Chiara Tremolanti

Neuroprotection contre les maladies de la myéline : études in vitro et in vivo pour explorer les rôles protecteurs des ligands TSPO et des stratégies de l'édition du génome: Dans le SNC, les oligodendrocytes (OLs) produisent de la myéline, la structure lipophile qui permet la bonne conduction de l’influx nerveux. La formation anormale ou les dommages de la myéline ou des OLs sous-tendent les pathologies de la myéline, caractérisées par de graves déficits neurologiques. Diverses données de la littérature suggèrent des effets anti-inflammatoires et neuroprotecteurs des ligands de la protéine translocatrice (TSPO) dans plusieurs modèles de neuroinflammation et de neurodégénérescence in vitro et in vivo. Dans la présente thèse, nous avons vérifié spécifiquement cette hypothèse en étudiant divers aspects des pathologies de la myéline afin d’identifier de nouvelles approches thérapeutiques. Nos résultats montrent que TSPO module négativement l’activation de la microglie vers le phénotype inflammatoire classique. Ensuite, nous avons démontré l’effet bénéfique de l’administration de deux ligands TSPO dans un modèle murin de SEP par l’évaluation de marqueurs pathologiques. Enfin, nous avons prouvé que TSPO peut également jouer un rôle important lors de la différenciation des OLs, ce qui fait de la protéine TSPO une cible prometteuse pour favoriser la remyélinisation.

Midbrain dopaminergic neurons (mDA) are selectively lost in Parkinson’s disease (PD), driving sustained efforts to generate bona fide mDA neurons from human-induced pluripotent stem cells (iPSCs) for replacement therapy. While morphogen gradients and transcription factors have been extensively studied, extracellular regulators remain largely overlooked. Here, we identify the heparan sulfate-modifying enzymes SULF1 and SULF2 as essential for establishing mDA neuron identity in vitro. Using CRISPR/Cas9-engineered iPSCs, we show that loss of SULF1/2 increases 6-O-sulfation of heparan sulfate chains and disrupts anterior-posterior and dorsoventral patterning in cells exposed to a midbrain differentiation protocol. Double-knockout cells fail to acquire midbrain fate and instead adopt caudal and neural crest-like identities, as revealed by single-nucleus RNA sequencing. Mechanistically, we find enhanced FGF signaling and demonstrate that FGF inhibition redirects cells toward midbrain progenitors, without fully restoring ventral identity. These findings establish a critical role for SULF1/2 in human mDA neuron development and uncover a previously unrecognized layer of extracellular control over neuronal patterning, opening for novel strategies to refine differentiation protocols for PD and beyond.

Parkinson’s disease (PD) is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons, leading to severe debilitating motor impairment. Recent single-cell analyses have revealed that specific mDA subpopulations, such as SOX6⁺ AGTR1⁺ neurons, are most vulnerable to degeneration. Current human pluripotent stem cell differentiation protocols fail to selectively generate these subtypes. Here, we describe a multiomic-guided strategy that enriches SOX6 ⁺ mDA neurons by combining enhancing Sonic Hedgehog agonism with prolonged Wnt activation. This approach accelerates floor plate specification, increases expression of mDA developmental markers, and substantially increases the yield of SOX6⁺ mature mDA neurons compared to prior attempts. Following intrastriatal transplantation into hemiparkinsonian mice, these cells restored motor function in approximately 4 months and generated grafts containing SOX6⁺ A9-like neurons. Our work thus establishes a reproducible differentiation platform for generating the PD-susceptible mDA subtype, providing a foundation for precision disease modelling and subtype-targeted cell replacement therapies.

An aberrant pro-inflammatory microglia response has been associated with most neurodegenerative disorders. Identifying microglia druggable checkpoints to restore their physiological functions is an emerging challenge. Recent data have shown that microglia produce de novo neurosteroids, endogenous molecules exerting potent anti-inflammatory activity. Here, the role of neurosteroidogenesis in the modulation of microgliosis was explored in human microglia cells. In particular, CYP11A1 inhibition or TSPO pharmacological stimulation, crucial proteins involved in the rate limiting step of the neurosteroidogenic cascade, were employed. CYP11A1 inhibition led microglia to acquire a dysfunctional and hyperreactive phenotype, while selective TSPO ligands promoted the establishment of an anti-inflammatory one. Analysis of specific neurosteroid levels (neurosteroidome) identified allopregnanolone/pregnanolone as crucial metabolites allowing controlled activation of microglia. Importantly, the neurosteroid shift towards a greater androgenic/estrogenic profile supported the transition from pro-inflammatory to neuroprotective microglia, suggesting the therapeutic potential of de novo microglial neurosteroidogenesis stimulation for neuroinflammatory-related disorders.

In the nervous system, inflammatory responses physiologically occur as defense mechanisms triggered by damaging events. If improperly regulated, neuroinflammation can contribute to the development of chronically activated states of glial cells, with the perpetuation of inflammation and neuronal damage, thus leading to neurological and neurodegenerative disorders. Interestingly, neuroinflammation is associated with the overexpression of the mitochondrial translocator protein (TSPO) in activated glia. Despite the precise role of TSPO in the immunomodulatory mechanisms during active disease states is still unclear, it has emerged as a promising target to promote neuroprotection. Indeed, TSPO ligands have been shown to exert beneficial effects in counteracting neuroinflammation and neuronal damage in several in vitro and in vivo models of neurodegenerative diseases. In particular, the regulation of neurosteroids’ production, cytokine release, metabolism of radical oxidative species, and cellular bioenergetics appear to be the main cellular events that underlie the observed effects. The present review aims to illustrate and summarize recent findings on the potential effect of TSPO ligands against neuroinflammation and related neurodegenerative mechanisms, taking into consideration some pathologies of the nervous system in which inflammatory events are crucial for the onset and progression of the disease and attempting to shed light onto the immunomodulatory effects of TSPO.

Neuroactive steroids are potent modulators of microglial functions and are capable of counteracting their excessive reactivity. This action has mainly been ascribed to neuroactive steroids released from other sources, as microglia have been defined unable to produce neurosteroids de novo. Unexpectedly, immortalized murine microglia recently exhibited this de novo biosynthesis; herein, de novo neurosteroidogenesis was characterized in immortalized human microglia. The results demonstrated that C20 and HMC3 microglial cells constitutively express members of the neurosteroidogenesis multiprotein machinery—in particular, the transduceosome members StAR and TSPO, and the enzyme CYP11A1. Moreover, both cell lines produce pregnenolone and transcriptionally express the enzymes involved in neurosteroidogenesis. The high TSPO expression levels observed in microglia prompted us to assess its role in de novo neurosteroidogenesis. TSPO siRNA and TSPO synthetic ligand treatments were used to reduce and prompt TSPO function, respectively. The TSPO expression downregulation compromised the de novo neurosteroidogenesis and led to an increase in StAR expression, probably as a compensatory mechanism. The pharmacological TSPO stimulation the de novo neurosteroidogenesis improved in turn the neurosteroid-mediated release of Brain-Derived Neurotrophic Factor. In conclusion, these results demonstrated that de novo neurosteroidogenesis occurs in human microglia, unravelling a new mechanism potentially useful for future therapeutic purposes.

Wearable devices now make it possible to record large quantities of physiological data, which can be used to obtain a clearer view of a person’s health status and behavior. However, to the best of our knowledge, there are no open datasets in the literature that provide psycho-physiological data. The Multilevel Monitoring of Activity and Sleep in Healthy people (MMASH) dataset presented in this paper provides 24 h of continuous psycho-physiological data, that is, inter-beat intervals data, heart rate data, wrist accelerometry data, sleep quality index, physical activity (i.e., number of steps per second), psychological characteristics (e.g., anxiety status, stressful events, and emotion declaration), and sleep hormone levels for 22 participants. The MMASH dataset will enable the investigation of possible relationships between the physical and psychological characteristics of people in daily life. Data were validated through different analyses that showed their compatibility with the literature.

A key role of the mitochondrial Translocator Protein 18 KDa (TSPO) in neuroinflammation has been recently proposed. However, little is known about TSPO-activated pathways underlying the modulation of reactive microglia. In the present work, the TSPO activation was explored in an in vitro human primary microglia model (immortalized C20 cells) under inflammatory stimulus. Two different approaches were used with the aim to (i) pharmacologically amplify or (ii) silence, by the lentiviral short hairpin RNA, the TSPO physiological function. In the TSPO pharmacological stimulation model, the synthetic steroidogenic selective ligand XBD-173 attenuated the activation of microglia. Indeed, it reduces and increases the release of pro-inflammatory and anti-inflammatory cytokines, respectively. Such ligand-induced effects were abolished when C20 cells were treated with the steroidogenesis inhibitor aminoglutethimide. This suggests a role for neurosteroids in modulating the interleukin production. The highly steroidogenic ligand XBD-173 attenuated the neuroinflammatory response more effectively than the poorly steroidogenic ones, which suggests that the observed modulation on the cytokine release may be influenced by the levels of produced neurosteroids. In the TSPO silencing model, the reduction of TSPO caused a more inflamed phenotype with respect to scrambled cells. Similarly, during the inflammatory response, the TSPO silencing increased and reduced the release of pro-inflammatory and anti-inflammatory cytokines, respectively. In conclusion, the obtained results are in favor of a homeostatic role for TSPO in the context of dynamic balance between anti-inflammatory and pro-inflammatory mediators in the human microglia-mediated inflammatory response. Interestingly, our preliminary results propose that the TSPO expression could be stimulated by NF-κB during activation of the inflammatory response.