Early acute necrosis, delayed apoptosis and cytoskeletal breakdown in cultured cerebellar granule neurons exposed to methylmercury

Abstract

Cerebellar granule cells (CGCs) are a sensitive target for methylmercury (MeHg) neurotoxicity. In vitro exposure of primary cultures of rat CGCs to MeHg resulted in a time- and concentration-dependent cell death. Within 1 hr exposure, MeHg at 5-10 microM caused impairment of mitochondrial activity, de-energization of mitochondria and plasma membrane lysis, resulting in necrotic cell death. Lower MeHg concentrations (0.5-1 microM) did not compromise cell viability, mitochondrial membrane potential and function at early time points. Later, however, the cells progressively underwent apoptosis and 100% cell death was reached by 18 hr treatment. Neuronal network fragmentation and microtubule depolymerization were detected as early as within 1.5 hr of MeHg (1 microM) exposure, long before the occurrence of nuclear condensation (6-9 hr). Neurite damage worsened with longer exposure time and proceeded to the complete dissolution of microtubules and neuronal processes (18 hr). Microtubule stabilization by taxol did not prevent MeHg-induced delayed apoptosis. Similarly ineffective were the caspase inhibitors z-VAD-fluoromethylketone and z-DEVD-chloromethylketone, the L-type calcium channel inhibitor nifedipine, the calcium chelator EGTA and BAPTA, and the NMDA receptor antagonist MK-801. On the other hand, insulin-like growth factor-I partially rescued CGCs from MeHg-triggered apoptosis. Altogether these results provide evidence that the intensity of MeHg insult is decisive in the time of onset and the mode of neuronal death that follows, i.e., necrosis vs. apoptosis, and suggest that cytoskeletal breakdown and deprivation of neurotrophic support play a role in MeHg delayed toxicity.

Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes

“…In human neural stem cells as well as in murine neural progenitor cells, MeHg was demonstrated to induce apoptosis at low cytotoxic concentrations and durations of exposure [ 1 0,50). In accordance to our data, Castaldi et al found an induction of apoptosis in mitotically inhibited cerebellar neurons at low MeHg hydroxide concentrations while at higher concentrations the compound induced necrosis [23). Additionally, incubation of thiomersal for 24 h in cultured human neurons induced apoptosis at low concentrations whereas high concentrations resulted in necrosis [19).…”

“…Nevertheless, numerous studies are limited to the use of immature, proliferating neurons [8,9,11,18,19] or neurons during differentiation [20][21][22] to investigate developmental neurotoxicity. Representing a differentiated neural cell culture model, Castoldi et al investigated effects of MeHg on primary cultures of mitotically inhibited rat granule neurons [23]. Furthermore, neurite outgrowth was affected in a differentiated PC12 cell clone [24].…”

Rescue of neuronal migration deficits in a mouse model of fetal Minamata disease by increasing neuronal Ca2+spike frequency

“…We used 10 μM MeHg, because the application of this amount of MeHg induces a significant deceleration of the speed of granule cell migration (Fig. 2 C and F), although prolonged application affects the viability of granule cells (36). In the case of Ca 2+ signaling, caffeine (stimulator of internal Ca 2+ release through ryanodine receptors, 1 mM) or NMDA (agonist of the NMDAtype glutamate receptors, 30 μM) markedly reduced the effect of MeHg on Ca 2+ spike frequency of granule cells (Fig.…”

Methylmercury inhibits electron transport chain activity and induces cytochrome c release in cerebellum mitochondria

“…Neuronal degeneration upon MeHg exposure has been reported to occur through apoptosis (Kunimoto, 1994;Toimela and Tähti, 2004;Castoldi et al, 2000). Nagashima et al (1996Nagashima et al ( , 1997 established that the degeneration patterns of cerebellar granule cells in MeHg-intoxicated rats were quite similar to the typical finding of apoptosis, which is one of the mechanisms of neuronal destruction in degenerative brain damage.…”