A brain organoid/ALL coculture model reveals the AP-1 pathway as critically associated with CNS involvement of BCP-ALL.

ABSTRACT: Central nervous system (CNS) involvement remains a clinical hurdle in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The disease mechanisms of CNS leukemia are primarily investigated using 2-dimensional cell culture and mouse models. Given the variations in cellular identity and architecture between the human and murine CNS, it becomes imperative to seek complementary models to study CNS leukemia. Here, we present a first-of-its-kind 3-dimensional coculture model combining human brain organoids and BCP-ALL cells. We noticed significantly higher engraftment of BCP-ALL cell lines and patient-derived xenograft (PDX) cells in cerebral organoids than non-ALL cells. To validate translatability between organoid coculture and in vivo murine models, we confirmed that targeting CNS leukemia-relevant pathways such as CD79a/Igα or C-X-C motif chemokine receptor 4-stromal cell-derived factor 1 reduced the invasion of BCP-ALL cells into organoids. RNA sequencing and functional validations of organoid-invading leukemia cells compared with the noninvaded fraction revealed significant upregulation of activator protein 1 (AP-1) transcription factor-complex members in organoid-invading cells. Moreover, we detected a significant enrichment of AP-1 pathway genes in PDX ALL cells recovered from the CNS compared with spleen blasts of mice that had received transplantation with TCF3::PBX1+ PDX cells, substantiating the role of AP-1 signaling in CNS disease. Accordingly, we found significantly higher levels of the AP-1 gene, jun proto-oncogene, in patients initially diagnosed as CNS-positive BCP-ALL compared with CNS-negative cases as well as CNS-relapse vs non-CNS-relapse cases in a cohort of 100 patients with BCP-ALL. Our results suggest CNS organoids as a novel model to investigate CNS involvement and identify the AP-1 pathway as a critical driver of CNS disease in BCP-ALL.

Cristae dynamics is modulated in bioenergetically compromised mitochondria.

Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are unclear. Here, we investigated whether and how cristae morphology and dynamics are dependent on oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential (ΔΨm), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy combined with in-depth quantification were employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A, and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not impair cristae dynamics. CCCP treatment leading to ΔΨm abrogation even enhanced cristae dynamics showing its ΔΨm-independent nature. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset. In sum, we provide quantitative data of cristae membrane remodelling under different conditions supporting an important interplay between OXPHOS, metabolite exchange, and cristae membrane dynamics.

Endo-lysosomal Aß concentration and pH trigger formation of Aß oligomers that potently induce Tau missorting.

Amyloid-ß peptide (Aß) forms metastable oligomers >50 kDa, termed AßOs, that are more effective than Aß amyloid fibrils at triggering Alzheimer's disease-related processes such as synaptic dysfunction and Tau pathology, including Tau mislocalization. In neurons, Aß accumulates in endo-lysosomal vesicles at low pH. Here, we show that the rate of AßO assembly is accelerated 8,000-fold upon pH reduction from extracellular to endo-lysosomal pH, at the expense of amyloid fibril formation. The pH-induced promotion of AßO formation and the high endo-lysosomal Aß concentration together enable extensive AßO formation of Aß42 under physiological conditions. Exploiting the enhanced AßO formation of the dimeric Aß variant dimAß we furthermore demonstrate targeting of AßOs to dendritic spines, potent induction of Tau missorting, a key factor in tauopathies, and impaired neuronal activity. The results suggest that the endosomal/lysosomal system is a major site for the assembly of pathomechanistically relevant AßOs.

The migration behavior of human glioblastoma cells is influenced by the redox-sensitive human macrophage capping protein CAPG.

The macrophage capping protein CAPG belongs to the gelsolin superfamily which modulates actin dynamics by capping the growing end of actin filaments in a Ca2+- and PIP2-dependent manner resulting in polymerization inhibition of actin filaments. In the last years, additional functions for CAPG in transcription regulation were described and higher CAPG amounts have been linked to increased invasiveness and migration behavior in different human tumor entities like e.g. glioblastoma. Nevertheless, there is a lack of knowledge how additional functions of CAPG are regulated. As CAPG contains several cysteine residues which may be accessible to oxidation we were especially interested to investigate how alterations in the cysteine oxidation state may influence the function, localization, and regulation of CAPG. In the present study, we provide strong evidence that CAPG is a redox-sensitive protein and identified two cysteines: C282 and C290 as reversibly oxidized in glioblastoma cell lines. Whereas no evidence could be found that the canonical actin capping function of CAPG is redox-regulated, our results point to a novel role of the identified cysteines in the regulation of cell migration. Along with this, we found a localization shift out of the nucleus of CAPG and RAVER1, a potential interaction partner identified in our study which might explain the observed altered cell migration properties. The newly identified redox sensitive cysteines of CAPG could perspectively be considered as new targets for controlling tumor invasive properties.

Deficiency of GABARAP but not its Paralogs Causes Enhanced EGF-induced EGFR Degradation.

The γ-aminobutyric acid type A receptor-associated protein (GABARAP) and its close paralogs GABARAPL1 and GABARAPL2 constitute a subfamily of the autophagy-related 8 (Atg8) protein family. Being associated with a variety of dynamic membranous structures of autophagic and non-autophagic origin, Atg8 proteins functionalize membranes by either serving as docking sites for other proteins or by acting as membrane tethers or adhesion factors. In this study, we describe that deficiency for GABARAP alone, but not for its close paralogs, is sufficient for accelerated EGF receptor (EGFR) degradation in response to EGF, which is accompanied by the downregulation of EGFR-mediated MAPK signaling, altered target gene expression, EGF uptake, and EGF vesicle composition over time. We further show that GABARAP and EGFR converge in the same distinct compartments at endogenous GABARAP expression levels in response to EGF stimulation. Furthermore, GABARAP associates with EGFR in living cells and binds to synthetic peptides that are derived from the EGFR cytoplasmic tail in vitro. Thus, our data strongly indicate a unique and novel role for GABARAP during EGFR trafficking.

Chlamydia-induced curvature of the host-cell plasma membrane is required for infection.

During invasion of host cells, Chlamydia pneumoniae secretes the effector protein CPn0678, which facilitates internalization of the pathogen by remodeling the target cell's plasma membrane and recruiting sorting nexin 9 (SNX9), a central multifunctional endocytic scaffold protein. We show here that the strongly amphipathic N-terminal helix of CPn0678 mediates binding to phospholipids in both the plasma membrane and synthetic membranes, and is sufficient to induce extensive membrane tubulations. CPn0678 interacts via its conserved C-terminal polyproline sequence with the Src homology 3 domain of SNX9. Thus, SNX9 is found at bacterial entry sites, where C. pneumoniae is internalized via EGFR-mediated endocytosis. Moreover, depletion of human SNX9 significantly reduces internalization, whereas ectopic overexpression of CPn0678-GFP results in a dominant-negative effect on endocytotic processes in general, leading to the uptake of fewer chlamydial elementary bodies and diminished turnover of EGFR. Thus, CPn0678 is an early effector involved in regulating the endocytosis of C. pneumoniae in an EGFR- and SNX9-dependent manner.

Soluble and membrane-bound protein carrier mediate direct copper transport to the ethylene receptor family.

The plant hormone ethylene is a key regulator of plant growth, development and stress adaption. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs) localized at the ER-Golgi network. The biological function of these receptors relies on a protein-bound copper cofactor. Nonetheless, molecular processes and structures controlling assembly and integration of the metal into the functional plant hormone receptor are still unknown. Here, we have explored the molecular pathways of copper transfer from the plant cytosol to the ethylene receptor family by analyzing protein-protein interactions of receptors with soluble and membrane-bound plant copper carriers. Our results suggest that receptors primarily acquire their metal cofactor from copper transporter RESPONSIVE-TO-ANTAGONIST-1 (RAN1) which has been loaded with the transition metal beforehand by soluble copper carriers of the ATX1-family. In addition, we found evidence for a direct interaction of ETRs with soluble chaperones ANTIOXIDANT-1 (ATX1) and COPPER TRANSPORT PROTEIN (CCH) raising the possibility of a direct copper exchange between soluble chaperones and receptors.

Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies.

Signal perception and transmission of the plant hormone ethylene are mediated by a family of receptor histidine kinases located at the Golgi-ER network. Similar to bacterial and other plant receptor kinases, these receptors work as dimers or higher molecular weight oligomers at the membrane. Sequence analysis and functional studies of different isoforms suggest that the ethylene receptor family is classified into two subfamilies. In Arabidopsis, the type-I subfamily has two members (ETR1 and ERS1) and the type-II subfamily has three members (ETR2, ERS2, and EIN4). Whereas subfamily-I of the Arabidopsis receptors and their interactions with downstream elements in the ethylene pathway has been extensively studied in the past; related information on subfamily-II is sparse. In order to dissect the role of type-II receptors in the ethylene pathway and to decode processes associated with this receptor subfamily on a quantitative molecular level, we have applied biochemical and spectroscopic studies on purified recombinant receptors and downstream elements of the ethylene pathway. To this end, we have expressed purified ETR2 as a prototype of the type-II subfamily, ETR1 for the type-I subfamily and downstream ethylene pathway proteins CTR1 and EIN2. Functional folding of the purified receptors was demonstrated by CD spectroscopy and autokinase assays. Quantitative analysis of protein-protein interactions (PPIs) by microscale thermophoresis (MST) revealed that ETR2 has similar affinities for CTR1 and EIN2 as previously reported for the subfamily-I prototype ETR1 suggesting similar roles in PPI-mediated signal transfer for both subfamilies. We also used in planta fluorescence studies on transiently expressed proteins in Nicotiana benthamiana leaf cells to analyze homo- and heteromer formation of receptors. These studies show that type-II receptors as well as the type-I receptors form homo- and heteromeric complexes at these conditions. Notably, type-II receptor homomers and type-II:type-I heteromers are more stable than type-I homomers as indicated by their lower dissociation constants obtained in microscale thermophoresis studies. The enhanced stability of type-II complexes emphasizes the important role of type-II receptors in the ethylene pathway.