Presenilin mutations are the main cause of familial Alzheimer's disease (FAD). Presenilins also play a key role in Ca ²⁺ homeostasis, and their FAD-linked mutants affect cellular Ca ²⁺ handling in several ways. We previously have demonstrated that FAD-linked presenilin 2 (PS2) mutants decrease the Ca ²⁺ content of the endoplasmic reticulum (ER) by inhibiting sarcoendoplasmic reticulum Ca ²⁺ -ATPase (SERCA) activity and increasing ER Ca ²⁺ leak. Here we focus on the effect of presenilins on mitochondrial Ca ²⁺ dynamics. By using genetically encoded Ca ²⁺ indicators specifically targeted to mitochondria (aequorin- and GFP-based probes) in SH-SY5Y cells and primary neuronal cultures, we show that overexpression or down-regulation of PS2, but not of presenilin 1 (PS1), modulates the Ca ²⁺ shuttling between ER and mitochondria, with its FAD mutants strongly favoring Ca ²⁺ transfer between the two organelles. This effect is not caused by a direct PS2 action on mitochondrial Ca ²⁺ -uptake machinery but rather by an increased physical interaction between ER and mitochondria that augments the frequency of Ca ²⁺ hot spots generated at the cytoplasmic surface of the outer mitochondrial membrane upon stimulation. This PS2 function adds further complexity to the multifaceted nature of presenilins and to their physiological role within the cell. We also discuss the importance of this additional effect of FAD-linked PS2 mutants for the understanding of FAD pathogenesis.

Ca ²⁺ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform ( w / a ) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler ( dfw ) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w / a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca ²⁺ pulse. The G293S and G283S mutations delayed the dissipation of Ca ²⁺ transients induced in CHO cells by InsP ₃ . In organotypic cultures, Ca ²⁺ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca ²⁺ transients induced by Ca ²⁺ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.

Autosomal dominant optic atrophy (ADOA), the commonest cause of inherited optic atrophy, is caused by mutations in the ubiquitously expressed gene optic atrophy 1 ( OPA1 ), involved in fusion and biogenesis of the inner membrane of mitochondria. Bioenergetic failure, mitochondrial network abnormalities and increased apoptosis have all been proposed as possible causal factors. However, their relative contribution to pathogenesis as well as the prominent susceptibility of the retinal ganglion cell (RGC) in this disease remains uncertain. Here we identify a novel deletion of OPA1 gene in the GTPase domain in three patients affected by ADOA. Muscle biopsy of the patients showed neurogenic atrophy and abnormal morphology and distribution of mitochondria. Confocal microscopy revealed increased mitochondrial fragmentation in fibroblasts as well as in myotubes, where mitochondria were also unevenly distributed, with clustered organelles alternating with areas where mitochondria were sparse. These abnormalities were not associated with altered bioenergetics or increased susceptibility to pro-apoptotic stimuli. Therefore, changes in mitochondrial shape and distribution can be independent of other reported effects of OPA1 mutations, and therefore may be the primary cause of the disease. The arrangement of mitochondria in RGCs, which degenerate in ADOA, may be exquisitely sensitive to disturbance, and this may lead to bioenergetic crisis and/or induction of apoptosis. Our results highlight the importance of mitochondrial dynamics in the disease per se , and point to the loss of the fine positioning of mitochondria in the axons of RGCs as a possible explanation for their predominant degeneration in ADOA.

CAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death.