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Adenophostin A is the most potent known agonist of inositol 1,4,5-trisphosphate (InsP(3)) receptors. Ca(2+) release from permeabilized hepatocytes was 9.9 +/- 1.6-fold more sensitive to adenophostin A (EC(50), 14.7 +/- 2.4 nM) than to InsP(3) (145 +/- 10 nM), consistent with the greater affinity of adenophostin A for hepatic InsP(3) receptors (K(d) = 0.48 +/- 0.06 and 3.09 +/- 0.33 nM, respectively). Here, we systematically modify the structures of the glucose, ribose, and adenine moieties of adenophostin A and use Ca(2+) release and binding assays to define their contributions to high-affinity binding. Progressive trimming of the adenine of adenophostin A reduced potency, but it fell below that of InsP(3) only after complete removal of the adenine. Even after substantial modifications of the adenine (to uracil or even unrelated aromatic rings, retaining the beta-orientation), the analogs were more potent than InsP(3). The only analog with an alpha-ribosyl linkage had massively decreased potency. The 2'-phosphate on the ribose ring of adenophostin A was essential and optimally active when present on a five-membered ring in a position stereochemically equivalent to its location in adenophostin A. Xylo-adenophostin, where xylose replaces the glucose ring of adenophostin A, was only slightly less potent than adenophostin A, whereas manno-adenophostin (mannose replacing glucose) had similar potency to InsP(3). These results are consistent with the relatively minor role of the 3-hydroxyl of InsP(3) (the equivalent is absent from xylo-adenophostin) and greater role of the equatorial 6-hydroxyl (the equivalent is axial in manno-adenophostin). This is the first comprehensive analysis of all the key structural elements of adenophostin A, and it provides a working model for the design of related high-affinity ligands of InsP(3) receptors.

Intracellular tau aggregates are the neuropathological hallmark of several neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and cases of frontotemporal dementia, but the link between these aggregates and neurodegeneration remains unclear. Neuronal models recapitulating the main features of tau pathology are necessary to investigate the molecular mechanisms of tau malfunction, but current models show little and inconsistent spontaneous tau aggregation. We show that dorsal root ganglion (DRG) neurons in transgenic mice expressing human P301S tau (P301S-htau) develop tau pathology similar to that found in brain and spinal cord and a significant reduction in mechanosensation occurs before detectable fibrillar tau formation. DRG neuronal cultures established from adult P301S-htau mice at different ages retained the pattern of aberrant tau found in vivo. Moreover, htau became progressively hyperphosphorylated over 2 months in vitro beginning with nonsymptomatic neurons, while hyperphosphorylated P301S-htau-positive neurons from 5-month-old mice cultured for 2 months died preferentially. P301S-htau-positive neurons grew aberrant axons, including spheroids, typically found in human tauopathies. Neurons cultured at advanced stages of tau pathology showed a 60% decrease in the fraction of moving mitochondria. SEG28019, a novel O-GlcNAcase inhibitor, reduced steady-state pSer396/ pSer404 phosphorylation over 7 weeks in a significant proportion of DRG neurons showing for the first time the possible beneficial effect of prolonged dosing of O-GlcNAcase inhibitor in vitro. Our system is unique in that fibrillar tau forms without external manipulation and provides an important new tool for understanding the mechanisms of tau dysfunction and for screening of compounds for treatment of tauopathies.

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