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.

ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca2+ Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons (MNs), probably by a combination of cell- and non-cell-autonomous processes. The past decades have brought many important insights into the role of astrocytes in nervous system function and disease, including the implication in ALS pathogenesis possibly through the impairment of Ca2+-dependent astrocyte-MN cross-talk. In this respect, it has been recently proposed that altered astrocytic store-operated Ca2+ entry (SOCE) may underlie aberrant gliotransmitter release and astrocyte-mediated neurotoxicity in ALS. These observations prompted us to a thorough investigation of SOCE in primary astrocytes from the spinal cord of the SOD1(G93A) ALS mouse model in comparison with the SOD1(WT)-expressing controls. To this purpose, we employed, for the first time in the field, genetically-encoded Ca2+ indicators, allowing the direct assessment of Ca2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca2+-ATPase and lower ER resting Ca2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage.

The Link of the Prion Protein with Ca2+ Metabolism and ROS Production, and the Possible Implication in Aß Toxicity.

The cellular prion protein (PrPC) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aß oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.

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.

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.

Generation and validation of novel adeno-associated viral vectors for the analysis of Ca2+ homeostasis in motor neurons.

A finely tuned Ca2+ homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca2+ oscillations direct the proper coordination of electro-chemical signals and overall neuronal metabolism. Once such a precise regulation is unbalanced, however, neuronal functions and viability are severely compromised. Accordingly, disturbed Ca2+ metabolism has often been claimed as a major contributor to different neurodegenerative disorders, such as amyotrophic lateral sclerosis that is characterised by selective motor neuron (MN) damage. This notion highlights the need for probes for the specific and precise analysis of local Ca2+ dynamics in MNs. Here, we generated and functionally validated adeno-associated viral vectors for the expression of gene-encoded fluorescent Ca2+ indicators targeted to different cell domains, under the transcriptional control of a MN-specific promoter. We demonstrated that the probes are specifically expressed, and allow reliable local Ca2+ measurements, in MNs from murine primary spinal cord cultures, and can also be expressed in spinal cord MNs in vivo, upon systemic administration to newborn mice. Preliminary analyses using these novel vectors have shown larger cytosolic Ca2+ responses following stimulation of AMPA receptors in the cytosol of primary cultured MNs from a murine genetic model of ALS compared to the healthy counterpart.

Structural and Antimicrobial Features of Peptides Related to Myticin C, a Special Defense Molecule from the Mediterranean Mussel Mytilus galloprovincialis.

Mussels (Mytilus spp.) have a large repertoire of cysteine-stabilized α,ß peptides, and myticin C (MytC) was identified in some hundreds of transcript variants after in vivo immunostimulation. Using a sequence expressed in Italian mussels, we computed the MytC structure and synthesized the mature MytC and related peptide fragments (some of them also prepared in oxidized form) to accurately assess their antibacterial and antifungal activity. Only when tested at pH 5 was the reduced MytC as well as reduced and oxidized fragments including structural ß-elements able to inhibit Gram-positive and -negative bacteria (MIC ranges of 4-32 and 8-32 µM, respectively). Such fragments caused selective Escherichia coli killing (MBC of 8-32 µM) but scarcely inhibited two fungal strains. In detail, the antimicrobial ß-hairpin MytC[19-40]SOX caused membrane-disrupting effects in E. coli despite its partially ordered conformation in membrane-mimetic environments. In perspective, MytC-derived peptides could be employed to protect acidic mucosal tissues, in cosmetic and food products, and, possibly, as adjuvants in aquaculture.