Thèse Identification des Mécanismes par Lesquels la Dyshoméostasie du Fer Lysosomal Altère la Protéostasie Neuronale et Induit la Ferroptose dans la Mps Iiib H/F Doctorat.Gouv.Fr

Iron homeostasis represents a plausible mechanistic link between lysosomal dysfunction, oxidative damage and neuronal death. Iron is essential for mitochondrial respiration, neurotransmitter synthesis and myelination, but excess labile ferrous iron catalyzes hydroxyl radical formation through Fenton chemistry and promotes the peroxidation of polyunsaturated membrane phospholipids. Cellular iron availability is controlled by coordinated uptake, storage and export mechanisms involving transferrin receptor 1, DMT1, ZIP14, ferritin and ferroportin, together with the hepcidin-ferroportin regulatory axis and IRP1/2 intracellular sensors. The lysosome occupies a central position in this network through endosomal iron release, autophagic degradation of iron-containing proteins and NCOA4-dependent ferritinophagy. Consequently, impaired lysosomal iron recycling may expand the redox-active iron pool and couple defective proteostasis to mitochondrial dysfunction and ferroptosis.
Clinical MRI observations have reported iron deposition in the globus pallidus and pulvinar in patients with MPS IIIB (Brady et al., 2013). Cerebral iron accumulation was also demonstrated in Naglu-/- mice (MPS IIIB mice), particularly in the parietal cortex (Puy et al., 2018; Larribau et al., 2025). Our recent work provided the first integrated evidence of a neuron-selective ferroptotic phenotype in this model. Iron accumulation was associated with increased ferritin heavy-chain expression, temporal dysregulation of iron import pathways, reduced expression of the cystine transporter xCT/SLC7A11 and glutathione peroxidase 4, induction of the pro-ferroptotic lipid-remodeling enzymes ACSL4 and LPCAT3, and neuronal accumulation of 4-hydroxynonenal, indicating enhanced membrane lipid peroxidation. Ferroportin abundance was increased but the protein exhibited marked intracellular vacuolar retention and reduced plasma-membrane localization. This defect was detected predominantly in NeuN-positive neurons and not in microglia or astrocytes, supporting a neuron-specific impairment of iron export (Larribau et al., 2025).
These findings identify iron dyshomeostasis and ferroptotic vulnerability as previously unrecognized components of MPS IIIB neuropathology. However, the identity of the ferroportin-containing vacuolar compartment, the presence of a redox-active lysosomal iron pool, the neuronal populations most susceptible to this process and the causal relationship between lysosomal iron retention, proteostasis failure and ferroptosis remain unresolved.
The aim of this doctoral project is to explore these mechanisms and determine whether defective lysosomal iron trafficking constitutes a mechanistic link between heparan sulfate storage, neuronal proteotoxicity and ferroptotic death. Elucidating this causal pathway will clarify the contribution of lysosomal iron dyshomeostasis to MPS IIIB neurodegeneration and support the identification of new biomarkers and therapeutic targets, with potential relevance to other lysosome-associated neurodegenerative disorders. Les objectifs :
L'objectif général est de déterminer comment les troubles du fer lysosomal contribue à la perte de protéostasie neuronale et à la ferroptose dans la MPS IIIB.
Les objectifs spécifiques sont :
1- Identifier les populations neuronales les plus vulnérables et caractériser leurs altérations lysosomales, ferroptotiques et antioxydantes.
2-Etablir la signature moléculaire du lysosome pathologique par des analyses protéomiques, métabolomiques et lipidomiques, et préciser le rôle de la ferritinophagie, des héparane-sulfates et de l'hepcidine cérébrale dans la rétention lysosomale du fer.
3- Démontrer le rôle causal des anomalies du trafic lysosomal du fer en modulant la ferritinophagie dépendante de NCOA4 et l'export du fer par la ferroportine, et évaluer, in vitro et in vivo, des approches capables de restaurer l'homéostasie lysosomale du fer, de préserver la protéostasie neuronale et de limiter la ferroptose.
A terme, le projet vise à identifier de nouveaux biomarqueurs et de nouvelles cibles thérapeutiques pour la MPS IIIB.

The overall objective is to determine how lysosomal iron dyshomeostasis contributes to neuronal proteostasis failure and ferroptosis in MPS IIIB.
The specific objectives are to:
1- Identify the most vulnerable neuronal populations and characterize their lysosomal alterations, ferroptotic phenotype and antioxidant responses.
2- Define the molecular signature of pathological lysosomes through proteomic, metabolomic and lipidomic analyses, and determine the respective contributions of ferritinophagy, heparan sulfate and brain-derived hepcidin to lysosomal iron retention.
3- Demonstrate the causal role of defective lysosomal iron trafficking by modulating NCOA4-dependent ferritinophagy and ferroportin-mediated iron export, and evaluate, both in vitro and in vivo, strategies capable of restoring lysosomal iron homeostasis, preserving neuronal proteostasis and limiting ferroptosis.
Ultimately, the project aims to identify new biomarkers and therapeutic targets for MPS IIIB. 1: Identification of vulnerable neuronal populations
Because studying the whole brain may mask differences between cell types, this work package will determine whether defects in iron trafficking and susceptibility to ferroptosis affect specific neuronal populations. For that, selected brain regions from control and MPS IIIB mice will be analyzed at the symptomatic stage. Glutamatergic, GABAergic, medium spiny and dopaminergic neurons, together with microglia and astrocytes, will be isolated by FACS. Cell-surface ferroportin will be measured during neuronal sorting. Sorted populations will undergo RNA sequencing and targeted RT-qPCR focused on ferroptosis, lysosomal function and iron homeostasis. These profiles will be correlated with lysosomal storage, autophagic flux, redox-active iron and lipid peroxidation, while confocal microscopy will assess ferroportin localization. The most affected neuronal populations and brain regions will be prioritized for Lyso-Tag profiling in Work Package 2 and used to evaluate causality and treatment response in Work Package 3.
2: Molecular profiling of pathological lysosomes and modulation by microglial hepcidin
The Lyso-Tag approach will be used to isolate intact lysosomes from control and NAGLU-deficient neuronal cell lines (MPSIIIB neurons) and patient-derived MPS IIIB fibroblasts. Following expression of TMEM192-3xHA, lysosomes will be rapidly isolated by anti-HA affinity immunopurification, and their integrity and purity will be assessed using organelle-specific markers. Purified lysosomes and corresponding whole-cell lysates will undergo proteomic, metabolomic and lipidomic analyses. These datasets will be combined with targeted measurements of redox-active iron and oxidized phospholipids to define the molecular signature of the pathological lysosome. Integrated bioinformatic analyses will identify alterations in iron trafficking, ferritinophagy, lysosomal degradation, lipid metabolism and ferroptotic pathways.
Once this signature has been established, its modulation by hepcidin will be examined in control and MPS IIIB neurons. Recombinant hepcidin will first be used to determine whether ferroportin internalization and lysosomal targeting amplify the disease-associated signature. The contribution of microglia-derived hepcidin will then be assessed in neuron-microglia co-cultures using HAMP silencing or hepcidin neutralization. This approach will distinguish the primary lysosomal iron-trafficking defect induced by MPS IIIB from its potential aggravation by the microglial hepcidin-ferroportin axis.
3: Mechanistic validation and therapeutic evaluation
The causal involvement of lysosomal iron trafficking in neuronal proteostasis failure and ferroptosis will be investigated by modulating two key regulatory mechanisms: NCOA4-dependent ferritinophagy and ferroportin-mediated iron export. A limited number of therapeutic approaches will be selected on the basis of the molecular signatures identified in Work Packages 1 and 2. Their precise modalities are not detailed here because they form part of a valorization strategy with potential for intellectual property protection. The selected approaches will first be evaluated in MPSIIIB neurons and neuron-microglia co-cultures. Their effects will be assessed by measuring ferritin turnover, cell-surface ferroportin, iron export, lysosomal redox-active iron, lipid peroxidation, ferroptosis, autophagic and lysosomal function, proteostasis, mitochondrial bioenergetics and neuronal viability. Only interventions producing a reproducible and concordant correction of these parameters without impairing essential cellular functions will progress to in vivo studies.
Lead approaches will then be evaluated in MPSIIIB mice. Therapeutic efficacy will be assessed through analyses of cerebral iron distribution, lipid peroxidation, ferroptotic markers, neuroinflammation, neuronal integrity and behavioral performance. Lyso-Tag-based analyses will determine whether these interventions correct the pathological lysosomal signature at the organelle level. This strategy will establish whether restoration of lysosomal iron homeostasis is sufficient to limit neuronal ferroptosis and improve the neurological phenotype.