Biology subjects

Belfiore, L.

Publications and source records attributed to Belfiore, L..

3 recordsLinked to original sources

bioRxiv2024

Glycolytic flux controls retinal progenitor cell differentiation via regulating Wnt signaling

Metabolic pathways are remodeled in response to energy and other homeostatic demands and are dynamically regulated during embryonic development, suggesting a role in guiding cellular differentiation. Here, we show that glycolytic flux is required and sufficient to bias multipotent retinal progenitor cells (RPCs) to acquire a rod photoreceptor fate in the murine retina. In RPC-specific Phosphatase and tensin homolog conditional knock-out (Pten-cKO) and RPC-specific conditional gain-of-function of dominant active PFKB3 (cytoPFKB3) mice, glycolytic gene expression and activity are elevated, correlating with precocious rod photoreceptor differentiation and outer segment maturation. Conversely, glycolytic inhibition in retinal explants suppresses RPC proliferation and photoreceptor differentiation, achieved either with 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism, by lowering media pH, which disables PKM2, a rate-limiting enzyme, or by inhibiting lactate/H+ symporters, which lowers intracellular pH. Mechanistically, we show that Wnt signaling, the top-upregulated pathway in Pten-cKO retinas, is a glycolysis-dependent pathway. Pharmacological and genetic perturbation of Wnt signaling by knocking-out Ctnnb1, encoding {beta}-catenin, phenocopies glycolytic inhibition, suppressing RPC proliferation, photoreceptor differentiation and outer segment maturation. Thus, developmental rewiring of glycolytic flux modulates Wnt signaling to drive rod photoreceptor differentiation and maturation, an instructive role that may be exploited therapeutically for cell replacement strategies. IMPACT STATEMENTTransgenic and pharmacological approaches reveal developmental elevations in glycolytic flux have an instructive role in promoting rod photoreceptor differentiation and maturation via activation of Wnt signaling.

developmental biology↗

bioRxiv2021

Enabling high throughput target-based drug discovery in 3D cell cultures through novel bioprinting workflow

Advanced three-dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter Rastrum with the high throughput screening assay AlphaLISA, we demonstrate the utility of the workflow in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3{beta} phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.

cell biology↗

bioRxiv2020

Etv5 is not required for Schwann cell development but is required to regulate the Schwann cell response to peripheral nerve injury

Schwann cells are the principal glial cells of the peripheral nervous system, and their development into myelinating glia is critically dependent on MEK/ERK signaling. Ets-domain transcription factors (Etv1, Etv4, Etv5) are common downstream effectors of MEK/ERK signalling, but so far, only Etv1 has been ascribed a role in Schwann cell development, and only in non-myelinating cells. Here, we examined the role of Etv5, which is expressed in Schwann cell precursors, including neural crest cells and satellite glia, in Schwann cell lineage development. We analysed Etv5tm1Kmm mutants (designated Etv5-/-) at embryonic days (E) 12.5, E15.5 and E18.5, focusing on dorsal root ganglia. At these embryonic stages, satellite glia (glutamine synthetase) and Schwann cell markers, including transcriptional regulators (Sox10, Sox9, Tfap2a, Pou3f1) and non-transcription factors (Ngfr, BFABP, GFAP), were expressed in the DRG of wild-type and Etv5-/- embryos. Furthermore, by E18.5, quantification of Sox10+ Schwann cells and NeuN+ neurons revealed that these cells were present in normal numbers in the Etv5-/- dorsal root ganglia. We next performed peripheral nerve injuries at postnatal day 21, revealing that Etv5-/- mice had an enhanced injury response, generating more Sox10+ Schwann cells compared to wild-type animals at five days post-injury. Thus, while Etv5 is not required for Schwann cell development, possibly due to genetic redundancy with Etv1 and/or Etv4, Etv5 is an essential negative regulator of the peripheral nerve injury repair response. SIGNIFICANCE STATEMENTOur study sought to determine whether the ets domain transcription factor, Etv5, plays a role in regulating Schwann cell development and nerve repair. By using an embryonically and postnatally viable hypomorphic Etv5 mutant allele, we demonstrated that Etv5 is not required for the development of Schwann cells or other neural crest derivatives in the dorsal root ganglia, including satellite glia and neurons. Surprisingly, loss of Etv5 had a direct impact on the Schwann cell repair response post-injury, resulting in more Schwann cells populating the distal injured nerve site compared to wild-type animals. Thus, this work describes for the first time a role for Etv5 in regulating the Schwann cell repair response after peripheral nerve injury.

developmental biology↗