Different ataxin-3 amyloid aggregates induce intracellular Ca 2+ deregulation by different mechanisms in cerebellar granule cells

Abstract

This 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 Ca(2+) levels and the abnormal Ca(2+) 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 Ca(2+) responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca(2+) 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.

Misfolding of Amyloidogenic Proteins and Their Interactions with Membranes

“…Colocalization experiments indicated that the interaction involved glutamate receptors, voltage-gated channels, and ganglioside-rich membrane domains. In contrast, the interaction with a more aged pre-fibrillar aggregate of the same protein caused Ca 2+ permeation by a mechanism involving only ganglioside-rich areas [17]. This result underlines the importance of the different amyloid conformations in determining the distinct and non–overlapping mechanisms by which Ca 2+ influx and neurotoxicity occur, consistent with what was observed for Aβ oligomers [232].…”

Antisense Oligonucleotide-Mediated Removal of the Polyglutamine Repeat in Spinocerebellar Ataxia Type 3 Mice

“…An ataxin-3 protein variant corresponding exactly to the Δ C terminus protein resulting from exon 10 skipping has been tested in a yeast model and rat cerebellar granule cells, where it was shown that this protein indeed did not aggregate, but in some tests showed a mild increase in toxicity compared to the wild-type ataxin-3 protein 47, 48. Whether these results can be extrapolated to the in vivo situation will have to be determined, but we did not observe overt signs of toxicity in the AON 10.4-treated mice expressing ataxin-3 Δ C terminus based on bodyweight and locomotor activity (Figures S3A and S3B).…”

Biochemical and Electrophysiological Modification of Amyloid Transthyretin on Cardiomyocytes

“…The lack of effective pharmacological treatments for these conditions is due, at least in part, to the lack of information regarding the key factors underlying TTR misfolding and aggregation in tissue, as well as the molecular features of the biochemical, functional, and viability impairments of cardiac cells exposed to aggregated TTR. Presently, the cytotoxicity of amyloid aggregates, particularly those that arise early in the aggregation path, is considered to be one of the main factors responsible for cell functional and viability impairments (15,47), although cell sufferance can also result from the physical barrier to nutrient exchange posed by fibrillar deposits (51).…”

“…It is known that amyloid oligomers permeabilize the plasma membrane of exposed cells, favoring uncontrolled Ca 2þ entry from the extracellular milieu either nonspecifically after disassembly of the membrane bilayer (46,47) and/or through the participation of specific Ca 2þ channels (48,49). Therefore, we removed Ca 2þ from the external solution to check whether and to what extent the CM plasma membrane, in addition to the SR one, was involved in the intracellular Ca 2þ increase induced by TTR aggregates.…”