Automated high-throughput high-content autophagy and mitophagy analysis platform

Abstract

Autophagic processes play a central role in cellular homeostasis. In pathological conditions, the flow of autophagy can be affected at multiple and distinct steps of the pathway. Current analyses tools do not deliver the required detail for dissecting pathway intermediates. The development of new tools to analyze autophagic processes qualitatively and quantitatively in a more straightforward manner is required. Defining all autophagy pathway intermediates in a high-throughput manner is technologically challenging and has not been addressed yet. Here, we overcome those requirements and limitations by the developed of stable autophagy and mitophagy reporter-iPSC and the establishment of a novel high-throughput phenotyping platform utilizing automated high-content image analysis to assess autophagy and mitophagy pathway intermediates.

Parkinson’s disease phenotypes in patient specific brain organoids are improved by HP-β-CD treatment

“…The mitophagy events observed in PINK1 patient-derived cells might be mediated by PINK1-Parkin-independent mitophagy pathways (McWilliams et al, 2018). In order to assess the autophagy pathway as a potential target for small molecules therapy, several known modulators as well as stressors were evaluated as we previously reported (Arias-Fuenzalida et al, 2019). Patient-derived cells treated with rapamycin showed an increase in the frequency of phagophores and autophagic vacuoles, to similar levels as those observed in controls ( Fig 2E).…”

“…To further understand the mitochondrial dynamics in these cells, we generated a pair of stable patient (PINK1, p.I368N) and a control line expressing the Rosella construct bound to microtubule-associated proteins 1A/1B light chain 3B (LC3) or to ATP synthase F1 subunit gamma (ATP5C1), to evaluate the autophagy status and mitochondrial degradation by mitophagy (Arias-Fuenzalida et al, 2019;Sargsyan et al, 2015) (Fig 2). Rosella cells were generated at the hiPSCs state, purified, and further differentiated into NESCs and neurons.…”

“…The pH sensor fluorescent protein pHluorin was fused to DsRed and the entire open reading frame of ATP5C1 or LC3 as described in (Arias-Fuenzalida et al, 2019;Sargsyan et al, 2015).…”

“…The image analysis was performed using MatLab, The MatWorks Inc. as previously described (Arias-Fuenzalida et al, 2019;Bolognin et al, 2018).…”

Mitochondrial and Clearance Impairment in p.D620N VPS35 Patient‐Derived Neurons

“…The generation of the lines carrying the Rosella reporter was performed as previously described. 21 Briefly, the tandem fluorescent proteins consisting of pH sensor fluorescent protein pHluorin (F64L, S65T, V193G, and H231Q) and DsRed were fused to the entire open reading frame of ATP5C1 serving as a mitochondrial targeting sequence, and placed in between the homology arms targeting the AAV1 safe harbour 22 (Addgene plasmid #22075). A double-strand break for triggering homologous recombination was performed with the px330 23 (Addgene plasmid #42230) carrying the sgRNA targeting sequence for the safe harbor as described by Mali et al 24 SmNPC from patients (VPS35 1_2) and control individuals (Control 1) were nucleofected (P3 Primary Cell 4D-Nucleofector, V4XP-3024; Lonza, Basel, Switzerland) with both constructs and expanded before purification by fluorescence-activated cell sorting (FACS) (Aria III; Beckton Dickinson, Franklin Lakes, NJ).…”