AMPA and kainate receptors mediate mutually exclusive effects on GABA(A) receptor expression in cultured mouse cerebellar granule neurones.

Studies on animal models of epilepsy and cerebellar ataxia, e.g., stargazer mice (stg) have identified changes in the GABAergic properties of neurones associated with the affected brain loci. Whether these changes contribute to or constitute homeostatic adaptations to a state of altered neuronal excitability is as yet unknown. Using cultured cerebellar granule neurones from control [+/+; alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor (AMPAR)-competent, Kainate receptor (KAR)-competent] and stg (AMPAR-incompetent, KAR-competent), we investigated whether non-NMDA receptor (NMDAR) activity regulates GABA(A) receptor (GABAR) expression. Neurones were maintained in 5 mmol/L KCl-containing basal media or depolarizing media containing either 25 mmol/L KCl or the non-NMDAR agonist kainic acid (KA) (100 micromol/L). KCl- and KA-mediated depolarization down-regulated GABAR alpha1, alpha6 and beta2, but up-regulated alpha4, beta3 and delta subunits in +/+ neurones. The KCl-evoked but not KA-evoked effects were reciprocated in stg neurones compatible with AMPAR-regulation of GABAR expression. Conversely, GABAR gamma2 expression was insensitive to KCl-mediated depolarization, but was down-regulated by KA-treatment in a 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-reversible manner in +/+ and stg neurones compatible with a KAR-mediated response. KA-mediated up-regulation of GABAR alpha4, beta3 and delta was inhibited by L-type voltage-gated calcium channel (L-VGCC) blockers and the Ca2+/calmodulin-dependent protein kinase inhibitor, 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinoline sulfonic acid ester (KN-62). Up-regulation of GABAR alpha4 and beta3 was also prevented by calcineurin (CaN) inhibitors, FK506 and cyclosporin A. Down-regulation of GABAR alpha1, alpha6 and beta2 was independent of L-VGCC activity, but was prevented by inhibitors of CaN. Thus, we provide evidence that a KAR-mediated and at least three mutually exclusive AMPAR-mediated signalling mechanisms regulate neuronal GABAR expression.

Aberrant GABA(A) receptor expression in the dentate gyrus of the epileptic mutant mouse stargazer.

Stargazer (stg) mutant mice fail to express stargazin [transmembrane AMPA receptor regulatory protein gamma2 (TARPgamma2)] and consequently experience absence seizure-like thalamocortical spike-wave discharges that pervade the hippocampal formation via the dentate gyrus (DG). As in other seizure models, the dentate granule cells of stg develop elaborate reentrant axon collaterals and transiently overexpress brain-derived neurotrophic factor. We investigated whether GABAergic parameters were affected by the stg mutation in this brain region. GABA(A) receptor (GABAR) alpha4 and beta3 subunits were consistently upregulated, GABAR delta expression appeared to be variably reduced, whereas GABAR alpha1, beta2, and gamma2 subunits and the GABAR synaptic anchoring protein gephyrin were essentially unaffected. We established that the alpha4 betagamma2 subunit-containing, flunitrazepam-insensitive subtype of GABARs, not normally a significant GABAR in DG neurons, was strongly upregulated in stg DG, apparently arising at the expense of extrasynaptic alpha4 betadelta-containing receptors. This change was associated with a reduction in neurosteroid-sensitive GABAR-mediated tonic current. This switch in GABAR subtypes was not reciprocated in the tottering mouse model of absence epilepsy implicating a unique, intrinsic adaptation of GABAergic networks in stg. Contrary to previous reports that suggested that TARPgamma2 is expressed in the dentate, we find that TARPgamma2 was neither detected in stg nor control DG. We report that TARPgamma8 is the principal TARP isoform found in the DG and that its expression is compromised by the stargazer mutation. These effects on GABAergic parameters and TARPgamma8 expression are likely to arise as a consequence of failed expression of TARPgamma2 elsewhere in the brain, resulting in hyperexcitable inputs to the dentate.

RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs.

The role of transmembrane AMPA receptor regulatory proteins (TARPs) in neurotransmission and receptor trafficking (Review).

AMPA receptors (AMPAR) mediate the majority of fast excitatory neurotransmission in the central nervous system (CNS). Transmembrane AMPAR regulatory proteins (TARPs) have been identified as a novel family of proteins which act as auxiliary subunits of AMPARs to modulate AMPAR trafficking and function. The trafficking of AMPARs to regulate the number of receptors at the synapse plays a key role in various forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). Expression of the prototypical TARP, stargazin/TARPgamma2, is ablated in the stargazer mutant mouse, an animal model of absence epilepsy and cerebellar ataxia. Studies on the stargazer mutant mouse have revealed that failure to express TARPgamma2 has widespread effects on the balance of expression of both excitatory (AMPAR) and inhibitory receptors (GABA(A) receptors, GABAR). The understanding of TARP function has implications for the future development of AMPAR potentiators, which have been shown to have therapeutic potential in both psychological and neurological disorders such as schizophrenia, depression and Parkinson's disease.

GABAA alpha6-containing receptors are selectively compromised in cerebellar granule cells of the ataxic mouse, stargazer.

Stargazer mice fail to express the gamma2 isoform of transmembrane alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 +/- 6% fewer inhibitory gamma-aminobutyric acid, type A (GABA(A)), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABA(A) receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (alpha6betaxdelta) alpha6-containing GABA(A) receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABA(A) receptor alpha6, delta (mature cerebellar granule cell-specific proteins), and beta3 subunit expression in stargazer. The subunit composition of the residual alpha6-containing GABA(A) receptors was unaffected by the stargazer mutation. However, we did find evidence of an approximately 4-fold up-regulation of alpha1betadelta receptors that may compensate for the loss of alpha6-containing GABA(A) receptors. PCR analysis identified a dramatic reduction in the steady-state level of alpha6 mRNA, compatible with alpha6 being the primary target of the stargazer mutation-mediated GABA(A) receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic alpha6-containing GABA(A) receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling.

Microtubule-associated protein light chain 2 is a stargazin-AMPA receptor complex-interacting protein in vivo.

The ataxic mutant mouse stargazer is a null mutant for stargazin, a protein involved in the regulation of cell surface trafficking and synaptic targeting of AMPA receptors. The extreme C terminus of stargazin (sequence, -TTPV), confers high affinity for PDZ domain-containing proteins e.g. PSD-95. Interaction with PDZ proteins enables stargazin to fulfill its role as an AMPA receptor synaptic targeting molecule but is not essential for its ability to influence AMPA receptor trafficking to the neuronal cell surface. Using the yeast-two hybrid approach we screened for proteins that interact with the intracellular C-terminal tail of stargazin. Positive interactors included PDZ domain-containing proteins e.g. SAP97, SAP102, and PIST. Interestingly, light chain 2 of microtubule-associated protein 1 (LC2), which does not contain a PDZ domain, was also a strong interactor. This was shown to be a direct interaction that occurred upstream of the -TTPV sequence of stargazin. Immunoprecipitations of Triton X-100 soluble cerebellar extracts revealed that LC2 is pulled down not only by anti-stargazin antibodies but also anti-GluR2 antibodies suggesting that stargazin and AMPA receptor subunits associate with LC2. Immunopurified full-length, native stargazin was shown to co-associate not only with GluR2 in vivo but also with full-length, native LC2. Indeed, LC2 co-associates with stargazin when part of a tripartite complex comprising LC2-stargazin-GluR2. Since this complex was extracted using Triton X-100 and was devoid of PSD95, SAP97, and actin we postulate that LC2 is involved in trafficking of AMPA receptors in cerebellar neurons before they are anchored at the synapse.