Mild acidosis enhances AMPA receptor-mediated intracellular zinc mobilization in cortical neurons.

Overactivation of glutamate receptors and subsequent deregulation of the intraneuronal calcium ([Ca2+]i) levels are critical components of the injurious pathways initiated by cerebral ischemia. Another hallmark of stroke is parenchymal acidosis, and we have previously shown that mild acidosis can act as a switch to decrease NMDAR-dependent neuronal loss while potentiating the neuronal loss mediated by AMPARs. Potentiation of AMPAR-mediated neuronal death in an acidotic environment was originally associated only with [Ca2+]i dyshomeostasis, as assessed by Ca2+ imaging; however, intracellular dyshomeostasis of another divalent cation, Zn2+, has recently emerged as another important co-factor in ischemic neuronal injury. Rises in [Zn2+]i greatly contribute to the fluorescent changes of Ca2+-sensitive fluorescent probes, which also have great affinity for Zn2+. We therefore revisited our original findings (Mcdonald et al., 1998) and investigated if AMPAR-mediated fura-2 signals we observed could also be partially due to [Zn2+]i increases. Fura-2 loaded neuronal cultures were exposed to the AMPAR agonist, kainate, in a physiological buffer at pH 7.4 and then washed either at pH 7.4 or pH 6.2. A delayed recovery of fura-2 signals was observed at both pHs. Interestingly this impaired recovery phase was found to be sensitive to chelation of intracellular Zn2+. Experiments with the Zn2+ sensitive (and Ca2+-insensitive) fluorescent probe FluoZin-3 confirmed the idea that AMPAR activation increases [Zn2+]i, a phenomenon that is potentiated by mild acidosis. Additionally, our results show that selective Ca2+ imaging mandates the use of intracellular heavy metal chelators to avoid confounding effects of endogenous metals such as Zn2+.

The first 17 amino acids of Huntingtin modulate its sub-cellular localization, aggregation and effects on calcium homeostasis.

A truncated form of the Huntington's disease (HD) protein that contains the polyglutamine repeat, Httex1p, causes HD-like phenotypes in multiple model organisms. Molecular signatures of pathogenesis appear to involve distinct domains within this polypeptide. We studied the contribution of each domain, singly or in combination, to sub-cellular localization, aggregation and intracellular Ca2+ ([Ca2+]i) dynamics in cells. We demonstrate that sub-cellular localization is most strongly influenced by the first 17 amino acids, with this sequence critically controlling Httex1p mitochondrial localization and also promoting association with the endoplasmic reticulum (ER) and Golgi. This domain also enhances the formation of visible aggregates and together with the expanded polyQ repeat acutely disrupts [Ca2+]i levels in glutamate-challenged PC12 cells. Isolated cortical mitochondria incubated with Httex1p resulted in uncoupling and depolarization of these organelles, further supporting the idea that Httex1p-dependent mitochondrial dysfunction could be instrumental in promoting acute Ca2+ dyshomeostasis. Interestingly, neither mitochondrial nor ER associations seem to be required to promote long-term [Ca2+]i dyshomeostasis.

Acidosis enhances toxicity induced by kainate and zinc exposure in aged cultured astrocytes.

A key feature of cerebral ischemia, one of the leading causes of death associated with ageing, is excessive accumulation of glutamate in the synaptic cleft. In some forms of cerebral ischemia, like transient global ischemia, high levels or synaptic glutamate are complemented by a concomitant increase in extracellular Zn(2+) as result of the release of the cation that is present in the pre-synaptic vesicles of glutamatergic neurons. Interestingly, while neurons are very sensitive to the toxicity triggered by exposure to either glutamate or Zn(2+), astrocytes show less vulnerability to these toxins. We examined the vulnerability of cortical type 1 astrocytes to a combined exposure to the AMPA/kainate receptor agonist kainate and Zn(2+). Astrocytes exposed to 1 mM kainate for 1 h did not exhibit any degeneration in the following 24 h, and addition of 50 microM Zn(2+) to the kainate exposure failed to produce any further glial loss. Another hallmark of cerebral ischemia is parechymal acidosis and therefore, we tested the susceptibility of our cultured astrocytes to a kainate/Zn(2+) exposure performed under acidotic conditions. We found that the combination of 1 h exposure to 1 mM kainate + 50 microM Zn(2+) at pH 6.2 produced a strong increase in intracellular free Zn(2+) ([Zn(2+)](i)), and extensive glial injury. Comparing [Zn(2+)](i) rises triggered by kainate/Zn(2+) exposure at pH 7.4 or pH 6.2 we found that acidosis promotes increased toxic [Zn(2+)](i) levels as a result of a lethal combination of both enhanced Zn(2+) influx through Zn(2+) permeable AMPA/kainate channels and impaired intracellular buffering of the cation.

SUMO modification of Huntingtin and Huntington's disease pathology.

Huntington's disease (HD) is characterized by the accumulation of a pathogenic protein, Huntingtin (Htt), that contains an abnormal polyglutamine expansion. Here, we report that a pathogenic fragment of Htt (Httex1p) can be modified either by small ubiquitin-like modifier (SUMO)-1 or by ubiquitin on identical lysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces its ability to form aggregates, and promotes its capacity to repress transcription. In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neurodegeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration. Lysine mutations that prevent both SUMOylation and ubiquitination of Httex1p reduce HD pathology, indicating that the contribution of SUMOylation to HD pathology extends beyond preventing Htt ubiquitination and degradation.