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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Succinic Semialdehyde Dehydrogenase Deficiency: GABAB receptor-mediated function Andrea Buzzi a,b,1 , Ying Wu a,b,1 , Marina V. Frantseva a,b,1 , Jose L. Perez Velazquez a,b , Miguel A. Cortez a,b , Chun C. Liu a,b , Li Q. Shen a,b , K. Michael Gibson d , O. Carter Snead III a,b,c,⁎ a
Brain and Behavior Program, Faculty of Medicine, University of Toronto, Toronto, ON, Canada Division of Neurology Hospital for Sick Children, Faculty of Medicine, University of Toronto, Toronto, ON, Canada c Department of Pediatrics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada d Children's Hospital Pittsburgh, Department of Pediatrics and Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA b
A R T I C LE I N FO
AB S T R A C T
The succinic semialdehyde dehydrogenase (SSADH) null mouse (SSADH−/−) represents a
Accepted 26 February 2006
viable animal model for human SSADH deficiency and is characterized by markedly
Available online 2 May 2006
elevated levels of both gamma-hydroxybutyric acid (GHB) and gamma-aminobutyric acid (GABA) in brain, blood, and urine. In physiological concentrations, GHB acts at the GHB
receptor (GHBR), but in high concentrations such as those observed in the brains of children
with SSADH deficiency, GHB is thought to be a direct agonist at the GABABR receptor
(GABABR). We tested the hypothesis that both GHBR and GABABR-mediated function are
perturbed in SSADH deficiency. Therefore, we examined the high affinity binding site for
SSADH knock-out mice
GHB as well as the expression and function of the GABABR in mutant mice made deficient in SSADH (SSADH−/−). There was a significant decrease in binding of the specific GABABR antagonist, [3H]CGP-54626A at postnatal day (PN)7 and PN14 in SSADH−/− when compared to wild type control animals (SSADH+/+), particularly in hippocampus. GABABR-mediated synaptic potentials were decreased in SSADH−/−. Immunoblot analysis of GABABR1a, R1b, and R2 in SSADH−/− indicated a trend towards a region-specific and time-dependent decrease of GABABR subunit protein expression. There was no difference between SSADH−/− and wild type in binding of either [3H]GHB or a specific GHBR antagonist to the GHBR. These data suggest that the elevated levels of GABA and GHB that occur in SSADH−/− lead to a usedependent decrease in GABABR-mediated function and raise the possibility that this GHBand GABA-induced perturbation of GABABR could play a role in the pathogenesis of the seizures and mental retardation observed in SSADH deficiency. © 2006 Elsevier B.V. All rights reserved.
⁎ Corresponding author. 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Fax: +1 416 813 7839. E-mail address: [email protected]
(O.C. Snead). 1 Contributed equally to this work. 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.02.131
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Succinic semialdehyde dehydrogenase (SSADH) deficiency is a rare autosomal recessive inborn error of γ-aminobutyric acid (GABA) metabolism (Fig. 1). GABA is derived from glutamic acid via glutamic acid decarboxylase and is converted to succinic semialdehyde (SSA) by GABA transaminase. The SSA either can be converted to succinic acid by SSADH or to γhydroxybutyric acid (GHB) by the enzyme SSA reductase (Maitre, 1997; Kelly et al., 2002). The major catabolic pathway for GHB degradation in mammals is the NAD(P)+-linked oxidation of GHB to SSA, the latter undergoing further metabolism to either GABA or succinate catalyzed by SSADH (Maitre, 1997; Snead et al., 1989). The absence of SSADH leads to a 30-fold increase of GHB and a 2–4-fold increase of GABA in the brains of patients with SSADH deficiency as compared to normal brain concentrations of these compounds (Gibson et al., 1998). The clinical phenotype of children with SSADH deficiency is manifested by a wide spectrum of neurological dysfunction, including language delay, ataxia, hypotonia, and mental retardation. Epilepsy occurs commonly in SSADH deficiency and is characterized by absence, myoclonic, and convulsive seizures, as well as convulsive status epilepticus (Gibson et al., 1998; Pearl et al., 2003; Dervent et al., 2004). Recently, we (KM Gibson) have generated SSADH-deficient mice (SSADH−/−) by standard gene targeting techniques. The mutant animal shows markedly elevated levels of both GABA and GHB in urine and homogenates of liver and brain, and has a phenotype that is characterized by ataxia and seizures (Hogema et al., 2001; Gibson et al., 2002; Gupta et al., 2002). The SSADH−/− manifest absence seizures at P10 and these became progressively more severe from P14–18 at which time they evolve into myoclonic and generalized convulsive seizures that progress into a lethal status epilepticus (Cortez et al., 2004). GHB is found in normal mammalian brain at a level <1% of its parent neurotransmitter, GABA (Doherty et al., 1978) and exerts its effects both through a specific GHB receptor
(Snead, 2000; Wu et al., 2004) and the GABAB receptor (GABABR) (Gervasi et al., 2003). The high brain levels of GHB in SSADH deficiency and SSADH−/− are significant because GHB has a multiplicity of pharmacological actions (Maitre, 1997; Wong et al., 2004) including the ability to induce absence-like seizures in a number of animal species (Snead, 2002). Therefore, GHB may be causative of the absence seizures that occur both in SSADH-deficient patients and SSADH−/−. GABA can activate ligand gated ion channels (GABAAR and GABACR) and G-protein coupled receptors (GABABR). The GABABR mediates a slow inhibitory postsynaptic potential (IPSP). Effector mechanisms associated with GABABR are the adenylate cyclase system, calcium channels, and G-protein-coupled inwardly rectifying potassium channels (GIRKS). The GABABR is a heterodimer composed of GABABR1 and GABABR2 subunits. Postsynaptic GABABRs are coupled to GIRKs. Presynaptic GABABRs are subdivided into those that control GABA release (autoreceptors) and those that inhibit all other neurotransmitter release (heteroreceptors). GABABR mediate their presynaptic effects through a voltage-dependent inhibition of high-voltage activated Ca2 channels of the N type or P/Q type (Bettler et al., 2004). The purpose of the following experiments was to test the hypothesis that GABABR-mediated function is perturbed in SSADH−/− mice. The rationale for this hypothesis is that the markedly elevated levels of GHB and GABA that are observed in SSADH−/− would be predicted to subject the GABABR to chronic hyperstimulation and therefore alter its function in the mutant animals.
SSADH−/− mice showed no changes in the GHB receptor
There were no significant differences observed between SSADH−/− and age-matched wild type control mice in binding
Fig. 1 – Metabolic interconversion of GABA. The X indicates the blockade of the interconversion of SSA to succinic acid in the absence of SSADH. The consequence of this inborn error, both in patients with SSADH deficiency and in SSADH−/− mice, is that SSA is converted preferentially to GABA and GHB with an accumulation of these compounds in brain.
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to the GHB receptor as measured either with [3H]GHB or the GHB receptor antagonist, [3H]NCS382 (data not shown).
SSADH−/− mice showed a decrease in GABABR binding
[3 H]CGP54626A binding showed an evolving pattern in SSADH−/− during development. Comparison of SSADH+/+ to SSADH−/− without seizures during the first three postnatal weeks of life revealed a significant (P < 0.01) decrease in binding at PN7 and PN14 (Fig. 2).
Intrinsic membrane properties (resting membrane potential and input resistance) and inhibitory neurotransmission were estimated using whole-cell patch clamp recordings of CA1 pyramidal hippocampal neurons in SSADH−/− and wild type control mice. While there was no difference in input resistance and resting membrane potential recorded at PN8; however, by PN14, a significant reduction in input resistance and depolarization of resting membrane potential had emerged in SSADH−/− mice (data not shown). Inhibitory GABABRmediated neurotransmission was measured from the intracellular postsynaptic responses in the presence of the glutamate receptor blockers CNQX and D-AP5 (to block excitatory postsynaptic potentials) and the GABAAR blocker bicuculline. Under these conditions, extracellular stimulation of the Schaffer collaterals evoked a GABABR-mediated inhibitory response. The GABAB potentials of SSADH−/− mice were significantly lower than those elicited from control mice at PN8 and PN14 days (Fig. 3).
Immunoblot analysis of GABABR1a, R1b, and R2 in SSADH−/− indicated a region-specific and time-dependent decrease of GABABR subunit protein expression (Fig. 4), but this difference between SSADH−/− and wild type failed to reach significance (Fig. 5).
We set out to prove a dual hypothesis: that GHB binding would be altered in the SSADH−/− and that GABABR-mediated function would be perturbed as well in the mutant animals. The data showing no alteration of [3H]GHB or [3H]NCS382 binding in SSADH−/− refute the first hypothesis. However, the GABABR data in these experiments lend credence to the hypothesis that there is altered GABABR-mediated function in the SSADH−/−. The data demonstrate a significant decrease in [3H]CGP54626A binding in developing SSADH−/− mice as well as electrophysiological evidence of decreased GABABR-mediated IPSPs with a qualitative decrease in GABABR subunit expression that failed to reach statistical significance. GHB exerts ubiquitous pharmacological effects in the brain (Wong et al., 2004) and also has many properties that suggest that this compound may play a role in the brain as a neurotransmitter or neuromodulator (Maitre, 1997). A specific GHB receptor (GHBR) is suggested by specific, high affinity [3H] GHB binding sites that occur in rat and human brain. [3H]GHB binding is saturable, reversible, and displays a high affinity binding site, 30 nM to 580 nM, as well as a low affinity binding site, 1.5 μM to 16 μM (Benavides et al., 1982). These kinetics comport with the concentration of 1–4 μM GHB found in mammalian brain (Doherty et al., 1978). Although there are some contradictory data (Castelli et al., 2003), there is evidence that the GHBR is G protein-coupled (Snead, 2000) and may be functionally coupled to a specific presynaptic GHBR to inhibit the release of GABA (Hu et al., 2000). The lack of any change in either agonist (GHB) or antagonist (NSC 382) binding in the SSADH−/− makes it unlikely that GHBR-mediated mechanisms are involved in the genesis of the seizures and all the complex clinical evidences associated to the SSADH deficiency. There are considerable biochemical, developmental, and molecular data to support the hypothesis that the GHBR is distinct from the GABABR (Wu et al., 2004). However, the
Fig. 2 – [3H]CGP54626A binding. (A) [3H]CGP54626A autoradiography in a PN14 SSADH−/− and age-matched, wild type control animal shows a decrease in [3H]CGP54626A binding throughout the brain of the mutant animal. (B) Quantification of the [3H]CGP54626A binding signal in hippocampus of a SSADH−/− vs. wild type control demonstrates a significant decrease at PN7 (**P < 0.01, Student's t test) and PN14 (*P < 0.05; Student's t test).
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yet present. They do not appear until the third postnatal week of life (Snead, 1994). Taken together, these data suggest that the inordinately high concentration of GHB in the brains of SSADH−/− provides a hyperstimulation of the GABABR which, over time could lead to a downregulation of the GABABR (Malcangio et al., 1995; Kroin et al., 1993), as indicated by the binding and electrophysiological data in the current experiments. The discordance between the immunoblotting and binding data suggests that the GABABR-mediated functional alteration observed in the SSADH−/− could be related to post-translational modification of the GABABR in the mutant animals. One posttranslational mechanism that could be operative in the SSADH−/− is that the chronically elevated levels of GHB and GABA in SSADH−/− might result in an alteration of GABABR trafficking. In support of this hypothesis are recently published data showing that prolonged agonist treatment results in increased endocytosis of GABABR with resultant decrease in GABABR-mediated function (Fairfax et al., 2004). This putative GHB and GABA-induced downregulation of GABABR-mediated activity observed in the SSADH+/+ may contribute to the evolution of generalized tonic–clonic seizures that occur in the mutant animals (Gassmann et al., 2004; Prosser et al., 2001). This could also be associated to our recent findings (Wu et al., 2005) about a change in GABAAR β2 subunits in SSADH−/− mice. Together, those alterations may explain the complex situation associated to the evolution of the seizures in the SSADH deficiency as a perturbation of the GABA receptors due to an imbalanced neurotransmitter concentration that lead to a pathological adjustment of the GABA inhibitory system.
Fig. 3 – GABAB receptor-mediated synaptic potential. At both time points chosen, PN7 and PN14, the GABAB receptor-mediated potential is significantly (*P < 0.05, paired Student's t test) decreased comparing SSADH knock out mice to slices prepared from an age-matched wild type control animal. The trace reported in the first graph is one of the GABAB receptor-mediated potential recorded during the experiments.
evidence is compelling that many, if not most, of the pharmacological, clinical, behavioral, and toxicological effects of exogenously administered GHB, are likely mediated via the GABABR (Wong et al., 2004). GHB has been proposed to be a weak GABABR agonist and can activate signaling pathways in recombinant GABABR heterodimers (Lingenhoehl et al., 1999). Similarly, GHB has a GABABR-like electrophysiologic effect that is blocked by a specific GABABR antagonist, but not the GHB antagonist, NCS 382 (Gervasi et al., 2003). Finally, while GHBR binding is retained in the GABABR1 knockout mouse (Kaupmann et al., 2003; Wu et al., 2004), GHB does not exert its usual pharmacological effects in these mutant animals (Kaupmann et al., 2003) indicating that while the GHBR is separate and distinct from the GABABR, most of the pharmacological effects of GHB are mediated via the GABABR. This is particularly likely to be true in the SSADH−/− because the seizures begin at PN10, a time when GHB binding sites are not
The SSADH knockout mice were generated by standard gene targeting and characterized by Hogema et al. (2001). Mice were genotyped by two-allele three-primer PCR using tail genomic DNA as previously described (Hogema et al., 2001). Experiments were conducted comparing SSADH−/− vs. wild type mice (SSADH+/+) used as control. The heterozygotes (SSADH+/−) were retained for breeding purposes, in order to propagate the colony. All mice used in experiments were C57BL6/129SV hybrids (50%/50%). Developing SSADH−/− and SSADH+/+ were used for the experiments described below at the times specified during the first three postnatal weeks of life. The present study was approved by the Animal Care and Use Committee of The Hospital For Sick Children in the University of Toronto.
[3H]CGP54626A (specific activity 40 Ci/mmol), a GABABR antagonist, was obtained from Tocris Cookson Ltd. (UK). [3H] GHB (specific activity 60 Ci/mmol) and [3H](2E)-(5-hydroxy5,7,8,9-tetrahydro-6H-benzo[a]annulen-6-y ethanoic acid ([3H]NCS-382) (specific activity 20 Ci/mM), a GHB receptor antagonist, were obtained from American Radiolabelled Chemicals Inc. (USA).
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Fig. 4 – GABABR immunoblotting. The immunoblotting analysis at the two time points chosen, PN 7 and PN 14, refers to the three main subunits of the GABAB receptor, GABAB R1a and b and GABAB R2. The areas used to perform the immunoblotting were hippocampus (H), cortex (CTX) (data shown), thalamus and cerebellum (data not shown).
For this and all autoradiographic binding experiments described below, SSADH−/− and age-matched wild type mice were sacrificed by decapitation, the brains removed and immediately immersed in isopentane at −35 °C. Coronal sections were cut at 20 μm at −20 °C and thaw-mounted onto gelatin-coated slides that were dried and stored at −80 °C until used. CGP54626A is a high affinity antagonist for the GABABR. [3H] CGP54626A binding was performed upon SSADH−/− and agematched wild type control mice as described (Bischoff et al., 1999). Before [3H]CGP54626A binding, brain sections were maintained at room temperature for 1 h and then preincubated for 15 min in Krebs–Henseleit buffer. Total binding was performed in Krebs–Henseleit buffer containing 2 nM [3H] CGP54626A for 2 h at room temperature. 10 mM of (−)baclofen was used to account for nonspecific binding which represented less than 10% of the total binding. The air-dried tissue sections were apposed to 3H-sensitive film along with 3H-standard radioactive microscales (Amersham, IL) for 2 weeks at room temperature.
[3H]GHB autoradiography was performed upon SSADH−/− and age-matched wild type control mice as described (Banerjee et al., 1993). Brain sections were thawed at room temperature for 1 h and preincubated in 100 mM phosphate buffer (pH 6.0) for 30 min at 4 °C and air-dried. Triplicate tissue sections were incubated in the same buffer containing 30 nM [3H]GHB for 30 min at 4 °C. The nonspecific binding represented less than 10% of the total binding. After three successive buffer washes (10 s each at 4 °C), sections were dipped in ice-cold deionized water and air-dried.
[3H]NCS-382 autoradiography was performed upon SSADH−/− and age-matched wild type control mice as described (Gould et
Fig. 5 – GABABRs immunoblotting analysis. Quantification was done in two of the four areas investigated (H: hippocampus; CTX: cortex) at the two time points chosen (PN 7 and PN 14) in mutant mice and age-matched, wild type control animals (n = 6). The value in SSADH−/− is expressed as percentage of SSADH+/+ used as control. Although there was a trend towards a decrease of the GABABR1b in mutant animals, statistical significance was not achieved (Student's t test).
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al., 2003). Brain sections were thawed at room temperature for 1 h and incubated in 50 mM Tris–HCl buffer, pH 7.4 at 4 °C containing 1 μM [3H]NCS-382 for 20 min. Nonspecific binding was determined in the presence of 500 μM unlabeled Na+-free GHB and represented less than 10% of the total binding. Following incubation, sections on slides were washed for 3 min in 50 mM Tris–HCl buffer, pH 7.4 at 4 °C, and dipped for 1 s into ice-cold de-ionized distilled water. The sections were air-dried and analyzed.
Analysis of binding
The autoradiographic data were analyzed as previously reported (Snead, 2000). Briefly, dried tissue sections were opposed to hyperfilm-bmax film (Amersham; Arlington IL) with [3H] microscale standards (Amersham, Arlington, IL) for 14 days at room temperature. The films were developed in D-19 (Kodak), fixed, and air-dried. Quantitative analysis of the resulting autoradiograms was performed densitometrically using a microcomputer-based densitometer system (MCID; Imaging Research; Ontario, Canada). Briefly, a standard curve between the optical density (o.d.) of [3H] standards and tissue radioactivity equivalents (pmol/mg of tissue) was constructed using a nonlinear regression analysis. The average o.d. values of the selected brain regions were in the linear portion of this standard curve. The pmol/ mg value in each brain region was calculated by interpolation using the image analyzer (Banerjee et al., 1998). Five to eight readings were determined and averaged for each anatomic area analyzed.
SSADH−/− and age-matched SSADH+/+ were sacrificed by decapitation under halothane anesthesia and brains were immediately excised and cortex, hippocampus, thalamus, and cerebellum dissected on ice-cold glass. The tissue samples were homogenized and suspended in samples buffer (62.5 mM Tris-base, 2% SDS, 10% glycerol; pH 6.8) and protein quantification was determined by BCA assay (Pierce). Samples were then added of 2.5% β-mercaptoethanol and 0.005% bromophenol blue, adjusted to a final concentration of 1 mg/ml for the thalamus and 3.5 mg/ml for all other brain regions and heated for 10 min at 37 °C. Protein separation was performed on 4–20% SDS-PAGE gels (Invitrogen) and transferred to nitrocellulose membrane in transfer buffer (25mM Tris-base, 192mM glycine, 20% methanol). A variety of primary antibodies were employed to probe the membranes. For the GABABR, anti-GABABR1 (1:1000, 2.5 h incubation at room temperature) and antiGABABR2 (1:2000, 1.5 h incubation at room temperature) guinea pig polyclonal antibodies (Chemicon, Temecula, CA, USA) were utilized. Appropriate guinea pig (Chemicon, Temecula, CA, USA) secondary horseradish-conjugated antisera were employed in the secondary incubation (1 h at room temperature) and then developed by ECL (Amersham Pharmacia). Quantification was obtained by a dosimeter. Each lane was loaded with 15 μl of samples; loading amount normalization was obtained by comparison to the anti-actin band on the same blot.
Brain slices and solutions
In order to obtain hippocampal slices, 8–14 days old mice were anesthetized with halothane (Fluorothane, Ayerst Laboratories, Montreal, Quebec, Canada) and decapitated. The brains were removed and placed in ice-cold artificial cerebrospinal fluid (ACSF), containing (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 25 NaHCO3, and 10 glucose at pH 7.4 when saturated with 95% O2–5% CO2. Transverse brain slices (450 μm) were obtained using a Vibratome (Series 1000, Technical Products International) and maintained in ACSF aerated with 95% O2–5% CO2 at room temperature for 1 h prior to the recordings. Slices were transferred to a superfusion chamber maintained at 36 °C (Medical Systems, model PDMI-2) and were superfused (4– 5 ml per minute) continuously with ACSF (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 25 NaHCO3, and 10 glucose, osmolarity 300 ± 5 mOsm. In order to investigate possible epileptiform activity (specifically, primary and/or secondary afterdischarges), KCl concentration was increased to 5 mM and the concentration of MgSO4 was decreased to 0.9 mM. When needed, drugs were applied by superfusion. D2-amino-5-phosphopentanoic acid [D-AP5, 20 mM, made of 50 mM stock solution in distilled water] and 6-cyano-7nitroquinoxaline-2,3-dione [CNQX, 100 μM, made of 50 mM stock in dimethyl sulfoxide (DMSO)] were obtained from Tocris Cookson and diluted daily.
Neuronal recordings were obtained with the use of the wholecell configuration of the patch-clamp technique from CA1 hippocampal pyramidal neurons. Patch pipettes were pulled from borosilicate capillary tubing (Word Precision Instruments, New Haven, CT). Electrodes, filled with internal solution [(in mM) 150 potassium gluconate, 10 N-2-hydroxyethylpiperazineN′-2-ethanesulfonic acid (HEPES), 2 Mg-ATP, 0.5 Li-GTP, 5 KCl, and 0.1 ethylene glycol-bis ((-aminoethyl ether)-N,N,N′,N′tetraacetic acid (EGTA), pH 7.2 adjusted with KOH, osmolarity 275 ± 5 mOsm] had tip resistances between 5 and 6 MΩ. The resistance to ground of the whole cell seal was 2–8 GΩ before breakthrough. Neuronal responses were recorded with the use of an Axoclamp 2-B amplifier in bridge mode. PCLAMP software (Clampex 8, Axon Instruments) was used for analysis of membrane potential (Vm), input resistance (Rn, which was measured from the linear part of the current-voltage plot), and amplitude of the postsynaptic responses. The amplitudes of the postsynaptic responses were measured from the peak to the baseline. To test for statistical significance, the paired Student's t test was used. Significance was considered at P < 0.05; numerical values are expressed as means ± SE. For extracellular stimulation, a bipolar stimulation electrode was placed in the Schaffer collaterals, input to CA1 area. A Grass 88K square-pulse stimulator was used for this purpose; current pulses of 150 ms duration were delivered at 0.66 Hz. Initial low-stimulus intensities were gradually increased to elicit stable postsynaptic responses and were maintained at this level for the duration of the experiment. For extracellular local field potential recordings, an extracellular recording electrode was filled with ACSF and placed in the CA1 cell body.
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Acknowledgments This work was supported by NIH grant NS 40270, CIHR, and members of the Partnership for Pediatric Epilepsy Research (including the American Epilepsy Society, the Epilepsy Foundation, Anna and Jim Fantaci, Fight Against Childhood Epilepsy and Seizures (F.A.C.E.S.), Neurotherapy Ventures Charitable Research Fund, and Parents Against Childhood Epilepsy (P.A.C.E.).
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