Phil Skolnick

Regulation of BDNF expression in primary neuron culture by LY392098, a novel AMPA receptor potentiator

The effects of a novel AMPA receptor potentiator (LY392098) on the expression of brain-derived neurotrophic factor (BDNF) were examined in primary neuron culture. The addition of either AMPA or LY392098 to cortical neurons elicited a time and concentration dependent increase in mRNA encoding BDNF. Moreover, co-addition of subeffective concentrations of AMPA (1 microM) and LY392098 (1 microM) resulted in dramatic increases in both BDNF mRNA (>25-fold) and protein ( approximately 7-fold) levels, whilst no changes in either NT-3 or NT-4 mRNA were detected. More modest ( approximately 1.5-2.5-fold) elevations in BDNF mRNA and protein expression were also produced by combinations of AMPA and LY392098 in cerebellar granule cell neurons. In contrast, AMPA and LY392098, either alone or in combination, did not elevate BDNF mRNA levels in primary astroglial cultures. Maximum elevations in BDNF mRNA and protein were produced by 6-12h of AMPA receptor activation 1-3h of AMPA receptor activation were required to elevate BDNF mRNA levels. AMPA receptor-mediated increases in BDNF mRNA and protein were abolished by the AMPA antagonist, NBQX, but were unaffected by the NMDA antagonist, MK-801. In cortical neuron cultures, activation of both L-type Ca(+2) channels and mitogen-activated protein (MAP) kinases contribute to AMPA receptor-mediated increases in BDNF mRNA. The ability of LY392098 to increase the expression of BDNF in primary neuron culture indicates this and related biarylpropylsulfonamides may be useful in the treatment of neuropsychiatric disorders.

Dizocilpine attenuates streptomycin-induced vestibulotoxicity in rats

NMDA receptor mediated excitotoxicity contributes substantially to aminoglycoside antibiotic-induced cochlear damage. Since vestibular as well as cochlear hair cells have glutamatergic synapses, aminoglycoside-induced vestibulotoxicity may also have an excitotoxic component. This hypothesis was tested by examining the effects of the uncompetitive NMDA receptor antagonist dizocilpine on streptomycin-induced vestibulotoxicity. Streptomycin-treated rats exhibited almost complete destruction of sensory hair cells in the crista ampullaris, vestibular impairment in the drop test, and hyperkinesia. Concurrent treatment with dizocilpine not only rescued a substantial population of sensory hair cells in the cristae, but prevented the attendant hyperkinesis and vestibular impairments. These results indicate that excitotoxic mechanisms contribute to aminoglycoside-induced vestibulotoxicity and that NMDA antagonists may be useful in attenuating aminoglycoside ototoxicity.

[3H]ATPA: a high affinity ligand for GluR5 kainate receptors

The pharmacological properties of [3H]ATPA ((RS)-2-amino-3(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid) are described. ATPA is a tert-butyl analogue of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) that has been shown to possess high affinity for the GluR5 subunit of kainate receptors. [3H]ATPA exhibits saturable, high affinity binding to membranes expressing human GluR5 (GluR5) kainate receptors (Kd approximately 13 nM). No specific binding was observed in membranes expressing GluR2 and GluR6 receptors. Several compounds known to interact with the GluR5 kainate receptor inhibited [3H]ATPA binding with potencies similar to those obtained for competition of [3H]kainate binding to GluR5. Saturable, high affinity [3H]ATPA binding (Kd approximately 4 nM) was also observed in DRG neuron (DRG) membranes isolated from neonatal rats. The rank order potency of compounds to inhibit [3H]ATPA binding in rat DRG and GluR5 membranes were in agreement. These finding demonstrate that [3H]ATPA can be used as a radioligand to examine the pharmacological properties of GluR5 containing kainate receptors.

The antibacterial and NMDA receptor activating properties of aminoglycosides are dissociable.

The use of aminoglycoside antibiotics is limited by side effects, the most critical of which are vestibular and cochlear toxicity. Recent evidence indicates that these effects result from an excitotoxic process mediated, at least in part, through a polyamine-like activation of NMDA receptors. This study investigated whether these positive modulatory effects of aminoglycosides at NMDA receptors are dissociable from their antibacterial properties. A group of structurally related apramycin derivatives was evaluated for the ability to enhance [3H]dizocilpine binding to rat brain membranes, and for the ability to augment agonist responses on recombinant (NR1A/2B) NMDA receptors expressed in Xenopus oocytes. Based on the antibacterial potencies of these derivatives against Staphylococcus aureus and Escherichia coli, it is concluded that there is no correlation between the ability of an aminoglycoside to produce a positive modulation of NMDA receptors and minimum inhibitory antibacterial concentrations. These findings indicate that it may be possible to develop an aminoglycoside antibiotic with reduced potential for ototoxicity.

AMPA receptor function is altered in GLUR2-deficient mice.

The GluR2 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor determines many of the biophysical properties of native AMPA receptors, including Ca++ permeability. Genetically engineered mice unable to edit the Q to R site of the GluR2 subunit die within 3 wk postpartum, presumably due to toxicity associated with enhanced Ca++ influx through AMPA receptors. In contrast, disruption of the gene encoding GluR2 is not necessarily lethal. The objective of this study was to explore potential mechanisms that permit survival of GluR2 (−/−) mice despite AMPA receptors that are highly Ca++ permeable. Whole-cell, patch-clamp recording of AMPA receptor responses in cortical pyramidal cells revealed that the kinetics of recovery from desensitization were significantly slower for receptors from GluR2 (−/−) mice compared to receptors from GluR2 (+/+) mice. The recovery time constants for AMPA receptors from GluR2 (−/−) and GluR2 (+/+) mice were 109.8±17 ms and 54.4±7.1 ms, respectively. The slower recovery kinetics would be expected to reduce Ca++ influx during repetitive stimulation. Because both RNA editing at the R/G site and alternative splicing of the flip and flp module affect AMPA receptor desensitization recovery rates, the possibility that these mechanisms were changed in GluR2 (−/−) mice was investigated. On a macroscopic level, neither editing nor splicing of the GluR-1, 3 or 4 subunits were changed in GluR2 (−/−) mice compared to GluR2 (+/+) mice. In summary, an increase in the time constant for recovery from desensitization may contribute to the ability of GluR2 (−/−) to survive.

A single residue contributes sensitivity to allosteric modulation of AMPA receptors by LY395153.

Previous studies have shown that a single point mutation (S(750)Q) in the splice variant region of rat Glu(1) subunits can eliminate positive allosteric modulation by cyclothiazide. The present study investigated the effects of mutating the equivalent residue (S(776)Q) in the human Glu(4) subunit on the activity and binding of a novel AMPA receptor potentiator, LY395153 (N-2-(4-benzamidophenylpropyl-2-propanesulfonamide)). The mutation markedly attenuated, but did not eliminate, potentiation by LY395153 and cyclothiazide. In addition, binding of [3H]LY395153 was significantly reduced by this mutation. These effects occurred in the absence of any change in the response to glutamate or the binding of a competitive AMPA receptor antagonist, [3H]Ro 48-8587 ([2,4,5-3H]9-imidazol-1-yl-8-nitro-2,3,5,6-tetrahydro[1,2,4]-triazolo[1,5-c]quinazoline-2,5-dione triethylammonium salt). Collectively, these results demonstrate that structurally diverse classes of potentiators are sensitive to mutations of this single Ser residue, suggesting that binding to this residue may be necessary for positive allosteric modulation of AMPA receptors.