Mitochondrial and Redox Modifications in Huntington Disease Induced Pluripotent Stem Cells Rescued by CRISPR/Cas9 CAGs Targeting

Carla Lopes; Yang Tang; Sandra I Anjo; Bruno Manadas; Isabel Onofre; Luís P de Almeida; George Q Daley; Thorsten M Schlaeger; Ana Cristina Carvalho Rego

doi:10.3389/fcell.2020.576592

Mitochondrial and Redox Modifications in Huntington Disease Induced Pluripotent Stem Cells Rescued by CRISPR/Cas9 CAGs Targeting

Front Cell Dev Biol. 2020 Sep 22:8:576592. doi: 10.3389/fcell.2020.576592. eCollection 2020.

Authors

Carla Lopes^{1

2}, Yang Tang^{3

4}, Sandra I Anjo^{1

5}, Bruno Manadas¹, Isabel Onofre^{1

2}, Luís P de Almeida^{1

6}, George Q Daley^{3

4

7

8}, Thorsten M Schlaeger^{3

4}, Ana Cristina Carvalho Rego^{1

5}

Affiliations

¹ CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
² IIIUC-Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal.
³ Division of Pediatric Hematology/Oncology, Children's Hospital Boston, Boston, MA, United States.
⁴ Harvard Stem Cell Institute, Boston, MA, United States.
⁵ Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
⁶ Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal.
⁷ Howard Hughes Medical Institute, Boston, MA, United States.
⁸ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States.

Abstract

Mitochondrial deregulation has gained increasing support as a pathological mechanism in Huntington's disease (HD), a genetic-based neurodegenerative disorder caused by CAG expansion in the HTT gene. In this study, we thoroughly investigated mitochondrial-based mechanisms in HD patient-derived iPSC (HD-iPSC) and differentiated neural stem cells (NSC) versus control cells, as well as in cells subjected to CRISPR/Cas9-CAG repeat deletion. We analyzed mitochondrial morphology, function and biogenesis, linked to exosomal release of mitochondrial components, glycolytic flux, ATP generation and cellular redox status. Mitochondria in HD cells exhibited round shape and fragmented morphology. Functionally, HD-iPSC and HD-NSC displayed lower mitochondrial respiration, exosomal release of cytochrome c, decreased ATP/ADP, reduced PGC-1α and complex III subunit expression and activity, and were highly dependent on glycolysis, supported by pyruvate dehydrogenase (PDH) inactivation. HD-iPSC and HD-NSC mitochondria showed ATP synthase reversal and increased calcium retention. Enhanced mitochondrial reactive oxygen species (ROS) were also observed in HD-iPSC and HD-NSC, along with decreased UCP2 mRNA levels. CRISPR/Cas9-CAG repeat deletion in HD-iPSC and derived HD-NSC ameliorated mitochondrial phenotypes. Data attests for intricate metabolic and mitochondrial dysfunction linked to transcriptional deregulation as early events in HD pathogenesis, which are alleviated following CAG deletion.

Keywords: huntington disease; induced pluripotent stem cells; mitochondrial dysfunction; neural stem cells; reactive oxygen species; transcriptional deregulation.