Assays of Monitoring and Measuring Autophagic Flux for iPSC-Derived Human Neurons and Other Brain Cell Types.

Autophagy is a highly conserved, cytoprotective, catabolic process induced in response to conditions of cellular stress and nutrient deprivation. It is responsible for the degradation of large intracellular substrates such as misfolded or aggregated proteins and organelles. This self-degradative mechanism is crucial for proteostasis in post-mitotic neurons, requiring its careful regulation. Due to its homeostatic role and the implications, it has for certain disease pathologies, autophagy has become a growing area of research. We describe here two assays that can be used as part of a tool kit for measuring autophagy-lysosomal flux in human iPSC-derived neurons.One way to measure autophagic flux is through a western blotting assay, which can be used to analyze two important autophagy proteins: microtubule-associated protein 1 light chain 3 (LC3) and p62. In this chapter, we describe a western blotting assay for use in human iPSC neurons that can be used to quantify these two proteins of interest to measure autophagic flux.In addition to conventional western blotting techniques, more sophisticated tools have come available to readout autophagic flux in a sensitive and high-throughput manner. In the latter portion of this chapter, we describe a flow cytometry assay which utilizes a pH-sensitive fluorescent reporter which can also be used to measure autophagic flux.

Potential role of chitinase-3-like protein 1 (CHI3L1/YKL-40) in neurodegeneration and Alzheimer's disease.

Chitinase-3-like protein 1 (CHI3L1/YKL-40) has long been known as a biomarker for early detection of neuroinflammation and disease diagnosis of Alzheimer's disease (AD). In the brain, CHI3L1 is primarily provided by astrocytes and heralds the reactive, neurotoxic state triggered by inflammation and other stress signals. However, how CHI3L1 acts in neuroinflammation or how it contributes to AD and relevant neurodegenerative conditions remains unknown. In peripheral tissues, our group and others have uncovered that CHI3L1 is a master regulator for a wide range of injury and repair events, including the innate immunity pathway that resembles the neuroinflammation process governed by microglia and astrocytes. Based on assessment of current knowledge regarding CHI3L1 biology, we hypothesize that CHI3L1 functions as a signaling molecule mediating distinct neuroinflammatory responses in brain cells and misfunctions to precipitate neurodegeneration. We also recommend future research directions to validate such assertions for better understanding of disease mechanisms.