GABAB Receptors in Neurodegeneration.

GABA is the main inhibitory neurotransmitter in the mammalian central nervous system (CNS) and acts via metabotropic GABAB receptors. Neurodegenerative diseases are a major burden and affect an ever increasing number of humans. The actual therapeutic drugs available are partially effective to slow down the progression of the diseases, but there is a clear need to improve pharmacological treatment thus find alternative drug targets and develop newer pharmaco-treatments. This chapter is dedicated to reviewing the latest evidence about GABAB receptors and their inhibitory mechanisms and pathways involved in the neurodegenerative pathologies.

The Sodium Channel B4-Subunits are Dysregulated in Temporal Lobe Epilepsy Drug-Resistant Patients.

Temporal lobe epilepsy (TLE) is the most common type of partial epilepsy referred for surgery due to antiepileptic drug (AED) resistance. A common molecular target for many of these drugs is the voltage-gated sodium channel (VGSC). The VGSC consists of four domains of pore-forming α-subunits and two auxiliary ß-subunits, several of which have been well studied in epileptic conditions. However, despite the ß4-subunits' role having been reported in some neurological conditions, there is little research investigating its potential significance in epilepsy. Therefore, the purpose of this work was to assess the role of SCN4ß in epilepsy by using a combination of molecular and bioinformatics approaches. We first demonstrated that there was a reduction in the relative expression of SCN4B in the drug-resistant TLE patients compared to non-epileptic control specimens, both at the mRNA and protein levels. By analyzing a co-expression network in the neighborhood of SCN4B we then discovered a linkage between the expression of this gene and K+ channels activated by Ca2+, or K+ two-pore domain channels. Our approach also inferred several potential effector functions linked to variation in the expression of SCN4B. These observations support the hypothesis that SCN4B is a key factor in AED-resistant TLE, which could help direct both the drug selection of TLE treatments and the development of future AEDs.

Quantitative expression and localization of GABAB receptor protein subunits in hippocampi from patients with refractory temporal lobe epilepsy.

This study investigates GABAB protein expression and mRNA levels in three types of specimens. Two types of specimens from patients with temporal lobe epilepsy (TLE), secondary to hippocampal sclerosis, sclerotic hippocampal samples (TLE-HS), and tissue from the structurally preserved non-spiking ipsilateral superior temporal gyrus (TLE-STG) removed from the same patient during epilepsy surgery; and third specimen is hippocampal tissue from individuals with no history of epilepsy (post-mortem controls, PMC). mRNA expression of GABAB subunits was quantified in TLE-HS, TLE-STG and PMC specimens by qRT-PCR. Qualitative and quantitative Western blot (WB) and immunohistochemistry techniques were employed to quantify and localize GABAB proteins subunits. qRT-PCR data demonstrated an overall decrease of both GABAB1 isoforms in TLE-HS compared to TLE-STG. These results were mirrored by the WB findings. GABAB2 mRNA and protein were significantly reduced in TLE-HS samples compared to TLE-STG; however they appeared to be upregulated in TLE-HS compared to the PMC samples. Immunohistochemistry (IHC) showed that GABAB proteins were widely distributed in PMC and TLE-HS hippocampal sections with regional differences in the intensity of the signal. The higher expression of mature GABAB protein in TLE-HS than PMC is in agreement with previous studies. However, these findings could be due to post-mortem changes in PMC specimens. The TLE-STG samples examined here represent a better 'control' tissue compared to TLE-HS samples characterised by lower than expected GABAB expression. This interpretation provides a better explanation for previous functional studies suggesting reduced inhibition in TLE-HS tissue due to attenuated GABAB currents. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

Transcriptome analysis suggests a role for the differential expression of cerebral aquaporins and the MAPK signalling pathway in human temporal lobe epilepsy.

Epilepsies are common disorders of the central nervous system (CNS), affecting up to 2% of the global population. Pharmaco-resistance is a major clinical challenge affecting about 30% of temporal lobe epilepsy (TLE) patients. Water homeostasis has been shown crucial for regulation of neuronal excitability. The control of water movement is achieved through a family of small integral membrane channel proteins called aquaporins (AQPs). Despite the fact that changes in water homeostasis occur in sclerotic hippocampi of people with TLE, the expression of AQPs in the epileptic brain is not fully characterised. This study uses microarray and ELISA methods to analyse the mRNA and protein expression of the human cerebral AQPs in sclerotic hippocampi (TLE-HS) and adjacent neocortex tissue (TLE-NC) of TLE patients. The expression of AQP1 and AQP4 transcripts was significantly increased, while that of the AQP9 transcript was significantly reduced in TLE-HS compared to TLE-NC. AQP4 protein expression was also increased while expression of AQP1 protein remained unchanged, and AQP9 was undetected. Microarray data analysis identified 3333 differentially regulated genes and suggested the involvement of the MAPK signalling pathway in TLE pathogenesis. Proteome array data validated the translational profile for 26 genes and within the MAPK pathway (e.g. p38, JNK) that were identified as differentially expressed from microarray analysis. ELISA data showed that p38 and JNK inhibitors decrease AQP4 protein levels in cultured human primary cortical astrocytes. Elucidating the mechanism of selective regulation of different AQPs and associated regulatory proteins may provide a new therapeutic approach to epilepsy treatment.

Detection of mRNA encoding receptors by in situ and Northern hybridization.

The ability to measure mRNA encoding protein is attractive, since, provided certain criteria are met, the investigator can be confident of highly selective detection. In addition, changes in mRNA expression as well as their precise cellular localization, provides the scientist with important information that may not be evident by the detection of the translated protein. Hence, the combined approach of assessing mRNA and protein expression will often allow a more precise hypothesis to be formulated. In the present chapter, we describe methods we have optimized for the detection of mRNA following extraction from tissue or cells (Northern hybridization) and the detection of specific mRNA transcripts within their synthesising cells (in situ hybridization).

Matrix-assisted laser desorption/ionization-ion mobility separation-mass spectrometry imaging of vinblastine in whole body tissue sections.

During early-stage drug development, drug and metabolite distribution studies are carried out in animal tissues using a range of techniques, particularly whole body autoradiography (WBA). While widely employed, WBA has a number of limitations, including the following: expensive synthesis of radiolabeled drugs and analyte specificity and identification. WBA only images the radiolabel. MALDI MSI has been shown previously to be advantageous for imaging the distribution of a range of drugs and metabolites in whole body sections. Ion mobility separation (IMS) adds a further separation step to imaging experiments; demonstrated here is MALDI-IMS-MS whole body imaging of rats dosed at 6 mg/kg i.v. with an anticancer drug, vinblastine and shown is the distribution of the precursor ion m/z 811.4 and several product ions including m/z 793, 751, 733, 719, 691, 649, 524, and 355. The distribution of vinblastine within the ventricles of the brain is also depicted. Clearly demonstrated in these data are the removal of interfering isobaric ions within the images of m/z 811.4 and also of the transition m/z 811-751, resulting in a higher confidence in the imaging data. Within this work, IMS has shown to be advantageous in both MS and MS/MS imaging experiments by separating vinblastine from an endogenous isobaric lipid.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging of lipids in rat brain tissue with integrated unsupervised and supervised multivariant statistical analysis.

To date matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) analysis has been largely concerned with mapping the distribution of known analytes in tissues. An important step in the progression of its applications is the determination of unknown variants for metabolite and protein profiling in both clinical studies and studies of disease. Principal component analysis (PCA) is a statistical approach which can be used as a means of determining latent variables in multivariate data sets. In the work reported here, PCA, in both unsupervised and supervised modes, has been used to differentiate brain regions based on their lipid composition determined by MALDI-MSI. PCA has been shown to be useful in the determination of hidden variables between spectra taken from six regions of brain tissue. It is possible to identify ions of interest from the loadings plot which are likely to be more prominent in the different regions of the brain and thus differentiating between white and grey matter. It is also possible to distinguish between the grey Cerebellar Cortex and the Hippocampal formation, due to the grey Cerebellar Cortex having a positive PC2 and the Hippocampal formation having a negative PC2 score; this is only possible in supervised PCA with this data set because with unsupervised PCA the two regions overlap.

Plasticity of GABA(B) receptor-mediated heterosynaptic interactions at mossy fibers after status epilepticus.

Several neurotransmitters, including GABA acting at presynaptic GABA(B) receptors, modulate glutamate release at synapses between hippocampal mossy fibers and CA3 pyramidal neurons. This phenomenon gates excitation of the hippocampus and may therefore prevent limbic seizure propagation. Here we report that status epilepticus, triggered by either perforant path stimulation or pilocarpine administration, was followed 24 hr later by a loss of GABA(B) receptor-mediated heterosynaptic depression among populations of mossy fibers. This was accompanied by a decrease in the sensitivity of mossy fiber transmission to the exogenous GABA(B) receptor agonist baclofen. Autoradiography revealed a reduction in GABA(B) receptor binding in the stratum lucidum after status epilepticus. Failure of GABA(B) receptor-mediated modulation of mossy fiber transmission at mossy fibers may contribute to the development of spontaneous seizures after status epilepticus.

Modification of GABA(B1) and GABA(B2) receptor subunits in the somatosensory cerebral cortex and thalamus of rats with absence seizures (GAERS).

In the present study, we have investigated GABA(B) receptor expression in somatosensory cortex (S1) and the ventrobasal (VB) and reticular (Rt) thalamic nuclei of Genetic Absence Epilepsy Rats from Strasbourg (GAERS), which represent an animal model for the human absence epilepsy. We focused our attention on the thalamocortical network because it has been demonstrated that absence seizures are generated in this specific circuit, which is under the control of several inhibitory, e.g. GABA, and excitatory systems. Autoradiography data obtained with the GABA(B) receptor antagonist [3H]CGP62349 did not show any differences in Kd or Bmax values between control rats and GAERS. In situ hybridisation (ISH) results showed a significant increase in messenger RNA for GABA(B1) in the S1 and a decrease in the VB thalamic nucleus but not in the Rt thalamic nucleus. By contrast the immunocytochemical data revealed an increased expression of both GABA(B1) and GABA(B2) receptor subunits in all the regions examined, somatosensory cerebral cortex, VB thalamus and Rt nucleus in GAERS compared to controls. The main finding was an up-regulation of GABA(B) receptor protein in the corticothalamic circuit in GAERS compared to controls.