Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons

Area of Science:

• Cell Biology
• Neuroscience
• Molecular Biology

Background:

• The endolysosomal system regulates plasma membrane protein levels, crucial for cell identity and function.
• This system is vital for neuronal specialization and implicated in neurodegenerative diseases like Parkinson's and Alzheimer's.
• Current understanding of neuronal endocytic pathways and cargo is limited by technical challenges.

Purpose of the Study:

• To develop and validate a toolkit for profiling endosomes and lysosomes in induced neurons (iNeurons).
• To identify endocytic cargo and protein landscapes in iNeurons and stem cells.
• To investigate the protein composition of endosomes and lysosomes in the context of neurodegenerative disease research.

Main Methods:

• Developed Endo-IP (Early Endosome Antigen 1-positive endosomes) and Lyso-IP (TMEM192-positive lysosomes) capture techniques.
• Applied these tools to stem cell-derived cortical-like iNeurons and stem cells.
• Performed global proteomic profiling of captured endosomes and lysosomes.

Main Results:

• Successfully profiled endolysosomal protein landscapes in iNeurons and stem cells.
• Identified hundreds of transmembrane proteins as endocytic cargo, including neurogenesis and synaptic proteins.
• Discovered endocytic cargo with predicted SNX17/SNX27 recognition motifs and characterized lysosomal protein repertoires.

Conclusions:

• The developed toolkit enables comprehensive analysis of neuronal endosomal and lysosomal pathways.
• This research provides novel insights into endocytic cargo and protein regulation in neurons.
• The findings facilitate mechanistic studies of endolysosomal components linked to neurodegenerative diseases.