Magnesium accumulation upon cyclin M4 silencing activates microsomal triglyceride transfer protein improving NASH

Background & Aims: Perturbations of intracellular magnesium (Mg) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). Methods: Serum Mg levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine® or conjugated to N-acetylgalactosamine. Results: Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs. Conclusions: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH. Lay summary: Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein.Ministerio de Ciencia e Innovación, Programa Retos-Colaboración RTC2019-007125-1 (for JS and MLM-C); Instituto de Salud Carlos III, Proyectos de Investigación en Salud DTS20/00138 (for JS and MLM-C); Departamento de Industria del Gobierno Vasco (for MLM-C); Ministerio de Ciencia, Innovación y Universidades MICINN: SAF2017-87301-R and RTI2018-096759-A-100 integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER (for MLM-C and TCD, respectively); BIOEF (Basque Foundation for Innovation and Health Research); EITB Maratoia BIO15/CA/014; Asociación Española contra el Cáncer (MLM-C, TCD); Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (for MLM); La Caixa Foundation Program (for MLM); Fundacion BBVA UMBRELLA project (for MLM); BFU2015-70067-REDC, BFU2016-77408-R, and BES-2017- 080435 (MINECO / FEDER, UE) and the FIGHT-CNNM2 project from the EJP RD Joint Transnational Call (JTC2019) (Ref. AC19/ 00073) (for LAM-C); RTI2018-095134-B-100 and Grupos de Investigación del Sistema Universitario Vasco (IT971-16) (for PA); National Institutes of Health under grant CA217817 (for DB); AGL2014-54585-R, AGL-2017-86927-R and EQC2018-004897-P from MINECO; PC0148-2016-0149 and PAI-BIO311 from Junta de Andalucía (for FM). Ciberehd_ISCIII_MINECO is funded by the Instituto de Salud Carlos III. We thank Silence Therapeutics plc. for the financial support provided. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV- 2016-0644)

A Fluorescent Activatable Probe for Imaging Intracellular Mg2+

An activatable BODIPY probe for in vitro detection and fluorescence cell imaging of free Mg2+ without interference from Ca2+ is described. Fluorescence amplification of the probe is observed upon detection of physiological concentrations of Mg2+ due to reduced rotation of the fluorophore and effective chelation by a quinolizine-based core

Visualizing Compartmentalized Cellular Mg²⁺ on Demand with Small-Molecule Fluorescent Sensors

The study of intracellular metal ion compartmentalization and trafficking involved in cellular processes demands sensors with controllable localization for the measurement of organelle-specific levels of cations with subcellular resolution. We introduce herein a new two-step strategy for in situ anchoring and activation of a fluorescent Mg²⁺ sensor within an organelle of choice, using a fast fluorogenic reaction between a tetrazine-functionalized pro-sensor, Mag-<i>S</i>-Tz, and a strained bicyclononyne conjugated to a genetically encoded HaloTag fusion protein of known cellular localization. Protein conjugation does not affect the metal-binding properties of the <i>o</i>-aminophenol-<i>N</i>,<i>N</i>,<i>O</i>-triacetic acid (APTRA)-based fluorescent indicator, which displays a dissociation constant <i>K</i>_d = 3.1 mM suitable for the detection of low millimolar concentrations of chelatable Mg²⁺ typical of the intracellular environment. We demonstrate the application of our sensing system for the ratiometric detection of Mg²⁺ in target organelles in HEK 293T cells, providing the first direct comparison of subcellular pools of the metal without interfering signal from other compartments. Activation of the fluorescence in situ through a fluorogenic conjugation step effectively constrains the fluorescence signal to the locale of interest, thus improving the spatial resolution in imaging applications and eliminating the need for washout of mislocalized sensor. The labeling strategy is fully compatible with live cell imaging, and provides a valuable tool for tracking changes in metal distribution that to date have been an unsolved mystery in magnesium biology