Biology subjects

Kao, A. W.

Publications and source records attributed to Kao, A. W..

10 recordsLinked to original sources

bioRxiv2026

The WD40 domain of autophagy protein ATG-16.2 is required for lysosomal regulation and longevity in germline-less C. elegans

Autophagy is a conserved cellular recycling pathway linked to longevity and regulated by autophagy-related (ATG) protein. Many of these proteins serve a key function in ATG8 lipidation onto autophagosomal membranes, a step that precedes subsequent lysosomal degradation. Recently, mammalian ATG16L1, a member of ATG8 lipidation machinery, have been shown to also direct ATG8 lipidation onto other membranes via its C-terminal WD40 domain in acutely stressed cells. However, whether such non-canonical functions of the ATG16L1 WD40 domain influence longevity remain largely unknown. Here, we endogenously deleted the WD40 domain of atg-16.2, the C. elegans ortholog of ATG16L1, and examined autophagy, healthspan and longevity. The ATG-16.2 WD40 domain was dispensable for healthspan measures and for ATG8 lipidation in whole animals and neurons but was required for autophagic flux across all major tissues examined. Notably, the ATG-16.2 WD40 domain was also critical in limiting lysosomal membrane damage after acute heat stress. Because long-lived C. elegans mutants are typically heat resistant, we asked whether this lysosomal function supported their thermotolerance and found the WD40 domain to be selectively required for thermotolerance induced by germline removal, but not by reduced insulin/IGF-1 signaling or dietary restriction. In germline-less animals, loss of the ATG-16.2 WD40 domain reduced nuclear TFEB/HLH-30, reduced neuronal autophagic degradation, altered neuronal lysosomal pH, and sensitized animals to heat-induced lysosomal damage. Collectively, these findings establish the ATG16 WD40 domain as a regulator of autophagic degradation and lysosomal integrity in vivo, and show that this role is associated with longevity in a pathway-selective manner.

genetics↗

bioRxiv2025

Drug-induced phospholipidosis as an artifact in antiviral drug repurposing

Drug repurposing in principle can speed antiviral drug discovery. Among the molecules most frequently advanced in such repurposing efforts are a group of structurally diverse cationic amphiphilic drugs (CADs). While CADs have shown micromolar to mid-nanomolar antiviral activity in cell-based assays, they can induce phospholipidosis, confounding in COVID-19 repurposing. A barrier to the identification of phospholipidosis inducers has been the involved nature of the microscopy assays used to characterize them. To ease the identification of these artifacts, we describe a rapid microplate-based assay to detect phospholipidosis. Leveraging this assay, we quantified the prevalence of phospholipidosis-inducers across several cell-based antiviral repurposing screens. We selected 40 drugs reported to have micromolar antiviral activities and found that 26 of them (65%) induced phospholipidosis within the same concentration range as their reported antiviral activities. Intriguingly, we identified four non-CADs that also induce phospholipidosis, revealing a new group of drugs that can lead to this toxic event and highlighting the importance of facile experimental assays to detect it. Understanding how phospholipidosis can confound antiviral drug discovery, and its rapid detection, will help prevent what is an apparently general artifact, active across viruses, from distracting investigators from potentially more useful candidates.

cell biology↗

bioRxiv2024

Prosaposin is cleaved into saposins by multiple cathepsins in a progranulin-regulated fashion

Prosaposin (PSAP) is a lysosomal protein that plays a key role in sphingolipid metabolism. PSAP is cleaved into four bioactive disulfide-rich peptides, saposins A, B, C, and D, that catalyze sphin-golipidases to promote sphingolipid breakdown. Considering the key role of PSAP and saposins in sphingolipid metabolism and the existence of genetic mutations in PSAP associated with juve-nile-onset lysosomal storage and adult-onset neurodegenerative diseases, maintaining optimal levels of PSAP and saposins is crucial for proper lysosomal function and sphingolipid homeosta-sis. Despite this, the mechanism by which saposins are released from PSAP, and thus available to modulate sphingolipidases, sphingolipid homeostasis, and downstream lysosomal function, is not well understood. Here, we performed a comprehensive study to identify lysosomal enzymes which regulate prosaposin cleavage into saposins. In vitro cleavage assays identified multiple enzymes that can process human prosaposin into multi- and single-saposin fragments in a pH-dependent manner. We confirmed the role of cathepsins D and B in PSAP processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, K, L, S, V, G and AEP/LGMN) are able to process PSAP in distinctive, pH-dependent manners. In addition, we have demonstrated a novel role for progranulin (PGRN) in the regulation of PSAP cleavage. We found that PGRN and multi-granulin fragments (MGFs) directly regulate the cleavage of PSAP by cathepsin D. With this study, we have identified that multiple cathepsins, PGRN and MGFs work in concert to produce saposins under different conditions, which could present novel opportunities to modulate saposin levels in disease.

cell biology↗

bioRxiv2023

Interaction with the membrane-anchored protein CHIC2 constrains the ubiquitin ligase activity of CHIP

Maintenance of cellular health requires the proper regulation of E3 ubiquitin ligases. The E3 ligase CHIP is canonically regulated by its interactions with the molecular chaperones Hsp70 and Hsp90, which focus CHIPs ubiquitination activity on misfolded proteins. Here, we report a chaperone-independent interaction of CHIP with the membrane-anchored protein CHIC2, which strongly attenuates CHIPs ligase activity. We show that CHIC2 outcompetes abundant, cytosolic chaperones through its exquisite CHIP selectivity, rather than through enhanced affinity. In proteomic experiments, we find that CHIC2 knockout phenocopies CHIP knockout in certain cell types, implying that chaperone-independent interactions can sometimes predominate CHIPs functions. Furthermore, loss of the CHIP-CHIC2 interaction induces neurodegeneration and shortens lifespan in C. elegans, demonstrating that formation of this chaperone-independent complex is important in animals. We propose that CHIC2 attenuates CHIP activity at the membrane, offering a novel mechanism by which this ubiquitin ligase can be regulated.

biochemistry↗

bioRxiv2021

mTOR controls neurotoxic lysosome exocytosis in inflammatory reactive astrocytes

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo - IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states, was upregulated in astrocytes in multiple sclerosis lesions, and preferentially co-localized with astrocytes in hypoxic-ischemic encephalopathy. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

cell biology↗

bioRxiv2021

A systematic comparison of fibroblasts derived from postmortem human dura mater versus dermal epithelium for neurodegenerative disease modeling

Patient-derived cells hold great promise for precision medicine approaches in human health. Fibroblast cells have been a major source of human cells for reprogramming and differentiating into specific cell types for disease modeling. Such cells can be isolated at various stages during life (presymptomatic, symptomatic, and postmortem) and thus can potentially be used to model different phases of disease progression. In certain circumstances, however, tissues are not collected during life and only postmortem tissues are the only available source of fibroblasts. Fibroblasts cultured from postmortem human dura mater of individuals with neurodegenerative diseases have been suggested as a primary source of cells for in vitro modeling of neurodegenerative diseases. Although fibroblast-like cells from human and mouse dura mater have been previously described, their utility for reprogramming and direct differentiation protocols requires further characterization. In this study, cells derived from dermal biopsies performed in living subjects were compared to cells derived from postmortem dura mater. In two instances, we have isolated and compared dermal and dural cell lines from the same subject. Notably, striking differences between the dermis and dura mater-derived cell lines were found. Compared to dermal fibroblasts, postmortem dura mater-derived cells demonstrated different morphology, exhibited slower growth rates, failed to express fibroblast protein markers, and exhibited significant differences in gene expression profiles. In addition, dura mater-derived cells were found to exhibit a high rate of chromosomal abnormalities, particularly in the loss of the Y chromosome. Our study highlights potential limitations of postmortem human dura mater-derived cells for disease modeling, argues for rigorous karyotyping prior to reprograming, and brings into question the identity of dura mater-derived cells as belonging to a fibroblast lineage.

cell biology↗

bioRxiv2020

TSC1 loss-of-function increases risk for tauopathy by inducing tau acetylation and preventing autophagy-mediated tau clearance

Age-associated neurodegenerative disorders demonstrating tau-laden intracellular inclusions, including Alzheimers disease (AD), frontotemporal lobar degeneration (FTLD) and progressive supranuclear palsy (PSP), are collectively known as tauopathies. The vast majority of human tauopathies accumulate non-mutant tau rather than mutant forms of the protein, yet cell and animal models for non-mutant tauopathies are lacking. We previously linked a monoallelic mutation in the TSC1 gene to tau accumulation and FTLD. Now, we have identified new variants in TSC1 that predisposed to other tauopathies such as AD and PSP. These new TSC1 risk variants significantly decreased the half-life of TSC1/hamartin in vitro. Cellular and murine models of TSC1 haploinsufficiency (TSC1+/-) accumulated tau protein that exhibited aberrant acetylation on six lysine residues. Tau acetylation hindered its lysosomal degradation via chaperone-mediated autophagy leading to neuronal tau accumulation. Enhanced tau acetylation in TSC1+/- models was achieved through both an increase in p300 acetyltransferase activity and a decrease in SIRT1 deacetylase levels. Pharmacological modulation of either enzyme restored tau levels. Together, these studies substantiate TSC1 as a novel tauopathy risk gene and advance TSC1 haploinsufficiency as a new genetic model for tauopathy. In addition, these results promote acetylated tau as a rational target for diagnostic and therapeutic modalities in multiple tauopathies.

neuroscience↗

bioRxiv2020

Genetically encoded ratiometric biosensor for probing lysosomal pH in mammalian cells and C. elegans

Lysosomes are important sites for macromolecular degradation, defined by an acidic lumenal pH of [~]4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP) based pH biosensor called FIRE-pHLy (Fluorescence Indicator REporting pH in Lysosomes). This biosensor was targeted to lysosomes with lysosomal-associated membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quantitative imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust and versatile lysosomal pH biosensor that has broad applications for investigating pH dynamics in aging and lysosome-related diseases, as well as in lysosome-based drug discovery.

cell biology↗

bioRxiv2019

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a novel target of NDGA and provide mechanistic insight into its role in longevity.

cell biology↗

bioRxiv2018

C. elegans granulins promote an age-associated decline in protein homeostasis via lysosomal protease inhibition

The progressive failure of protein homeostasis is a hallmark of aging and a common feature in neurodegenerative disease. As the enzymes executing the final stages of autophagy, lysosomal proteases (or cathepsins) are key contributors to maintenance of protein homeostasis with age. Here, we identify the cysteine-rich granulin peptides as a new class of regulators of lysosomal aspartyl protease activity. Granulins are produced in an age and stress-dependent manner through cleavage of the neurodegenerative disease protein, progranulin. Once liberated, granulins selectively interact with the aspartyl protease ASP-3/cathepsin D to impair enzymatic activity. Consequently, protein homeostasis and lysosome function is disrupted, prompting cells to activate a compensatory transcriptional program. Our results support a model in which granulin production modulates a critical transition between the normal, physiological regulation of protease activity and the impairment of lysosomal function that can occur with age and disease.

neuroscience↗