Biology subjects

Makrides, N.

Publications and source records attributed to Makrides, N..

8 recordsLinked to original sources

bioRxiv2026

A synthetic PGC1α co-activator reprograms microglial immunometabolism to preserve cone function in retinal degeneration

Retinitis pigmentosa (RP) is genetically heterogeneous, yet initiating mutations converge on primary rod degeneration followed by cone dysfunction and loss. Activated microglia are implicated in this secondary phase, but whether selective immunometabolic reprogramming of microglia preserves retinal function remains unknown. In human iPSC-derived microglia and retinal CD11b cells, PGC1 induction suppressed LPS-driven interferon signaling and cytokine release, yet Seahorse analysis and isotopologue tracing showed that energy metabolism remained altered. We therefore fused PGC1 to a multivalent p65-HSF1 module, yielding ~2.5-fold higher nuclear receptor output than native PGC1 and coordinated engagement of oxidative and anti-inflammatory programs in mouse retinal CD11b cells. Microglia-restricted expression in humanized RHOP347L mice preserved photopic responses through six months, increased retinal thickness and promoted ramified subretinal IBA1 morphology. Microglial immunometabolism thus represents a potentially genotype-independent neuroprotective target in RP, and engineered PGC1 co-activators provide a means to sustain oxidative metabolism under inflammatory pressure.

immunology↗

bioRxiv2026

FGF Signaling Potentiates Müller Glia for Mammalian Retinal Regeneration

Muller glia possess latent regenerative potential that could be harnessed to restore retinal neurons lost to injury or disease. Although fibroblast growth factor (FGF) signaling is upregulated following retinal damage, its role in mammalian retinal regeneration remains unclear. Here, we investigated the function of FGF signaling in Muller glial reprogramming using genetic, pharmacological, and single-cell transcriptomic approaches. Activation of FGF signaling alone was insufficient to induce Muller glial proliferation in the mouse retina. However, conditional deletion of FGFR1/2 in Muller glia abolished regenerative responses induced by multiple independent pathways, demonstrating that FGF signaling is essential for regenerative competence. Mechanistically, loss of FGF signaling impaired sustained ERK/MAPK activation following injury, while MEK/ERK inhibition phenocopied the regenerative defect. Conversely, constitutive MEK activation induced limited Muller glial proliferation in the absence of injury. Although STAT3/5 inhibition synergized with Activin-A to promote robust proliferation and neurogenic gene expression, it failed to rescue regeneration in FGF-deficient Muller glia. Single-cell RNA sequencing revealed that FGF signaling suppresses multiple anti-regenerative programs, including Hes1, S1pr1, p27, NF-{kappa}B, and p300/CBP activity. Together, these findings identify FGF signaling as a critical permissive regulator of mammalian retinal regeneration that potentiates Muller glia through sustained ERK activation and suppression of transcriptional barriers to regeneration.

Developmental Biology↗

bioRxiv2025

Allosteric inhibition rescues hydrocephalus caused by catalytically inactive Shp2

SHP2, a protein tyrosine phosphatase (PTP) crucial in Ras-MAPK signaling, is associated with various human congenital diseases and cancers. Here, we show that the catalytically inactive Shp2C459S mutation results in communicating hydrocephalus, similar to the catalytically activating Shp2E76K and Mek1DD mutants. Unlike previous mutants, however, Shp2C459S/+ mutation uniquely affects ciliary development rather than neurogenesis, leading to reduced cilia density and impaired ciliary motility. Differential scanning fluorimetry revealed that SHP2C459S, SHP2E76K and SHP2C459S/E76K mutations all induce an open SHP2 conformation, but only SHP2C459S leads to aberrant GAB1 phosphorylation in cells expressing wild-type SHP2. This distinctive signaling pattern correlates with our observations in brain ventricular tissues of Shp2C459S/+ mice, where Erk and Stat3 activities remain normal but Gab1 phosphorylation is elevated. Critically, we show that the hydrocephalus phenotype in Shp2C459S mice can be mitigated by allosteric inhibition of Shp2. These findings suggest that Shp2-associated hydrocephalus is driven by conformational changes rather than altered catalytic activity. Our results underscore the therapeutic potential of conformation-specific allosteric inhibitors in targeting both catalytically active and inactive SHP2 mutants.

pathology↗

bioRxiv2025

Ptpn14 Knockout Mice Reveal Critical Female-Specific Roles for the Hippo Pathway

The Hippo pathway regulates many physiological processes, including development, tumor suppression, and wound healing. One understudied Hippo pathway component is PTPN14, an evolutionarily conserved tyrosine-phosphatase that inhibits YAP/TAZ. While an established tumor suppressor, PTPN14s role in tissue homeostasis has remained unclear. We thus generated Ptpn14-deficient mice and found that only [~]60% of Ptpn14-/- mice survived postnatally, highlighting the importance of PTPN14 for viability, while also enabling the discovery of PTPN14 physiological functions. Ptpn14-/-mice developed debilitating corneal lesions and the uterus defect, hydrometra, as well as heart and kidney abnormalities. Ptpn14-deficiency precipitated an impaired injury response in the cornea and dysregulated YAP signaling in the uterus. Notably, these phenotypes were female-specific, revealing sexually-dimorphic Hippo pathway function through PTPN14. Finally, analysis of human PTPN14 variants suggested that PTPN14s essential roles are conserved in humans, underscoring the importance of our insights for designing therapies to improve womens health.

developmental biology↗

bioRxiv2024

Abl kinases regulate FGF signaling independent of Crk phosphorylation to prevent Peters anomaly

Peters anomaly, the most common cause of congenital corneal opacity, stems from corneal-lenticular adhesion. Despite numerous identified mutations, a cohesive molecular framework of the diseases etiology remains elusive. Here, we identified Abl kinases as pivotal regulators of FGF signaling, as genetic ablation of Abl kinases restores lens induction even in the absence of FGF signaling. Intriguingly, both Abl kinase deficiency and increased FGF-Ras activity result in Peters anomaly independent of ERK signaling, which can be rescued by allelic deletion of Abl substrate, Crk. However, contrary to the prevailing belief that Abl kinases regulate Crk proteins by direct phosphorylation, mutations at Abl kinase phosphorylation sites on Crk and CrkL did not yield any observable effects. Instead, our findings reveal that Abl kinases phosphorylate Ptpn12, which in turn inhibits p130Cas phosphorylation and Crk recruitment, crucial for Rho GTPases activation and cytoskeletal dynamics. Consequently, Abl kinase deficiency reduces actomyosin contractility within the lens vesicle and genetically interacts with RhoA inhibition. Conversely, Rac1 deletion mitigates Peters anomaly in models with aberrant FGF, Abl kinase and RhoA signaling. Our results demonstrate that Abl kinases regulate FGF signaling to balance RhoA and Rac1 activity via the Ptpn12-p130Cas pathway, suggesting that targeting tension-mediated lens vesicle separation could be a therapeutic strategy for Peters anomaly.

developmental biology↗

bioRxiv2024

Crk mediates Csk-Hippo signaling independently of Yap tyrosine phosphorylation to induce cell extrusion

Src family kinases (SFKs), including Src, Fyn and Yes, play important roles in development and cancer. Despite being first discovered as the Yes-associated protein, the regulation of Yap by SFKs remains poorly understood. Here, through single-cell analysis and genetic lineage tracing, we show that the pan-epithelial ablation of C-terminal Src kinase (Csk) in the lacrimal gland unleashes broad Src signaling but specifically causes extrusion and apoptosis of acinar progenitors at a time when they are shielded by myoepithelial cells from the basement membrane. Csk mutants can be phenocopied by constitutively active Yap and rescued by deleting Yap or Taz, indicating a significant functional overlap between Src and Yap signaling. Although Src-induced tyrosine phosphorylation has long been believed to regulate Yap activity, we find that mutating these tyrosine residues in both Yap and Taz fails to perturb mouse development or alleviate the Csk lacrimal gland phenotype. In contrast, Yap loses Hippo signaling-dependent serine phosphorylation and translocates into the nucleus in Csk mutants. Further chemical genetics studies demonstrate that acute inhibition of Csk enhances Crk/CrkL phosphorylation and Rac1 activity, whereas removing Crk/CrkL or Rac1/Rap1 ameliorates the Csk mutant phenotype. These results show that Src controls Hippo-Yap signaling through the Crk/CrkL-Rac/Rap axis to promote cell extrusion.

developmental biology↗

bioRxiv2022

Chondroitin sulfates enhances the barrier function of basement membrane assembled by heparan sulfates

Glycosaminoglycans (GAGs) are ubiquitously expressed polysaccharides attached to proteoglycans, but their functions in the retina are poorly understood. Here we generated conditional knockouts of biosynthetic enzymes for heparan sulfate (HS) and chondroitin sulfate (CS) in retinal progenitor cells. We showed that ablation of HS polymerase Ext1 did not affect initial progression of retinal angiogenesis, but it disrupted the pruning of blood vessels and establishment of arterioles and venules. In the absence of retinal HS, blood vessels were also vulnerable to high oxygen tension in early postnatal stages, which can be rescued by exogenous VEGF, consistent with the role of retinal HS in the fine-tuning of VEGF signaling. Furthermore, we observed that the retinal inner limiting membrane (ILM) was disrupted by deletion of Ext1 in a timing specific manner, suggesting that retinal HS is required for the assembly but not the maintenance of the ILM. Lastly, we showed that further deletion of C4st1, a CS sulfation enzyme, did not affect the assembly of the ILM, but aggravated the ILM permeability when combined with Ext1 deletion. These results demonstrated an important role of CS and HS in establishing the barrier function of basement membrane.

developmental biology↗

bioRxiv2021

Phase transition specified by a binary code patterns the vertebrate eye cup

The developing vertebrate eye cup is partitioned into the neural retina (NR), the retinal pigmented epithelium (RPE) and the ciliary margin (CM). By single cell analysis, we showed that a gradient of FGF signaling regulates demarcation and subdivision of the CM and controls its stem cell-like property of self-renewal, differentiation and survival. This regulation by FGF is balanced by an evolutionarily conserved Wnt signaling gradient induced by the lens ectoderm and the periocular mesenchyme, which specifies the CM and the distal RPE. These two morphogen gradients converge in the CM where FGF signaling promotes Wnt signaling by stabilizing {beta}-catenin in a GSK3{beta}-independent manner. We further showed that activation of Wnt signaling converts the NR to either the CM or the RPE depending on the level of FGF signaling. Conversely, activation of FGF transforms the RPE to the NR or CM dependent on Wnt activity. We demonstrated that the default fate of the eye cup is the NR, but synergistic FGF and Wnt signaling promotes CM formation both in vivo and in retinal organoid culture of human iPS cells. Our study reveals that the vertebrate eye develops through phase transition determined by a combinatorial code of FGF and Wnt signaling.

developmental biology↗