experiments in Fig. 5, the cultured microglia (see Supplementary Figure) that had been preincubated with or without ATP (50 mM) were injected intrathecally in normal rats (see Supplementary Methods for full details). ImmunohistochemistryTransverse L5 spinal cord sections (30 mm) were cut and processed for immunohistochemistry with anti-P2X4R antibody (Alomone). Identification of the type of P2X 4 R-positive cells was performed with the following markers: for microglia, OX42 (Chemicon) and iba1 (a gift from S. Kohsaka); for astrocytes, GFAP (Boehringer Mannheim); for spinal cord neurons, NeuN (Chemicon) and MAP2 (Chemicon). To assess immunofluorescence staining of cells quantitatively, we measured the immunofluorescence intensity of the P2X 4 R or OX42 as the average pixel intensity within each cell (see also Supplementary Methods). Western blottingWestern blot analysis of P2X 4 R expression in the membrane fraction from L4-L6 spinal cord was performed with anti-P2X4R polyclonal antibody (Oncogene) as described in detail in the Supplementary Methods. Microglial cultureRat primary cultured microglia were prepared in accordance with the method described previously 28 . In brief, mixed glial culture was prepared from neonatal Wistar rats and maintained for 10-16 days in DMEM medium with 10% fetal bovine serum. Immediately before experiments, microglia were collected by a gentle shake as the floating cells over the mixed glial culture. The microglia were transferred to coverslips or to Eppendorf tubes for subsequent intrathecal administration. StatisticsStatistical analyses of the results were made with Student's t-test, Student's paired t-test or the Mann-Whitney U-test. Lancet 353, 1959Lancet 353, -1964Lancet 353, (1999. 2. Woolf, C. J. & Salter, M. W. Neuronal plasticity: Increasing the gain in pain. Science 288, 1765Science 288, -1769Science 288, (2000. 3. Bo, X., Zhang, Y., Nassar, M., Burnstock, G. & Schoepfer, R. A P2X purinoceptor cDNA conferring a novel pharmacological profile. FEBS Lett. 375, 129-133 (1995). 4. Buell, G., Lewis, C., Collo, G., North, R. A. & Surprenant, A. An antagonist-insensitive P2X receptor expressed in epithelia and brain. EMBO J. 15, 55-62 (1996). 5. Seguela, P., Haghighi, A., Soghomonian, J. J. & Cooper, E. A novel neuronal P2X ATP receptor ion channel with widespread distribution in the brain. J. Neurosci. 16, 448-455 (1996). 6. Soto, F. et al. P2X4: an ATP-activated ionotropic receptor cloned from rat brain. Proc. Natl Acad. Sci. USA 93, 3684-3688 (1996). 7. Wang, C. Z., Namba, N., Gonoi, T., Inagaki, N. & Seino, S. Cloning and pharmacological characterization of a fourth P2X receptor subtype widely expressed in brain and peripheral tissues including various endocrine tissues. Biochem. Biophys. Res. Commun. 220, 196-202 (1996 Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues 1-3 , often with serious pathological consequences. Non-neuropathic systemic...

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The unfolding and refolding properties of human lysozyme and two amyloidogenic variants (Ile56Thr and Asp67His) have been studied by stopped-flow fluorescence and hydrogen exchange pulse labeling coupled with mass spectrometry. The unfolding of each protein in 5.4 M guanidine hydrochloride (GuHCl) is well described as a two-state process, but the rates of unfolding of the Ile56Thr variant and the Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wild type. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistent intermediates, revealed by multistep kinetics in fluorescence experiments and by the detection of well-defined populations in quenched-flow hydrogen exchange experiments. These findings are consistent with a predominant mechanism for refolding of human lysozyme in which one of the structural domains (the alpha-domain) is formed in two distinct steps and is followed by the folding of the other domain (the beta-domain) prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67His variant are closely similar to those of the wild-type protein, consistent with the location of this mutation in an outer loop of the beta-domain which gains native structure only toward the end of the refolding process. By contrast, the Ile56Thr mutation is located at the base of the beta-domain and is involved in the domain interface. The refolding of the alpha-domain is unaffected by this substitution, but the latter has the effect of dramatically slowing the folding of the beta-domain and the final assembly of the native structure. These studies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in the stability of the native fold relative to partially folded intermediates. The origin of this instability is different in the two variants, being caused in one case primarily by a reduction in the folding rate and in the other by an increase in the unfolding rate. In both cases this results in a low population of soluble partially folded species that can aggregate in a slow and controlled manner to form amyloid fibrils.