Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons

Abstract

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimer's disease-related amyloid precursor protein (APP)-derived Aβ42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC-derived neurons for the investigation of neuronal cell biology and pathology. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

AP-4 regulates neuronal lysosome composition, function, and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network

“…In Control i 3 Neurons, consistent with previous reports (Gowrishankar et al, 2021), most LAMP1 vesicles are largely concentrated in the soma with relatively fewer vesicles observed in neurites (Fig. 5 A, B).…”

“…We next examined the distribution of different lysosomal proteins in these i 3 Neurons at 14 days differentiation. Staining with LAMP1, a marker of late endosomes as well as degradative lysosomes (Lie et al, 2021;Gowrishankar et al, 2021;Cheng et al, 2018;Yap et al, 2018), revealed that there was a significant increase in size of LAMP1-positive vesicles in the cell bodies of AP-4 i 3 Neurons (Fig. 1 C, D; Fig.…”

“…This system allows simple and rapid generation of glutamatergic cortical neurons (i 3 Neurons) upon culture of the iPSCs in the presence of doxycycline for a brief period. These i 3 Neurons exhibit morphological and biochemical properties of neurons 14 days post-induction (Gowrishankar et al, 2021;Wang et al, 2017) and are electrically active after 21 days (Wang et al, 2017). We have employed a modified i 3 iPSC system, in which CRISPR-inhibition (CRISPRi) machinery is integrated into a safe harbor locus (Wu et al, 2021;Tian et al, 2019).…”

“…Cells were transduced for 16 h and selected using 1 μg/mL puromycin 24 h later. iPSC culture and neuronal differentiation iPSCs were cultured and i 3 Neurons were differentiated from them as previously described (Fernandopulle et al, 2018;Gowrishankar et al, 2021), with slight modifications. Briefly, on day 0 iPSCs were dissociated into single cells using Accutase (Thermo Fisher Scientific) and seeded at a density of 100,000 cells/well on a Matrigel-coated 6-well plate in Induction Medium (IM) composed of KO DMEM, 1× N-2 Supplement, 1× MEM Non-Essential Amino Acids Solution, 1× GlutaMAX Supplement (Thermo Fisher Scientific), 10 μM Y-27632 (Tocris Biosciences), and 2 μg/mL doxycycline hydrochloride (Sigma-Aldrich).…”

“…Immunofluorescence analysis of i 3 Neurons i 3 Neurons were differentiated for one to six weeks on 35 mm glass bottom dishes (MatTek Life Sciences) and processed for immunostaining as described previously (Gowrishankar et al, 2021). See Table S1 for antibody information.…”