Glycinergic tonic inhibition of hippocampal neurons with depolarizing GABAergic transmission elicits histopathological signs of temporal lobe epilepsy

Abstract

An increasing number of epilepsy patients are afflicted with drug-resistant temporal lobe epilepsy (TLE) and require alternative therapeutic approaches. High-affinity glycine receptors (haGlyRs) are functionally adapted to tonic inhibition due to their response to hippocampal ambient glycine, and their synthesis is activity-dependent. Therefore, in our study, we scanned TLE hippocampectomies for expression of haGlyRs and characterized the effects mediated by these receptors using primary hippocampal neurons. Increased haGlyR expression occurred in TLE hippocampi obtained from patients with a severe course of disease. Furthermore, in TLE patients, haGlyR and potassium chloride cotransporter 2 (KCC2) expressions were inversely regulated. To examine this potential causal relationship with respect to TLE histopathology, we established a hippocampal cell culture system utilising tonic inhibition mediated by haGlyRs in response to hippocam-pal ambient glycine and in the context of a high Cl equilibrium potential, as is the case in TLE hippocampal neurons. We showed that hypoactive neurons increase their ratio between glutamatergic and GABAergic synapses, reduce their dendrite length and finally undergo excitotoxicity. Pharmacological dissection of the underlying processes revealed ionotropic glutamate and TrkB receptors as critical mediators between neuronal hypoactivity and the emergence of these TLE-characteristic histopathological signs. Moreover, our results indicate a beneficial role for KCC2, because decreasing the Cl− equilibrium potential by KCC2 expression also rescued hypoactive hippocampal neurons. Thus, our data support a causal relationship between increased haGlyR expression and the emergence of histopathological TLE-characteristic signs, and they establish a pathophysiological role for neuronal hypoactivity in the context of a high Cl− equilibrium potential.

Changes in neural network homeostasis trigger neuropsychiatric symptoms

“…A cDNA minigene coding for a floxed allele of the disease-relevant RNA variant GlyR α3L 185L (24)(25)(26) was recombined with the Hprt gene locus on the X chromosome and allowed neuron type-specific targeted (ectopic) gain-of-function receptor expression in hemizygous males, i.e., in addition to Glra3 gene promoter-dependent GlyR α3 expression. Ectopic GlyR α3L 185L expression has several advantages: (a) It more closely matches the situation in patients with epilepsy, because RNA-edited and nonedited GlyRs coexist in neurons due to the enzymatic nature of RNA editing; (b) replacement of the Glra3 gene with the GlyR HA-3L 185L knockin cassette would knock out Glra3 gene function, yield a fraction of total RNA-edited Glra3 mRNA, and considerably overrepresent GlyR α3L 185L function; (c) Glra3 gene replacement with our cDNA copy of GlyR α3L 185L would reflect Glra3 gene promoter regulation and preclude analysis of the spatiotemporal (neuron type-specific) effects of RNA-edited GlyR α3L, which depends on the regulation of neuronal C-to-U RNA-editing enzymes (47,48).…”

“…Nonetheless, impaired neural network homeostasis is associated with a plethora of clinical symptoms of neuropsychiatric disorders (1,(19)(20)(21) including cognitive dysfunction and various symptoms of mood disorders in patients with epilepsy (22,23), which raises the question of identifying the responsible molecular and cellular mechanisms that are able to subvert homeostatic control of synaptic transmission and neural network excitability in disease conditions. Glycine receptor (GlyR) α3 is a pathogenic molecular candidate, because changes in RNA processing of this subunit are associated with the pathophysiology of epilepsy (24)(25)(26). In fact, expression of RNA-edited GlyR α3 is increased in patients with temporal lobe epilepsy and leads to P185L amino acid substitution and neurotransmitter receptor gain of function.…”

Intracellular glycine receptor function facilitates glioma formation in vivo

“…cDNA was obtained by reverse transcription of 2 mg RNA with an equimolar mixture of 39-anchored poly(T) oligonucleotides (T 18 V, T 15 V, T 13 V) and Superscript II (Invitrogen), according to the manufacturer's protocol. For PCR amplification of cDNA obtained from human glioma cells, oligonucleotides binding to specific sequences of GlyR a1 (59-CAGTTTGCCTGCTCTTTGTGT-39 and 59-GTTGAAAATGAGGA-AGGCCATG-39), a2 (59-ATCAACAGTTTTGGATCAGTCA-39 for the a2A splice variant or 59-TCAACAGCTTTGGGTCAATAG-39 for a2B and 59-CCTTCAGCAACTTGCACTGG-39 for both) and a3 (59-GGGTACACAATGAATGATCTC-39 and 59-AGAGACTTAATCTTG-CTGCTGATG-39) were designed as described previously (Eichler et al, 2008;Eichler et al, 2009;Meier et al, 2002) and used in combination with GAPDH-specific primers (59-ATGGCACCGTCAAGGCTGAG-39 and 59-CGACGCCTGCTTCACCACC-39) (Eichler et al, 2009). The ratio between GlyR a-and GAPDH-specific oligonucleotides was adjusted to 15:1 (a1 and a2) and 20:1 (a3).…”

Glycine receptor α3 and α2 subunits mediate tonic and exogenous agonist-induced currents in forebrain

“…For example, polymorphisms resulting in single amino acid mutations in both GlyR α and β subunits have been identified in human hyperekplexia (startle disease) (33). In addition, differential splicing of the human GlyR α3 subunit gene (Glra3) and changes in the expression of high-affinity (RNA-edited) GlyR α2 and α3 subunits have been observed in cases of intractable temporal lobe epilepsy (32,34). More recently, the Glra3 gene has been genetically linked to alcoholism (35), and Glra3 −/− mice have been found to show increased ethanol preference in a two-bottle choice model, while Glra2 −/Y animals display the opposite behavior (36).…”