Dodecyl creatine ester improves cognitive function and identifies key protein drivers including KIF1A and PLCB1 in a mouse model of creatine transporter deficiency

Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8−/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8−/y mice and wild type and DCE-treated Slc6a8−/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD

Développement d'organoïdes cérébraux de patients atteints du déficit en transporteur de la créatine comme outil d'évaluation de l'efficacité d'agents thérapeutiques

Creatine transporter deficiency (CTD) is a rare genetic and metabolic disorder in which loss of functionality of the creatine transporter (CRT, SLC6A8) leads to an absence of creatine in the brain. CTD patients present with a neurodevelopmental disorder manifesting as intellectual disability with language delay, as well as behavioral disorders and epilepsy in some cases. At present, no effective therapy is available. The absence of a functional CRT represents a major obstacle to delivering creatine to the brain and improving the clinical picture of CTD patients. In addition, our knowledge of the molecular and cellular mechanisms in the brain affected by creatine depletion has yet to be clarified. Current studies are based on the use of conventional human cell cultures, often considered non-physiological, and on animal models, whose inter-species differences complicate the extrapolation of results to humans. To clarify the affected mechanisms and identify potential new targets for pharmacological treatments, it is important to develop new, relevant human CTD models. The combined use of these different models would enhance CTD biomarker discovery and provide solid convergent data to support clinical trials.This thesis project is based on the development of CTD brain organoids, a human cellular model mimicking the brain of CTD patients, to better understand the role of creatine in brain dysfunctions in affected patients.After obtaining and characterizing this new in vitro human CTD model, comparison of the proteomes of healthy vs CTD organoids identified several molecular and cellular alterations linked to neurogenesis and synaptogenesis processes, energy metabolism and methylation processes. These observations were validated using CTD brain organoids re-expressing functional CRT, as well as Slc6a8-/y mouse models treated or untreated with dodecyl creatine ester (DCE), a prodrug used to increase brain creatine content.The results obtained highlight that the absence of cerebral creatine leads to overactivation of the GSK3β pathway, gene expression of factors, microtubule stability, glucose incorporation deficiency, mitochondrial deficiency, and DNA hypomethylation. These changes appear to alter the processes of neurogenesis and synaptogenesis responsible for the cognitive impairment in patients. These different dysregulated pathways, described for the first time in CTD thanks to the combined use of human CTD brain organoids and Slc6a8-/y mouse models, highlight new biomarkers that can be used to test the therapeutic efficacy of drug candidates in preclinical and clinical studies.Le déficit en transporteur de la créatine (CTD) est une maladie génétique et métabolique rare dans laquelle la perte de fonctionnalité du transporteur de la créatine (CRT, SLC6A8) conduit à une absence de créatine dans le cerveau. Les patients CTD présentent un trouble neurodéveloppemental qui se manifeste par une déficience intellectuelle avec un retard de langage, ainsi que des troubles du comportement et de l'épilepsie dans certains cas. A l'heure actuelle, aucune thérapie efficace n'est disponible. L'absence de CRT fonctionnel représente un obstacle majeur à l'administration de créatine dans le cerveau et à l'amélioration du tableau clinique des patients CTD. De plus, les connaissances sur les mécanismes moléculaires et cellulaires cérébraux affectés par la diminution en créatine restent encore à préciser. Les études actuelles sont basées sur l'utilisation de cultures cellulaires humaines classiques, souvent considérées comme non physiologiques, et sur des modèles animaux dont les différences inter-espèces compliquent l'extrapolation des résultats à l'être humain. Pour préciser les mécanismes affectés et identifiés de nouvelles cibles potentielles pour les traitements pharmacologiques, il est important de développer de nouveaux modèles CTD humains pertinents. L'utilisation combinée de ces différents modèles permettrait de renforcer la découverte de biomarqueurs du CTD et de fournir des données convergentes solides pour soutenir les essais cliniques.Ce projet de thèse repose sur le développement d'organoïdes cérébraux CTD, un modèle cellulaire humain mimant le cerveau des patients CTD pour mieux appréhender le rôle de la créatine dans les dysfonctionnements cérébraux des patients atteints.Après avoir obtenu et caractérisé ce nouveau modèle in vitro humain CTD, la comparaison des protéomes d'organoïdes sains vs CTD a permis d'identifier plusieurs altérations moléculaires et cellulaires en lien avec les processus de neurogenèse et de synaptogenèse, le métabolisme énergétique et les processus de méthylation. Ces observations ont été validées par l'utilisation d'organoïdes cérébraux CTD ré-exprimant le CRT fonctionnel, ainsi que par des modèles murins Slc6a8-/y traités ou non avec l'ester dodécylique de créatine (DCE), une prodrogue permettant d'augmenter la teneur en créatine du cerveau.Les résultats obtenus mettent en exergue que l'absence de créatine cérébrale entraîne une suractivation de la voie GSK3β, l'expression génique de facteurs, la stabilité des microtubules, un déficit d'incorporation du glucose, un déficit mitochondrial et une hypométhylation de l'ADN. Ces modifications semblent altérer les processus de neurogenèse et de synaptogenèse responsables des altérations des fonctions cognitives chez les patients. Ces différentes voies dérégulées, décrites pour la première fois dans le CTD grâce à l'utilisation conjointe d'organoïdes cérébraux CTD humains et de modèles murins Slc6a8-/y, mettent en évidence de nouveaux biomarqueurs qui peuvent être utilisés pour tester l'efficacité thérapeutique de candidats médicaments dans des études précliniques et cliniques

Deciphering neuronal deficit and protein profile changes in human brain organoids from patients with creatine transporter deficiency

International audienceCreatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3β, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3β, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy

Dodecyl Creatine Ester Improves Cognitive Function and Identifies Drivers of Creatine Deficiency

Abstract Creatine transporter deficiency prevents creatine uptake into the brain, leading to mental retardation. To better understand the pathophysiology, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparisons of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8-/y mice and wild type and DCE-treated Slc6a8-/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins associated with CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Functional analysis confirmed their key roles and associated molecules in CTD pathogenesis

Stromal cells regulate malignant B-cell spatial organization, survival, and drug response in a new 3D model mimicking lymphoma tumor niche

Non-Hodgkin B-cell lymphomas (B-NHL) mainly develop within lymph nodes as densely packed aggregates of tumor cells and their surrounding microenvironment, creating a tumor niche specific to each lymphoma subtypes. Until now, in vitro preclinical models mimicking biomechanical forces, cellular microenvironment, and 3D organization of B lymphomas remain scarce while all these parameters constitute key determinants of lymphomagenesis and drug resistance. Using a microfluidic method based on the encapsulation of cells inside permeable, elastic, and hollow alginate microspheres, we developed a new tunable 3D-model incorporating extracellular matrix and/or stromal cells. Lymphoma B cells and stromal cells dynamically formed self-organized 3D spheroids, thus initiating a coevolution of these two cell types, reflecting their bidirectional crosstalk, and recapitulating the heterogeneity of B-NHL subtypes. In addition, this approach makes it suitable to assess in a relevant in vitro model the activity of new therapeutic agents in B-NHL

A novel 3D culture model recapitulates primary FL B cell features and promotes their survival

Non-Hodgkin B-cell lymphomas (B-NHL) mainly develop within lymph nodes (LN) as densely packed aggregates of tumor cells and their surrounding microenvironment, creating a tumor niche specific to each lymphoma subtypes. In vitro preclinical models mimicking biomechanical forces, cellular microenvironment, and 3D organization of B-cell lymphomas remain scarce, while all these parameters constitute key determinants of lymphomagenesis and drug resistance. Using a microfluidic method based on cell encapsulation inside permeable, elastic, and hollow alginate microspheres, we developed a new tunable 3D-model incorporating lymphoma B cells, extracellular matrix (ECM), and/or tonsil stromal cells (TSC). We revealed that under 3D confinement lymphoma B cells were able to form cohesive spheroids resulting from overexpression of ECM components. Moreover, lymphoma B cells and TSC dynamically formed self-organized 3D spheroids favoring spheroid growth. 3D culture induced resistance to classical chemotherapeutic agent doxorubicin, but not to BCL2 inhibitor ABT-199, identifying this approach as a relevant in vitro model to assess the activity of therapeutic agents in B-NHL. RNAseq analysis highlighted the synergy of 3D, ECM, and TSC in upregulating similar pathways in malignant B cells in vitro than those overexpressed in primary lymphoma cells in situ. Finally, our 3D model including ECM and TSC allowed long-term in vitro survival of primary follicular lymphoma B cells. In conclusion, we propose a new high throughput 3D model mimicking lymphoma tumor niche and making it possible to study the dynamic relationship between lymphoma B cells and their microenvironment and to screen new anti-cancer drugs