Background. Our previously data suggest that FAD-linked PS2 mutants (M239I, T122R and N141I) cause a different type of Ca2+ dysregulation compared to the majority of FAD-linked PS1 mutants. In fact, at the store level, “Ca2+ reduction” instead of “Ca2+ overload” was invariably reported with these mutants, when studied in different experimental systems ranging from endogenous expression in fibroblasts from FAD patients to both stable and transient expression in cell lines and primary rat neuronal cultures (1-3). Methods. By employing recombinant aequorins or cameleons, specifically targeted to the ER and the Golgi apparatus, we here monitor the Ca2+ concentration inside their lumen in different cell types including SH-SY5Y, MEFs - either wt or devoid of endogenous PSs (DKO MEFs) – and primary neurons and investigate the mechanisms by which PS2 variants alter store calcium handling. Results. We provide evidence that: i) not only over-expression of wt and mutant PS2 but also the endogenous level of PS2 reduces the store calcium content mainly by reducing the ER calcium uptake due to SERCA pumps (4); ii) the full-length (FL) form of the protein is required to interfere with store calcium handling (4); iii) at variance with the ER, the trans-Golgi compartment is not as much as affected by PS2, indicating that the secretory pathway Ca2+/Mn2+ ATPase type (SPCA) is likely not a target of PS2; iv) mitochondria Ca2+ uptake is also affected by mutant PS2; whether this is a direct effect or it is mediated by the ER-mitochondria cross-talk is now under investigation. Conclusions. At variance with the majority of PS1 mutants that leave unchanged or even overloaded the intracellular calcium stores, making the cells more susceptible to toxic stimuli, PS2 mutants, by depressing the store Ca2+ content and altering the ER-mitochondria cross-talk might play a completely different role on cellular Ca2+ homeostasis that needs a careful reconsideration. 1. Zatti et al., Neurobiology of Disease 15, 269-278, 2004. 2.Giacomello et al., Neurobiology of Disease 18, 638-648, 2005. 3. Zatti et al., Cell Calcium 39, 539-550, 2006 4. Brunello et al., 2009 (submitted)

Organelle-targeted Ca2+ probes help to visualize store Ca2+ handling by wild-type and mutant presenilin-2.

ZAMPESE, ENRICO;POZZAN, TULLIO;PIZZO, PAOLA;FASOLATO, CRISTINA
2009

Abstract

Background. Our previously data suggest that FAD-linked PS2 mutants (M239I, T122R and N141I) cause a different type of Ca2+ dysregulation compared to the majority of FAD-linked PS1 mutants. In fact, at the store level, “Ca2+ reduction” instead of “Ca2+ overload” was invariably reported with these mutants, when studied in different experimental systems ranging from endogenous expression in fibroblasts from FAD patients to both stable and transient expression in cell lines and primary rat neuronal cultures (1-3). Methods. By employing recombinant aequorins or cameleons, specifically targeted to the ER and the Golgi apparatus, we here monitor the Ca2+ concentration inside their lumen in different cell types including SH-SY5Y, MEFs - either wt or devoid of endogenous PSs (DKO MEFs) – and primary neurons and investigate the mechanisms by which PS2 variants alter store calcium handling. Results. We provide evidence that: i) not only over-expression of wt and mutant PS2 but also the endogenous level of PS2 reduces the store calcium content mainly by reducing the ER calcium uptake due to SERCA pumps (4); ii) the full-length (FL) form of the protein is required to interfere with store calcium handling (4); iii) at variance with the ER, the trans-Golgi compartment is not as much as affected by PS2, indicating that the secretory pathway Ca2+/Mn2+ ATPase type (SPCA) is likely not a target of PS2; iv) mitochondria Ca2+ uptake is also affected by mutant PS2; whether this is a direct effect or it is mediated by the ER-mitochondria cross-talk is now under investigation. Conclusions. At variance with the majority of PS1 mutants that leave unchanged or even overloaded the intracellular calcium stores, making the cells more susceptible to toxic stimuli, PS2 mutants, by depressing the store Ca2+ content and altering the ER-mitochondria cross-talk might play a completely different role on cellular Ca2+ homeostasis that needs a careful reconsideration. 1. Zatti et al., Neurobiology of Disease 15, 269-278, 2004. 2.Giacomello et al., Neurobiology of Disease 18, 638-648, 2005. 3. Zatti et al., Cell Calcium 39, 539-550, 2006 4. Brunello et al., 2009 (submitted)
2009
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2484645
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