Investigating the mechanisms underlying ATG7 related neurological disease

Abstract

The key catabolic process of autophagy relies on a set of core autophagy related (ATG) genes, of which very few have been directly associated with human disease. Genetic variants in ATG4D, ATG5, ATG7 and ATG9B have been associated with developmental delay, intellectual disability, ataxia, cerebellar and corpus callosum abnormalities. Here the first de novo heterozygous missense ATG7 subject is described, presenting similarly to the previously described ATG7 cohort, with impairment of the LC3-conjugation system and accumulation of the autophagy adapter p62 (PMID: 34161705). Also described is the first foetal ATG7 subject, harbouring segregating loss-of-function variants. Immunohistochemical and immunofluorescence analyses of subject brain tissue shows clusters of unmigrated neurons and p62 accumulation in neurons and cerebellar proliferative zone. To investigate these findings further, I have developed and characterised an induced pluripotent stem cell derived neural model of ATG7 pathology. CRISPR/Cas9 genome editing was used to engineer ATG7+/+, ATG7+/- and ATG7-/- iPSCs. Differentiation of these iPSCs into neuron-astrocyte co-cultures showed that ATG7-/- co-cultures recapitulated the biochemical phenotype of ATG7 primary fibroblasts. ATG7+/- and ATG7-/- co-cultures also demonstrated altered proliferation and morphology. Astrocyte specific changes were observed, with differing astrocyte differentiation and cell death among the ATG7+/- and ATG7-/- cultures. Functional Ca2+ live cell imaging showed increased intracellular Ca2+ in ATG7+/- neurons, with both ATG7+/- and ATG7-/- neurons having impaired Ca2+ recovery. Finally, a cohort of 3 subjects from 2 unrelated families harbouring rare, damaging ATG12 variants is also described, with affected individuals presenting with developmental delay, ataxia and cerebellar abnormalities. Biochemically they had mild impairment of either the ATG5-ATG12 or LC3-II conjugation pathway. The equivalent atg12 variants were also unable to fully recover autophagy in atg12-null Saccharomyces cerevisiae models. Together these data expand the existing cohorts of ATG related congenital disorders, with the ATG7 iPSC derived neural model elucidating disease mechanisms in addition to ATG7’s core role in autophagy.