Rescue of astrocyte activity by the calcium sensor STIM1 restores long-term synaptic plasticity in female mice modelling Alzheimer's disease.

Calcium dynamics in astrocytes represent a fundamental signal that through gliotransmitter release regulates synaptic plasticity and behaviour. Here we present a longitudinal study in the PS2APP mouse model of Alzheimer's disease (AD) linking astrocyte Ca2+ hypoactivity to memory loss. At the onset of plaque deposition, somatosensory cortical astrocytes of AD female mice exhibit a drastic reduction of Ca2+ signaling, closely associated with decreased endoplasmic reticulum Ca2+ concentration and reduced expression of the Ca2+ sensor STIM1. In parallel, astrocyte-dependent long-term synaptic plasticity declines in the somatosensory circuitry, anticipating specific tactile memory loss. Notably, we show that both astrocyte Ca2+ signaling and long-term synaptic plasticity are fully recovered by selective STIM1 overexpression in astrocytes. Our data unveil astrocyte Ca2+ hypoactivity in neocortical astrocytes as a functional hallmark of early AD stages and indicate astrocytic STIM1 as a target to rescue memory deficits.

ER Morphology in the Pathogenesis of Hereditary Spastic Paraplegia.

The endoplasmic reticulum (ER) is the most abundant and widespread organelle in cells. Its peculiar membrane architecture, formed by an intricate network of tubules and cisternae, is critical to its multifaceted function. Regulation of ER morphology is coordinated by a few ER-specific membrane proteins and is thought to be particularly important in neurons, where organized ER membranes are found even in the most distant neurite terminals. Mutation of ER-shaping proteins has been implicated in the neurodegenerative disease hereditary spastic paraplegia (HSP). In this review we discuss the involvement of these proteins in the pathogenesis of HSP, focusing on the experimental evidence linking their molecular function to disease onset. Although the precise biochemical activity of some ER-related HSP proteins has been elucidated, the pathological mechanism underlying ER-linked HSP is still undetermined and needs to be further investigated.

Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus.

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.

Lighting Up Ca2+ Dynamics in Animal Models.

Calcium (Ca2+) signaling coordinates are crucial processes in brain physiology. Particularly, fundamental aspects of neuronal function such as synaptic transmission and neuronal plasticity are regulated by Ca2+, and neuronal survival itself relies on Ca2+-dependent cascades. Indeed, impaired Ca2+ homeostasis has been reported in aging as well as in the onset and progression of neurodegeneration. Understanding the physiology of brain function and the key processes leading to its derangement is a core challenge for neuroscience. In this context, Ca2+ imaging represents a powerful tool, effectively fostered by the continuous amelioration of Ca2+ sensors in parallel with the improvement of imaging instrumentation. In this review, we explore the potentiality of the most used animal models employed for Ca2+ imaging, highlighting their application in brain research to explore the pathogenesis of neurodegenerative diseases.

Mt-fura-2, a Ratiometric Mitochondria-Targeted Ca2+ Sensor. Determination of Spectroscopic Properties and Ca2+ Imaging Assays.

Ca2+ handling by mitochondria is implicated in energy production, shaping of cytosolic Ca2+ rises, and determination of cell fate. It is therefore of crucial interest for researchers to directly measure mitochondrial Ca2+ concentration [Ca2+] in living cells. Synthetic fluorescent Ca2+ indicators, providing a straightforward loading technique, represents a tempting strategy. Recently, we developed a new highly selective mitochondria-targeted Ca2+ indicator named mt-fura-2 , obtained by coupling two triphenylphosphonium cation-containing groups to the molecular backbone of the cytosolic ratiometric Ca2+ indicator fura-2 .The protocols we describe here cover all the significant steps that are necessary to characterize the probe and apply it to biologically relevant contexts. The procedures reported are referred to mt-fura-2 but could in principle be applied to characterize other mitochondria-targeted Ca2+ probes . More in general, with the due modifications, this chapter can be considered as a handbook for the characterization and/or application of mitochondria-targeted chemical Ca2+ probes .

Familial Alzheimer's disease presenilin-2 mutants affect Ca2+ homeostasis and brain network excitability.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aß) peptides, providing genetic support to the hypothesis of Aß pathogenicity. However, clinical trials focused on the Aß pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca2+ signaling. By generating novel tools for measuring Ca2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca2+ signaling and brain network activity, as summarized below.

A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals.

Mitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila.

The endoplasmic reticulum (ER) is a highly dynamic network whose shape is thought to be actively regulated by membrane resident proteins. Mutation of several such morphology regulators cause the neurological disorder Hereditary Sp astic Paraplegia (HSP), suggesting a critical role of ER shape maintenance in neuronal activity and function. Human Atlastin-1 mutations are responsible for SPG3A, the earliest onset and one of the more severe forms of dominant HSP. Atlastin has been initially identified in Drosophila as the GTPase responsible for the homotypic fusion of ER membrane. The majority of SPG3A-linked Atlastin-1 mutations map to the GTPase domain, potentially interfering with atlastin GTPase activity, and to the three-helix-bundle (3HB) domain, a region critical for homo-oligomerization. Here we have examined the in vivo effects of four pathogenetic missense mutations (two mapping to the GTPase domain and two to the 3HB domain) using two complementary approaches: CRISPR/Cas9 editing to introduce such variants in the endogenous atlastin gene and transgenesis to generate lines overexpressing atlastin carrying the same pathogenic variants. We found that all pathological mutations examined reduce atlastin activity in vivo although to different degrees of severity. Moreover, overexpression of the pathogenic variants in a wild type atlastin background does not give rise to the loss of function phenotypes expected for dominant negative mutations. These results indicate that the four pathological mutations investigated act through a loss of function mechanism.

Presenilin-2 and Calcium Handling: Molecules, Organelles, Cells and Brain Networks.

Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer's disease (FAD). It forms the catalytic core of the γ-secretase complex-a function shared with its homolog presenilin-1 (PS1)-the enzyme ultimately responsible of amyloid-ß (Aß) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca2+ signalling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca2+ handling to Ca2+ entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca2+ homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer's disease (AD).

Calcium Imaging in Drosophila melanogaster.

Drosophila melanogaster, colloquially known as the fruit fly, is one of the most commonly used model organisms in scientific research. Although the final architecture of a fly and a human differs greatly, most of the fundamental biological mechanisms and pathways controlling development and survival are conserved through evolution between the two species. For this reason, Drosophila has been productively used as a model organism for over a century, to study a diverse range of biological processes, including development, learning, behavior and aging. Ca2+ signaling comprises complex pathways that impact on virtually every aspect of cellular physiology. Within such a complex field of study, Drosophila offers the advantages of consolidated molecular and genetic techniques, lack of genetic redundancy and a completely annotated genome since 2000. These and other characteristics provided the basis for the identification of many genes encoding Ca2+ signaling molecules and the disclosure of conserved Ca2+ signaling pathways. In this review, we will analyze the applications of Ca2+ imaging in the fruit fly model, highlighting in particular their impact on the study of normal brain function and pathogenesis of neurodegenerative diseases.

Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon.

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.

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 Förster 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.

A Synthetic Fluorescent Mitochondria-Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells.

Ca2+ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca2+ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca2+ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca2+ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca2+ indicator fura-2. Mt-fura-2 binds Ca2+ with a dissociation constant of ≈1.5 µm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca2+ ] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.

Proteasome dysfunction induces excessive proteome instability and loss of mitostasis that can be mitigated by enhancing mitochondrial fusion or autophagy.

The ubiquitin-proteasome pathway (UPP) is central to proteostasis network (PN) functionality and proteome quality control. Yet, the functional implication of the UPP in tissue homeodynamics at the whole organism level and its potential cross-talk with other proteostatic or mitostatic modules are not well understood. We show here that knock down (KD) of proteasome subunits in Drosophila flies, induced, for most subunits, developmental lethality. Ubiquitous or tissue specific proteasome dysfunction triggered systemic proteome instability and activation of PN modules, including macroautophagy/autophagy, molecular chaperones and the antioxidant cncC (the fly ortholog of NFE2L2/Nrf2) pathway. Also, proteasome KD increased genomic instability, altered metabolic pathways and severely disrupted mitochondrial functionality, triggering a cncC-dependent upregulation of mitostatic genes and enhanced rates of mitophagy. Whereas, overexpression of key regulators of antioxidant responses (e.g., cncC or foxo) could not suppress the deleterious effects of proteasome dysfunction; these were alleviated in both larvae and adult flies by modulating mitochondrial dynamics towards increased fusion or by enhancing autophagy. Our findings reveal the extensive functional wiring of genomic, proteostatic and mitostatic modules in higher metazoans. Also, they support the notion that age-related increase of proteotoxic stress due to decreased UPP activity deregulates all aspects of cellular functionality being thus a driving force for most age-related diseases. Abbreviations: ALP: autophagy-lysosome pathway; ARE: antioxidant response element; Atg8a: autophagy-related 8a; ATPsynß: ATP synthase, ß subunit; C-L: caspase-like proteasomal activity; cncC: cap-n-collar isoform-C; CT-L: chymotrypsin-like proteasomal activity; Drp1: dynamin related protein 1; ER: endoplasmic reticulum; foxo: forkhead box, sub-group O; GLU: glucose; GFP: green fluorescent protein; GLY: glycogen; Hsf: heat shock factor; Hsp: Heat shock protein; Keap1: kelch-like ECH-associated protein 1; Marf: mitochondrial assembly regulatory factor; NFE2L2/Nrf2: nuclear factor, erythroid 2 like 2; Opa1: optic atrophy 1; PN: proteostasis network; RNAi: RNA interference; ROS: reactive oxygen species; ref(2)P: refractory to sigma P; SQSTM1: sequestosome 1; SdhA: succinate dehydrogenase, subunit A; T-L: trypsin-like proteasomal activity; TREH: trehalose; UAS: upstream activation sequence; Ub: ubiquitin; UPR: unfolded protein response; UPP: ubiquitin-proteasome pathway.

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

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/Förster 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.

Microtubules Stabilization by Mutant Spastin Affects ER Morphology and Ca2+ Handling.

The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an in vivo approach involving the manipulation of MT dynamics in Drosophila melanogaster by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.

Manipulation of Mitochondria Dynamics Reveals Separate Roles for Form and Function in Mitochondria Distribution.

Mitochondria shape is controlled by membrane fusion and fission mediated by mitofusins, Opa1, and Drp1, whereas mitochondrial motility relies on microtubule motors. These processes govern mitochondria subcellular distribution, whose defects are emphasized in neurons because of their polarized structure. We have studied how perturbation of the fusion/fission balance affects mitochondria distribution in Drosophila axons. Knockdown of Marf or Opa1 resulted in progressive loss of distal mitochondria and in a distinct oxidative phosphorylation and membrane potential deficit. Downregulation of Drp1 rescued the lethality and bioenergetic defect caused by neuronal Marf RNAi, but induced only a modest restoration of axonal mitochondria distribution. Surprisingly, Drp1 knockdown rescued fragmentation and fully restored aberrant distribution of axonal mitochondria produced by Opa1 RNAi; however, Drp1 knockdown did not improve viability or mitochondria function. Our data show that proper morphology is critical for proper axonal mitochondria distribution independent of bioenergetic efficiency. The health of neurons largely depends on mitochondria function, but does not depend on shape or distribution.

Mitofusin 2: from functions to disease.

Mitochondria are highly dynamic organelles whose functions are essential for cell viability. Within the cell, the mitochondrial network is continuously remodeled through the balance between fusion and fission events. Moreover, it dynamically contacts other organelles, particularly the endoplasmic reticulum, with which it enterprises an important functional relationship able to modulate several cellular pathways. Being mitochondria key bioenergetics organelles, they have to be transported to all the specific high-energy demanding sites within the cell and, when damaged, they have to be efficiently removed. Among other proteins, Mitofusin 2 represents a key player in all these mitochondrial activities (fusion, trafficking, turnover, contacts with other organelles), the balance of which results in the appropriate mitochondrial shape, function, and distribution within the cell. Here we review the structural and functional properties of Mitofusin 2, highlighting its crucial role in several cell pathways, as well as in the pathogenesis of neurodegenerative diseases, metabolic disorders, cardiomyopathies, and cancer.

Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins.

Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔµH+) across the inner membrane. Although several drugs can modulate ΔµH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic ß-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.