Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity.

Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.

The newly discovered Parkinson's disease associated Finnish mutation (A53E) attenuates α-synuclein aggregation and membrane binding.

α-Synuclein (α-Syn) oligomerization and amyloid formation are associated with Parkinson's disease (PD) pathogenesis. Studying familial α-Syn mutants associated with early onset PD has therapeutic importance. Here we report the aggregation kinetics and other biophysical properties of a newly discovered PD associated Finnish mutation (A53E). Our in vitro study demonstrated that A53E attenuated α-Syn aggregation and amyloid formation without altering the major secondary structure and initial oligomerization tendency. Further, A53E showed reduced membrane binding affinity compared to A53T and WT. The present study would help to delineate the role of A53E mutation in early onset PD pathogenesis.

Induction of pluripotency.

The molecular and phenotypic irreversibility of mammalian cell differentiation was a fundamental principle of developmental biology at least until the 1980s, despite numerous reports dating back to the 1950s of the induction of pluripotency in amphibian cells by nuclear transfer (NT). Landmark reports in the 1980s and 1990s in sheep progressively challenged this dogmatic assumption; firstly, embryonic development of reconstructed embryos comprising whole (donor) blastomeres fused to enucleated oocytes, and famously, the cloning of Dolly from a terminally differentiated cell. Thus, the intrinsic ability of oocyte-derived factors to reverse the differentiated phenotype was confirmed. The concomitant elucidation of methods for human embryonic stem cell isolation and cultivation presented opportunities for therapeutic cell replacement strategies, particularly through NT of patient nuclei to enucleated oocytes for subsequent isolation of patient-specific (autologous), pluripotent cells from the resulting blastocysts. Associated logistical limitations of working with human oocytes, in addition to ethical and moral objections prompted exploration of alternative approaches to generate autologous stem cells for therapy, utilizing the full repertoire of factors characteristic of pluripotency, primarily through cell fusion and use of pluripotent cell extracts. Stunningly, in 2006, Japanese scientists described somatic cell reprogramming through delivery of four key factors (identified through a deductive approach from 24 candidate genes). Although less efficient than previous approaches, much of current stem cell research adopts this focused approach to cell reprogramming and (autologous) cell therapy. This chapter is a quasi-historical commentary of the various aforementioned approaches for the induction of pluripotency in lineage-committed cells, and introduces transcriptional and epigenetic changes occurring during reprogramming.

Development of polyvinyl alcohol-gelatin membranes for antibiotic delivery in the eye.

AIM: The aim of the present study was to develop biosynthetic hybrid polymer-based ocular insert for topical administration of antibiotics for treatment of ocular infections. METHODS: Ciprofloxacin hydrochloride-loaded three different inserts were prepared by solution casting method by esterification of a biopolymer (gelatin) with a synthetic polymer (polyvinyl alcohol, PVA). Esterification between PVA and gelatin was confirmed by Fourier transform infrared spectroscopy. RESULTS: Inserts were found to be wettable and swellable with simulated tear fluid and had a contact angle <50° with simulated tear fluid. Mechanical properties of PVA-gelatin (10:3 wt%) inserts included a maximal tensile strength of 8.6 ± 2 MPa and the inserts showed adequate mucoadhesion with reconstituted mucin. In vitro drug release of ciprofloxacin hydrochloride for up to 24 hours was observed from the inserts. Inserts were found to be biocompatible using SIRC rabbit corneal epithelial cell line by sulforhodamine B assay and by Draize test in albino rabbits. Further inserts showed higher ocular penetration of sodium fluorescein in goat eye as compared to eyedrop solution. CONCLUSIONS: In brief, the study suggests that PVA-gelatin polymeric blends are promising as ocular inserts for prolonged release of antibiotic in the eye as compared to eyedrops. Such inserts may also be therapeutically beneficial for treatment of corneal ulcers and external ocular infections.

Regulation of Ca2+ exchanges and signaling in mitochondria.

Mitochondrial calcium (mCa2+) homeostasis also plays a key role in the buffering of cytosolic calcium (cCa2+) and calcium transported into the mitochondrial matrix regulates cellular metabolism, migration and cell fate decisions. Recent work has highlighted the importance of mCa2+ homeostasis in regulating cellular function. The discovery of the mCa2+ uptake complex has shed new light on the role of mCa2+ dynamics in cytoskeletal remodeling, mitochondrial shape and motility in cellular dynamics. Here we attempt to decipher the vast landscape of calcium regulatory effects of the mitochondria, the underlying mechanisms and the dynamics that control cellular function.

Liposome-encapsulated fish oil protein-tagged gold nanoparticles for intra-articular therapy in osteoarthritis.

AIM: To provide multilayered combination therapies encompassing nanoparticles and organic peptides and to assess their efficacy in the treatment of arthritis. MATERIALS & METHODS: Fish oil protein (FP) was isolated from fish oil glands and tagged with spherical gold nanoparticles (GNPs). Tagged GNPs were encapsulated in DPPC liposomes (FP-GNP-DPPC) and characterized. RESULTS & CONCLUSION: FP increased the hydrophilicity of GNP, while encapsulation of FP-GNP within liposomes increased the hydrophobicity. In vitro release studies of FP-GNP-DPPC exhibited sustained release of FP in simulated synovial fluid. FP-GNP-DPPC injected into intra-articular joints of rats displayed anti-osteoarthritic effects in osteoarthritic rat model. This is the first study to report the anti-osteoarthritic activity of FP and DPPC encapsulated FP-GNP liposomes.

A Selective and Cell-Permeable Mitochondrial Calcium Uniporter (MCU) Inhibitor Preserves Mitochondrial Bioenergetics after Hypoxia/Reoxygenation Injury.

Mitochondrial Ca2+ (mCa2+) uptake mediated by the mitochondrial calcium uniporter (MCU) plays a critical role in signal transduction, bioenergetics, and cell death, and its dysregulation is linked to several human diseases. In this study, we report a new ruthenium complex Ru265 that is cell-permeable, minimally toxic, and highly potent with respect to MCU inhibition. Cells treated with Ru265 show inhibited MCU activity without any effect on cytosolic Ca2+ dynamics and mitochondrial membrane potential (ΔΨm). Dose-dependent studies reveal that Ru265 is more potent than the currently employed MCU inhibitor Ru360. Site-directed mutagenesis of Cys97 in the N-terminal domain of human MCU ablates the inhibitory activity of Ru265, suggesting that this matrix-residing domain is its target site. Additionally, Ru265 prevented hypoxia/reoxygenation injury and subsequent mitochondrial dysfunction, demonstrating that this new inhibitor is a valuable tool for studying the functional role of the MCU in intact biological models.

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (mCa2+) uptake, delays cytosolic Ca2+ (cCa2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid mCa2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism.

FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation.

Although many factors contribute to cellular differentiation, the role of mitochondria Ca2+ dynamics during development remains unexplored. Because mammalian embryonic epiblasts reside in a hypoxic environment, we intended to understand whether mCa2+ and its transport machineries are regulated during hypoxia. Tissues from multiple organs of developing mouse embryo evidenced a suppression of MICU1 expression with nominal changes on other MCU complex components. As surrogate models, we here utilized human embryonic stem cells (hESCs)/induced pluripotent stem cells (hiPSCs) and primary neonatal myocytes to delineate the mechanisms that control mCa2+ and bioenergetics during development. Analysis of MICU1 expression in hESCs/hiPSCs showed low abundance of MICU1 due to its direct repression by Foxd1. Experimentally, restoration of MICU1 established the periodic cCa2+ oscillations and promoted cellular differentiation and maturation. These findings establish a role of mCa2+ dynamics in regulation of cellular differentiation and reveal a molecular mechanism underlying this contribution through differential regulation of MICU1.

MIRO-1 Determines Mitochondrial Shape Transition upon GPCR Activation and Ca2+ Stress.

Mitochondria shape cytosolic calcium ([Ca2+]c) transients and utilize the mitochondrial Ca2+ ([Ca2+]m) in exchange for bioenergetics output. Conversely, dysregulated [Ca2+]c causes [Ca2+]m overload and induces permeability transition pore and cell death. Ablation of MCU-mediated Ca2+ uptake exhibited elevated [Ca2+]c and failed to prevent stress-induced cell death. The mechanisms for these effects remain elusive. Here, we report that mitochondria undergo a cytosolic Ca2+-induced shape change that is distinct from mitochondrial fission and swelling. [Ca2+]c elevation, but not MCU-mediated Ca2+ uptake, appears to be essential for the process we term mitochondrial shape transition (MiST). MiST is mediated by the mitochondrial protein Miro1 through its EF-hand domain 1 in multiple cell types. Moreover, Ca2+-dependent disruption of Miro1/KIF5B/tubulin complex is determined by Miro1 EF1 domain. Functionally, Miro1-dependent MiST is essential for autophagy/mitophagy that is attenuated in Miro1 EF1 mutants. Thus, Miro1 is a cytosolic Ca2+ sensor that decodes metazoan Ca2+ signals as MiST.

Mechanically Stiff, Zinc Cross-Linked Nanocomposite Scaffolds with Improved Osteostimulation and Antibacterial Properties.

Nanocomposite scaffolds are studied widely due to their resemblance with the natural extracellular matrix of bone; but their use as a bone tissue engineered scaffold is clinically hampered due to low mechanical stiffness, inadequate osteoconduction, and graft associated infections. The purpose of the current study was the development of a mechanically stiff nanocomposite scaffold using biodegradable gellan and xanthan polymers reinforced with bioglass nanoparticles (nB) (Size: 20-120 nm). These nanocomposite scaffolds were cross-linked with zinc sulfate ions to improve their osteoconduction and antibacterial properties for the regeneration of a functional bone. The compressive strength and modulus of the optimized nanocomposite scaffold (1% w/v polymer reinforced with 4%w/v nB nanoparticles, cross-linked with 1.5 mM zinc sulfate) was 1.91 ± 0.31 MPa and 20.36 ± 1.08 MPa, respectively, which was comparable to the trabecular bone and very high compared to nanocomposite scaffolds reported in earlier studies. Further, in vitro simulated body fluid (SBF) study suggested deposition of biomimetic apatite on the surface of zinc cross-linked nanocomposite scaffolds confirming their bioactivity. MG 63 osteoblast-like cells cultured with the nanocomposite scaffolds responded to matrix stiffness with better adhesion, spreading and cellular interconnections compared to the polymeric gellan and xanthan scaffolds. Incorporation of bioglass nanoparticles and zinc cross-linker in nanocomposite scaffolds demonstrated 62% increment in expression of alkaline phosphatase activity (ALP) and 150% increment in calcium deposition of MG 63 osteoblast-like cells compared to just gellan and xanthan polymeric scaffolds. Furthermore, zinc cross-linked nanocomposite scaffolds significantly inhibited the growth of Gram-positive Bacillus subtilis (70% reduction) and Gram-negative Escherichia coli (81% reduction) bacteria. This study demonstrated a facile approach to tune the mechanical stiffness, bioactivity, osteoconduction potential and bacteriostatic properties of scaffolds, which marked it as a potential bone tissue engineered scaffold.

Structural Insights into Mitochondrial Calcium Uniporter Regulation by Divalent Cations.

Calcium (Ca(2+)) flux into the matrix is tightly controlled by the mitochondrial Ca(2+) uniporter (MCU) due to vital roles in cell death and bioenergetics. However, the precise atomic mechanisms of MCU regulation remain unclear. Here, we solved the crystal structure of the N-terminal matrix domain of human MCU, revealing a ß-grasp-like fold with a cluster of negatively charged residues that interacts with divalent cations. Binding of Ca(2+) or Mg(2+) destabilizes and shifts the self-association equilibrium of the domain toward monomer. Mutational disruption of the acidic face weakens oligomerization of the isolated matrix domain and full-length human protein similar to cation binding and markedly decreases MCU activity. Moreover, mitochondrial Mg(2+) loading or blockade of mitochondrial Ca(2+) extrusion suppresses MCU Ca(2+)-uptake rates. Collectively, our data reveal that the ß-grasp-like matrix region harbors an MCU-regulating acidic patch that inhibits human MCU activity in response to Mg(2+) and Ca(2+) binding.

Myocardial infarction: stem cell transplantation for cardiac regeneration.

It is estimated that by 2030, almost 23.6 million people will perish from cardiovascular disease, according to the WHO. The review discusses advances in stem cell therapy for myocardial infarction, including cell sources, methods of differentiation, expansion selection and their route of delivery. Skeletal muscle cells, hematopoietic cells and mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs)-derived cardiomyocytes have advanced to the clinical stage, while induced pluripotent cells (iPSCs) are yet to be considered clinically. Delivery of cells to the sites of injury and their subsequent retention is a major issue. The development of supportive scaffold matrices to facilitate stem cell retention and differentiation are analyzed. The review outlines clinical translation of conjugate stem cell-based cellular therapeutics post-myocardial infarction.

Cytotoxic helix-rich oligomer formation by melittin and pancreatic polypeptide.

Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson's associated α-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail.

Biodegradable hybrid polymeric membranes for ocular drug delivery.

Ophthalmic delivery systems such as ocular inserts are useful strategies to improve the ocular bioavailability of topically administered drugs. In the present study polyvinyl alcohol and sodium carboxymethylcellulose based ocular inserts were prepared by solution casting for sustained drug delivery of ciprofloxacin for treatment of topical infections. The polymers were esterified and the formation of ester bonds was confirmed by Fourier transform infrared spectroscopy. The inserts had a smooth structure with a surface roughness of 7.3 nm. Inserts were found to be wettable by simulated tear fluid with contact angle <45 degrees . Mechanical testing results indicated that the tensile strength of polyvinyl alcohol-sodium carboxymethylcellulose (10:2 wt.%) inserts was up to 8.9 + or - 1.9 MPa, which is adequate to resist the pressure likely to be exerted during application. In vitro drug release kinetics showed sustained release of ciprofloxacin for up to 48 h from the inserts. Sodium fluorescein-loaded inserts showed higher penetration of the dye in the posterior segment tissues of explanted goat eye balls as compared with an eye drop solution of sodium fluorescein. The inserts were non-toxic to corneal epithelial cells and showed no signs of acute ocular toxicity in in vivo studies in albino rabbits.