Different ataxin-3 amyloid aggregates induce intracellular Ca2+ deregulation by different mechanisms in cerebellar granule cells

AbstractThis work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca2+ levels and the abnormal Ca2+ signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca2+ responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca2+ response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells

Copper-Triggered Aggregation of Ubiquitin

Neurodegenerative disorders share common features comprising aggregation of misfolded proteins, failure of the ubiquitin-proteasome system, and increased levels of metal ions in the brain. Protein aggregates within affected cells often contain ubiquitin, however no report has focused on the aggregation propensity of this protein. Recently it was shown that copper, differently from zinc, nickel, aluminum, or cadmium, compromises ubiquitin stability and binds to the N-terminus with 0.1 micromolar affinity. This paper addresses the role of copper upon ubiquitin aggregation. In water, incubation with Cu(II) leads to formation of spherical particles that can progress from dimers to larger conglomerates. These spherical oligomers are SDS-resistant and are destroyed upon Cu(II) chelation or reduction to Cu(I). In water/trifluoroethanol (80∶20, v/v), a mimic of the local decrease in dielectric constant experienced in proximity to a membrane surface, ubiquitin incubation with Cu(II) causes time-dependent changes in circular dichroism and Fourier-transform infrared spectra, indicative of increasing β-sheet content. Analysis by atomic force and transmission electron microscopy reveals, in the given order, formation of spherical particles consistent with the size of early oligomers detected by gel electrophoresis, clustering of these particles in straight and curved chains, formation of ring structures, growth of trigonal branches from the rings, coalescence of the trigonal branched structures in a network. Notably, none of these ubiquitin aggregates was positive to tests for amyloid and Cu(II) chelation or reduction produced aggregate disassembly. The early formed Cu(II)-stabilized spherical oligomers, when reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and in POPC planar bilayers, form annular and pore-like structures, respectively, which are common to several neurodegenerative disorders including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis, and prion diseases, and have been proposed to be the primary toxic species. Susceptibility to aggregation of ubiquitin, as it emerges from the present study, may represent a potential risk factor for disease onset or progression while cells attempt to tag and process toxic substrates

Cogan syndrome in children: early diagnosis and treatment is critical to prognosis

Am J Ophthalmol. 2004 Apr;137(4):757-8. Cogan syndrome in children: early diagnosis and treatment is critical to prognosis. Orsoni JG, Zavota L, Vincenti V, Pellistri I, Rama P. SourceOphthalmology Department, University of Parma, Parma, Italy. [email protected] Abstract PURPOSE: To present two cases of pediatric Cogan Syndrome and to highlight the differences between the adult and pediatric forms of the disease, as well as the importance of early diagnosis and treatment. DESIGN: Interventional case report. METHODS: Institutional setting. RESULTS: Corneal lesions were much more diffuse than those observed in adult Cogan syndrome. Immunosuppressive drug combination therapy successfully resolved systemic and ocular inflammation, but the involvement of the pupillary area caused permanent low vision in one case and amblyopia in the other. CONCLUSION: When chronic ocular inflammation is observed in association with sensory neural hearing loss and any systemic signs of autoimmune inflammation, a diagnosis of Cogan syndrome should be suspected. If immunosuppressive treatment is not initiated as soon as possible, permanent low vision and deafness can result

GabaA Receptors of Cerebellar Granule Cells in Culture: Explanation of Overall Insensitivity to Ethanol.

GABA-activated chloride currents were studied in cerebellar granule cells put in culture from neonatal rats. As previously described, 10 \ub5M GABA perfusion of these cells recorded by whole cell patch-clamp elicits chloride currents displaying a peak and a steady-state component. The two components were studied in the presence of 1 mM furosemide,1 \ub5M Zn2+ and a combination of the two in order to evaluate the contribution of the different types of GABAA receptors. Furosemide inhibits \u3b16 containing receptors whereas low levels of Zn2+ specifically block incomplete GABAA receptors made up of \u3b1 and \u3b2 subunits only. The results show that the peak component involves the following receptors: \u3b1x \u3b2y, 25%; \u3b11 \u3b2y \u3b32, 45%; \u3b16 \u3b2y \u3b32 plus \u3b11 \u3b16 \u3b2y \u3b32, 30%. The steady state component is made up by \u3b1x \u3b2y, 38%; \u3b11 \u3b2y \u3b4, 62%. Ethanol at relatively high concentration, 100 mM, slows further down the desensitization of \u3b11 \u3b2y \u3b4 receptors. The results indicate that the relative insensitivity to ethanol of GABAA receptors of neonatal cerebellar granule cells in culture is due to the absence of mature \u3b16 \u3b2y \u3b4 receptors, a major receptor brand involved in tonic inhibition

Only High Concentrations Of Ethanol Affect Gabaa Receptors Of Rat Cerebellum Granule Cells In Culture

In the experiments described in the present report, we evaluated the effects of ethanol on the activity of GABA(A) receptors of cerebellar granule cells in culture. Only very high ethanol concentrations (100-300 mM) showed a clear and significant stimulatory effect on the activity of such receptors. This result was unexpected. In fact, previous reports from other groups would have suggested high ethanol sensitivity of at least one population of GABA(A) receptors expressed by granule cells

Gabor\u2019s hologram in a modern perspective

We review Dennis Gabor\u2019s early results in light of more than fifty years of technological achievements, including the advent of CCD cameras and fast computers. By applying digital reading to one of the first holograms, we demonstrate the continuity between the classical technique and the digital implementation. This experiment can be used as a demonstration without needing the instrumentation of an optics laboratory