citations

                    A novel CRISPR/Cas9-based iduronate-2-sulfatase (IDS) knockout human neuronal cell line reveals earliest pathological changes
                

Authors:

Badenetti L, Manzoli R, Trevisan M, D'Avanzo F, Tomanin R, Moro E.

In:

Source: Scientific Reports
Publication Date: (2023)
Issue: 13(1): 10289

Research Area:

Neurobiology

Gene Expression

Basic Research

Cells used in publication:

LUHMES: Species: human; Tissue Origin: brain

Platform:

4D-Nucleofector® X-Unit

Experiment

Undifferentiated LUHMES cells were trypsinized with trypsin/EDTA 0.025% (Thermofisher, Italy), counted and diluted with phosphate buffered solution (PBS). One million cells were washed with PBS and re-suspended in 100 µl of Amaxa P3 solution (Lonza, Basel, Switzerland) together with a mixture of sgRNAs/Cas9 (120 pmol and 104 pmol, respectively). Cells were then electroporated with a 4D Nucleofector device (Lonza) using program CA-137 and allowed to immediately recover for 5 min at 37 °C,
after adding 500 µl of medium supplemented with N2, glutamine and FGF.

Abstract

Multiple complex intracellular cascades contributing to Hunter syndrome (mucopolysaccharidosis type II) pathogenesis have been recognized and documented in the past years. However, the hierarchy of early cellular abnormalities leading to irreversible neuronal damage is far from being completely understood. To tackle this issue, we have generated two novel iduronate-2-sulfatase (IDS) loss of function human neuronal cell lines by means of genome editing. We show that both neuronal cell lines exhibit no enzymatic activity and increased GAG storage despite a completely different genotype. At a cellular level, they display reduced differentiation, significantly decreased LAMP1 and RAB7 protein levels, impaired lysosomal acidification and increased lipid storage. Moreover, one of the two clones is characterized by a marked decrease of the autophagic marker p62, while none of the two mutants exhibit marked oxidative stress and mitochondrial morphological changes. Based on our preliminary findings, we hypothesize that neuronal differentiation might be significantly affected by IDS functional impairment.

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