Development of new tools to explore organelle calcium dynamics in vivo: a new fret-based calcium sensor and a mitochondria targeted channelrhodopsin

The first part of this thesis aims to develop new tools to explore mitochondrial Ca2+ dynamic in vivo by the improvement of a mitochondria targeting Cameleon probes and by creating a mitochondria targeted Channelrhodopsin. Genetically encoded calcium indicators (GECIs) allow quantitative Ca2+ measurements in different experimental models. Organelle-specific targeting signals are fused with the GECI’s sequence, achieving selective targeting to a specific organelle or cytoplasmic domain. Moreover, GECI’s coding sequences can be placed under the control of tissue specific or inducible promoters, allowing spatial and temporal control of their expression. Different types of GECIs have been created: in our laboratory we use a class of FRET-based Ca2+ sensors, called Cameleons. FRET (Förster resonance energy transfer) microscopy detects the direct transfer of energy from a donor to an acceptor fluorescence protein (FP) in a living cell. Cameleon structure consists of two Ca2+-responsive elements that alter the efficiency of FRET between two FPs: a cyan fluorescent protein (CFP), the donor, and a yellow fluorescent protein (cpV), the acceptor. The two Ca2+-responsive elements are Calmodulin (CaM) and the CaM-binding domain of myosin light chain kinase. As reported in the beginning, the aim of this study is to develop novel molecular sensors and new methodologies to express the probes in vivo in intact tissues as well as in organisms in order to explore organelle Ca2+ dynamics in vivo (in particularly in brain and heart). One of the limitations of Cameleon probes, especially critical for in vivo applications, is the low fluorescence of CFP, reducing the maximal obtainable signal-to-noise ratio, and a multi-exponential lifetime, indicating the presence of multiple excited-state decay pathways. Recently, a brighter and more stable FP, compared to CFP, has been developed: mCerulean3. It has high fluorescence quantum yield and high photostability, making this protein a good donor candidate in Cameleon probes. For this reason, CFP has been replaced with mCerulean3 in two different Cameleons: the cytosolic D3cpv and the mitochondria-targeted probe named 4mtD3cpv. The new probes have been tested in different cell types: HeLa, neonatal rat cardiomyocytes and neonatal mouse neurons. The brightness, the photostability, the pH-sensitivity and the dissociation constant (Kd) of the new probes have also been measured in situ and the data show a clear improvement in brightness and in photostability, compared to the original Cameleons, in both cytosolic and mitochondrial probes. The only drawback of the new probes is a reduced amplitude (about 20-30%) in the maximum change in the fluorescence emission ratio due to Ca2+ binding (dynamic range). In order to extend the dynamic range different approaches have been used. The addition of 16 glycines between the two Ca2+ responsive elements allowed us to generate a new cytosolic probe (D3mCerulean3+16) with an increased dynamic range (+ 20%). Similarly, we have modified the mitochondrial probe, starting from the targeting sequence, each N-terminal sequence of COX-VIII (subunit VIII of human cytochrome C oxidase) was elongated of 5 aminoacids. Indeed, 24 h after transfection a significant mis-localization in the cytosol was observed with the original probe, while the mis-targeting of the modified targeting sequence containing probe was decreased. Moreover, the reduction in the dynamic range due to the mCerulean3 presence was almost fully recovered adding the 16 glycines linker. We than analysed the effect of pH, observing a general stability of all the probe variants in the pH range tested. Finally, we evaluated the Ca2+ affinity of all the mitochondrial variants generated so far, obtaining a Kd of 0.06 µM and one of 10.84 µM in 4mtD3cpV, a Kd of 6,5 µM in 4mtD3mCerulean3 and a Kd of 0.03 µM and one of 10.7 µM in 4mtD3mCerulean3+16. To better characterize the new cytosolic and mitochondrial Cameleon probes, we purified the proteins expressed in E.Coli in order to measure also in vitro all the parameters described above. Up to now only preliminary data have been obtained, however a comparison of in situ and in vitro data suggests that the DR calculated for cytosolic probes is about 3, while that for a mitochondria like environment is about 2.5. Concerning the Ca2+ affinity, just few [Ca2+] concentrations have been tested so far, therefore all the mitochondrial and cytosolic probes display a single Kd. However, further experiments are required to confirm the in vitro calculated Kd. In parallel, we are also assessing another method to measure Ca2+ with FRET-GECIs, as an alternative of the classical intensity-based method. We are indeed employing FLIM technique that measures the lifetime of FPs (the time the molecule spend in the exited state), which is totally independent from phenomena such as probe photobleaching, expression level, image shading. Preliminary results in cells expressing all the Camelon probes described so far, suggest the presence of homo-FRET phenomena and mCerulean3-based Ca2+ sensors seem to be less affected by them. Finally, to express the new probes in vivo we are testing three strategies: i) adeno-associated virus (serotype 9) with cardiac tropism viral vectors; ii) adeno-associated virus (serotype 9) with neuron-specific promoters (synapsin promoter) for intracranial injection; iii) transgenic mice. These strategies will allow us to perform in vivo Ca2+ measurements in different tissues and subcellular compartments. We have just generated the AAV9 for the expression of the D3mCerulean3 and 4mD3mCerulean3 under the control of CMV promoter. A good expression levels in heart was obtained through intraperiotneal injection of neonatal mice. In conclusion, the results obtained so far make the mCerulean3-based Cameleons an attractive choice for in vivo experiments. To study the role of mitochondria in situ and in vivo, we also took advantage of an optogenetic tool: Channel Rhodopsins (ChRs). ChRs represent the only type of ion channels directly gated by light. When excited by light, ChRs open and depolarize the plasma membrane. For activation, opsins require binding of retinal, a vitamin A–related organic cofactor. In collaboration with Prof. Sekler’s group (Ben-Gurion University-Israel), we have developed a new mitochondria-targeted ChR, called mitochondrial Stabilized Step Function Opsin (4mt-SSFO). This ChR variant was modified to stabilize the conducting state of the channel: SSFO deactivation occurs in 30 minutes after a brief pulse of activating blue light (460-480 nm). It has also been reported the possibility to terminate the photocurrents with red light. To create a non-functional control, a truncated form Chr2(TR) lacking retinal binding site was generated. The targeting of proteins to the inner mitochondrial membrane (IMM) was obtained fusing four signal sequences, derived from human cytochrome C oxidase subunit VIII sequence, to the N-terminus of ChR. To verify the correct topology of the probe, confocal microscopy, elecrtophisiological recordings and fluorescence quenching experiments were carried out. When 4mtSSFO-YFP is properly inserted in the IMM, its C-terminal YFP tag should face the mitochondrial matrix. Indeed, the application of proteinase K to Digitonin permeabilized cells did not cause a significant reduction of 4mtChR2B-YFP signal, while that of N33D3cpv (a probe in which the YFP is located on the cytoplasmic surface of the OMM) is completely abolished. Trypan blue addition, that is permeable across the OMM, but not the IMM, did not affect the fluorescence of 4mtSSFO-YFP or that of the matrix located Cameleon 4mtD3cpv. The 4mtD3cpv fluorescence however was totally lost after alamethicin application that permeabilizes both mitochondrial membranes and releases all matrix proteins into the medium, while the fluorescence of 4mtSSFO-YFP was not affected by the latter treatment, as expected for a membrane bound protein. Taken together these data are consistent with a proper IMM localization. The new mitochondrial 4mtSSFO-YFP constructs were then tested in situ for their ability to change the mitochondrial membrane potential in response to light, resulting in a significant organelle depolarization in cells expressing 4mtSSFO-YFP, while no effect was induced by blue light in control cells. We then analysed in more details the effects of mitochondrial depolarization on mitochondrial Ca2+ uptake using two genetically encoded probes, 4mtD3cpv and mt-aequorin. Both approaches demonstrate that the depolarization of mitochondria triggered by photo-activation of the channel causes, as predicted, a significant reduction in the amplitude of the mitochondrial Ca2+ rise observed upon stimulation of HeLa cells with an IP3-generating agonist. Thus, we generated a tool able to modulate diverse mitochondria activities in a temporary-controlled, reversible (at least in principle) and cell-specific manner, offering an approach to quantitatively investigate mitochondrial role in a large variety of critical cellular processes. In the second part of the thesis, the investigation was focused on the role of Presenilins in Ca2+dyshomeostasis associated to Alzheimer's Disease (AD) using a single cell analysis approach and focusing on the role of AD Presenilin 1 and Presenilin 2 mutations in the modulation of Capacitative Calcium Entry (CCE). AD is the most frequent form of dementia. A small percentage of cases is inherited (Familial AD, FAD) and is due to dominant mutations on three genes, coding for Amyloid Precursor Protein (APP), Presenilin-1 (PS1) and Presenilin-2 (PS2). Mutations in these genes cause alterations in the cleavage of APP by a PS1- or PS2- containing enzyme, named y-secretase complex, thus leading to an increase in the ratio between Abeta42 and Abeta40, the two main peptides eventually derived from APP maturation. This event, in turn, leads to an increased deposition of amyloid plaques, one of the main histopathological feature of AD. To date, the generation of A42 peptides, its oligomers and amyloid plaques is the core of the most widely accepted pathogenic hypothesis for AD, the “Amyloid Cascade Hypothesis”. PS1 and PS2 are ubiquitous 9 transmembrane domains homologous proteins localized mainly in intracellular membranes (Endoplasmic Reticulum, ER, Golgi apparatus, and endosomes) and plasma membrane. Despite being the catalytic core of y-secretase complex, PSs display also some specialized, y-secretase independent activities. On this line, numerous studies reported a role for FAD-linked PS mutations in cellular Ca2+ alterations. Ca2+ is a key second messenger in living cells and it regulates a multitude of cell functions; thus, alterations in its signalling cascade can be detrimental for cell fate. Ca2+ mishandling has been proposed as a causative mechanism for different neurodegenerative diseases and in particular for AD. Although supported by several groups for many years, the Ca2+ hypothesis for AD pathogenesis has never been undisputedly accepted, since some data were clearly conflicting, especially those considering FAD-PS2 mutations. One of the Ca2+ pathway reported to be modulated by different FAD-PSs mutation is the so called CCE or Store Operated Calcium Entry (SOCE). CCE is the mechanism responsible for Ca2+ entry in response to ER Ca2+ depletion. The key molecules responsible for this Ca2+ entry have been identified only recently: STIM and Orai. Basically, Orai forms the channels located in the PM, while STIM is the protein that can “sense” the [Ca2+] in the ER lumen. Upon store depletion, STIM1 changes its distribution from diffuse to clusterized “puncta” and interacts with plasma membrane-located Orai1. Employing a cytosolic Cameleon probe (D3cpV), the CCE variation in SH-SY5Y cells overexpressing PSs or in human FAD and control fibroblasts was investigated. In particular, measuring the effect of PS1-A246, PS2-T122R (in overexpression) and PS1-A246 and PS2N141I (in fibroblasts) FAD-linked mutations, a decrease in both peak and rate of CCE was observed. This phenomenon could be explained by a decrease in STIM1 protein levels, while Orai1 level was not analysed because no antibody sufficiently specific is available. In the over-expression system of SH-SY5Y cells also wild type forms of PS1 and PS2 cause a decrease in CCE and this could be due to the accumulation of the full-length form of the proteins that is thought to be the mediator of the effec

Presenilin 2 Modulates Endoplasmic Reticulum-Mitochondria Coupling by Tuning the Antagonistic Effect of Mitofusin 2

Communication between organelles plays key roles in cell biology. In particular, physical and functional coupling of the endoplasmic reticulum (ER) and mitochondria is crucial for regulation of various physiological and pathophysiological processes. Here, we demonstrate that Presenilin 2 (PS2), mutations in which underlie familial Alzheimer’s disease (FAD), promotes ER-mitochondria coupling only in the presence of mitofusin 2 (Mfn2). PS2 is not necessary for the antagonistic effect of Mfn2 on organelle coupling, although its abundance can tune it. The two proteins physically interact, whereas their homologues Mfn1 and PS1 are dispensable for this interplay. Moreover, PS2 mutants associated with FAD are more effective than the wild-type form in modulating ER-mitochondria tethering because their binding to Mfn2 in mitochondrial-associated membranes is favored. We propose a revised model for ER-mitochondria interaction to account for these findings and discuss possible implications for FAD pathogenesis

Familial Alzheimer's disease-linked presenilin mutants and intracellular Ca2+ handling: A single-organelle, FRET-based analysis

Abstract An imbalance in Ca2+ homeostasis represents an early event in the pathogenesis of Alzheimer's disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca2+ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca2+ content is still debated and the mechanism of action of mutant proteins is unclear. To fulfil the need of a direct investigation of intracellular stores Ca2+ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca2+ handling using specifically targeted, FRET (Fluorescence/Forster Resonance Energy Transfer)-based Ca2+ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca2+ concentration within the main intracellular Ca2+ stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca2+ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca2+ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) activity. Moreover, by using a cytosolic Ca2+ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca2+ influx activated by stores depletion (Store-Operated Ca2+ Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway. Our data indicate that FAD-linked PSs mutants differentially modulate the Ca2+ content of intracellular stores yet leading to a complex dysregulation of Ca2+ homeostasis, which represents a common disease phenotype of AD

Presenilin 2 Modulates Endoplasmic Reticulum-Mitochondria Coupling by Tuning the Antagonistic Effect of Mitofusin 2

Communication between organelles plays key roles in cell biology. In particular, physical and functional coupling of the endoplasmic reticulum (ER) and mitochondria is crucial for regulation of various physiological and pathophysiological processes. Here, we demonstrate that Presenilin 2 (PS2), mutations in which underlie familial Alzheimer's disease (FAD), promotes ER-mitochondria coupling only in the presence of mitofusin 2 (Mfn2). PS2 is not necessary for the antagonistic effect of Mfn2 on organelle coupling, although its abundance can tune it. The two proteins physically interact, whereas their homologues Mfn1 and PS1 are dispensable for this interplay. Moreover, PS2 mutants associated with FAD are more effective than the wild-type form in modulating ER-mitochondria tethering because their binding to Mfn2 in mitochondria-associated membranes is favored. We propose a revised model for ER-mitochondria interaction to account for these findings and discuss possible implications for FAD pathogenesis

mCerulean3-Based Cameleon Sensor to Explore Mitochondrial Ca2+ Dynamics In Vivo

Genetically Encoded Ca2+ Indicators (GECIs) are extensively used to study organelle Ca2+ homeostasis, although some available probes are still plagued by a number of problems, e.g., low fluorescence intensity, partial mistargeting, and pH sensitivity. Furthermore, in the most commonly used mitochondrial F\uf6rster Resonance Energy Transfer based-GECIs, the donor protein ECFP is characterized by a double exponential lifetime that complicates the fluorescence lifetime analysis. We have modified the cytosolic and mitochondria-targeted Cameleon GECIs by (1) substituting the donor ECFP with mCerulean3, a brighter and more stable fluorescent protein with a single exponential lifetime; (2) extensively modifying the constructs to improve targeting efficiency and fluorescence changes caused by Ca2+ binding; and (3) inserting the cDNAs into adeno-associated viral vectors for in vivo expression. The probes have been thoroughly characterized in situ by fluorescence microscopy and Fluorescence Lifetime Imaging Microscopy, and examples of their ex vivo and in vivo applications are described

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases

Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER

Calcium ion (Ca2+) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+]) within its lumen ([Ca2+]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2). The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls

The elusive importance of being a mitochondrial Ca2+ uniporter

AbstractThe molecular components of the mitochondrial Ca2+ uptake machinery have been only recently identified. In the last months, in addition to the pore forming subunit and of one regulatory protein (named MCU and MICU1, respectively) other four components of this complex have been described. In addition, a MCU KO mouse model has been generated and a genetic human disease due to missense mutation of MICU1 has been discovered. In this contribution, we will first summarize the recent findings, discussing the roles of the different subunits of the mitochondrial Ca2+ uptake complex, pointing to the current contradictions in the published data, as well as possible explanations. Finally we will speculate on the recent, totally unexpected, results obtained in the MCU knock-out (KO) mice

Highlighting the Endoplasmic Reticulum-Mitochondria connection: focus on Mitofusin 2

The endoplasmic reticulum (ER) and the mitochondrial network are two highly interconnected cellular structures. By proteinaceous tethers, specialized membrane domains of the ER are tightly associated with the outer membrane of mitochondria, allowing the assembly of signaling platforms where different cell functions take place or are modulated, such as lipid biosynthesis, Ca2+ homeostasis, inflammation, autophagy and apoptosis. The ER-mitochondria coupling is highly dynamic and contacts between the two organelles can be modified in their number, extension and thickness by different stimuli. Importantly, several pathological conditions, such as cancer, neurodegenerative diseases and metabolic syndromes show alterations in this feature, underlining the key role of ER-mitochondria crosstalk in cell physiology. In this contribution, we will focus on one of the major modulator of ER-mitochondria apposition, Mitofusin 2, discussing the structure of the protein and its debated role on organelles tethering. Moreover, we will critically describe different techniques commonly used to investigate this crucial issue, highlighting their advantages, drawbacks and limits