Investigation of metabolic enzymes in glial cells and their implication in the development of neuroinflammatory diseases

Author(s)

Rafaela Mano Guimarães

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-12-01

Examining board members

Thiago Mattar Cunha; Alexandre Salgado Basso; Lucila Leico Kagohara Elias

• Advisor: Thiago Mattar Cunha

Abstract

Astrocytes are the most abundant glial cells in the central nervous system (CNS) and essential for maintaining brain homeostasis. However, in pathological conditions such as those observed in the experimental model of multiple sclerosis (EAE), it is suggested that its activation is deleterious as it amplifies the local inflammatory response. Recently, an interesting correlation has been demonstrated between inflammation and the promotion of alternative metabolic pathways, such as the hexosamine biosynthesis pathway (HBP). The final product of HBP is UDP-N-acetylglucosamine which, by the action of the enzyme O-GlycosylTransferase (OGT), generates post-translational modifications called O-GlcNAcylation. The occurrence of O-GlcNAcylation has been associated with the activation of immune system cells such as macrophages and T lymphocytes, and with the progression of certain neurodegenerative diseases. However, the relationship between BPH and O-GlcNAcylation in astrocytes remains unexplored in the context of neuroinflammation. Through the use of in vitro and in vivo studies, flow cytometry, largescale transcriptional analyses, imaging techniques and protein quantification, this project aimed to better understand these mechanisms. After establishing primary cultures of astrocytes obtained from newborn mice, we characterize an alternative method for studying these cells in an isolated system. Under stimulus of molecules that mimic some aspects of neuroinflammation (TNF, IL-1alpha and C1q) we observed a significant induction of IL-6, CCL2, CXCL1 and CXCL10. Under these conditions, analysis of the expression of crucial enzymes in the BPH pathway showed a positive correlation with the induction of Ogt, Oga, Gfpt11/2 transcripts. A series of functional experiments using pharmacological inhibitors of these enzymes revealed that the blockade of OGA (OGliconAcase - catalyzes the removal of the UDP-N-acetylglucosamine group), via Thiamet G, negatively impacted the promotion of IL-6 and CXCL1 against stimuli inflammatory. In order to explore these mechanisms in vivo, we generated Aldh1l1CreER/Ogtflox/flox animals in which OGT can be genetically depleted in astrocytes by the use of tamoxifen. After the satisfactory validation of this experimental strategy, we submitted these animals to the EAE model and investigated the functional role of OGT in the clinical parameters of the disease. In the absence of OGT in astrocytes, a significant improvement in disease development was observed, suggesting a deleterious role for BPH in neuroinflammation. By flow cytometry, we observed a significant correlation between OGT deficiency and a lower infiltration of immune cells in the CNS of these animals. Both the proportion and the absolute number of leukocytes, microglia, T lymphocytes (CD4+, Treg, Th17), macrophages and dendritic cells were affected in the absence of OGT. These results suggest that OGT expression and, virtually, OGlcNAcylation of proteins is responsible for the communication of astrocytes with immune cells that infiltrate the CNS of animals with EAE. The non-involvement of OGT in the production of all inflammatory mediators in culture suggests an independent effect of cytokines and chemokines classically described in EAE. Further studies investigating these mechanisms in more detail are underway in our research group and may identify potential targets for the treatment of diseases that affect the CNS. (AU)

• FAPESP's process: 19/13829-0 - Role of O-GlcNAcylation in the neuroinflammatory functions of astrocytes
• Grantee: Rafaela Mano Guimarães
• Support Opportunities: Scholarships in Brazil - Doctorate