Focal increases of axoplasmic Ca2+, aggregation of sodium-calcium exchanger, N-type Ca2+ channel, and actin define the sites of spheroids in axons undergoing oxidative stress.

Axonal spheroids occur as part of the pathology of a variety of neurologic diseases. Reactive oxygen species (ROS) trigger formation of spheroids, axonal severing, and Ca(2+) overload. The mechanisms by which ROS lead to the spheroid formation at specific axonal sites remain elusive. Here, using adult mouse primary neurons, we investigate the role of Ca(2+), its regulating systems, and cytoskeletal changes in formation of axonal spheroids triggered by ROS. The results reveal that dramatically higher axoplasmic Ca(2+) levels occur at the sites of axonal spheroids than in the rest of the axon. High focal axoplasmic Ca(2+) levels correlate with focal aggregation of the reverse Na(+)/Ca(2+) exchanger 1, voltage-gated N-type Ca(2+) channel α1B subunit, and actin at the sites of spheroids in individual axons. This study provides new insights into the mechanism of a spheroid formation at specific sites along axons undergoing oxidative stress and a basis for new neuroprotective strategies.

Mitochondrial calcium and its regulation in neurodegeneration induced by oxidative stress.

A proposed mechanism of neuronal death associated with a variety of neurodegenerative diseases is the response of neurons to oxidative stress and consequent cytosolic Ca(2+) overload. One hypothesis is that cytosolic Ca(2+) overload leads to mitochondrial Ca(2+) overload and prolonged opening of the permeability transition pore (PTP), resulting in mitochondrial dysfunction. Elimination of cyclophilin D (CyPD), a key regulator of the PTP, results in neuroprotection in a number of murine models of neurodegeneration in which oxidative stress and high cytosolic Ca(2+) have been implicated. However, the effects of oxidative stress on the interplay between cytosolic and mitochondrial Ca(2+) in adult neurons and the role of the CyPD-dependent PTP in these dynamic processes have not been examined. Here, using primary cultured cerebral cortical neurons from adult wild-type (WT) mice and mice missing cyclophilin D (CyPD-KO), we directly assess cytosolic and mitochondrial Ca(2+) , as well as ATP levels, during oxidative stress. Our data demonstrate that during acute oxidative stress mitochondria contribute to neuronal Ca(2+) overload by release of their Ca(2+) stores. This result contrasts with the prevailing view of mitochondria as a buffer of cytosolic Ca(2+) under stress conditions. In addition, we show that CyPD deficiency reverses the release of mitochondrial Ca(2+) , leading to lower of cytosolic Ca(2+) levels, attenuation of the decrease in cytosolic and mitochondrial ATP, and a significantly higher viability of adult CyPD-knockout neurons following exposure of neurons oxidative stress. The study offers a first insight into the mechanism underlying CyPD-dependent neuroprotection during oxidative stress.

Estrogen facilitates neurite extension via apolipoprotein E in cultured adult mouse cortical neurons.

Literature review suggests a close relationship between estrogen and apolipoprotein E (ApoE) in the central nervous system. Epidemiology studies show that estrogen replacement therapy (ERT) decreases the morbidity from several chronic neurological diseases. Alleles of ApoE modify the risk for and progression of the same diseases. ApoE levels in the rodent brain vary during the estrous cycle and increase after 17beta-estradiol administration. Both estradiol and ApoE3, the most common isoform of human ApoE, increase the extent of neurite outgrowth in culture. Combined, these observations suggest a common mechanism whereby estrogen may increase ApoE levels to facilitate neurite growth. We tested this hypothesis by characterizing the effects of estradiol and ApoE isoforms on neurite outgrowth in cultured adult mouse cortical neurons. Estradiol increased ApoE levels and neurite outgrowth. ApoE2 increased neurite length more so than ApoE3 in the presence of estradiol. Estradiol had no effect on neurite outgrowth from mice lacking the ApoE gene or when only ApoE4, the isoform of ApoE that is associated with increased risk of neurological disease, was exogenously supplied. Cultures from mice transgenic for human ApoE3 or ApoE4 showed the same isoform-specific effect. Neuronal internalization of recombinant human ApoE3 was greater than ApoE4, and ApoE3 was more effective than ApoE4 in facilitating neuronal uptake of a fatty acid. We conclude that estradiol facilitates neurite growth through an ApoE-dependent mechanism. The effects of ERT on chronic neurological diseases may vary with ApoE genotype. The clinical use of ERT may require ApoE genotyping for optimal efficacy.