Dissertation committee:
1. Ευθυμιόπουλος Σπυρίδων, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ (Επιβλέπων)
2. Παπαζαφείρη Παναγιώτα, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
3. Σιδεράς Πασχάλης, Ερευνητής Α’, Κέντρο Κλινικής, Πειραματικής Χειρουργικής & Μεταφραστικής Έρευνας, ΙΙΒΕΑΑ
4. Τσιτσιλώνη Ουρανία, Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
5. Μουστάκας Αριστείδης, Καθηγητής, Τμήμα Ιατρικής Βιοχημείας και Μικροβιολογίας, Πανεπιστήμιο της Ουψάλα, Σουηδία
6. Θωμαΐδου Δήμητρα, Ερευνήτρια Α’, Τομέας Νευροβιολογίας, Ελληνικό Ινστιτούτο Παστέρ
7. Πολίτης Παναγιώτης, Ερευνητής Γ’, Κέντρο Βασικής Έρευνας, ΙΙΒΕΑΑ
Summary:
The Transforming Growth Factor beta (TGFβ) superfamily comprises more than 30 structurally related members including, among others, TGFβs, Bone Morphogenetic Proteins (BMPs) and Activins. Signaling by members of this superfamily has been implicated in fundamental processes regulating the homeostasis of the Central Nervous System (CNS), including neurogenesis, differentiation of glia cells, formation of blood-brain barrier and maintenance of its integrity and others. In addition, numerous studies have linked TGFβ superfamily to CNS pathologies affecting a large proportion of the world's population, such as neuroinflammation, tissue damage following mechanical traumatic injuries and neurodegenerative diseases. In fact, several components of this signaling system, including ligands, receptors and intracellular mediators, are upregulated at mRNA and/or protein levels in experimental models of mechanical and/or biochemical – through neurotoxin administration – CNS injuries. Furthermore, increased protein levels of these components have been found in human spinal cord injuries, as well as in the cerebrospinal fluid or the striatum of patients with Parkinson's disease.
Despite the extensive characterization of individual components of the TGFβ superfamily system during CNS homeostasis and pathophysiology, data concerning the identity of recipient cells of this action, as well as the way by which these cells decode the complex TGFβ-superfamily system generated signaling-input to produce the desired physiological response at any given time, are still lacking. Τhe precise role of the two branches of this signaling system, the TGFβ/Activin- and the BMP-induced pathways, under CNS pathological conditions is still not fully understood. Is there a functional interplay between these two signaling branches in physiology and pathophysiology?
The aim of this thesis was to clarify the above questions using a double transgenic reporter mice system, developed in BRFAA by crossing two transgenic animal lines carrying the genes of Red (RFP) and Green Fluorescent Proteins (eGFP) in their genome, under the control of well-characterized sequences responding selectively to TGFβs/Activins (TGFβ Responsive Elements, TRE-RFP) or BMPs (BMP Responsive Elements, BRE-eGFP), respectively. The potential cellular targets of canonical TGFβ or BMP signaling express the RFP or eGFP protein, respectively. Using this animal model in previous experiments, we found that signaling by members of the TGFβ superfamily is active and confined to various regions of neonatal and adult brain, such as the olfactory bulbs, the cortex, the ventricle walls, the choroid plexus and the vasculature, under homeostatic conditions. Actually, neurons, astrocytes and structural cells of the blood-brain barrier, such as endothelial cells and pericytes, were included among the cellular targets of TGFβ and/or BMP signaling.
To investigate the contribution of both TGFβ and BMP signaling branches in CNS pathophysiology, we used a mouse model of penetrating focal brain injury. More specifically, adult wild-type mice were subjected to unilateral acute stab wound injury and brain tissues encompassing injured (ipsilateral) and corresponding intact (contralateral) areas were collected, at different time-points post-injury. Hallmarks of TGFβ-superfamily system activation, such as levels of phosphorylated Smads, ligands and target genes for both TGFβ and BMP pathways, were found upregulated within injured tissues, the first days post-injury. Next, using double transgenic reporter mice we showed that activation of both TGFβ and BMP signaling branches involved primarily reactive astrocytes that demarcated the wound area.
In order to gain indirectly insights regarding the role of TGFβ and/or BMP signaling activation in glia cells within the injured tissues, we developed primary glia culture systems, as well as fluorescence-activated cell-sorting protocols for the establishment of purified astrocytic and microglial cultures. The latter were treated with recombinant TGFβ1 and BMP4 and their transcriptomes were analyzed by next-generation sequencing (RNA-Seq). Astrocytes responded substantially to both ligands with considerably overlapping transcriptional profiles. Although microglia responded remarkably well to TGFβ1, they failed to respond robustly to BMP4, a finding that is probably related to the relatively lower mRNA expression levels for BMP-related receptors and Smads, compared to the astrocytic ones.
Bioinformatics analysis of the transcriptomic profiles of TGFβ1- or BMP4-treated astrocytes revealed novel pathways crucial for tissue-injury repair and blood-brain barrier restoration, such as activation of cholesterol biosynthesis and transport, production of axonal guidance and extracellular matrix components and regulation of glutamate homeostasis. Moreover, both ligands in astrocytes and TGFβ1 in microglia shifted the phenotype of reactive glia cells towards the anti-inflammatory and tissue-reparatory A2-like and M0/M2-like phenotypes, respectively. Increased expression of selected key components of the in vitro modulated pathways and markers of A2-like astrocytes was confirmed in vivo within the wound area, suggesting that these processes could also be modulated in situ by the integrated action of TGFβ- and/or BMP-mediated signaling.
Besides the mechanical penetrating injury, a biochemical brain injury model was also applied, through administration of the mitochondrial neurotoxin MPTP that simulates a parkinsonian phenotype in adult mice. Substantial activation of the TGFβ superfamily signaling system was observed across the nigrostriatal pathway in MPTP-treated animals. Also in this brain injury model, the majority of TRE-RFP and BRE-eGFP positive cells in the striatum and the substantia nigra were identified as astrocytes. Then, stereotactic overexpression of activators or inhibitors of BMP signaling was performed in the substantia nigra of animals, prior to MPTP administration, followed by assessment of their locomotor activity. We observed a relative improvement in some behavioral parameters of animals subjected to Noggin overexpression, suggesting that focal inhibition of BMP signaling in the substantia nigra is probably exerting a neuroprotective action in the ipsilateral nigrostriatal pathway.
To sum up, using the murine brain as model system, we uncovered important aspects of the interaction between TGFβ and BMP branches of the TGFβ superfamily signaling system and highlighted the importance of the relative equilibrium of cytokines/members of this system for the establishment of homeostasis and progression of pathology in the CNS. In addition, we identified not only key neuroprotective and/or neurotoxic processes under pathophysiological conditions, but also specific molecular events/mechanisms, which involve glia cells and could be potentially used towards the development of more effective therapeutic approaches in the context of neuroinflammatory disorders and/or traumatic CNS injuries.
Keywords:
Transforming growth factor-beta signaling, TGF-beta, Bone morphogenetic protein, brain, astrocytes, microglia, traumatic brain injury, TBI, Parkinson's disease, neuroprotection, tissue repair, neuroinflammation, MPTP
