Publications by authors named "Antonietta Franco"

16 Publications

  • Page 1 of 1

Burst mitofusin activation reverses neuromuscular dysfunction in murine CMT2A.

Elife 2020 10 19;9. Epub 2020 Oct 19.

Department of Internal Medicine, Pharmacogenomics, Washington University School of Medicine, St Louis, United States.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an untreatable childhood peripheral neuropathy caused by mutations of the mitochondrial fusion protein, mitofusin (MFN) 2. Here, pharmacological activation of endogenous normal mitofusins overcame dominant inhibitory effects of CMT2A mutants in reprogrammed human patient motor neurons, reversing hallmark mitochondrial stasis and fragmentation independent of causal mutation. In mice expressing human T105M, intermittent mitofusin activation with a small molecule, MiM111, normalized CMT2A neuromuscular dysfunction, reversed pre-treatment axon and skeletal myocyte atrophy, and enhanced axon regrowth by increasing mitochondrial transport within peripheral axons and promoting in vivo mitochondrial localization to neuromuscular junctional synapses. MiM111-treated T105M mouse neurons exhibited accelerated primary outgrowth and greater post-axotomy regrowth, linked to enhanced mitochondrial motility. MiM111 is the first pre-clinical candidate for CMT2A.
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http://dx.doi.org/10.7554/eLife.61119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655101PMC
October 2020

Discovery of 6-Phenylhexanamide Derivatives as Potent Stereoselective Mitofusin Activators for the Treatment of Mitochondrial Diseases.

J Med Chem 2020 07 18;63(13):7033-7051. Epub 2020 Jun 18.

Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, Missouri 63110, United States.

Mutations in the mitochondrial fusion protein mitofusin (MFN) 2 cause the chronic neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A), for which there is currently no treatment. Small-molecule activators of MFN1 and MFN2 enhance mitochondrial fusion and offer promise as therapy for this condition, but prototype compounds have poor pharmacokinetic properties. Herein, we describe a rational design of a series of 6-phenylhexanamide derivatives whose pharmacokinetic optimization yielded a 4-hydroxycyclohexyl analogue, , with the potency, selectivity, and oral bioavailability of a preclinical candidate. Studies of - and -4-hydroxycyclohexyl isostereomers unexpectedly revealed functionality and protein engagement exclusively for the trans form, . Preclinical absorption, distribution, metabolism, and excretion (ADME) and target engagement studies of support further development of 6-phenylhexanamide derivatives as therapeutic agents for human CMT2A.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731667PMC
July 2020

Pharmacological inhibition of GRK2 improves cardiac metabolism and function in experimental heart failure.

ESC Heart Fail 2020 08 30;7(4):1571-1584. Epub 2020 Apr 30.

Department of Advanced Biomedical Sciences, 'Federico II' University of Naples, Naples, Italy.

Aims: The effects of GRK2 inhibition on myocardial metabolism in heart failure (HF) are unchartered. In this work, we evaluated the impact of pharmacological inhibition of GRK2 by a cyclic peptide, C7, on metabolic, biochemical, and functional phenotypes in experimental HF.

Methods And Results: C7 was initially tested on adult mice ventricular myocyte from wild type and GRK2 myocardial deficient mice (GRK2-cKO), to assess the selectivity on GRK2 inhibition. Then, chronic infusion of 2 mg/kg/day of C7 was performed in HF mice with cryogenic myocardial infarction. Cardiac function in vivo was assessed by echocardiography and cardiac catheterization. Histological, biochemical, and metabolic studies were performed on heart samples at time points. C7 induces a significant increase of contractility in wild type but not in adult ventricle myocytes from GRK2-cKO mice, thus confirming C7 selectivity for GRK2. In HF mice, 4 weeks of treatment with C7 improved metabolic features, including mitochondrial organization and function, and restored the biochemical and contractile responses.

Conclusions: GRK2 is a critical molecule in the physiological regulation of cardiac metabolism. Its alterations in the failing heart can be pharmacologically targeted, leading to the correction of metabolic and functional abnormalities observed in HF.
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http://dx.doi.org/10.1002/ehf2.12706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373898PMC
August 2020

The tethering function of mitofusin2 controls osteoclast differentiation by modulating the Ca-NFATc1 axis.

J Biol Chem 2020 05 12;295(19):6629-6640. Epub 2020 Mar 12.

Division of Bone and Mineral Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

Dynamic regulation of the mitochondrial network by mitofusins (MFNs) modulates energy production, cell survival, and many intracellular signaling events, including calcium handling. However, the relative importance of specific mitochondrial functions and their dependence on MFNs vary greatly among cell types. Osteoclasts have many mitochondria, and increased mitochondrial biogenesis and oxidative phosphorylation enhance bone resorption, but little is known about the mitochondrial network or MFNs in osteoclasts. Because expression of each MFN isoform increases with osteoclastogenesis, we conditionally deleted MFN1 and MFN2 (double conditional KO (dcKO)) in murine osteoclast precursors, finding that this increased bone mass in young female mice and abolished osteoclast precursor differentiation into mature osteoclasts Defective osteoclastogenesis was reversed by overexpression of MFN2 but not MFN1; therefore, we generated mice lacking only MFN2 in osteoclasts. MFN2-deficient female mice had increased bone mass at 1 year and resistance to Receptor Activator of NF-κB Ligand (RANKL)-induced osteolysis at 8 weeks. To explore whether MFN-mediated tethering or mitophagy is important for osteoclastogenesis, we overexpressed MFN2 variants defective in either function in dcKO precursors and found that, although mitophagy was dispensable for differentiation, tethering was required. Because the master osteoclastogenic transcriptional regulator nuclear factor of activated T cells 1 (NFATc1) is calcium-regulated, we assessed calcium release from the endoplasmic reticulum and store-operated calcium entry and found that the latter was blunted in dcKO cells. Restored osteoclast differentiation by expression of intact MFN2 or the mitophagy-defective variant was associated with normalization of store-operated calcium entry and NFATc1 levels, indicating that MFN2 controls mitochondrion-endoplasmic reticulum tethering in osteoclasts.
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http://dx.doi.org/10.1074/jbc.RA119.012023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212632PMC
May 2020

Restoring mitofusin balance prevents axonal degeneration in a Charcot-Marie-Tooth type 2A model.

J Clin Invest 2019 03 18;129(4):1756-1771. Epub 2019 Mar 18.

Center for Neural Science and Medicine, and.

Mitofusin-2 (MFN2) is a mitochondrial outer-membrane protein that plays a pivotal role in mitochondrial dynamics in most tissues, yet mutations in MFN2, which cause Charcot-Marie-Tooth disease type 2A (CMT2A), primarily affect the nervous system. We generated a transgenic mouse model of CMT2A that developed severe early onset vision loss and neurological deficits, axonal degeneration without cell body loss, and cytoplasmic and axonal accumulations of fragmented mitochondria. While mitochondrial aggregates were labeled for mitophagy, mutant MFN2 did not inhibit Parkin-mediated degradation, but instead had a dominant negative effect on mitochondrial fusion only when MFN1 was at low levels, as occurs in neurons. Finally, using a transgenic approach, we found that augmenting the level of MFN1 in the nervous system in vivo rescued all phenotypes in mutant MFN2R94Q-expressing mice. These data demonstrate that the MFN1/MFN2 ratio is a key determinant of tissue specificity in CMT2A and indicate that augmentation of MFN1 in the nervous system is a viable therapeutic strategy for the disease.
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http://dx.doi.org/10.1172/JCI124194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436852PMC
March 2019

G-protein receptor kinases 2, 5 and 6 redundantly modulate Smoothened-GATA transcriptional crosstalk in fetal mouse hearts.

J Mol Cell Cardiol 2018 08 30;121:60-68. Epub 2018 Jun 30.

Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, United States. Electronic address:

G-protein receptor kinases (GRKs) regulate adult hearts by modulating inotropic, chronotropic and hypertrophic signaling of 7-transmembrane spanning neurohormone receptors. GRK-mediated desensitization and downregulation of β-adrenergic receptors has been implicated in adult heart failure; GRKs are therefore a promising therapeutic target. However, germ-line (but not cardiomyocyte-specific) GRK2 deletion provoked lethal fetal heart defects, suggesting an unexplained role for GRKs in heart development. Here we undertook to better understand the consequences of GRK deficiency on fetal heart development by creating mice and cultured murine embryonic fibroblasts (MEFs) having floxed GRK2 and GRK5 alleles on the GRK6 null background; simultaneous conditional deletion of these 3 GRK genes was achieved using Nkx2-5 Cre or adenoviral Cre, respectively. Phenotypes were related to GRK-modulated gene expression using whole-transcriptome RNA sequencing, RT-qPCR, and luciferase reporter assays. In cultured MEFs the atypical 7-transmembrane spanning protein and GRK2 substrate Smoothened (Smo) stimulated Gli-mediated transcriptional activity, which was interrupted by deleting GRK2/5/6. Mice with Nkx2-5 Cre mediated GRK2/5/6 ablation died between E15.5 and E16.5, whereas mice expressing any one of these 3 GRKs (i.e. GRK2/5, GRK2/6 or GRK5/6 deleted) were developmentally normal. GRK2/5/6 triple null mice at E14.5 exhibited left and right heart blood intermixing through single atrioventricular valves or large membranous ventricular septal defects. Hedgehog and GATA pathway gene expression promoted by Smo/Gli was suppressed in GRK2/5/6 deficient fetal hearts and MEFs. These data indicate that GRK2, GRK5 and GRK6 redundantly modulate Smo-GATA crosstalk in fetal mouse hearts, orchestrating transcriptional pathways previously linked to clinical and experimental atrioventricular canal defects. GRK modulation of Smo reflects convergence of conventional neurohormonal signaling and transcriptional regulation pathways, comprising an unanticipated mechanism for spatiotemporal orchestration of developmental gene expression in the heart.
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http://dx.doi.org/10.1016/j.yjmcc.2018.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178805PMC
August 2018

MFN2 agonists reverse mitochondrial defects in preclinical models of Charcot-Marie-Tooth disease type 2A.

Science 2018 04;360(6386):336-341

Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.

Mitofusins (MFNs) promote fusion-mediated mitochondrial content exchange and subcellular trafficking. Mutations in cause neurodegenerative Charcot-Marie-Tooth disease type 2A (CMT2A). We showed that MFN2 activity can be determined by Met and His interactions with Asp and Leu and controlled by PINK1 kinase-mediated phosphorylation of adjacent MFN2 Ser Small-molecule mimics of the peptide-peptide interface of MFN2 disrupted this interaction, allosterically activating MFN2 and promoting mitochondrial fusion. These first-in-class mitofusin agonists overcame dominant mitochondrial defects provoked in cultured neurons by CMT2A mutants MFN2 Arg→Gln and MFN2 Thr→Met, as demonstrated by amelioration of mitochondrial dysmotility, fragmentation, depolarization, and clumping. A mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of MFN2 Thr→Met mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism and/or trafficking.
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http://dx.doi.org/10.1126/science.aao1785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109362PMC
April 2018

GRK2 moderates the acute mitochondrial damage to ionizing radiation exposure by promoting mitochondrial fission/fusion.

Cell Death Discov 2018 Dec 14;4:25. Epub 2018 Feb 14.

3Department of Medicine, Surgery and Dentistry, University of Salerno, Baronissi, Italy.

The modern understanding of the G protein-coupled receptor kinase 2 has grown towards the definition of a stress protein, for its ability to rapidly compartmentalize within the cell in response to acute stimulation. Also, mitochondria can be regulated by GRK2 localization. We show that Ionizing Radiation (IR) exposure acutely damages mitochondria regarding mass, morphology, and respiration, with recovery in a framework of hours. This phenomenon is actively regulated by GRK2, whose overexpression results to be protective, and reciprocally, deletion accelerates degenerative processes. The regulatory effects of the kinase involve a new interactome that includes binding HSP90 and binding and phosphorylation of the key molecules involved in the process of mitochondrial fusion and recovery: MFN-1 and 2.
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http://dx.doi.org/10.1038/s41420-018-0028-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841414PMC
December 2018

Abrogating Mitochondrial Dynamics in Mouse Hearts Accelerates Mitochondrial Senescence.

Cell Metab 2017 Dec 26;26(6):872-883.e5. Epub 2017 Oct 26.

Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA. Electronic address:

Mitochondrial fusion and fission are critical to heart health; genetically interrupting either is rapidly lethal. To understand whether it is loss of, or the imbalance between, fusion and fission that underlies observed cardiac phenotypes, we engineered mice in which Mfn-mediated fusion and Drp1-mediated fission could be concomitantly abolished. Compared to fusion-defective Mfn1/Mfn2 cardiac knockout or fission-defective Drp1 cardiac knockout mice, Mfn1/Mfn2/Drp1 cardiac triple-knockout mice survived longer and manifested a unique pathological form of cardiac hypertrophy. Over time, however, combined abrogation of fission and fusion provoked massive progressive mitochondrial accumulation that severely distorted cardiomyocyte sarcomeric architecture. Mitochondrial biogenesis was not responsible for mitochondrial superabundance, whereas mitophagy was suppressed despite impaired mitochondrial proteostasis. Similar but milder defects were observed in aged hearts. Thus, cardiomyopathies linked to dynamic imbalance between fission and fusion are temporarily mitigated by forced mitochondrial adynamism at the cost of compromising mitochondrial quantity control and accelerating mitochondrial senescence.
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http://dx.doi.org/10.1016/j.cmet.2017.09.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5718956PMC
December 2017

Correcting mitochondrial fusion by manipulating mitofusin conformations.

Nature 2016 12 24;540(7631):74-79. Epub 2016 Oct 24.

Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

Mitochondria are dynamic organelles that exchange contents and undergo remodelling during cyclic fusion and fission. Genetic mutations in MFN2 (the gene encoding mitofusin 2) interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A). It has not yet been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is not completely understood. Here we show that mitofusins adopt either a fusion-constrained or a fusion-permissive molecular conformation, directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated in mouse cells by targeting these conformational transitions. On the basis of this model, we engineered a cell-permeant minipeptide to destabilize the fusion-constrained conformation of mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons that harbour CMT2A-associated genetic defects. The relationship between the conformational plasticity of mitofusin 2 and mitochondrial dynamism reveals a central mechanism that regulates mitochondrial fusion, the manipulation of which can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.
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http://dx.doi.org/10.1038/nature20156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315023PMC
December 2016

Integrating GRK2 and NFkappaB in the Pathophysiology of Cardiac Hypertrophy.

J Cardiovasc Transl Res 2015 Nov 30;8(8):493-502. Epub 2015 Jul 30.

Department of Medicine and Surgery, University of Salerno, Via Salvador Allende, 84081, Baronissi, SA, Italy.

G protein coupled receptor kinase type 2 (GRK2) plays an important role in the development and maintenance of cardiac hypertrophy and heart failure even if its exact role is still unknown. In this study, we assessed the effect of GRK2 on the regulation of cardiac hypertrophy. In H9C2 cells, GRK2 overexpression increased atrial natriuretic factor (ANF) activity and enhanced phenylephrine-induced ANF response, and this is associated with an increase of NFκB transcriptional activity. The kinase dead mutant and a synthetic inhibitor of GRK2 activity exerted the opposite effect, suggesting that GRK2 regulates hypertrophy through upregulation of NFκB activity in a phosphorylation-dependent manner. In two different in vivo models of left ventricle hypertrophy (LVH), the selective inhibition of GRK2 activity prevented hypertrophy and reduced NFκB transcription activity. Our results suggest a previously undisclosed role for GRK2 in the regulation of hypertrophic responses and propose GRK2 as potential therapeutic target for limiting LVH.
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http://dx.doi.org/10.1007/s12265-015-9646-0DOI Listing
November 2015

Good at Heart: Preserving Cardiac Metabolism during aging.

Curr Diabetes Rev 2015 ;12(2):90-9

Department of Medicine and Surgery, University of Salerno, Italy.

The natural process of aging determinates several cardiac modifications with increased susceptibility to heart diseases and ultimately converging on development of chronic heart failure as final stage. These changes mainly include left ventricular hypertrophy, diastolic dysfunction, valvular degeneration, increased cardiac fibrosis, increased prevalence of atrial fibrillation, and decreased maximal exercise capacity, as demonstrated in several humans and animal models of aging. While different theories have been proposed to explain the natural process of aging, it is clear that most of the alterations affect mechanisms involved in cell homeostasis and maintenance. Latest research studies have in particular focused on role of mitochondrial oxidative stress, energy production and mitochondria quality control. This article reviews the central role played by this organelle in aging and the role of new molecular players involved into the progression toward heart failure and potentially susceptible of new "anti-aging" strategies.
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http://dx.doi.org/10.2174/1573399811666150722124958DOI Listing
September 2016

Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy.

PLoS One 2015 25;10(6):e0130477. Epub 2015 Jun 25.

Department of Translational and Medical Sciences, Federico II University, Naples, Italy.

Aims: Activation of Ca2+/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization.

Methods And Results: In H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.

Conclusion: These results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130477PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4481531PMC
March 2016

Endothelial G protein-coupled receptor kinase 2 regulates vascular homeostasis through the control of free radical oxygen species.

Arterioscler Thromb Vasc Biol 2013 Oct 15;33(10):2415-24. Epub 2013 Aug 15.

From the Department of Medicine and Surgery, University of Salerno, Salerno, Italy (M.C., E.C., G.l.); Department of Pharmacology, Center for Translational Medicine, Temple University, Philadelphia, PA (M.C.); Department of Biomedical Sciences, University of Naples Federico II, Naples, Italy (D.S., A.F., C.D.G., R.A., M.G.M., B.T.); Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO (G.W.D.); and IRCCS Multimedica, Milan, Italy (G.I.).

Objective: The role of endothelial G protein-coupled receptor kinase 2 (GRK2) was investigated in mice with selective deletion of the kinase in the endothelium (Tie2-CRE/GRK2(fl/fl)).

Approach And Results: Aortas from Tie2-CRE/GRK2(fl/fl) presented functional and structural alterations as compared with control GRK2(fl/fl) mice. In particular, vasoconstriction was blunted to different agonists, and collagen and elastic rearrangement and macrophage infiltration were observed. In primary cultured endothelial cells deficient for GRK2, mitochondrial reactive oxygen species was increased, leading to expression of cytokines. Chronic treatment with a reactive oxygen species scavenger in mice corrected the vascular phenotype by recovering vasoconstriction, structural abnormalities, and reducing macrophage infiltration.

Conclusions: These results demonstrate that GRK2 removal compromises vascular phenotype and integrity by increasing endothelial reactive oxygen species production.
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http://dx.doi.org/10.1161/ATVBAHA.113.302262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262246PMC
October 2013