No abstract

Amyloid-β (Aβ) neurotoxicity is believed to contribute to the pathogenesis of Alzheimer's disease (AD). Previously we found that E2-25K/Hip-2, an E2 ubiquitin-conjugating enzyme, mediates Aβ neurotoxicity. Here, we report that E2-25K/Hip-2 modulates caspase-12 activity via the ubiquitin/proteasome system. Levels of endoplasmic reticulum (ER)–resident caspase-12 are strongly up-regulated in the brains of AD model mice, where the enzyme colocalizes with E2-25K/Hip-2. Aβ increases expression of E2-25K/Hip-2, which then stabilizes caspase-12 protein by inhibiting proteasome activity. This increase in E2-25K/Hip-2 also induces proteolytic activation of caspase-12 through its ability to induce calpainlike activity. Knockdown of E2-25K/Hip-2 expression suppresses neuronal cell death triggered by ER stress, and thus caspase-12 is required for the E2-25K/Hip-2–mediated cell death. Finally, we find that E2-25K/Hip-2–deficient cortical neurons are resistant to Aβ toxicity and to the induction of ER stress and caspase-12 expression by Aβ. E2-25K/Hip-2 is thus an essential upstream regulator of the expression and activation of caspase-12 in ER stress–mediated Aβ neurotoxicity.

Abstract. The precise causative factors in neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's disease remain elusive, but mechanisms implicated comprise excitotoxicity, mitochondrial dysfunction, and in the case of AD, the amyloid beta peptide (Aβ). Current therapeutic strategies for such disorders are very limited; thus, traditional herbal medicines currently receive increased attention. The seeds of Cassia obtisufolia have long been used in traditional eastern medicine and more recently the ethanolic fraction of the seeds (COE) has been shown to attenuate memory impairments in mice. In this study, we set out to determine the effect of COE (range: 0.1 -10 μg / ml) on calcium dysregulation and cell death models in mouse primary hippocampal cultures implicated in general neurodegenerative processes and in the pathogenesis of AD: excitotoxicity, mitochondrial dysfunction, and Aβ toxicity. It was found that treatment with COE attenuated secondary Ca 2+ dysregulation induced by NMDA (700 μM), while a pre-application of COE also reduced NMDA-induced cell death. Furthermore, COE was neuroprotective against the mitochondrial toxin 3-NP (1 mM), while having no significant effect on cell death induced by incubation with naturally-secreted oligomers of Aβ (8.2 pg / ml). Collectively, these results are important for the therapeutic use of COE in the treatment of neurodegenerative disorders.

The non-competitive NMDA receptor antagonist memantine, currently prescribed for the treatment of Alzheimer's disease, is assumed to prevent the excitotoxicity implicated in neurodegenerative processes. Here, we investigated the actions of memantine on hippocampal function and signalling. In behavioural experiments using the water maze, we observed that memantine (at 2 mg/kg) reversed scopolamine-induced learning deficits in mice. When acutely applied to mouse hippocampal slices, memantine caused a significant upward shift in the population spike input-output relationship at 10 and 100 µM, and a corresponding downward shift in latency, indicative of overall enhanced synaptic transmission. This action was blocked by the muscarinic antagonist scopolamine (10 µM) but not by the NMDA antagonist MK-801 (10 µM) or the GABA antagonist bicuculline (20 µM). Further, memantine occluded potentiation induced by 50 nM carbachol (CCh), while enhancing inhibitory actions of CCh at 1 µM, suggesting additive actions. As anticipated for an NMDA antagonist, 100 µM (but not 10 µM) memantine also inhibited tetanus-induced long-term potentiation (LTP), and NMDA-induced Ca 2+ signals were blocked in cultured hippocampal neurones at 10 µM (by 88%). Overall, our data suggest actions of memantine beyond NMDA receptor antagonism, including stimulating effects on cholinergic signalling via muscarinic receptors. These interactions with the cholinergic system are likely to contribute to memantine's therapeutic potential.

In contrast to conventional signaling by growth factors that requires their continual presence, a 1-min pulse of nerve growth factor (NGF) is sufficient to induce electrical excitability in PC12 cells due to induction of the peripheral nerve type 1 (PN1) sodium channel gene. We have investigated the mechanism for this triggered signaling pathway by NGF in PC12 cells. Mutation of TrkA at key autophosphorylation sites indicates an essential role for the phospholipase C-γ (PLC-γ) binding site, but not the Shc binding site, for NGF-triggered induction of PN1. In concordance with results with Trk mutants, drug-mediated inhibition of PLC-γ activity also blocks PN1 induction by NGF. Examination of the kinetics of TrkA autophosphorylation indicates that triggered signaling does not result from sustained activation and autophosphorylation of the TrkA receptor kinase, whose phosphorylation state declines rapidly after NGF removal. Rather, TrkA triggers an unexpectedly prolonged phosphorylation and activation of PLC-γ signaling that is sustained for up to 2 h. Prevention of the elevation of intracellular Ca2+ levels using BAPTA-AM results in a block of PN1 induction by NGF. Sustained signaling by PLC-γ provides a means for differential neuronal gene induction after transient exposure to NGF.