Chronic exposure to elevated levels of manganese (Mn2+) causes neuronal injury and inflammatory activation of glia. Astrocytes selectively accumulate Mn2+, which inhibits mitochondrial respiration and increases production of reactive oxygen species. We previously reported that sub-acute exposure to low micromolar levels of Mn2+ in primary astrocytes inhibited ATP-induced calcium (Ca2+) signaling, associated with decreased levels of endoplasmic reticulum Ca2+ and increased mitochondrial Ca2+ loads. In the present studies, we postulated that the mechanism underlying the capacity of Mn2+ to inhibit these purinergic signals in astrocytes could be due to competition with Ca2+ for entry through a plasma membrane channel. These data demonstrate that acutely applied Mn2+ rapidly inhibited ATP-induced Ca2+ waves and transients in primary striatal astrocytes. Mn2+ also decreased influx of extracellular Ca2+ induced by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a direct activator of the transient receptor potential channel, TRPC3. The TRPC3 inhibitor, pyrazole-3, prevented ATP- and OAG-dependent transport of Mn2+ from extracellular stores, demonstrated by a dramatic reduction in the rate of fluorescence quenching of Fura-2. These data indicate that Mn2+ can acutely inhibit ATP-dependent Ca2+ signaling in astrocytes by blocking Ca2+ entry through the receptor-operated cation channel, TRPC3. Loss of normal astrocytic responses to purinergic signals due to accumulation of Mn2+ could therefore comprise critical homeostatic functions necessary for metabolic and trophic support of neurons.

Dopaminergic nuclei in the basal ganglia are highly sensitive to damage from oxidative stress, inflammation, and environmental neurotoxins. Disruption of adenosine triphosphate (ATP)-dependent calcium (Ca2+) transients in astrocytes may represent an important target of such stressors that contributes to neuronal injury by disrupting critical Ca2+-dependent trophic functions. We therefore postulated that plasma membrane cation channels might be a common site of inhibition by structurally distinct cationic neurotoxicants that could modulate ATP-induced Ca2+ signals in astrocytes. To test this, we examined the capacity of two dopaminergic neurotoxicants to alter ATP-dependent Ca2+ waves and transients in primary murine striatal astrocytes: MPP+, the active metabolite of 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 6-hydroxydopamine (6-OHDA). Both compounds acutely decreased ATP-induced Ca2+ transients and waves in astrocytes and blocked OAG-induced Ca2+ influx at micromolar concentrations, suggesting the transient receptor potential channel, TRPC3, as an acute target. MPP+ inhibited 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ca2+ transients similarly to the TRPC3 antagonist, pyrazole-3, whereas 6-OHDA only partly suppressed OAG-induced transients. RNAi directed against TRPC3 inhibited the ATP-induced transient as well as entry of extracellular Ca2+, which was augmented by MPP+. Whole-cell patch clamp experiments in primary astrocytes and TRPC3-overexpressing cells demonstrated that acute application of MPP+ completely blocked OAG-induced TRPC3 currents, whereas 6-OHDA only partially inhibited OAG currents. These findings indicate that MPP+ and 6-OHDA inhibit ATP-induced Ca2+ signals in astrocytes in part by interfering with purinergic receptor mediated activation of TRPC3, suggesting a novel pathway in glia that could contribute to neurotoxic injury.

Background Cryptosporidium is a genus of apicomplexan parasites, the causative agents of cryptosporidiosis in humans and/or animals. Although most apicomplexans parasitize within the host cell cytosols, Cryptosporidium resides on top of host cells, but it is embraced by a double-layer parasitophorous vacuole membrane derived from host cell. There is an electron-dense band to separate the parasite from host cell cytoplasm, making it as an intracellular but extracytoplasmic parasite. However, little is known on the molecular machinery at the host cell-parasite interface. Methods Cryptosporidium parvum at various developmental stages were obtained by infecting HCT-8 cells cultured in vitro. Immunofluorescence assay was used to detect CpEF1α with a polyclonal antibody and host cell F-actin with rhodamine-phalloidin. Recombinant CpEF1α protein was used to evaluate its effect on the invasion by the parasite. Results We discovered that a C parvum translation elongation factor 1α (CpEF1α) was discharged from the invading sporozoites into host cells, forming a crescent-shaped patch that fully resembles the electron-dense band. At the same time, host cell F-actin aggregated to form a globular-shaped plug beneath the CpEF1α patch. The CpEF1α patch remained for most of the time but became weakened and dissolved upon the completion of the invasion process. In addition, recombinant CpEF1α protein could effectively interfere the invasion of sporozoites into host cells. Conclusions CpEF1α plays a role in the parasite invasion by participating in the formation of electron-dense band at the base of the parasite infection site.

2Phagocytosis and autophagy play critical roles in immune defense. Cryptococcus neoformans (Cn), 3 a fungal pathogen that causes fatal infection, subverts the host autophagy initiation complex (AIC) 4 and its upstream regulatory proteins, to promote its phagocytosis and intracellular parasitism of 5 host cells. The mechanisms by which the pathogen engages host AIC proteins remain obscure. 6Here, we show that the recruitment of host AIC proteins to forming phagosomes is dependent 7 upon the activity of CD44, a host cell surface receptor that engages fungal hyaluronic acid (HA). 8This interaction elevates intracellular Ca 2+ concentrations and activates CaMKKb and its 9 downstream target AMPKα, which results in activation of ULK1 and the recruitment of AIC 10 components. Moreover, we demonstrate that HA-coated beads efficiently recruit AIC components 11 to phagosomes. Taken together, these findings show that fungal HA plays a critical role in 12 directing the internalization and productive intracellular membrane trafficking of a fungal 13 pathogen of global importance. 14 15 16 Ganley, I.G., Lam, D.H., Wang, J., Ding, X., Chen, S., and Jiang, X. (2009). ULK1· ATG13· FIP200 complex 1 mediates mTOR signaling and is essential for autophagy.