Androniki Kretsovali

Background Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, characterized by amyloid deposition, neurofibrillary tangles, neuroinflammation and synaptic dysfunction. The Promyelocytic leukemia protein (PML) and the cognate nuclear bodies (PML-NB) have emerged as critical regulators of the nervous system, regulating neocortex development, neuronal survival, protein homeostasis and protection from stress. PML-NB have been implicated in the solubility of pathological aggregates in Neurodegenerative Diseases (NDD). However, the impact of PML on AD progression and whether its loss affects amyloid pathology remain unknown. Methods To investigate the role of PML in neuroinflammation we used intracerebroventricular (ICV) injections of oligomeric amyloid beta 1–42 (oAβ ₁₋₄₂ ), in WT and Pml-/- mice and primary microglia cultures derived from these genotypes. To explore the role of PML in AD pathology we employed phenotypic, transcriptomic and behavioral analyses of WT, Pml-/- , 5xFAD and 5xFAD Pml-/- mice. Results Pml-/- mice displayed reduced recruitment and activation of microglia in the vicinity of οΑβ ₁₋₄₂ injection, accompanied by deregulated expression of disease-associated microglia (DAM) genes. Consistently, Pml-/- primary microglial cultures exhibit reduced phagocytosis, activation, viability and impaired cytokine responsiveness following β-amyloid challenge. PML depletion in 5xFAD mice accelerates Aβ accumulation, impairs microglial activation, lysosomal acidification and recruitment to amyloid plaques while enhances astrocyte reactivity and neuronal degeneration. Hippocampal transcriptomic analyses reveal sex-dependent effects of PML loss, with downregulation of pathways related to cell migration, axonogenesis and synapse organization in 5xFAD Pml-/- females and peroxisomal functions, DNA repair and immune responses, in 5xFAD Pml-/- males. Both sexes show suppression of immune response genes and deregulated expression of DAM genes. PML depletion increases impulsivity and hippocampus-dependent behavioral abnormalities in the context of Aβ pathology, highlighting a role for PML in maintaining cognitive function. Conclusions PML loss exacerbates multiple aspects of AD pathophysiology including amyloid deposition, impaired anti-inflammatory responses, neurotoxicity and cognitive performance. Our findings identify PML as a key regulator for microglial homeostasis and neuroprotective functions in amyloid pathology. Through its actions in microglia, PML emerges as an effector and a marker of aging and neurodegeneration. Restoring or enhancing its activity may represent a promising therapeutic strategy to preserve neuronal function in AD.

The promyelocytic leukemia protein (PML) and its associated nuclear bodies have recently emerged as critical regulators of embryonic stem (ES) cell identity. Despite their recognized importance, the complete spectrum of PML-mediated molecular events in ES cells remains unclear. In this report, we study how PML is shaping the proteomic and SUMO proteomic landscape in ES cells. Proteomic profiling of PML-depleted ES cells uncovered a downregulation of self-renewal factors and an upregulation of proteins associated with translation and proteasomal activity, reflecting a cellular transition from pluripotency to differentiation. Importantly, PML promotes the sumoylation of pluripotency-related factors, chromatin organizers, and cell cycle regulators. We identified SALL1 and CDCA8 as novel PML-directed sumoylation targets, both critical for ES cell maintenance. SALL1 sumoylation increases the activation of the Wnt pathway, contributing to its ability to inhibit ES cell differentiation. Similarly, CDCA8 sumoylation enhances its capacity to promote cell proliferation. Collectively, our findings demonstrate that PML regulates ES cell identity by modulating the abundance or sumoylation of key regulators involved in pluripotency and cell cycle progression.

The Promyelocytic Leukemia protein (PML) and its associated nuclear bodies have recently emerged as critical regulators of embryonic stem (ES) cell identity. Despite their recognized importance, the complete spectrum of PML-mediated molecular events in ES cells remains unclear. In this report we have studied how PML is shaping the proteomic and SUMO proteomic landscape in ES cells. Proteomic profiling of PML-depleted ES cells uncovered a downregulation of self-renewal factors and an upregulation of proteins associated with translation and proteasomal activity, reflecting a cellular transition from pluripotency to differentiation. Importantly, PML promotes the sumoylation of pluripotency-related factors, chromatin organizers and cell cycle regulators. We identify SALL1 and CDCA8 as novel PML-directed sumoylation targets, both critical for ES cell maintenance. SALL1 sumoylation increases the activation of the Wnt pathway, contributing to its ability to inhibit ES cell differentiation. Similarly, CDCA8 sumoylation enhances its capacity to promote cell proliferation. Collectively, our findings demonstrate that PML regulates ES cell identity by modulating the abundance or sumoylation of key regulators involved in pluripotency and cell cycle progression.

Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor-suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.

Promyelocytic Leukemia Protein (PML) is the core protein of nuclear bodies (NBs) that regulate a large number of cellular processes, including, context dependent, tumor –suppressor and pro-oncogenic effects. PML knockdown (KD) in breast cancer lines MDA-MB-231, but not MCF7, cells showed higher cell proliferation, increased migration properties and prolonged stem cell –like survival in line with gene enrichment results from RNA sequencing analysis. MDA-MB-231 PML KD cells showed an increase of hypoxic and mesenchymal characteristics manifested by higher HIF1a and the EMT-TWIST2 protein levels respectively. Mechanistically, PML loss caused an increase of HIF1a and TWIST2 RNA levels. Interestingly, TWIST2 binds to PML. Moreover, PML directly opposed the action of HIF1a and TWIST2 on VEGFa and CD24 reporters, respectively. Tumor xenografts of MDA-MB-231 PML KD cells showed a higher micro vessel content, grew faster and had higher metastatic ability with a preference for lung. Thus, PML opposes the aggressive cancer phenotype by multiple mechanisms that antagonize the HIF-hypoxia-angiogenic and EMT axis.

Protein-based carriers are promising vehicles for the intracellular delivery of therapeutics. In this study, we designed and studied adenovirus protein fiber constructs with potential applications as carriers for the delivery of protein and nanoparticle cargoes. We used as a basic structural framework the fibrous shaft segment of the adenovirus fiber protein comprising of residues 61–392, connected to the fibritin foldon trimerization motif at the C-terminal end. A fourteen-amino-acid biotinylation sequence was inserted immediately after the N-terminal, His-tagged end of the construct in order to enable the attachment of a biotin moiety in vivo. We report herein that this His-tag biotinylated construct folds into thermally and protease-stable fibrous nanorods that can be internalized into cells and are not cytotoxic. Moreover, they can bind to proteins and nanoparticles through the biotin–streptavidin interaction and mediate their delivery to cells. We demonstrate that streptavidin-conjugated gold nanoparticles can be transported into NIH3T3 fibroblast and HeLa cancer cell lines. Furthermore, two streptavidin-conjugated model proteins, alkaline phosphatase and horseradish peroxidase can be delivered into the cell cytoplasm in their enzymatically active form. This work is aimed at establishing the proof-of-principle for the rational engineering of diverse functionalities onto the initial protein structural framework and the use of adenovirus fiber-based proteins as nanorods for the delivery of nanoparticles and model proteins. These constructs could constitute a stepping stone for the development of multifunctional and modular fibrous nanorod platforms that can be tailored to applications at the sequence level.

Cell-penetrating peptides are used extensively to deliver molecules into cells due to their unique characteristics such as rapid internalization, charge, and non-cytotoxicity. Amyloid fibril biomaterials were reported as gene transfer or retroviral infection enhancers; no cell internalization of the peptides themselves is reported so far. In this study, we focus on two rationally and computationally designed peptides comprised of β-sheet cores derived from naturally occurring protein sequences and designed positively charged and aromatic residues exposed at key residue positions. The β-sheet cores bestow the designed peptides with the ability to self-assemble into amyloid fibrils. The introduction of positively charged and aromatic residues additionally promotes DNA condensation and cell internalization by the self-assembled material formed by the designed peptides. Our results demonstrate that these designer peptide fibrils can efficiently enter mammalian cells while carrying packaged luciferase-encoding plasmid DNA, and they can act as a protein expression enhancer. Interestingly, the peptides additionally exhibited strong antimicrobial activity against the enterobacterium Escherichia coli.

Groucho related gene 5 (GRG5) is a multifunctional protein that has been implicated in late embryonic and postnatal mouse development. Here, we describe a previously unknown role of GRG5 in early developmental stages by analyzing its function in stem cell fate decisions. By both loss and gain of function approaches we demonstrate that ablation of GRG5 deregulates the Embryonic Stem Cell (ESC) pluripotent state whereas its overexpression leads to enhanced self-renewal and acquisition of cancer cell-like properties. The malignant characteristics of teratomas generated by ESCs that overexpress GRG5 reveal its pro-oncogenic potential. Furthermore, transcriptomic analysis and cell differentiation approaches underline GRG5 as a multifaceted signaling regulator that represses mesendodermal-related genes. When ESCs exit pluripotency, GRG5 promotes neuroectodermal specification via Wnt and BMP signaling suppression. Moreover, GRG5 promotes the neuronal reprogramming of fibroblasts and maintains the self-renewal of Neural Stem Cells (NSCs) by sustaining the activity of Notch/Hes and Stat3 signaling pathways. In summary, our results demonstrate that GRG5 has pleiotropic roles in stem cell biology functioning as a stemness factor and a neural fate specifier.