Armato Ubaldo, Chiarini Anna, Chakravarthy Balu, Chioffi Franco, Pacchiana Raffaella, Colarusso Enzo, Whitfield James F, Dal Prà Ilaria
Department of Life and Reproduction Sciences, University of Verona Medical School, Verona, Italy.
Biochim Biophys Acta. 2013 Oct;1832(10):1634-52. doi: 10.1016/j.bbadis.2013.04.020. Epub 2013 Apr 26.
The "amyloid-β (Aβ) hypothesis" posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ25-35, an Aβ42 proxy, evokes a surplus endogenous Aβ42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAβ25-35 and soluble (s)Aβ25-35 on Aβ42 and Aβ40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aβs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAβ25-35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aβ42, while Aβ40 also accrued but its secretion was unchanged. Accordingly, secreted Aβ42/Aβ40 ratio values rose for astrocytes and neurons. While slightly enhancing Aβ40 secretion by fAβ25-35-treated astrocytes, NPS 2143 specifically suppressed the fAβ25-35-elicited surges of endogenous Aβ42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aβ42/Aβ40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/β-secretase and γ-secretase activity in fAβ25-35-treated astrocytes. Compared to fAβ25-35, sAβ25-35 also stimulated Aβ42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aβ/CaSR-induced surplus secretion of endogenous Aβ42 and hence further vicious cycles of Aβ self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.
“淀粉样蛋白-β（Aβ）假说”认为，神经元产生的Aβ肽（Aβs）不断积累会导致阿尔茨海默病（AD）。然而，数量更多的星形胶质细胞对Aβ的贡献仍未确定。此前我们发现，Aβ42的替代物纤维状（f）Aβ25 - 35会引发成年人类皮质星形胶质细胞内源性Aβ42产生/积累过剩。在此，我们通过免疫细胞化学、免疫印迹、酶活性测定以及高灵敏度夹心ELISA试剂盒，研究了fAβ25 - 35和可溶性（s）Aβ25 - 35对人类皮质星形胶质细胞和HCN - 1A神经元Aβ42和Aβ40积累/分泌的影响，并且由于钙敏感受体（CaSR）能结合Aβs，我们还研究了CaSR变构拮抗剂（钙溶解剂）NPS 2143对它们的调节作用。暴露于fAβ25 - 35的星形胶质细胞和存活的神经元产生、积累并分泌了更多的Aβ42，而Aβ40也有所增加但其分泌未变。因此，星形胶质细胞和神经元分泌的Aβ42/Aβ40比值升高。虽然fAβ25 - 35处理的星形胶质细胞对Aβ40分泌略有增强，但NPS 2143特异性抑制了fAβ25 - 35引发的星形胶质细胞和神经元内源性Aβ42分泌激增。因此，添加NPS 2143总能使Aβ42/Aβ40值保持在基线水平或更低。从机制上讲，NPS 2143降低了CaSR总蛋白量，短暂提高了蛋白酶体胰凝乳蛋白酶活性，并阻断了过量一氧化氮的产生，而不影响fAβ25 - 35处理的星形胶质细胞中持续增加的β-分泌酶1（BACE1）/β-分泌酶和γ-分泌酶活性。与fAβ25 - 35相比，sAβ25 - 35也刺激了星形胶质细胞和神经元的Aβ42分泌，而NPS 2143特异性且完全抑制了这种作用。因此，由于NPS 2143能阻止任何Aβ/CaSR诱导的内源性Aβ42过剩分泌，从而阻止Aβ自我诱导/分泌/扩散的进一步恶性循环，钙溶解剂可能有效预防/阻止向全面AD的进展。
