GABAA-receptor mRNA expression in the prefrontal and temporal cortex of ALS patients

Journal of the Neurological Sciences 250 (2006) 124 – 132 www.elsevier.com/locate/jns

GABAA-receptor mRNA expression in the prefrontal and temporal cortex of ALS patients Susanne Petri a,⁎, Katja Kollewe a , Claudia Grothe b , Akira Hori c , Reinhard Dengler a , Johannes Bufler a , Klaus Krampf l a a

Department of Neurology, Hannover Medical School, Carl-Neuberg-Str. 1, 30623 Hannover, Germany b Department of Neuroanatomy, Hannover Medical School, Hannover, Germany c Department of Neuropathology, Hannover Medical School, Hannover, Germany Received 9 March 2006; received in revised form 4 August 2006; accepted 8 August 2006 Available online 2 October 2006

Abstract There is evidence that excitotoxic cell death is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Electrophysiological and histological studies support the pathophysiological concept of an impaired inhibitory, namely GABAergic, control of the motoneurons in the cerebral cortex of ALS patients. Recently, pathological, neuropsychological and functional imaging data have challenged the view that ALS is a disorder restricted to the motor system. The aim of our study was to investigate the expression of the most abundant GABAAreceptor subunit mRNAs and the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) in the prefrontal, temporal, occipital and cerebellar cortex of ALS patients compared to tissue of control persons. We performed in situ hybridization histochemistry (ISH) on human post-mortem cortex sections of ALS patients (n = 5) and age-matched controls with no history of neurological disease (n = 5). In the prefrontal and temporal cortex of ALS patients, we detected significantly reduced mRNA expression of the α1-subunit, while the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) was significantly upregulated in these regions. In the occipital and cerebellar cortex, we did not see disease-specific differences of the mRNA expression of the investigated subunits. © 2006 Published by Elsevier B.V. Keywords: Amyotrophic laterals sclerosis; Inhibitory neurotransmission; GABAA-receptors; In situ hybridization histochemistry; Extramotor brain regions

1. Introduction ALS is the most common adult motor neuron disorder which becomes manifest in a rapidly progressive paralysis due to degeneration of motor neurons in the primary motor cortex, brainstem and spinal cord. Ninety percent of cases occur sporadically, 10% are familial. Among the familial cases, 10– 20% can be attributed to point mutations in the gene coding for superoxide dimutase1 (SOD)1 [1]. Besides the most impressive degeneration of motor neurons in the cerebral cortex, brainstem and the anterior horn cells of the spinal cord, the involvement of other neuronal systems like the frontal and temporal lobes has ⁎ Corresponding author. Tel.: +49 511 532 6677; fax: +49 511 532 3115. E-mail address: [email protected] (S. Petri). 0022-510X/$ - see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.jns.2006.08.005

been shown as well [2,3]. While clinically manifest dementia can occur in 5–10% of ALS patients, precise neuropsychological testing of frontal lobe function shows more widespread cognitive impairment also in non-demented ALS patients suggesting a continuum between discrete cognitive alterations at one and obvious dementia at the other extreme [3–11]. Many different pathomechanisms such as mitochondrial dysfunction, inflammatory processes, protein aggregation and oxidative stress are discussed in ALS [2,12–18]. The common final pathway of neuronal death appears to be related to excitotoxicity as chronic dysregulation of the intracellular calcium homeostasis [19–21]. This could be influenced by a dysregulation of inhibitory and excitatory synaptic neurotransmission. The most widely distributed inhibitory neurotransmitter in the mammalian central nervous system (CNS) is gamma-

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amino butyric acid A (GABAA) [22]. Its receptors are formed by members of at least three distinct subunit families (α1–6, β1–3, γ1–3, δ, ε, ρ1–2). The subunit composition of the receptor channels defines their physiological and pharmacological properties. The most abundant subunit stoichiometry of GABA-receptors seems to be two α-, two β- and one γsubunit [23,24]. We could previously show differences in the mRNA expression pattern of GABAA-receptor subunits, in particular an increased expression of the β1- and a reduced expression of the α1-subunit in the primary motor cortex of ALS patients which could lead to decreased formation of

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GABAA-receptors with highest agonist affinity [25]. This is consistent with positron emission tomography (PET) studies showing that binding of the benzodiazepine antagonist flumazenil was reduced in the motor cortex [3]. However, besides the primary motor cortex, PET-studies also revealed a much more widespread cortical involvement including frontal, temporal and parietal regions in sporadic ALS patients without clinical signs of dementia [3,26]. The primary interest of the present in situ hybridization (ISH) study was therefore to investigate whether the alterations in flumazenil binding patterns in sporadic ALS in extramotor brain regions could be

Fig. 1. X-ray autoradiograms of GABAA receptor subunit mRNA expression in the prefrontal cortex of controls (left) and ALS patients (right). Levels of subunitspecific mRNA expression in nCi/g (abscissae) across the six cortical layers (ordinates). GABAA receptor mRNA expression in the motor cortex of ALS patients and controls was assessed semiquantitatively by densitometry of the film autoradiograms. Abscissae indicate density readings converted to measures of radioactivity (nCi/g) by reference to [14C] standards exposed on the same sheet of film. Layers have been added by matching to digitized images of adjacent Nissl-stained sections. Data are shown as mean values ± S.D. ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001. Grey bars, controls; black bars, patients.

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attributed to distinct mRNA expression patterns of GABAAreceptor subunits. We studied the distribution of the mRNA of the GABAA-receptor subunits α1, β2 and γ2 which form the A1a GABAA-receptor subtype [27] with highest affinity for classical benzodiazepines and zolpidem, of the β1-subunit which had been elevated in our study in ALS motor cortex [25], and of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) in the frontal and temporal cortex of ALS patients, to investigate whether we could see similar alterations in the mRNA expression pattern as in our previous study done in the primary motor cortex, corresponding to the

flumazenil PET data. We further investigated the mRNA of these subunits in the occipital and cerebellar cortex, i.e. in brain regions for which no ALS-specific changes have been described histopathologically or via imaging techniques. 2. Materials and methods Post-mortem brain specimens of five patients aged between 61 and 82 years (mean age 67 years) with a definite diagnosis of amyotrophic lateral sclerosis according to the El Escorial criteria [28] and of five control patients aged between 65 and

Fig. 2. X-ray autoradiograms and densitometrical semiquantitative analysis of GABAA receptor subunit mRNA expression in the temporal cortex of controls (left) and ALS patients (right). Data are shown as mean values ± S.D. ⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001. Grey bars, controls; black bars, patients.

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84 years (mean age 65 years) with no evidence of neurological or psychiatric disease were available from autopsies. The postmortem delay ranged from 8 to 32 h. Brains were cut into coronal slices of 1 cm thickness and rapidly frozen on dry-ice. Before starting the in situ hybridization experiments, cryostat sections of the asserved and shock frozen tissue blocks from ALS patients and controls were Nissl-stained and their cytoarchitecture was studied histologically by a neuropathologist. Then, 20 μm sections of the prefrontal, temporal, occipital and cerebellar cortex were cut on a cryostat, thawmounted onto poly-L-lysine-coated slides, fixed for 5 min in

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4% phosphate-buffered paraformaldehyde and stored in 96% ethanol at 4 °C until use. In situ hybridization histochemistry was performed as previously described [25]. Oligonucleotide probes (40 to 45 mers) complementary to coding parts of the cDNAs of the subunits GABAA α1: 5′-ggg tcg ccc ctg gcc aaa tta ggg gtg tag ctg gtt ggt-3′, β1: 5′-ctt gcg gta ctg gat gct ggc gct gtc ata gga gta cat ggt ggc-3′, β2: 5′-tcg tcc aga gca ttt cgg cca aaa cta tgc ctg ggc aac cca gc-3′ and γ2: 5′-cat ttg aat ggt tgc tga tct tgg gcg gat atc aat ggt agg ggc-3′ as well as to glutamic acid decarboxylase (GAD): 5′-ctc tgt ttt taa tct tgg cat aga ggt att cag cca gtt cc-3′ were synthesized (MWG

Fig. 3. X-ray autoradiograms and densitometrical semiquantitative analysis of GABAA receptor subunit mRNA expression in the occipital cortex of controls (left) and ALS patients (right). Data are shown as mean values ± S.D. Grey bars, controls; black bars, patients.

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Biotech, Ebersberg, Germany) and 3′-end labelled with α-35SdATP (Perkin Elmer, Zaventem, Belgium). Only labels between 250,000 and 350,000 cpm/μl of the eluate were used for experiments. For each oligonucleotide probe, tissue sections were processed in parallel to obtain X-ray- and emulsion-dipped autoradiograms in adjacent slices. Slides were hybridized for 48 h at 42 °C with labelled probe diluted in hybridization buffer (50% formamide, 4× SSC, 5× Denhardt's solution, 10% dextran sulphate) to a final concentration of 0.07 pmol/ml. After washing in 1× SSC at 56 °C for 30 min, neighbouring sections were either exposed to Biomax X-ray film (Kodak, Stuttgart, Germany) for 4 weeks or dipped in NTB 2 nuclear track emulsion (Kodak, Stuttgart, Germany). Dipped sections were counterstained with 0.05% thionin, dehydrated and coverslipped. For negative control, a 100-fold excess of non-labelled oligonucleotides was added to the radioactive probe and applied to the adjacent section, leading to a complete suppression of the signal (not shown). Semiquantitative analysis of film autoradiograms of the sixlayered neocortical regions (prefrontal, temporal, occipital cortex) was performed according to the method of Akbarian et al. as previously described [25,29]: Optical density readings were taken in vertical strips 0.5 mm wide across the full thickness of the cortex, and optical densities were determined for the thickness of each cortical layer separately, by matching the autoradiogram readings to digitized images of the adjacent Nissl-stained section. Optical density over white matter more than 3 mm deep to overlaying cortex was used for background subtraction. Absolute values of radioactivity were determined from 14C plastic standards (Amersham, Freiburg, Germany) on the same sheet of film (NIH image software). Quantified hybridization signals for the patient and the control group were compared using Student's t-test (Figs. 1, 2, and 3). Cellular expression patterns of the mRNA transcripts were determined microscopically throughout the cortical layers by classifying the amount of silver grains over individual cells at the Nissl-counterstained sections as strong, moderate or weak according to a visual analogue scale (Fig. 5, Table 1).

3. Results We first investigated the distribution of GABAA-receptor subunits (α1, β1, β2, γ2) and of the GABA synthesising enzyme glutamic acid decarboxylase (GAD) in the frontal and temporal cortex of ALS patients and controls by in situ hybridization techniques using film autoradiograms and, for a cellular localization of the hybridization signal, by emulsion dipped tissue sections. For semiquantitative analysis of film autoradiograms, optical density readings were converted to measures of radioactivity (nCi/g) and attributed to the cortical layers that were identified microscopically as previously described. We found a distinct expression pattern of GABAA-receptor subunit RNA in the cortical layers of the temporal and prefrontal cortex of ALS patients and controls (Figs. 1 and 2). While the β1-, β2- and γ2-subunits had similar expression patterns throughout the layers of the temporal and frontal cortex in patients and controls, the α1subunit was expressed significantly less in all layers in the ALS patients tissue (Figs. 1 and 2). The GABA synthesizing enzyme GAD showed a different expression in ALS prefrontal and temporal cortex, too: While it was weakly to moderately (up to 400 nCi/g) expressed throughout all layers of the prefrontal and temporal cortex of the controls, in ALS prefrontal and temporal cortex, expression levels were significantly higher in layers III to V (up to 700 nCi/g) (Figs. 1 and 2). In the occipital and cerebellar sections, we did detect similar expression levels as in the prefrontal and temporal sections with no significant differences in mRNA expression between patients and controls (Figs. 3 and 4) with moderate to strong signals of the α1-, β2- and γ2-subunits, weak signals of the β1-subunit and weak to moderate signals of GAD. To get information about the mRNA expression of GABAA-receptors at the cellular level, emulsion dipped slides of the prefrontal and temporal cortex of patients and controls were Nissl-counterstained and analysed (Fig. 5). This allowed a more detailed study of the cortical architecture, the attribution of distinct expression patterns to the cortical layers

Table 1 Cellular distribution of the GABAA receptor subunit and GAD mRNA expression according to a visual analogue scale

Lamina I (patients) Lamina I (controls) Lamina II (patients) Lamina II (controls) Lamina III (patients) Lamina III (controls) Lamina IV (patients) Lamina IV (controls) Lamina V (patients) Lamina V (controls) Lamina VI (patients) Lamina VI (controls)

α1 prefrontal

α1 temporal

β1 prefrontal

β1 temporal

β2 prefrontal

β2 temporal

γ2 prefrontal

γ2 temporal

GAD prefrontal

GAD temporal

+ + +/++ ++ ++/+++ +++ ++ +++ ++/+++ +++ +/++ ++

+ + +/++ ++ ++/+++ +++ ++ +++ ++/+++ +++ ++ ++

+ + +/++ +/++ +/++ +/++ +/++ +/++ ++ ++ +/++ +/++

+ + +/++ +/++ ++ ++ +/++ +/++ ++ ++ +/++ +/++

+ + +/++ +/++ +++ +++ +++ +++ +++ +++ ++ ++

+ + +/++ +/++ +++ +++ +++ +++ +++ +++ ++ ++

(+) (+) +/++ +/++ ++/+++ ++/+++ ++ ++ ++ ++ +/++ +/++

(+) (+) +/++ +/++ ++/+++ ++/+++ ++ ++ ++ ++ +/++ +/++

(+) (+) + + ++/+++ ++ ++ + ++/+++ ++ + (+)

(+) (+) + + ++/+++ ++ ++ + ++/+++ ++ + +

−, not detectable; (+), weak, if any; +, weak; ++, moderate; +++, high.

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(Table 1, Fig. 5). Pyramidal cells were labelled weekly to moderately by GAD in both groups. 4. Discussion

Fig. 4. X-ray autoradiograms of GABAA receptor subunit mRNA expression in the cerebellar cortex of controls (left) and ALS patients (right). Data are shown as mean values ± S.D. Grey bars, controls; black bars, patients.

and a differentiation between pyramidal cells and other cellular elements. We found a good correlation with the data from film autoradiograms (Table 1, Fig. 5): In the external and internal pyramidal layers III and Vand in the internal granular layer IV, a strong cellular hybridization signal of the mRNA of the α1subunit was seen in the control sections, while in the patients tissue, labelling of pyramidal cells alternated between moderate and strong with a higher proportion of cells labelled moderately (Table 1, Fig. 5), corresponding to a decrease in optical density as measured in the film autoradiograms (Figs. 1 and 2). The GAD-specific oligonucleotide probe labelled mostly medium-sized round neurons in layers III to V which appear to be GABAergic interneurons, moderately in the patients sections, significantly stronger in the ALS tissues

In the present study, we investigated the mRNA expression of the GABAA-receptor subunits α1, β1, β2 and γ2, and of the GABA synthesizing enzyme GAD in the frontal, temporal, occipital and cerebellar cortex of ALS patients without dementia as compared to normal controls. So far, the human prefrontal cortex (area 46 according to Brodmann) has been investigated by ISH for the expression of the GABAA-receptor subunits α1, α2, α5, β1, β2 and γ2 in sections of schizophrenics compared to control tissue [29]. Concerning the values of mRNA levels expressed in nCi/g as well as the relative abundances between the different receptor subunit mRNAs, the results of our study are quite similar [29] besides the fact that, in our semiquantitaitve analysis, the β2-subunit had slightly higher expression levels than the α1-subunit, while in the study of Akbarian and coworkers, the α1-subunit showed the highest signal intensity. In a previous study done in our lab, we investigated 16 GABAA-receptor subunits in the primary motor cortex of ALS patients and controls [25]. As in the present study, we observed a reduction of the α1-subunits in the primary motor cortex of the patients group. In addition, the β1-subunit was upregulated in ALS, while the GAD mRNA levels were unaltered as compared to the control group [25]. Besides most obvious alterations in the primary motor cortex, neuropathological, neuropsychological and imaging studies also revealed ALS-related changes in extramotor brain regions: Patients with ALS and clinical signs of dementia often have neuronal loss in the frontotemporal cortical regions [9,30]. Frontotemporal white matter changes have been observed in ALS patients with and to a lesser extent also in patients without cognitive impairment using volumetric voxel based analysis of structural magnetic resonance imaging (MRI) [4,5,31–33]. Frontal lobe function has been impaired in event-related EEG potential measurements in ALS patients without obvious signs of dementia [34]. Single photon emission tomography (SPECT) showed reduced global resting blood flow throughout the brain in ALS patients which were most prominent in the frontal cortex [35]. Several positron emission tomography (PET) studies showed alterations in the regional cerebral blood flow (rCBF), microglial activation and significantly decreased 11C-flumazenil binding in the primary sensory, premotor, prefrontal, thalamic and parietal regions [2,7,26]. [36]. The decrease in flumazenil binding as a marker of benzodiazepine affinity correlates with reduction of intracortical inhibition as measured by transcranial magnetic stimulation, therefore supporting the hypothesis that increased cortical excitability plays a role in ALS [37,38]. In correlation with these data, our study reveals alterations in the GABAA-receptor subunit mRNA expression levels in the prefrontal and temporal regions of ALS patients. The most abundant GABAA-receptor of the vertebrate brain

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Fig. 5. Photomicrographs of emulsion-dipped sections exemplarily depicting the mRNA expression of the GABAA receptor subunits in cellular resolution in pyramidal cells (arrows) and GABAergic interneurons identified by glutamate decarboxylase (GAD) labelling (arrowheads) in layer Vof the prefrontal cortex of patients and controls. ×400.

contains two α-, two β- and one γ- or δ-subunit [39,40]. The high mRNA levels of the α1-, β2- and γ2-subunits in the frontal and temporal cortex of ALS patients and controls (Figs. 1, 2, and 5) indicate a preferred expression of BDZ sensitive GABAA-receptors [23,41–43]. Comparing the results of normal controls with ALS patients, we found a significant reduction of the mRNA levels of the α1-subunit in the prefrontal and temporal cortex of the patients group (Figs. 1, 2, and 5). At the cellular level, this decreased α1subunit mRNA expression was most impressive in pyramidal neurons of layers III and V (Table 1, Fig. 5). As the BDZ sensitivity of GABAA-receptors depends mainly on the α1-

subunit, this indicates that GABAA-receptors with high BDZ affinity are specifically reduced in large pyramidal neurons in the prefrontal and temporal cortex of ALS patients, similar to our finding in the primary motor cortex of ALS and control tissue [25]. Therefore, these data are consistent with the results in the PET studies showing decreased binding of the BDZ-receptor antagonist flumazenil. Furthermore, we also detected an increase of the mRNA of the GABA synthesizing enzyme GAD in frontal and temporal section of ALS patients in contrast to unaltered GAD levels in ALS motor cortex as previously described [25]. At the cellular level, this is most probably due to an

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increase of GAD mRNA at the interneuron level and not to changes of the total number of GAD expressing cells (Fig. 5, Table 1). This is consistent with immunohistochemical investigations showing no alterations of parvalbumin and calbindin as markers of GABAergic interneurons in ALS post-mortem sensorimotor cortex [44] and with stereological quantifications of cell counts which did not detect neuronal loss in the motor and extramotor cortical regions in ALS [45]. An inverse relationship of GABAA-receptors and GAD mRNA has previously been shown by ISH in the prefrontal cortex of schizophrenics: a decrease of GAD mRNA without significant cell loss could be attributed to an activitydependent down-regulation due to an increase of GABAAreceptors [46]. We therefore conclude that the disturbed cortical inhibition seen in ALS is not due to a loss of inhibitory interneurons but rather to a different expression pattern of postsynaptic inhibitory receptor channels. The mRNA upregulation of the GABA synthesizing enzyme GAD could be explained as a compensatory reaction to counteract altered GABA-receptor profiles and excitotoxicity in ALS. Even though it is not clear whether these alterations really are pathogenic factors or disease-related adaptations, they may provide a basis for the development of a more disease-specific pharmacotherapy. Acknowledgements We thank Prof. G. Meyer (Department of Nuclear Medicine, Medical School Hannover) for his support with the radioactive labelling of the oligonucleotide probes, S. Henke and H. Streich for excellent help with preparation of the fresh frozen sections and in situ hybridization experiments, and J. Kilian and A. Niesel for expert technical support. This study was supported by a grant of the Medizinische Hochschule Hannover to KK and SP, and by a grant of the Deutsche Gesellschaft für Muskelkranke to SP. References [1] Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993;362 (6415):59–62. [2] Abrahams S, Goldstein LH, Kew JJ, Brooks DJ, Lloyd CM, Frith CD, et al. Frontal lobe dysfunction in amyotrophic lateral sclerosis. A PET study. Brain 1996;119(Pt 6):2105–20. [3] Lloyd CM, Richardson MP, Brooks DJ, Al Chalabi A, Leigh PN. Extramotor involvement in ALS: PET studies with the GABA(A) ligand [(11)C]flumazenil. Brain 2000;123(Pt 11):2289–96. [4] Abrahams S, Leigh PN, Harvey A, Vythelingum GN, Grise D, Goldstein LH. Verbal fluency and executive dysfunction in amyotrophic lateral sclerosis (ALS). Neuropsychologia 2000;38(6):734–47. [5] Abrahams S, Leigh PN, Goldstein LH. Cognitive change in ALS: a prospective study. Neurology 2005;64(7):1222–6. [6] Gallassi R, Montagna P, Morreale A, Lorusso S, Tinuper P, Daidone R, et al. Neuropsychological, electroencephalogram and brain computed tomography findings in motor neuron disease. Eur Neurol 1989;29 (2):115–20.

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