Imipridones affect tumor bioenergetics and promote cell lineage differentiation in diffuse midline gliomas.

BACKGROUND: Pediatric diffuse midline gliomas (DMGs) are incurable childhood cancers. The imipridone ONC201 has shown early clinical efficacy in a subset of DMGs. However, the anticancer mechanisms of ONC201 and its derivative ONC206 have not been fully described in DMGs. METHODS: DMG models including primary human in vitro (n = 18) and in vivo (murine and zebrafish) models, and patient (n = 20) frozen and FFPE specimens were used. Drug-target engagement was evaluated using in silico ChemPLP and in vitro thermal shift assay. Drug toxicity and neurotoxicity were assessed in zebrafish models. Seahorse XF Cell Mito Stress Test, MitoSOX and TMRM assays, and electron microscopy imaging were used to assess metabolic signatures. Cell lineage differentiation and drug-altered pathways were defined using bulk and single-cell RNA-seq. RESULTS: ONC201 and ONC206 reduce viability of DMG cells in nM concentrations and extend survival of DMG PDX models (ONC201: 117 days, P = .01; ONC206: 113 days, P = .001). ONC206 is 10X more potent than ONC201 in vitro and combination treatment was the most efficacious at prolonging survival in vivo (125 days, P = .02). Thermal shift assay confirmed that both drugs bind to ClpP, with ONC206 exhibiting a higher binding affinity as assessed by in silico ChemPLP. ClpP activation by both drugs results in impaired tumor cell metabolism, mitochondrial damage, ROS production, activation of integrative stress response (ISR), and apoptosis in vitro and in vivo. Strikingly, imipridone treatment triggered a lineage shift from a proliferative, oligodendrocyte precursor-like state to a mature, astrocyte-like state. CONCLUSION: Targeting mitochondrial metabolism and ISR activation effectively impairs DMG tumorigenicity. These results supported the initiation of two pediatric clinical trials (NCT05009992, NCT04732065).

Publisher Correction: Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.

Although structural nuclear pore proteins (nucleoporins) are seemingly required in every cell type to assemble a functional nuclear transport machinery, mutations or deregulation of a subset of them have been associated with specific human hereditary diseases. In particular, previous genetic studies of patients with nephrotic syndrome identified mutations in Nup107 that impaired the expression or the localization of its direct partner at nuclear pores, Nup133. In the present study, we characterized the zebrafish nup133 orthologous gene and its expression pattern during larval development. Using a morpholino-mediated gene knockdown, we show that partial depletion of Nup133 in zebrafish larvae leads to the formation of kidney cysts, a phenotype that can be rescued by co-injection of wild type mRNA. Analysis of different markers for tubular and glomerular development shows that the overall kidney development is not affected by nup133 knockdown. Likewise, no gross defect in nuclear pore complex assembly was observed in these nup133 morphants. On the other hand, nup133 downregulation results in proteinuria and moderate foot process effacement, mimicking some of the abnormalities typically featured by patients with nephrotic syndrome. These data indicate that nup133 is a new gene required for proper glomerular structure and function in zebrafish.

Comparative transcriptomic analysis identifies evolutionarily conserved gene products in the vertebrate renal distal convoluted tubule.

Understanding the molecular basis of the complex regulatory networks controlling renal ion transports is of major physiological and clinical importance. In this study, we aimed to identify evolutionarily conserved critical players in the function of the renal distal convoluted tubule (DCT) by a comparative transcriptomic approach. We generated a transgenic zebrafish line with expression of the red fluorescent mCherry protein under the control of the zebrafish DCT-specific promoter of the thiazide-sensitive NaCl cotransporter (NCC). The mCherry expression was then used to isolate from the zebrafish mesonephric kidneys the distal late (DL) segments, the equivalent of the mammalian DCT, for subsequent RNA-seq analysis. We next compared this zebrafish DL transcriptome to the previously established mouse DCT transcriptome and identified a subset of gene products significantly enriched in both the teleost DL and the mammalian DCT, including SLCs and nuclear transcription factors. Surprisingly, several of the previously described regulators of NCC (e.g., SPAK, KLHL3, ppp1r1a) in the mouse were not found enriched in the zebrafish DL. Nevertheless, the zebrafish DL expressed enriched levels of related homologues. Functional knockdown of one of these genes, ppp1r1b, reduced the phosphorylation of NCC in the zebrafish pronephros, similar to what was seen previously in knockout mice for its homologue, Ppp1r1a. The present work is the first report on global gene expression profiling in a specific nephron portion of the zebrafish kidney, an increasingly used model system for kidney research. Our study suggests that comparative analysis of gene expression between phylogenetically distant species may be an effective approach to identify novel regulators of renal function.

Highly efficient baculovirus-mediated multigene delivery in primary cells.

Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters. MultiPrime viruses efficiently transduce a wide range of cell types, including non-dividing primary neurons and induced-pluripotent stem cells (iPS). We show that MultiPrime can be used for reprogramming, and for genome editing and engineering by CRISPR/Cas9. Moreover, we implemented dual-host-specific cassettes enabling multiprotein expression in insect and mammalian cells using a single reagent. Our experiments establish MultiPrime as a powerful and highly efficient tool, to deliver multiple genes for a wide range of applications in primary and established mammalian cells.

Histone deacetylase inhibitor enhances recovery after AKI.

At present, there are no effective therapies to ameliorate injury, accelerate recovery, or prevent postinjury fibrosis after AKI. Here, we sought to identify candidate compounds that accelerate recovery after AKI by screening for small molecules that increase proliferation of renal progenitor cells in zebrafish embryos. One compound identified from this screen was the histone deacetylase inhibitor methyl-4-(phenylthio)butanoate, which we subsequently administered to zebrafish larvae and mice 24-48 hours after inducing AKI. In zebrafish, treatment with the compound increased larval survival and proliferation of renal tubular epithelial cells. In mice, treatment accelerated recovery, reduced postinjury tubular atrophy and interstitial fibrosis, and increased the regenerative capacity of actively cycling renal tubular cells by decreasing the number of cells in G2/M arrest. These data suggest that accelerating recovery may be a viable approach to treating AKI and provide proof of concept that a screen in zebrafish embryos can identify therapeutic candidates for kidney injury.

Apical targeting and endocytosis of the sialomucin endolyn are essential for establishment of zebrafish pronephric kidney function.

Kidney function requires the appropriate distribution of membrane proteins between the apical and basolateral surfaces along the kidney tubule. Further, the absolute amount of a protein at the cell surface versus intracellular compartments must be attuned to specific physiological needs. Endolyn (CD164) is a transmembrane protein that is expressed at the brush border and in apical endosomes of the proximal convoluted tubule and in lysosomes of more distal segments of the kidney. Endolyn has been shown to regulate CXCR4 signaling in hematopoietic precursor cells and myoblasts; however, little is known about endolyn function in the adult or developing kidney. Here we identify endolyn as a gene important for zebrafish pronephric kidney function. Zebrafish endolyn lacks the N-terminal mucin-like domain of the mammalian protein, but is otherwise highly conserved. Using in situ hybridization we show that endolyn is expressed early during development in zebrafish brain, eye, gut and pronephric kidney. Embryos injected with a translation-inhibiting morpholino oligonucleotide targeted against endolyn developed pericardial edema, hydrocephaly and body curvature. The pronephric kidney appeared normal morphologically, but clearance of fluorescent dextran injected into the common cardinal vein was delayed, consistent with a defect in the regulation of water balance in morphant embryos. Heterologous expression of rat endolyn rescued the morphant phenotypes. Interestingly, rescue experiments using mutant rat endolyn constructs revealed that both apical sorting and endocytic/lysosomal targeting motifs are required for normal pronephric kidney function. This suggests that both polarized targeting and postendocytic trafficking of endolyn are essential for the protein's proper function in mammalian kidney.

Requirement for a uroplakin 3a-like protein in the development of zebrafish pronephric tubule epithelial cell function, morphogenesis, and polarity.

Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na(+)/K(+)-ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression.

Intravenous microinjections of zebrafish larvae to study acute kidney injury.

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant. The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography. We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.