Neurosteroids block AβP neurotoxicity Figure 3 Effects of DHEA, DHEA-S, and pregnenolone on [Ca2+]i increase induced by AβP. A. After a 20-min incubation period at 37 °C in 25 µ M DHEA-S, DHEA, and preg- nenolone (PG), the average increase in [Ca2+]i induced by 20 µ M AβP[25–35] was determined. As a control, the same DMSO concentration (0.5%) was added to the cultured neurons by the same protocol. Data are expressed as mean ± SEM, n = 200, ** p 0.001. B. Percentage of cells responding to AβP under same conditions as those in (A). Data are expressed as mean ± SEM, n = 5. membrane ﬂuidity and thus inhibits the assembly of peptides (Fujii et al 1997). The addition of cholesterol to membrane lipids inhibits the formation of ion channels by prion fragment peptides (Mirzabekov 1994) or Staphylococcus aureus α-toxin (Tomita et al 1992). Numerous epidemiological studies have suggested that a genotype of apolipoprotein E (E4 allele), which plays important roles in the transfer and metabolism of cholesterol, is a risk factor for AD (Corder et al 1993) and that cholesterol is signiﬁcant in AD pathology (Hartmann 2001). Cholesterol has been reported to block the elevation of [Ca2+]i induced by AβP (Hartmann et al 1994; Kawahara et al 2000), inhibit AβP neurotoxicity (Zhou and Richardson 1996), and affect the secretion of AβP (Frears et al 1999). It is also possible that the percentage of neurons that are resistant to AβP neurotoxicity changes during in vitro development. In particular, gamma-aminobutyric acid (GABA)-immunoposi- tive neurons may be important in the developmental change in susceptibility to AβP because of their resistance to AβP neurotoxicity (Pike and Cotman 1993). We have demonstrated that the developmental changes of GABAergic synapses occur at 7–14 DIV (Kato-Negishi et al 2004). The search for protective agents against AβP neurotoxicity is of great importance. Our system for observing Ca2+ inﬂux through AβP channels has contributed to the search for such substances (Kawahara and Kuroda 2001). In line with the search for protective agents, we found that neurosteroids Neuropsychiatric Disease and Treatment 2008:4(1) including DHEA, DHEA-S, and pregnenolone signiﬁcantly inhibit the [Ca2+]i elevation induced by AβP. DHEA and DHEA-S have neuroprotective effects against excitotoxicity (Kimonides et al 1998). Considering that plasma DHEA-S level is reduced in healthy individuals in an age-dependent manner and in AD patients (Hillen et al 2000), the implication of DHEA-S in the pathogenesis of AD may be important. Although the mechanism that DHEA-S inhibits AβP-induced elevation of [Ca2+] is still under investigation, it is possible i that DHEA-S modulates membrane ﬂuidity or membrane potential, and inﬂuences the afﬁnity of AβP to membranes. Further research about the inﬂuences of DHEA-S on the AβP binding to membrane surfaces is necessary. Moreover, it is interesting that DHEA-S can modulate GABA receptors (Meyer et al 1999). Further studies of the mechanism under- lying the differences in AβP susceptibility and the mechanism of inhibition by neurosteroids are necessary to conﬁrm the above possibilities. Our results are the ﬁrst to suggest the role of neurosteroids in AβP neurotoxicity and the develop- ment of neurosteroids for AD treatment. In conclusion, our results may aid in improving our understanding of AD and the development of drugs for AD treatment. Acknowledgments We thank Ms M Sekiguchi, Ms R Hosoda-Yabe, and Mr M Yanagita for technical assistance. This work was 215