brains (see Table 1). To determine whether the gene expression changes were apparent at the protein level, we compared the protein levels of the NMDA receptor subunits, NR1 and NR2A/B, in whole brain cell lysates of 2- to 4-month-old and 8- to 10-month-old mice via western blotting (Fig. 2). The 8- to 10 month-old T brains showed
2-fold higher levels of NR1 subunit, which was consistent with the 2-fold increased levels observed in the microarray studies (Fig. 2B). Levels of NR2A and NR2B subunits were detected using an antibody that does not distinguish between these two subunits. Western blotting experiments indicated that T brains also showed 1.5-fold higher levels of the NR2A/B subunits than age-matched NT brains (Fig. 2). Increased protein levels of NMDA receptor subunits were also detected in 2- to 4-month-old T brains (Fig. 2A).
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Immunolocalization of NR1 and NR2A/B
To identify specific brain regions expressing increased levels of NR1 and NR2A/B, IHC experiments were performed. T brains at both young and old age points showed increased levels of the NR1 protein in the cerebral cortex (Fig. 3A and B) and hippocampus (not shown) as well as in other brain regions including the cerebellum and thalamus (not shown). Increased staining was observed in neuronal cell bodies as well as in the dendritic processes of the cortex in T brains. Increased NR2A/B staining was also dramatically increased in the cortex and hippocampus regions of the T brains (Fig. 3C–F).
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Radioligand binding studies
Radioligand binding experiments were carried out to determine NMDA, GABAA and GABAB receptor densities (Table 5). GABAB receptor levels were not significantly different in both 2- to 4- and 10- to 24-month-old T brains when compared with their age-matched controls. GABAA receptor levels, however, were significantly decreased by
50% in 10- to 24-month-old T brains (Table 5), which corresponded to the decrease in transcript levels as observed in the microarray studies (see Table 1). Increased NMDA receptor levels (3- to 7-fold) were also confirmed in both 2- to 4- and 10- to 24-month-old T brains compared with age-matched control brains. No significant differences in affinity (Kd) was detected in T brains for all ligands used in the saturation experiments. To determine whether the T mice increased the NR2A or NR2B subunits of the NMDA receptor, competition ligand binding studies were performed using [125I]MK801 and the NR2B-selective antagonist, ifenprodil. T mice had an increase in the percentage of high affinity sites (Fig. 4, Table 5) compared with age-matched controls.
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Confirmation of apoptosis
Since many different types of apoptotic genes were upregulating in the younger mice and some of these pathways appeared to be regulated through transforming growth factor-ß (TGF-ß), we chose to use antibodies against the activated form of caspase 3, a pathway converging effector of apoptosis for the TGF-ß pathway. T brains from 2- to 3-month-old mice had high levels of activated caspase 3 in the white matter tracts of the cerebellum (Fig. 5B), the corpus callosum and the myelinated tracts of the caudate/putamen (Fig. 5D). Upon higher magnification of the medulla, activated caspase 3 was seen only in the nuclei (Fig. 5F).
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Localization of the
1BAR to hippocampal and cerebral cortex regionsTo determine if the
1BAR protein is located in the same regions of the hippocampus and cerebral cortex that are also upregulating NMDA receptor subunits, we performed immunofluorescence studies on T mice expressing a WT 1BAR tagged at the C-terminal tail with EGFP (Fig. 6). Due to the low endogenous expression of the T mice with this housekeeping promoter, the green fluorescent images were present but are very low intensity. Therefore, we employed an antibody against EGFP to visualize the receptor. The WT 1BAR does have neurodegeneration (Zuscik et al., 2000) but is only present later in life. For the localization studies, we employed animals that were 2–3 months old. We found that the 1BAR does indeed localize to regions that control the molecular areas of the dentate gyrus and hippocampal associational fibres, and regions of the cerebral cortex which correlate to the distribution of glutamatergic systems (Fig. 6).
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The functional roles of distinct
1AR subtypes have remained relatively elusive primarily due to the lack of sufficiently powerful tools with which to study their functional differences. Thus to attempt to gain greater insight into the physiological contribution of 1BARs, we previously generated a mouse model that systemically overexpresses the 1BAR (Zuscik et al., 2000). Interestingly, overactivity of the 1BAR led to impaired hindlimb mobility that became apparent starting at 3 months and progressively worsened with age. Additionally, neurodegeneration was observed predominantly in the cerebellum and medulla, but progressed to virtually all brain areas as the mice grew older. By 7 months of age, T mice had epileptic seizures that could be induced by stresses (e.g. handling) that became spontaneous and more frequent as the mice grew older. To gain insight into the potential pathways that may be involved in initiating or propagating these symptoms, we have carried out microarray analyses of NT and T brains at various ages.
One class of genes that were differentially expressed in T brains were those involved in glutamate/calcium regulation (Table 1). Changes in the expression of these genes primarily occurred in 8 month or older T mice. Of note, genes coding for three different ionotropic glutamate receptor subunits, NR1, -amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid 1 (AMPA1) and AMPA3, were upregulated in older T brains. Similarly, mRNA levels of GABAA receptor subunits were also decreased in older T brains (Table 1). These changes coincided with the manifestation and progression of epileptic seizures in the T mice and were confirmed by ligand binding (Table 5), western blotting (Fig. 2) and IHC (Fig. 3).
Glutamate is the major excitatory neurotransmitter of the CNS (Petralia et al., 1994). A key factor in the mechanism of epileptogenesis is the ratio of inhibitory versus excitatory synapses (Najm et al., 2001). Excessive excitation or inadequate inhibition results in seizures. Previous studies have indicated increased NMDA receptor binding in cortex of some human epilepsies (Geddes et al., 1990; McDonald et al., 1991) as well as increased mRNA levels of both NR1 and NR2 subunits in temporal lobe epilepsy patients in in situ hybridization experiments (Mathern et al., 1997). Additionally, previous studies also associated increased glutamatergic AMPA receptor density and mRNA expression with focal epileptic human cortex and epileptic hippocampus (McDonald et al., 1991; García-Ladona et al., 1994; Zilles et al., 1999).
For a functional NMDA receptor, the NR1 subunit must partner with one or more NR2 subunits. The various NR2 subunits, NR2A–D, are differentially expressed in a spatiotemporal manner. Embryonic and neonatal forebrains show relatively high levels of NR2B, with increasing NR2A levels from the time of birth through the second postnatal week. At this time, an adult expression pattern is reached (Takahashi et al., 1996). The NR2A/B subunit has previously been associated with epilepsy. Dysplastic cortical neurons express both NR1 and NR2A/B proteins; in contrast, non-dysplastic neurons express only NR1 proteins. The co-expression of NR1 and NR2A/B in dysplastic neurons suggests that heteromeric NR1–NR2A/B receptors in these neurons could contribute to focal seizure onset (Ying et al., 1999). IHC studies further indicate an increase in the densities of NR2A/B subunits in human epileptic cortex (Najm et al., 2000).
The NR2A/B subunit in our studies is likely to be the NR2B as determined by the competition ligand binding studies using the NR2B-selective ligand, ifenprodil (Fig. 4, Table 5). The identity of the NR2 subunit can be distinguished since recombinant NR1–NR2B receptors exhibit a 10- to 100-fold higher affinity for ifenprodil than NR1–NR2A receptors (Grimwood et al., 2000). T mice displayed an 16% increase in the amount of high affinity sites for ifenprodil compared with age-matched normals. Since the increased NR2B may be highly localized to cortex and hippocampus (Fig. 3), ligand binding using whole brain membranes may not be as sensitive as using membranes from these distinct regions. Nonetheless, even under these conditions, the T brain displayed higher amounts of the NR2B subunits.
Our studies show an increase in NR1 and NR2A/B protein in whole brain lysates from T brains (Fig. 2). While there are differences in penetrance (two out of three samples are increasing), this result is consistent with previous data in which the seizure phenotype also has a 60% penetrance (Zuscik et al., 2000). Moreover, these increases in both NR1 and NR2A/B were detected in both the cortex and the hippocampus, two brain regions that are highly associated with epileptogenesis (Wahnschaffe et al., 1993). Radioligand binding studies further showed that T mice had increased membrane expression of NMDA receptors, confirming an increase in functional NMDA receptors in T brains, consistent with a state of excessive excitation. Overall, 60% of the T mice tested showed increased NR2A/B levels via IHC and western studies, which is most probably due to the penetrance of the transgene.
Changes in gene expression that code for glutamate receptors responsible for excitatory signals were also accompanied by a decrease in mRNA of two GABAA receptor subunits which resulted in reduced GABAA receptor density as measured by receptor binding. The inter-regulation of the NMDA and GABA receptors is common since a homeostatic balance is needed to maintain proper neurotransmission. Fast synaptic inhibition is mediated primarily by the GABA neurotransmitter acting upon the GABAA receptors; therefore, decreased GABA transmission can also lead to a hyperexcitable state that results in epileptogenesis. While gene expression of glutamate decarboxylase does increase in the older T mice, this may be a compensatory mechanism to counteract the decreased GABAA signalling. Our results are consistent with previous findings, in which human epileptic patients and experimental epileptic models have also shown reduced or mutated GABAA receptors (Savic et al., 1988; McDonald et al., 1991; Kapur et al., 1994; DeLorey et al., 1998). 1AR stimulation is known to excite the inhibitory GABAergic interneurons of the hippocampus (Bergles et al., 1996), but the 1AR subtype remains unknown. While these results do not prove that the epilepsy in these T mice is mediated through the NMDA/GABA dysregulation, they are suggestive. T mice expressing an EGFP-tagged WT 1BAR do indeed show that this receptor subtype is localized to areas co-existent with the glutamatergic and NMDA receptors, such as the CA1–3 regions and the dentate gyrus of the epileptic-prone hippocampus (Fig 6). Also consistent with epilepsy and distribution of the glutamatergic system is the localization of the 1BAR to the neurons in the cerebral cortex. In fact, it seems that the areas that increased in NR1 and NR2B subunits (Fig 3) also co-express the 1BAR (Fig 6). We are currently performing patch-clamp experiments in in vitro brain slices that include the hippocampal formation of the T mice.
Contrary to our findings, noradrenaline is known to be an anticonvulsant, and direct stimulation of 1ARs shows anticonvulsant effects in many seizure models, including amygdala kindling (Löscher and Czuczwar, 1987), audiogenic (Yan et al., 1998) and spontaneous petit mal seizures (Micheletti et al., 1987). However, strychnine-induced convulsions in mice are potentiated by noradrenaline, possibly through 1ARs (Amabeoku and Chandomba, 1994). Our findings indicate that chronic activation of the 1BAR can lead to seizures through regulation of the NMDA and GABA receptors. Previous reports show that noradrenaline modulates NMDA (Kirkwood et al., 1999) and GABA signalling (Beani et al., 1986) in the cortex. However, our results are likely to be due to the secondary effects of neurodegeneration since glutamate dysregulation was a late-onset event.
Expression of genes with protein products involved in apoptosis and neurodegeneration was also found to be changed in T brains (Table 1). Pro-apoptotic proteins such as the Bik-like killer protein and TGF-ß were upregulated in the 2- to 4-month-old T brains, indicating that apoptotic cell death begins early in life and may be mediated directly through 1BAR stimulation. Previous TUNEL staining showed increased labelling of fragmented DNA in discrete T brain regions (Zuscik et al., 2000). A common effector of the TGF-ß pathway is caspase 3. Using an antibody that recognizes only the activated form, immunostaining experiments in 2- to 3-month-old T brains show increased labelling of activated caspase 3 proteins (Fig 5), indicating caspase-mediated cell death in T brains. These findings are consistent with our previous microarray studies of T hearts (Yun et al., 2003), where TGF-ß-mediated apoptotic genes were differentially expressed in 2-month-old T hearts rather than older hearts. The large amount of white matter apoptosis is consistent with our original assessment that the neurodegenerative phenotype in this T mouse resembles the white matter disease caused by multiple system atrophy (Zuscik et al., 2000). Additionally, G protein-coupled receptor-mediated apoptosis has been shown previously to occur in the parathyroid hormone/parathyroid hormone-related protein signalling pathway via Gq/phospholipase C (PLC) signalling (Turner et al., 2000), which is the same pathway that is activated by the 1BAR. Heart-targeted Gq overexpression also induced apoptosis (Adams et al., 1998). Because of the excitatory effects of glutamate transmission and the widespread expression of glutamate receptors in the mammalian CNS, glutamate neurotransmission has been implicated in the pathogenesis of a number of central defects including ischaemia, and neurodegenerative diseases, as well as epilepsy (Choi et al., 1990; Reynolds et al., 1994; Ulas et al., 1994; Glass and Dragunow, 1995). Thus excessive glutamate signalling could also be excitotoxic, contributing to the widespread neurodegeneration apparent in older T brains. On the other hand, the excessive activation of the 1BAR itself could directly mediate the apoptosis.
1BAR activation also seems to regulate the expression of genes involved in synaptic or vesicular transport (Table 2). Proteins that are associated with neurotransmitter release include the synaptotagmins, SNARE and complexin, which are all involved in Ca2+-dependent exocytosis and vesicle docking. Other regulated proteins include the synaptophysins, which form fusion channels to allow neurotransmitter release and the synapsins, which are responsible for regulating the reserve pool of synaptic vesicles (reviewed in Hou and Dahlström, 2000). Kinesin proteins that are involved in axonal transport of synaptic vesicles were also differentially expressed in T brains, which may indicate a significant role for 1BARs in the regulation of neurotransmitter release. In fact, previous studies showed that 1AR activation is involved in a presynaptic reduction in glutamate release in the dentate gyrus (Scanziani et al., 1993) and may be involved in the release of noradrenaline (Murphy and Majewski, 1989; Forray et al., 1999). However, because there is little evidence for presynaptic 1ARs, more experiments should be carried out to examine these potential actions further.
Knowledge of the transcriptional regulation by the 1ARs is limited. 1AR stimulation is well known to activate the fetal gene programme in cardiac myocytes, such as ß-myosin heavy chain and skeletal -actin (Stewart et al., 1998). Activation of the transcription of c-fos and c-jun is also regulated by the 1AR (Garcia-Sainz et al., 1998). In the brain, nothing is known about 1AR effects on transcription let alone the role of the individual subtypes. To understand 1AR subtype-specific gene transcription on a wide scale, we performed an oligonucleotide microarray study in transfected rat-1 fibroblasts, stably expressing each of the three 1AR subtypes (Gonzalez-Cabrera et al, 2003). Common to all three subtypes were genes involved in transcriptional regulation such as c-Fos and c-Jun, and cytokines and growth factors. Fos did increase in the T brain (Table 4) as well as jun kinase (Table 3). Two genes that were specific to the 1BAR subtype in the rat-1 fibroblasts that also changed in the T brains were tau and TGF-ß. While the expression of many other gene families was conserved in the microarray studies of fibroblasts versus brain, the exact genes were different, which is likely to be due to cell type differences. For instance, in fibroblasts, STAT 3 was actively transcribed while, in the brain, STAT 6 and STAT 1 were affected. From the same T mice used in this study, a microarray analysis from the heart was also performed (Yun et al, 2003) which also showed TGF-ß pathways being activated, along with apoptosis.
In summary, we have carried out a microarray study to identify the functional and transcriptional consequences of 1BAR overexpression in the mouse brain. Notably, T mice that systemically overexpress a constitutively activated 1BAR showed changes in the expression of genes involved in glutamate and calcium regulation, apoptosis and neurodegeneration, growth and synaptic vesicle transport. Age-dependent changes in gene expression were correlated with distinct neurological phenotypes observed in T mice, thus, this study should serve to aid in the delineation of the specific functional roles of 1BARs in the CNS.
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This work was funded by RO1HL61438 (D.M.P.), a T32 HL07914 training grant in Vascular Cell Biology (J.Y.) and a local American Heart Association fellowship (P.J.G.-C.).
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