Disruption of mitochondrial complex I induces progressive parkinsonism

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson’s disease1. Yet, whether this change contributes to Parkinson’s disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism—which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson’s disease paradigm.Electron microscopy tissue processing and imaging was performed at the Northwestern University Center for Advanced Microscopy, supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. This study was supported by grants from the Michael J. Fox Foundation (to D.J.S.), the JPB Foundation (to D.J.S.), the IDP Foundation (to D.J.S.), the Flanagan Fellowship (to P.G.-R.) and the European Research Council ERC Advanced Grant PRJ201502629 (to J.L.-B.)

Author Correction: Disruption of mitochondrial complex I induces progressive parkinsonism

In the version of this article initially published, the two bottom-left panels in Extended Data Fig. 8b duplicated the top-left and bottom-right panels of Fig. 4d presenting open field traces in mice. The panels have now been replaced with new images. The errors have been corrected in the online version of the article.Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson’s disease1. Yet, whether this change contributes to Parkinson’s disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism—which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson’s disease paradigm.Electron microscopy tissue processing and imaging was performed at the Northwestern University Center for Advanced Microscopy, supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. This study was supported by grants from the Michael J. Fox Foundation (to D.J.S.), the JPB Foundation (to D.J.S.), the IDP Foundation (to D.J.S.), the Flanagan Fellowship (to P.G.-R.) and the European Research Council ERC Advanced Grant PRJ201502629 (to J.L.-B.).Peer reviewe

PRESENILIN-2 AND CALCIUM HANDLING IN FAMILIAL ALZHEIMER'S DISEASE: A GENETICALLY ENCODED Ca2+ PROBES-BASED STUDY ROLE OF PS2 STRUCTURE, ER Ca2+-LEAK PATHWAYS AND ER-MITOCHONDRIA INTERPLAY

Calcium (Ca2+) is a key second messenger in living cells and it regulates a multitude of cell functions; this means as well that a dysregulation in its signaling cascade can be detrimental for cell fate. Ca2+ mishandling has been proposed as a causative mechanism for most neurodegenerative diseases and in particular for Alzheimer’s Disease (AD). From the middle ‘80s, alterations in Ca2+ dynamics were noticed in fibroblasts from AD patients, but extensive studies on AD and Ca2+ homeostasis started only after the identification of mutations linked to Familial Alzheimer’s Disease (FAD) in three genes, app, psen-1 and psen-2, coding for Amyloid Precursors Protein (APP), Presenilin-1 and Presenilin-2 (PS1, PS2). Mutations in these genes caused alterations in the cleavage of APP by a PS1- or PS2- containing enzyme, thus leading to an increase in the ratio between the two main peptides finally derived from APP maturation, called Ab40 and Ab42, in favor of the latter, the most toxic and most prone to aggregation specie; this in turn would increase the deposition of “Amyloid Plaques”, one of the principal histopathological feature of AD. Up to now, the generation of Ab42 peptides, its oligomers and finally amyloid plaques is the core of the most widely accepted pathogenic hypothesis for AD, the “Amyloid Cascade Hypothesis”. Considering Ca2+ homeostasis, most of the attention has been paid to the effect of PS1 (and only lately of PS2) mutations. Initially, most works reported an increase in the Ca2+ released in the cytosolic compartment from ER upon stimulation in cells expressing FADPS( 1), thus suggesting an increase in ER Ca2+ loading, the “Ca2+ Overload Hypothesis”. Although supported by several groups for many years, this hypothesis has never been undisputedly accepted, since some data were clearly in contrast with it, especially those considering PS2 mutations. Recently a strong evidence in support of “Ca2+ overload hypothesis” came from works suggesting that wt PSs can form low conductance Ca2+ channels in the ER membrane, providing most of the constitutive Ca2+ leak from the organelle; this function would be compromised by FAD mutations, that would thus lead to an ER Ca2+ overload. Again, these data have not been collectively accepted and other groups provided alternative explanations for the enhanced Ca2+ release in FAD-PSs (mostly PS1) expressing cells, such as enhanced Ryanodine Receptor (RyR) activation, augmented IP3 Receptor (IP3R) opening probability or potentiated SERCA activity. Interestingly, some of these data were no more suggesting an increased ER Ca2+ content (and so what is properly named “Ca2+ overload”) but rather an exaggerated Ca2+ release from the store, with unchanged (or even reduced) ER Ca2+ concentration. The effect of PS2 mutations has been less studied and is more controversial. Fibroblasts from patients bearing PS2 mutations showed reductions in Ca2+ release upon stimulation and actually data obtained by employing ER-targeted Ca2+ probes (e.g. ER-targeted aequorin) in FAD-PS2 stable clones, as well as cell lines transiently-transfected with different FAD-PS2 mutations, showed a reduction in ER Ca2+ concentration. Starting from these results, demonstrating that the expression of PS2 bearing FAD-linked mutations dampens the intracellular Ca2+ stores, the molecular mechanisms behind this phenomenon have been taken under investigation. In particular, three different aspects of PS2 effect on Ca2+ handling have been analyzed, mostly employing organelle-targeted aequorin Ca2+ probes: (i) the role of PS2 conformation on its effect in reducing the ER Ca2+ level; PSs are in fact synthesized as holoprotein but soon after their incorporation into g-secretase complex undergo a maturation cleavage to their active, dimeric form; ii) the involvement of the Ca2+ leak pathway across the ER membrane in PS2’s effect and more specifically of three different possible leak pathways proposed by literature, RyR, IP3R and the Ribosomal-Translocon complex (RTC); (iii) the role of PS2 in regulating the interplay between ER and mitochondria, a critical feature in cell life. Experiments on cells devoid of endogenous PS1 and PS2 and transfected with different PS2 constructs (resulting in the expression of the full length PS2, the dimeric PS2 or both) demonstrated that the full length conformation of this protein is necessary for the reduction in ER Ca2+ level linked to its expression; moreover, it has been shown that increasing, by different approaches, the endogenous level of the full length PS2 decreases ER Ca2+ content. Data aimed at measuring the decay rate of ER Ca2+ in control cells compared with FADPS2 expressing cells revealed an increased Ca2+ leak in the latter, and so the possible involvement of RyR, IP3R and RTC was investigated both by pharmacological (application of RyR inhibitor Dantrolene, IP3R antagonist Heparin, RTC opener/closer Puromycin/Anisomycin) or genetic (siRNA against IP3R-1 and -3 isoforms) approaches, showing that the PS2-induced ER Ca2+ reduction is at least partially mediated by RyR and IP3R, but not by RTC. Mitochondrial uptake of Ca2+ released from ER was evaluated. When mitochondrial Ca2+ peaks were measured in PS2-expressing and control cells in a condition in which their cytosolic peaks were comparable (by partially pre-emptying control cells), an increase in mitochondrial Ca2+ uptake was observed for cells over-expressing PS2 wt and, more prominently, FAD-PS2. This was not due to a direct effect of PS2 on mitochondrial uptake machinery but to an increased interaction between these organelles and the ER, as demonstrated by evaluating the two organelles proximity by confocal microscopy. By downregulating PS2 with siRNA, it was also shown that endogenous PS2 can control ERmitochondria interaction. These results were confirmed by employing FRET-based “Cameleon” cytosolic and mitochondrial Ca2+ probes on single-cells experiments. Altogether these findings provide new insights into the PS2 effect on Ca2+ homeostasis in Familial Alzheimer’s Disease and, more specifically, on the role of PS2 conformation and on the involvement of RyR and IP3R in its effect. A new aspect of the PS2 control of cell (and in particular Ca2+) dynamics is also emerging since it is here shown, for the first time, that this protein can influence the interplay between ER and mitochondria and that FAD-mutations affect this interaction, opening new directions for the investigation of the effects of PSs’ mutations in Familial Alzheimer’s Disease.Il calcio (Ca2+) è un secondo messaggero chiave nelle cellule e regola una moltitudine di funzioni cellulari; questo significa che una dis-regolazione nella sua cascata di trasduzione del segnale può essere deleteria per il destino della cellula. Difetti nella regolazione del Ca2+ sono stati proposti come possibili cause della maggior parte delle malattie neurodegenerative e in particolare della Malattia di Alzheimer (AD). Sin dalla metà degli anni ’80 furono notate alterazioni nelle dimamiche intracellulari del Ca2+ in fibroblasti ottenuti da pazienti affetti da AD, ma studi sistematici sulla relazione tra AD e omeostasi del Ca2+ cominciarono solo dopo l’identificazione di mutazioni legate alle forme familiari di AD (FAD) a carico di tre geni, app, psen-1 e psen-2, codificanti per la Proteina Precursone dell’Amiloide (APP), Presenilina-1 e Presenilina-2 (PS1, PS2). Mutazioni in questi geni causano alterazioni nel taglio di APP ad opera di un enzima contenente PS1 o PS2, le quali comportano quindi un aumento nel rapporto tra i due principali peptidi derivanti dalla maturazione di APP, chiamati Ab40 e Ab42, in favore di quest’ultimo, la specie più tossica e con maggiore tendenza all’aggregazione; questo d’altra parte aumenta la deposizioni delle “Placche Amiloidi”, una delle caratteristiche istopatologiche dell’AD. Attualmente, la generazione dei peptidi Ab42, dei suoi oligomeri e infine delle placche amiloidi è alla base della principale ipostesi sulla patogenesi dell’AD, l’“Amyloid Cascade Hypothesis”. Riguardo l’omeostasi del Ca2+, la maggior parte dell’attenzione è stata rivolta all’effetto delle mutazioni in PS1 (e solo successivamente in PS2). Inizialmente la maggior parte dei lavori riportava un incremento degli aumenti citosolici di Ca2+ indotti dalla stimolazione del rilascio dal reticolo endoplasmatico (ER) in cellule esprimenti mutazioni in PS1 associate a FAD (FAD-PS1), e perciò suggerivano un aumento nel contenuto di Ca2+ del ER, “Ca2+ Overload Hypothesis”. Nonostante sia stata sostenuta da molti gruppi per diversi anni questa ipotesi non è mai stata indiscutibilmente accettata, poiché esistevano alcuni dati evidentemente in contrasto con essa, soprattutto considerando mutazioni a carico di PS2. Recentemente l’ipotesi del “Ca2+ Overload” ha ricevuto forte supporto dalla dimostrazione che le PS nella loro forma wild type (wt) formano canali a bassa conduttanza per il Ca2+ nella membrana del ER, rappresentando la maggior parte della via di fuga (leak) costitutiva del Ca2+ dallo stesso organulo; questa funzione sarebbe compromessa dalle mutazioni associate a FAD, che quindi porterebbero ad un sovraccarico di Ca2+ nel ER. Anche in questo caso, questi dati non sono stati universalmente accettati e altri gruppi hanno fornito spiegazioni alternative all’aumentato rilascio di Ca2+ nelle cellule esprimenti mutazioni associate a FAD a carico delle PS (soprattutto PS1), cioè un’aumentata attivazione del Recettore Rianodinico (RyR), una maggiore probabilità di apertura del Recettore dell’IP3 (IP3R) o un potenziamento dell’attività della Ca2+-ATPasi del ER (SERCA). È interessante osservate che alcuni di questi dati non riportano più un aumento nel contenuto di Ca2+ del ER (cioè quello che è propriamente definito “Ca2+ Overload”) ma piuttosto un rilascio esagerato di Ca2+ dallo stesso deposito, con una concentrazione al suo interno inalterata o persino diminuita. L’effetto delle mutazioni in PS2 è stato meno studiato ed è più dibattuto. Fibroblasti ottenuti da pazienti affetti da mutazioni FAD in PS2 hanno rivelato un ridotto rilascio di Ca2+ dal ER in seguito a stimolazione, e dati ottenuti utilizzando sonde in grado di misurare direttamente la concentrazione di Ca2+ nel ER (in particolare basate su equorina modificata indirizzata al ER) hanno dimostrato una diminuzione nella concentrazione di Ca2+ nel ER sia in cloni stabili che in linee cellulari esprimenti mutazioni FAD in PS2. A partire da questi risultati, i quali dimostrano che l’espressione di PS2 recanti mutazioni associate a FAD riduce il contenuto di Ca2+ dei depositi intracellulari, si è deciso di investigare i meccanismi molecolari alla base di questo fenomeno. In particolare, sono stati investigati, soprattutto mediante l’impiego di equorine indirizzate a diversi compartimenti cellulari, tre diversi aspetti dell’effetto di PS2 sull’omeostasi del Ca2+: i) il ruolo della conformazione di PS2 nel suo effetto di riduzione del livello di Ca2+ del ER; le PS sono infatti sintetizzate come proteine intere ma subiscono rapidamente una maturazione mediante taglio ad una forma dimerica quando sono incorporate nel complesso g- secretasico; ii) il coinvolgimento del leak di Ca2+ attraverso la membrane del ER nell’effetto di PS2, e più in particolare di tre diverse possibili “vie di fuga” suggerite in letteratura, RyR, IP3R e il complesso Ribosoma-Traslocone (RTC); iii) il ruolo di PS2 nell’interazione tra ER e mitocondri, un aspetto centrale nella fisiologia della cellula. Esperimenti su cellule prive di PS1 e PS2 endogene transfettate con diversi costrutti di PS2 (in grado di dare l’espressione di PS2 intera, dimerica o di entrambe) hanno dimostrato che la sua conformazione intera è necessaria per la riduzione del contenuto di Ca2+ del ER indotta da PS2; inoltre, è stato dimostrato che un aumento nel livello endogeno di PS2 intera diminuisce il livello di Ca2+ nel ER. Dati mirati a misurare la velocità di uscita del Ca2+ attraverso la membrana del ER hanno evidenziato un leak di ioni Ca2+ aumentato in cellule esprimenti PS2 associate a FAD rispetto ai controlli, e quindi si è investigato il possibile coinvolgimento di RyR, IP3R e RTC sia con approcci di tipo farmacologico (impiego dell’inibitore di RyR Dantrolene, dell’antagosista di IP3 Eparina, di Puromicina e Anisomicina, due agenti che bloccano RTC in conformazione aperta o chiusa rispettivamente) che di tipo genetico (siRNA contro le isoforme 1 e 3 di IP3R al fine di diminuirne il livello proteico), arrivando a dimostrare che la riduzione del contenuto di Ca2+ del ER indotta da PS2 è almeno parzialmente mediata da RyR e IP3R ma non da RTC. È stato inoltre valutato l’accumulo da parte dei mitocondri del Ca2+ rilasciato dal ER. Quando sono stati misurati i picchi di Ca2+ mitocondriale in cellule esprimenti PS2 e cellule di controllo in condizioni in cui il rilascio citosolico era confrontabile (le cellule di controllo venivano parzialmente pre-svuotate) si è osservato un aumento nella captazione di Ca2+ da parte dei mitocondri in cellule esprimenti PS2 wt e, in modo più pronunciato, PS2 associata a FAD. Questo fenomeno non è dovuto a un effetto diretto di PS2 sul meccanismo di ingresso di Ca2+ nei mitocondri, bensì ad un aumento nell’interazione tra i mitocondri stessi e il ER, come è stato dimostrato misurando la prossimità dei due organuli mediante microscopia confocale. L’abbattimento dei livelli proteici di PS2 grazie a siRNA ha inoltre evidenziato che la PS2 endogena può controllare il grado di interazione tra ER e mitocondri. Questi risultati sono stati infine confermati da esperimenti fatti con sonde “Cameleon” per il Ca2+, basate sul fenomeno del FRET. Complessivamente questi dati forniscono nuovi elementi sull’effetto di PS2 sull’omeostasi del Ca2+ nelle forme familiari della Patologia di Alzheimer e, più in dettaglio, sul ruolo della conformazione di PS2 e sul coinvolgimento di RyR e IP3R nel suo effeto. Un nuovo aspetto del controllo da parte di PS2 sulle dinamiche cellulari (e del Ca2+ in particolare) viene inoltre riportato per la prima volta, cioè che questa proteina può regolare la relazione tra ER e mitocondri, e che mutazioni FAD a suo carico influenzano questa interazione; questo suggerisce ovviamente nuove possibili vie di indagine sull’effetto delle mutazioni a carico delle PS nelle forme familiari di AD

Intracellular organelles in the saga of Ca(2+) homeostasis: different molecules for different purposes?

An increase in the concentration of cytosolic free Ca(2+) is a key component regulating different cellular processes ranging from egg fertilization, active secretion and movement, to cell differentiation and death. The multitude of phenomena modulated by Ca(2+), however, do not simply rely on increases/decreases in its concentration, but also on specific timing, shape and sub-cellular localization of its signals that, combined together, provide a huge versatility in Ca(2+) signaling. Intracellular organelles and their Ca(2+) handling machineries exert key roles in this complex and precise mechanism, and this review will try to depict a map of Ca(2+) routes inside cells, highlighting the uniqueness of the different Ca(2+) toolkit components and the complexity of the interactions between them

Source data for "Feed-forward metabotropic signaling by Cav1 Ca2+ channels supports pacemaking in pedunculopontine cholinergic neurons"

<p><strong>Fig.1A_ChAT.tif</strong></p><p>Confocal image (green channel, anti-ChAT staining) for Fig.1A</p><p> </p><p><strong>Fig.1A_tdTomato.tif </strong></p><p>Confocal image (red channel, tdTomato) for Fig.1A</p><p> </p><p><strong>Fig.1B_ChAT.tif</strong></p><p>Confocal image (green channel, anti-ChAT staining) for Fig.1B</p><p> </p><p><strong>Fig.1B_tdTomato.tif</strong></p><p>Confocal image (red channel, tdTomato) for Fig.1B</p><p> </p><p><strong>Fig.1C_DIC.png</strong></p><p>Differential interference contrast micrograph for Fig.1C left</p><p> </p><p><strong>Fig.1C_Fluo.png</strong></p><p>Epifluorescent illumination micrograph for Fig. 1C right</p><p> </p><p><strong>Fig.1DEH.xlsx</strong></p><p>Numerical data for the charts in Fig. 1D, Fig.1E, Fig.1H</p><p> </p><p><strong>Fig.1F.tif</strong></p><p>MAX projection of z-stack of 2PLSM images (red channel, Alexa 594) used to generate Fig.1F </p><p> </p><p><strong>Fig.1F_inset.tif</strong></p><p>2PLSM image (green channel, Fura-2) for the right inset of Fig.1F</p><p> </p><p><strong>Fig.2A_inset.tif</strong></p><p>Confocal image (green channel, GFP) for the higher magnification inset of Fig.2A</p><p> </p><p><strong>Fig.2A.tif</strong></p><p>Confocal image (green channel, GFP) for Fig.2A</p><p> </p><p><strong>Fig.2B_bottom.tif</strong></p><p>Confocal image (green channel, GFP) for Fig.2B (bottom and overlay panels)</p><p> </p><p><strong>Fig.2B_top.tif</strong></p><p>Confocal image (red channel, td Tomato) for Fig.2B (top and overlay panels)</p><p> </p><p><strong>Fig.2CE.xlsx</strong></p><p>Numerical data for the charts in Fig. 2C, Fig. 2E</p><p> </p><p><strong>Fig.3B.tif</strong></p><p>Confocal image (green channel, MitoGCaMP6) for Fig.3B and overlay in Fig.3D</p><p> </p><p><strong>Fig.3C.tif</strong></p><p>Confocal image (red channel, tdTomato) for Fig.3C and overlay in Fig.3D</p><p> </p><p><strong>Fig.3E.tif</strong></p><p>2PLSM image (green channel, MitoGCaMP6) for Fig.3E</p><p> </p><p><strong>Fig.3GIJ.xlsx</strong></p><p>Numerical data for the charts in Fig. 3G, Fig. 3I, Fig.3J</p><p> </p><p><strong>Fig.4B.tif</strong></p><p>2PLSM image (green channel, MitoGCaMP6) for Fig.4B</p><p> </p><p><strong>Fig.4DFG.xlsx</strong></p><p>Numerical data for the charts in Fig.4D, Fig.4F, Fig.4G</p><p> </p><p><strong>Fig.5A.tif</strong></p><p>Confocal image (green channel, PercevalHR) for Fig.5A and overlay in Fig.5C</p><p> </p><p><strong>Fig.5B.tif</strong></p><p>Confocal image (red channel, tdTomato) for Fig.5B and overlay in Fig.5C</p><p> </p><p><strong>Fig.5D.tif</strong></p><p>2PLSM image (green channel, PercevalHR) for Fig.5D</p><p> </p><p><strong>Fig.5GHJ.xlsx</strong></p><p>Numerical data for the charts in Fig.5G, Fig.5H, Fig.5J</p><p> </p><p><strong>Fig.6BCD.xlsx</strong></p><p>Numerical data for the charts in Fig.6b, Fig.6C, Fig.6D</p><p> </p><p><strong>Fig.7A.tif</strong></p><p>Confocal image (green channel, mito-roGFP) for Fig.7A and overlay in Fig.7C</p><p> </p><p><strong>Fig.7B.tif</strong></p><p>Confocal image (red channel, tdTomato) for Fig.7B and overlay in Fig.7C</p><p> </p><p><strong>Fig.7D.tif</strong></p><p>2PLSM image (green channel, mito-roGFP) for Fig.7D</p><p> </p><p><strong>Fig.7F.xlsx</strong></p><p>Numerical data for the charts in Fig.7F</p&gt

Calcium, Bioenergetics, and Parkinson’s Disease

Degeneration of substantia nigra (SN) dopaminergic (DAergic) neurons is responsible for the core motor deficits of Parkinson’s disease (PD). These neurons are autonomous pacemakers that have large cytosolic Ca2+ oscillations that have been linked to basal mitochondrial oxidant stress and turnover. This review explores the origin of Ca2+ oscillations and their role in the control of mitochondrial respiration, bioenergetics, and mitochondrial oxidant stress

Ca 2+ dysregulation in neurons from transgenic mice expressing mutant presenilin 2

Summary: Mutations in amyloid precursor protein (APP), and presenilin-1 and presenilin-2 (PS1 and PS2) have causally been implicated in Familial Alzheimer's Disease (FAD), but the mechanistic link between the mutations and the early onset of neurodegeneration is still debated. Although no consensus has yet been reached, most data suggest that both FAD-linked PS mutants and endogenous PSs are involved in cellular Ca 2+ homeostasis. We here investigated subcellular Ca 2+handling in primary neuronal cultures and acute brain slices from wild type and transgenic mice carrying the FAD-linked PS2-N141I mutation, either alone or in the presence of the APP Swedish mutation. Compared with wild type, both types of transgenic neurons show a similar reduction in endoplasmic reticulum (ER) Ca 2+ content and decreased response to metabotropic agonists, albeit increased Ca 2+ release induced by caffeine. In both transgenic neurons, we also observed a higher ER-mitochondria juxtaposition that favors increased mitochondrial Ca 2+ uptake upon ER Ca 2+ release. A model is described that integrates into a unifying hypothesis the contradictory effects on Ca 2+ homeostasis of different PS mutations and points to the relevance of these findings in neurodegeneration and aging. © 2012 The Authors. Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.Peer Reviewe

Endoplasmic Reticulum-mitochondria connections, calcium cross-talk and cell fate: a closer inspection.

In eukaryotic cells, calcium (Ca2+) stores form a complex web where the capability to take up and release the cation is spread among different but highly interconnected structures that are physically based on the most abundant intracellular membranes: i.e., those forming the endoplasmic reticulum (ER) and the mitochondrial networks. Main hubs of these infra-structures are the Mitochondria-Associated Membranes (MAMs), ER and mitochondria juxtaposed membrane domains whose precise composition and functionality are now emerging. Understanding how these intracellular networks control Ca2+ dynamics under physiological and pathological conditions is fundamental to life sciences. The relevance of this issue is documented by the extraordinarily large number of qualified contributions that can offer both extensive reviews and in-depth examinations of specific aspects. Here we update the ER-mitochondria connection, with a special glance at the Ca2+ cross-talk, from different points of view: the molecules that are involved, either as essential building blocks or as modulators; the messages that travel between the two networks; the most novel technical approaches that allow to answer old questions and open new perspectives

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

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