A splice variant of the TATA-box binding protein encoding the polyglutamine-containing N-terminal domain that accumulates in Alzheimer's disease.

Previously we have reported the accumulation of an N-terminal fragment of the TATA-box binding protein (TBP) in Alzheimer's disease brain tissue and here we report the identification of a naturally occurring TBP splice variant as a likely mechanism for its production. The splice variant described here encodes the polyglutamine-containing N-terminal domain of this key transcription factor. We demonstrate the expression of the splice variant mRNA in a variety of human tissues and that the resulting protein forms inclusions in cell culture transfection studies. The unusual properties of the variant protein suggest that it may be functionally relevant in late onset neurodegenerative diseases.

P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea.

Type I and type II spiral ganglion neurons (SGN) innervate the inner and outer hair cells of the cochlea, respectively. This neural system is established by reorganization of promiscuous innervation of the hair cells, immediately before hearing is established. The mechanism for this synaptic reorganization is unresolved but probably includes regulation of trophic support between the hair cells and the neurons. We provide evidence that P2X receptors (ATP-gated ion channels) contribute such a mechanism in the neonatal rat cochlea. Single-cell quantitative RT-PCR identified the differential expression of two P2X receptor subunits, splice variant P2X(2)(-3) and P2X(3), in a 1:2 transcript ratio. Downregulation of this P2X(2-3/3) receptor coincided with maturation of the SGN innervation of the hair cells. When the P2X(2-3) and P2X(3) subunits were co-expressed in Xenopus oocytes, the resultant P2X receptor properties corresponded to the SGN phenotype. This included enhanced sensitivity to ATP and extended agonist action. In P4 spiral ganglion explants, activation of the P2X receptor signaling pathway by ATPgammaS or alpha,betaMeATP inhibited BDNF-induced neurite outgrowth and branching. These findings indicate that P2X receptor signaling provides a mechanism for inhibiting neurotrophin support of SGN neurites when synaptic reorganization is occurring in the cochlea.

TRPC-like conductance mediates restoration of intracellular Ca2+ in cochlear outer hair cells in the guinea pig and rat.

Ca2+ signalling is central to cochlear sensory hair cell physiology through its influence on sound transduction, membrane filter properties and neurotransmission. However, the mechanism for establishing Ca2+ homeostasis in these cells remains unresolved. Canonical transient receptor potential (TRPC) Ca2+ entry channels provide an important pathway for maintaining intracellular Ca2+ levels. TRPC3 subunit expression was detected in guinea pig and rat organ of Corti by RT-PCR, and localized to the sensory and neural poles of the inner and outer hair cells (OHCs) by confocal immunofluorescence imaging. A cation entry current with a TRPC-like phenotype was identified in guinea pig and rat OHCs by whole-cell voltage clamp. This slowly activating current was induced by the lowering of cytosolic Ca2+ levels ([Ca2+]i) following a period in nominally Ca2+-free solution. Activation was dependent upon the [Ca2+]o and was sustained until [Ca(2+)]i was restored. Ca2+ entry was confirmed by confocal fluorescence imaging, and rapidly recruited secondary charybdotoxin- and apamin-sensitive K(Ca) currents. Dual activation by the G protein-coupled receptor (GPCR)-phospholipase C-diacylglycerol (DAG) second messenger pathway was confirmed using the analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Ion substitution experiments showed that the putative TRPC Ca2+ entry current was selective for Na+ > K+ with a ratio of 1: 0.6. The Ca2+ entry current was inhibited by the TRPC channel blocker 2-aminoethyl diphenylborate (2APB) and the tyrosine kinase inhibitor, erbstatin analogue. We conclude that TRPC Ca2+ entry channels, most likely incorporating TRPC3 subunits, support cochlear hair cell Ca2+ homeostasis and GPCR signalling.

Developmental regulation of neuron-specific P2X3 receptor expression in the rat cochlea.

ATP-gated ion channels assembled from P2X3 receptor (P2X3R) subunits contribute to neurotransmission and neurotrophic signaling, associated with neurite development and synaptogenesis, particularly in peripheral sensory neurons. Here, P2X3R expression was characterized in the rat cochlea from embryonic day 16 (E16) to adult (P49-56), using RT-PCR and immunohistochemistry. P2X3R mRNA was strongly expressed in the cochlea prior to birth, declined to a minimal level at P14, and was absent in adult tissue. P2X3R protein expression was confined to spiral ganglion neurons (SGN) within Rosenthal's canal of the cochlea. At E16, immunolabeling was detected in the SGN neurites, but not the distal neurite projection within the developing sensory epithelium (greater epithelial ridge). From E18, the immunolabeling was observed in the peripheral neurites innervating the inner hair cells but was reduced by P6. However, from P2-8, immunolabeling of the SGN neurites extended to include the outer spiral bundle fiber tract beneath the outer hair cells. This labeling of type II SGN afferent fiber declined after P8. By P14, all synaptic terminal immunolabeling in the organ of Corti was absent, and SGN cell body labeling was minimal. In adult cochlear tissue, P2X3R immunolabeling was not detected. Noise exposure did not induce P2X3R expression in the adult cochlea. These data indicate that ATP-gated ion channels incorporating P2X3R subunit expression are specifically targeted to the afferent terminals just prior to the onset of hearing, and likely contribute to the neurotrophic signaling which establishes functional auditory neurotransmission.

Activation of purinergic receptor subtypes modulates odor sensitivity.

Purinergic nucleotides, including ATP and adenosine, are important neuromodulators of peripheral auditory and visual sensory systems (Thorne and Housley, 1996). ATP released by the olfactory epithelium (OE) after noxious stimuli provides a physiological source for a neuromodulatory substance independent of efferent innervation. Here we show that multiple subtypes of purinergic receptors are differentially expressed in olfactory receptor neurons and sustentacular support cells. Activation of purinergic receptors evoked inward currents and increases in intracellular calcium in cultured mouse olfactory receptor neurons. A mouse olfactory epithelial slice preparation and confocal imaging were used to measure changes in intracellular calcium in response to odors, purinergic receptor (P2R) agonists, or combined odor + P2R agonists. Pharmacological studies show that both P2Y and P2X receptor activation by exogenous and endogenous ATP significantly reduces odor responsiveness. Moreover, purinergic receptor antagonists increase the odor-evoked calcium transient, providing direct evidence that endogenous ATP modulates odor sensitivity via activation of multiple purinergic receptor subtypes in olfactory receptor neurons. Odor activation of G-protein-coupled receptors results in increased cAMP production, opening of cyclic nucleotide-gated channels, influx of Ca2+ and Na+, depolarization of the membrane, and activation of voltage- and Ca2+-gated ion channels. On-cell current-clamp recordings of olfactory receptor neurons from neonatal mouse slices revealed that ATP reduced cyclic nucleotide-induced electrical responses. These data also support the idea that ATP modulates odor sensitivity in mammalian olfactory neurons. Peripheral ATP-mediated odor suppression is a novel mechanism for reduced olfactory sensitivity during exposure to olfactotoxins and may be a novel neuroprotective mechanism.

Allosteric modulation of native cochlear P2X receptors: insights from comparison with recombinant P2X2 receptors.

Extracellular adenosine 5'-triphosphate (ATP)-gated ion channels assembled from P2X receptor subunits exhibit subunit-selective allosteric modulation by protons and divalent cations. In voltage-clamped guinea-pig cochlear outer hair cells (OHC) and Deiters' cells (DC), H(+) and Cu(2+), but not Zn(2+), enhanced the P2X receptor-mediated inward currents. Acid pH (6.5) potentiated OHC ATP-gated currents by 45%. Co-application of Cu(2+) (1-40 microM) with ATP increased the response by 20%. In DCs, ATP-gated currents were potentiated 85% by acid pH, and 70% by Cu(2+). Alkaline pH inhibited ATP-gated inward currents by 73% in OHCs and 85% in DCs. Zn(2+) was either ineffective (1-10 microM) or inhibitory (40-400 microM). Recombinant rat P2X(2) receptor-mediated inward currents in XENOPUS oocytes displayed allosteric modulation that was different from the native guinea-pig cochlear P2X receptors. The oocyte ATP-gated inward current was potentiated 450% by shifting from pH 7.5 to pH 6.5, and 130% with 40 microM Cu(2+). The enhanced response to ATP with acid pH and Cu(2+) is a signature of the P2X(2) subunit. In contrast to native guinea-pig cochlear cells, extracellular Zn(2+) (40 microM) increased the recombinant ATP-gated inward current by 200% in oocytes. These results suggest that the positive allosteric modulation of cochlear OHC and DC ATP-gated ion channels by protons and Cu(2+) arises in part from the P2X(2) receptor subunit, with additional regulatory elements.

Potential role of purinergic signalling in cochlear pathology.

Adenosine triphosphate (ATP) is a major intercellular signalling molecule that is involved in neurotransmission in the central and autonomic nervous systems, regulation of blood flow, and neuroendocrine function. It is also a key signalling molecule involved in normal cochlear homoeostasis, regulating hearing sensitivity, controlling vascular tone and acting as a candidate neurotransmitter at the hair cell afferent synapses. It has also been established that extracellular ATP mediates some pathological processes such as inflammation, apoptosis and cell proliferation. Evidence for a profound influence of extracellular ATP on normal cochlear function offers the tantalizing possibility that extracellular purine nucleotides may play a role in disease processes in the inner ear. This review draws on the current understanding of the pathophysiological role of extracellular ATP in tissues, and the evidence for the functional expression of purinergic signalling elements in the inner ear, to speculate on the potential role of purine nucleotides in cochlear pathology.