β25-35 alters calcium homeostasis and induces neurotoxicity in cerebellar granule cells

We studied the neurotoxic effects of β25-35 amyloid fragment (β25-35) on cerebellar granule cells and the intracellular mechanisms involved. Treatment for 3 days with peptide greatly reduced the survival of 1 day in vitro (DIV) cultures kept in 5 mM KCl but slightly modified the survival of 25 mM KCl- cultured cerebellar granule cells. We also studied the effect of glutamate on survival of undifferentiated cerebellar granules. We report no neurotoxic effect of glutamate on 3-DIV-treated cultures; whereas in β25-35-pretreated cells, a significant glutamate toxicity was observed. Treatment of 6-DIV cells with β25-35, performed with 25 mM KCl, induced a late but significant neurotoxic effect after 5 days of exposure, and death occurred within 8 days. Differentiated cerebellar granule cells were also sensitive to glutamate- related neurotoxicity, and this effect was enhanced by β25-35 pretreatment. To study the molecular mechanisms underlying the neurotoxic effects of β25- 35, changes in calcium homeostasis after glutamate stimulation were evaluated in control and β25-35-treated cells. β25-35 did not affect basal [Ca²⁺](i) but modified glutamate-induced [Ca²⁺](i) increase, causing a sustained plateau phase that persisted even after the removal of the agonist. These results show that β25-35 induces neurotoxicity in cerebellar granule cells and that this effect is related to modifications in the control of calcium homeostasis.