Quantitative assessment of cell fate decision between autophagy and apoptosis

Abstract Autophagy and apoptosis are cellular processes that regulate cell survival and death, the former by eliminating dysfunctional components in the cell, the latter by programmed cell death. Stress signals can induce either process, and it is unclear how cells ‘assess’ cellular damage and make a ‘life’ or ‘death’ decision upon activating autophagy or apoptosis. A computational model of coupled apoptosis and autophagy is built here to analyze the underlying signaling and regulatory network dynamics. The model explains the experimentally observed differential deployment of autophagy and apoptosis in response to various stress signals. Autophagic response dominates at low-to-moderate stress; whereas the response shifts from autophagy (graded activation) to apoptosis (switch-like activation) with increasing stress intensity. The model reveals that cytoplasmic Ca2+ acts as a rheostat that fine-tunes autophagic and apoptotic responses. A G-protein signaling-mediated feedback loop maintains cytoplasmic Ca2+ level, which in turn governs autophagic response through an AMP-activated protein kinase (AMPK)-mediated feedforward loop. Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ) emerges as a determinant of the competing roles of cytoplasmic Ca2+ in autophagy regulation. The study demonstrates that the proposed model can be advantageously used for interrogating cell regulation events and developing pharmacological strategies for modulating cell decisions

Mechanisms of action of autophagy modulators dissected by quantitative systems pharmacology analysis

Autophagy plays an essential role in cell survival/death and functioning. Modulation of autophagy has been recognized as a promising therapeutic strategy against diseases/disorders associated with uncontrolled growth or accumulation of biomolecular aggregates, organelles, or cells including those caused by cancer, aging, neurodegeneration, and liver diseases such as α1-antitrypsin deficiency. Numerous pharmacological agents that enhance or suppress autophagy have been discovered. However, their molecular mechanisms of action are far from clear. Here, we collected a set of 225 autophagy modulators and carried out a comprehensive quantitative systems pharmacology (QSP) analysis of their targets using both existing databases and predictions made by our machine learning algorithm. Autophagy modulators include several highly promiscuous drugs (e.g., artenimol and olanzapine acting as activators, fostamatinib as an inhibitor, or melatonin as a dual-modulator) as well as selected drugs that uniquely target specific proteins (~30% of modulators). They are mediated by three layers of regulation: (i) pathways involving core autophagy-related (ATG) proteins such as mTOR, AKT, and AMPK; (ii) upstream signaling events that regulate the activity of ATG pathways such as calcium-, cAMP-, and MAPK-signaling pathways; and (iii) transcription factors regulating the expression of ATG proteins such as TFEB, TFE3, HIF-1, FoxO, and NF-κB. Our results suggest that PKA serves as a linker, bridging various signal transduction events and autophagy. These new insights contribute to a better assessment of the mechanism of action of autophagy modulators as well as their side effects, development of novel polypharmacological strategies, and identification of drug repurposing opportunities

CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism

Impaired degranulation but enhanced cytokine production after FcɛRI stimulation of diacylglycerol kinase ζ–deficient mast cells

Calcium and diacylglycerol are critical second messengers that together effect mast cell degranulation after allergen cross-linking of immunoglobulin (Ig)E-bound FcɛRI. Diacylglycerol kinase (DGK)ζ is a negative regulator of diacylglycerol-dependent signaling that acts by converting diacylglycerol to phosphatidic acid. We reported previously that DGKζ−/− mice have enhanced in vivo T cell function. Here, we demonstrate that these mice have diminished in vivo mast cell function, as revealed by impaired local anaphylactic responses. Concordantly, DGKζ−/− bone marrow–derived mast cells (BMMCs) demonstrate impaired degranulation after FcɛRI cross-linking, associated with diminished phospholipase Cγ activity, calcium flux, and protein kinase C–βII membrane recruitment. In contrast, Ras-Erk signals and interleukin-6 production are enhanced, both during IgE sensitization and after antigen cross-linking of FcɛRI. Our data demonstrate dissociation between cytokine production and degranulation in mast cells and reveal the importance of DGK activity during IgE sensitization for proper attenuation of FcɛRI signals

Dominant negative variants in KIF5B cause osteogenesis imperfecta via down regulation of mTOR signaling

BACKGROUND: Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown. METHODS: To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy. RESULTS: C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells. CONCLUSION: We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development

An analog of glibenclamide selectively enhances autophagic degradation of misfolded α1-antitrypsin Z

The classical form of α1-antitrypsin deficiency (ATD) is characterized by intracellular accumulation of the misfolded variant α1-antitrypsin Z (ATZ) and severe liver disease in some of the affected individuals. In this study, we investigated the possibility of discovering novel therapeutic agents that would reduce ATZ accumulation by interrogating a C. elegans model of ATD with high-content genome-wide RNAi screening and computational systems pharmacology strategies. The RNAi screening was utilized to identify genes that modify the intracellular accumulation of ATZ and a novel computational pipeline was developed to make high confidence predictions on repurposable drugs. This approach identified glibenclamide (GLB), a sulfonylurea drug that has been used broadly in clinical medicine as an oral hypoglycemic agent. Here we show that GLB promotes autophagic degradation of misfolded ATZ in mammalian cell line models of ATD. Furthermore, an analog of GLB reduces hepatic ATZ accumulation and hepatic fibrosis in a mouse model in vivo without affecting blood glucose or insulin levels. These results provide support for a drug discovery strategy using simple organisms as human disease models combined with genetic and computational screening methods. They also show that GLB and/or at least one of its analogs can be immediately tested to arrest the progression of human ATD liver disease.</div

Macrocephaly and developmental delay caused by missense variants in RAB5C

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway

Functional analysis of a novel de novo variant in PPP5C associated with microcephaly, seizures, and developmental delay

We describe a proband evaluated through the Undiagnosed Diseases Network (UDN) who presented with microcephaly, developmental delay, and refractory epilepsy with a de novo p.Ala47Thr missense variant in the protein phosphatase gene, PPP5C. This gene has not previously been associated with a Mendelian disease, and based on the population database, gnomAD, the gene has a low tolerance for loss-of-function variants (pLI = 1, o/e = 0.07). We functionally evaluated the PPP5C variant in C. elegans by knocking the variant into the orthologous gene, pph-5, at the corresponding residue, Ala48Thr. We employed assays in three different biological processes where pph-5 was known to function through opposing the activity of genes, mec-15 and sep-1. We demonstrated that, in contrast to control animals, the pph-5 Ala48Thr variant suppresses the neurite growth phenotype and the GABA signaling defects of mec-15 mutants, and the embryonic lethality of sep-1 mutants. The Ala48Thr variant did not display dominance and behaved similarly to the reference pph-5 null, indicating that the variant is likely a strong hypomorph or complete loss-of-function. We conclude that pph-5 Ala48Thr is damaging in C. elegans. By extension in the proband, PPP5C p.Ala47Thr is likely damaging, the de novo dominant presentation is consistent with haplo-insufficiency, and the PPP5C variant is likely responsible for one or more of the proband\u27s phenotypes