Differential expression of GABA A receptor (GABR) subunits has been demonstrated in hippocampus from patients and animals with temporal lobe epilepsy (TLE), but whether these changes are important for epileptogenesis remains unknown. Previous studies in the adult rat pilocarpine model of TLE found reduced expression of GABR ␣1 subunits and increased expression of ␣4 subunits in dentate gyrus (DG) of epileptic rats compared with controls. To investigate whether this altered subunit expression is a critical determinant of spontaneous seizure development, we used adeno-associated virus type 2 containing the ␣4 subunit gene (GABRA4) promoter to drive transgene expression in DG after status epilepticus (SE). This novel use of a condition-dependent promoter upregulated after SE successfully increased expression of GABR ␣1 subunit mRNA and protein in DG at 1-2 weeks after SE. Enhanced ␣1 expression in DG resulted in a threefold increase in mean seizure-free time after SE and a 60% decrease in the number of rats developing epilepsy (recurrent spontaneous seizures) in the first 4 weeks after SE. These findings provide the first direct evidence that altering GABR subunit expression can affect the development of epilepsy and suggest that ␣1 subunit levels are important determinants of inhibitory function in hippocampus.

Altered function of ␥-aminobutyric acid type A receptors (GABA A Rs) in dentate granule cells of the hippocampus has been associated with temporal lobe epilepsy (TLE) in humans and in animal models of TLE. Such altered receptor function (including increased inhibition by zinc and lack of modulation by benzodiazepines) is related, in part, to changes in the mRNA levels of certain GABA A R subunits, including ␣4, and may play a role in epileptogenesis. The majority of GABA A Rs that contain ␣4 subunits are extra-synaptic due to lack of the ␥2 subunit and presence of ␦. However, it has been hypothesized that seizure activity may result in expression of synaptic receptors with altered properties driven by an increased pool of ␣4 subunits. Results of our previous work suggests that signaling via protein kinase C (PKC) and early growth response factor 3 (Egr3) is the plasticity trigger for aberrant ␣4 subunit gene (GABRA4) expression after status epilepticus. We now report that brain derived neurotrophic factor (BDNF) is the endogenous signal that induces Egr3 expression via a PKC/MAPK-dependent pathway. Taken together with the fact that blockade of tyrosine kinase (Trk) neurotrophin receptors reduces basal GABRA4 promoter activity by 50%, our findings support a role for BDNF as the mediator of Egr3-induced GABRA4 regulation in developing neurons and epilepsy and, moreover, suggest that BDNF may alter inhibitory processing in the brain by regulating the balance between phasic and tonic inhibition.The type A ␥-aminobutyric acid (GABA) 5 receptor (GABA A R) is an integral ligand gated ion channel that mediates the majority of inhibition in the central nervous system. Being a hetero-oligomeric complex, it is composed of five membrane spanning subunits that are chosen from the products of 19 different genes (␣ 1-6 , ␤ 1-3 , ␥ 1-3 , ␦, ⑀, , 1-3 , and ). These genes are differentially transcribed during development and in various regions of the adult brain and spinal cord (1-5). Alteration in the function of GABA A Rs has been associated with a variety of diseases whose etiology leads to an imbalance between inhibition and excitation in specific populations of neurons (6 -8).For instance, changes in certain GABA A R subunit levels occur in dentate granule cells (DGCs) of both humans with temporal lobe epilepsy (TLE) and in animal models of TLE (6, 9). These molecular responses have been hypothesized to underlie persistent changes in GABA A R function associated with epileptogenesis. Most notably, individual DGCs display an elevation of ␣4 subunit mRNAs and a decrease in ␣1 (6). Receptors that contain ␣4 subunits have unique pharmacological properties that include heightened blockade of receptor function by zinc (11-13) and decreased benzodiazepine modulation (14). In addition, the majority of GABA A Rs that contain ␣4 subunits (co-assembled with a ␤ and ␦) are located extrasynaptically and mediate tonic GABA currents, while those containing ␣(1, 2, 3, or 5) without ␦ and with ␥2 are targeted to the synapse (1, 15). Although...

Fig. 1. Protein-protein interactions between gp32 and gp59 on fDNA. (A) The fluorescence from individual molecules of fDNA with the proteins bound in the order as indicated at the side of each row. The gp32 protein is labeled with A488 (gp32 D ) and the gp59 protein is labeled with A555 (gp59 A ). The filter sets are described in Experimental Methods: F1 is for A488 emission, F2 for FRET between A488 and A555, and F3 for A555 emission. (B) Ensemble FRET studies of Oregon-green-488-maleimide-labeled gp59 titrated into a solution of 400 nM CPM-labeled gp32 and 100 nM fDNA. The fluorescence spectra of 400 nM CPM-gp32 alone (black line), the endpoint of the titration at 1 M Oregon-green-488-maleimide-gp59 (dark gray line), and several intermediate spectra (light gray lines) are shown. (C) Analysis of the donor quenching and acceptor sensitization plotted against the gp59 concentration determines the stoichiometry among gp32, gp59, and fDNA to be 1:1:1 with a calculated binding constant of Ϸ40 nM.

Patients over the age of 80 years had lower speech perception scores than other adult CI recipients but did not have higher rates of dizziness or vertigo after surgery. A family history of hearing loss was associated with a trend toward better speech recognition, possibly representing a new prognostic variable. These findings provide important information that will aid clinicians in counseling older CI candidates.

Hypothesis We hypothesize that the severity of hearing loss (HL) associated with sporadic vestibular schwannomas (VS) is correlated with tumor secretion of proteins with ototoxic or otoprotective potential. Background Since the recognition that HL associated with VS is not solely due to compression of the auditory nerve, elucidating the mechanism by which VS cause HL has been an important task. We previously showed that VS stratified by hearing have differential gene expression. We now focus on identifying differentially expressed proteins in tumor secretions. Methods Fresh surgical specimens of VS were incubated in sterile PBS at 37°C to collect secretions. The specimens were divided into a group associated with good hearing (GH, word recognition ≥70% and pure-tone average ≤30 dB, n=11) or poor hearing (PH, n=10). The groups were compared using a customized cytokine array. Statistically significant results were verified with ELISA on a different set of secretions (n=8 for GH and n=10 for PH group). Results Of the 37 molecules we studied, 9 were significantly expressed in secretions from VS compared to secretions from control nerves. Secretion of fibroblast growth factor 2 (FGF2) was 3.5-fold higher in VS associated with GH versus PH based on cytokine array analysis (p=0.02), which was validated with ELISA. Conclusions This study highlights FGF2, a mitogen known to protect the auditory nerve, as a potential tumor-secreted mediator of hearing protection in VS. If FGF2's significant role in hearing protection in patients with VS is validated, then FGF2 could be utilized as a biomarker for HL in VS and therapeutic targeting of the FGF2 signaling pathway may reduce HL due to VS.

Regulation of gene expression via brain-derived neurotrophic factor (BDNF) is critical to the development of the nervous system and may well underlie cognitive performance throughout life. We now describe a mechanism by which BDNF can exert its effects on postsynaptic receptor populations that may have relevance to both the normal and diseased brain where BDNF levels either rise or fall in association with changes in excitatory neurotransmission. Increased levels of NMDA receptors (NMDARs) occur in rat cortical neurons via synthesis of new NMDA receptor 1 (NR1) subunits. The majority of synthesis is controlled by binding of cAMP response element binding protein (CREB) and early growth response factor 3 (Egr3) to the core NR1 promoter (NR1-p) region. BDNF-mediated NR1 transcription depends upon induction of the mitogen-activated protein kinase (MAPK) pathway through activation of the TrK-B receptor. Taken together with the fact that NMDAR activation stimulates BDNF synthesis, our results uncover a feed-forward gene regulatory network that may enhance excitatory neurotransmission to change neuronal behavior over time.

2b.

Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology.