Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver

Luis González-Moreno; Andrea Santamaría-Cano; Alberto Paradela; María Luz Martínez-Chantar; Miguel Á Martín; Mercedes Pérez-Carreras; Alberto García-Picazo; Jesús Vázquez; Enrique Calvo; Gloria González-Aseguinolaza; Takeyori Saheki; Araceli Del Arco; Jorgina Satrústegui; Laura Contreras

doi:10.1016/j.ymgmr.2023.100967

Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver

Mol Genet Metab Rep. 2023 Mar 16:35:100967. doi: 10.1016/j.ymgmr.2023.100967. eCollection 2023 Jun.

Authors

Luis González-Moreno^{1

2

3}, Andrea Santamaría-Cano^{1

2

3}, Alberto Paradela⁴, María Luz Martínez-Chantar^{5

6}, Miguel Á Martín^{7

8

9}, Mercedes Pérez-Carreras¹⁰, Alberto García-Picazo¹¹, Jesús Vázquez^{12

13}, Enrique Calvo^{12

13}, Gloria González-Aseguinolaza^{14

15}, Takeyori Saheki¹⁶, Araceli Del Arco^{2

3

17

18}, Jorgina Satrústegui^{1

2

3}, Laura Contreras^{1

2

3}

Affiliations

¹ Departamento de Biología Molecular, Universidad Autónoma de Madrid, 28049 Madrid, Spain.
² Instituto Universitario de Biología Molecular, (IUBM), and Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid, Spain.
³ Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD), Universidad Autónoma de Madrid, 28049 Madrid, Spain.
⁴ Centro Nacional de Biotecnología (CNB), CSIC. C/Darwin 3, 28049 Madrid, Spain.
⁵ Liver Disease Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), 48160 Derio, Bizkaia, Spain.
⁶ Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, 28029 Madrid, Spain.
⁷ Grupo Enfermedades Mitocondriales y Neuromusculares, Instituto de Investigación Hospital 12 de Octubre (imas12), Madrid, Spain.
⁸ Servicio de Genética, Hospital Universitario 12 de Octubre, Madrid, Spain.
⁹ Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain.
¹⁰ Servicio del Aparato Digestivo, Hospital Universitario 12 de Octubre, Madrid, Spain.
¹¹ Departamento de Cirugía General Aparato Digestivo, Hospital Universitario 12 de Octubre, Madrid, Spain.
¹² Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.
¹³ CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain.
¹⁴ Gene Therapy and Regulation of Gene Expression Program, Center for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain.
¹⁵ IdiSNA Navarra Institute for Health Research, 31008 Pamplona, Spain.
¹⁶ Ito Memorial Hospital, Ibusuki City, Japan.
¹⁷ Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla la Mancha, Toledo 45071, Spain.
¹⁸ Centro Regional de Investigaciones Biomédicas, Unidad Asociada de Biomedicina, Toledo 45071, Spain.

Abstract

The deficiency of CITRIN, the liver mitochondrial aspartate-glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD⁺ ratio increases in hepatocytes from citrin(-/-) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD⁺ observed in these cells. Liver mitochondria from citrin (-/-) mice expressing liver specific transgenic aralar had a small (~ 4-6 nmoles x mg prot^-1 x min^-1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(-/-) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(-/-) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficiency.

Keywords: (BNGE), Blue native gel electrophoresis; AGC, aspartate-glutamate carrier; AQUA, Absolute Quantification methods; Aspartate-glutamate carrier; CD, CITRIN Deficiency; CTNL2, citrullinemia type II; Citrin deficiency; DAB, 3,3-diaminobenzidine; FBS, Fetal Bovine serum; FTTDCD, failure to thrive and dyslipidemia caused by CITRIN Deficiency; GOT, aspartate transaminase; GPD2, mitochondrial glycerol phosphate dehydrogenase; GPS, glycerol phosphate shuttle; Hepatocyte; IM, imaging medium; LC-MS, liquid chromatography mass spectrometry; LNP, lipid nanoparticles; MAS, malate aspartate shuttle; Malate-aspartate shuttle; Mitochondria; NAA, N-Acetyl-aspartate; NICCD, neonatal intrahepatic cholestasis caused by CITRIN Deficiency; OXPHOS, oxidative phosphorylation; PFA, paraformaldehyde; PRM, parallel reaction monitoring; SDS, sodium dodecyl sulfate; TBS, Tris-Buffered saline.; hCitrin, human citrin.