Cell-specific vulnerability to metabolic failure: the crucial role of parvalbumin expressing neurons in creatine transporter deficiency

Mutations in the solute carrier family 6-member 8 (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with intellectual disability, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood, hindering the development of therapies. In this study, we generated an extensive transcriptomic profile of CTD showing that Cr deficiency causes perturbations of gene expression in excitatory neurons, inhibitory cells, and oligodendrocytes which result in remodeling of circuit excitability and synaptic wiring. We also identified specific alterations of parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional electrophysiological phenotype. Mice lacking Slc6a8 only in PV+ interneurons recapitulated numerous CTD features, including cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ interneurons is sufficient to determine the neurological phenotype of CTD. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses significantly improved cortical activity in Slc6a8 knock-out animals. Altogether, these data demonstrate that Slc6a8 is critical for the normal function of PV+ interneurons and that impairment of these cells is central in the disease pathogenesis, suggesting a novel therapeutic venue for CTD.This work has been supported by grant GR-2017–02364378 funded by the Italian Ministry of Health and by Telethon grant GGP19177 to LB; Italian Ministry of Health, RC 2021; grant from Fondazione Cassa di Risparmio di Firenze “Human Brain Optical Mapping” to TP; grants from the Spanish Ministry of Science and Innovation (MICINN) co-financed by ERDF (grant no. RTI2018-102260-B-I00; Generalitat Valenciana, project no. PROMETEO/2020/007; and CSIC Interdisciplinary Thematic Platform (PTI +) NEURO-AGINGl + (PTI-NEURO-AGING +). C.M.N-I. was the recipient of a FPI fellowship from the MICINN. The Instituto de Neurociencias (UMH-CSIC) is a “Centre of Excellence Severo Ochoa” (grant no. SEV-2017–0723).Peer reviewe

Deficit del trasportatore della creatina: un viaggio sulla strada della terapia genica

La sindrome da deficienza del trasportatore di creatina (CTD) è un disordine metabolico ereditato dal cromosoma X (CTD, OMIM # 300352), che causa deplezione di creatina cerebrale, deficit cognitivi di apprendimento e memoria, disordini del movimento ed alterazioni comportamentali, difficoltà di linguaggio e di articolazione, ed un’elevata suscettibilità a crisi epilettiche in circa l’1% dei soggetti di sesso maschile con ritardo mentale di eziologia sconosciuta. Attualmente non esiste una cura per questo disturbo. Lo scopo di questo progetto di tesi è stato quello di capire se la terapia genica potesse essere un potenziale trattamento per la CTD. Per affrontare questo, sono stati usati modelli in vitro e un modello murino che porta la delezione degli esoni 5-7 del gene Slc6a8, che codifica per il trasportatore della creatina (CrT). Precedentemente, in laboratorio, è stato dimostrato che la sovra-espressione di CrT nel cervello si traduce in effetti tossici e morte di animali iniettati a PND0-1 con AAV9 contenente una copia funzionale del gene SLC6A8, sotto il controllo del promotore CAG. È stato ipotizzato che la tossicità fosse dovuta a un sovraccarico cellulare di Cr e che un'ottimizzazione del costrutto virale potesse superare i problemi di tossicità, fornendo un miglior compromesso tra i livelli di espressione esogena di SLC6A8 in singole cellule e il numero di cellule infettate dal virus nel cervello. Così, è stato sostituito il promotore forte CAG con JeT, un promotore sintetico che mostra un'attività più debole, e sono state eseguite iniezioni intraventricolari del virus a diverse diluizioni per costruire una curva dose-risposta. Tuttavia, l'espressione esogena di CrT ha ancora provocato la morte dei topi, con effetti tossici che diminuiscono progressivamente in parallelo alla riduzione del titolo virale senza differenze evidenti nella sopravvivenza tra gli animali iniettati WT e KO. A seguito dell’iniezione del virus a basso titolo, si sono determinati i livelli di espressione della proteina esogena, la sua distribuzione nel tessuto nervoso, la funzionalità del trasportatore esogeno in termini di incremento dei livelli cerebrali di creatina e gli effetti di questo trattamento a livello neurocomportamentale. Infine, per ottimizzare ulteriormente il costrutto virale, sono stati sviluppati nuovi plasmidi finalizzati a indurre una minore espressione del gene esogeno in singole cellule: JeT è stato sostituito con promotori più deboli e, come strategia alternativa, è stato aggiunto un dominio destabilizzante ligando-dipendente (DDs) all'estremità 3' del gene SLC6A8. I DDs aumentano la cinetica di ubiquitinazione e degradazione da parte del proteasoma della proteina transgenica, mantenendo così basso il livello di espressione del CrT esogena. Creatine transporter deficiency (CrT) is a metabolic disorder inherited from the X chromosome (CTD, OMIM #300352), which causes depletion of brain creatine, cognitive learning and memory deficits, movement disorders and behavioral alterations, language and articulation difficulties, and high susceptibility to seizures in about 1% of male subjects with mental retardation of unknown etiology. There is currently no cure for this disorder. The aim of this thesis project was to understand whether gene therapy might be a potential disease-modifying treatment for CTD. To address this issue, we used in vitro models and a mouse carrying the deletion of exons 5, 6, 7 of Slc6a8 gene, encoding for the creatine transporter (CrT) protein. It has been previously shown in the lab that the overexpression of CrT in the brain results in overt toxic effects, with cellular degeneration, enlargement of brain ventricles, neuroinflammation and death in animals injected at PND0-1 with AAV9 carrying a functional copy of SLC6A8 gene under the control of CAG promoter. We hypothesized that the toxicity was due to a cellular overload of Cr and that an optimization of the viral construct might overcome the toxicity issues, providing a better compromise between the expression levels of exogenous SLC6A8 in single cells and the number of cells infected by the virus in the whole brain. Thus, we replaced the strong CAG promoter with JeT, a synthetic promoter showing a weaker activity with respect to CAG, and we performed intraventricular injections of the virus at different dilutions in order to build a dose-response curve. However, CrT exogenous expression still resulted in the death of mice, with toxic effects progressively decreasing in parallel to the reduction of the viral titer with no obvious differences in survival between injected WT and KO animals. Following injection of the low-titer virus, we determined the expression levels of the exogenous protein, its distribution in nervous tissue, the functionality of the exogenous transporter in terms of increasing brain levels of creatine, and the effects of this treatment at the neurobehavioral level. Finally, to further optimize the viral construct, we created new plasmids with the aim of inducing a lower expression of the exogenous gene at single-cell level: we replaced JeT with weaker promoters and, as alternative strategy, we attached a ligand-dependent destabilizing domains (DDs) at the 3' end of SLC6A8 gene. DDs increase the kinetic of ubiquitination and degradation by the proteasome of the transgene protein, thus keeping down the expression level of the exogenous CrT

Cell-specific vulnerability to metabolic failure: the crucial role of parvalbumin expressing neurons in creatine transporter deficiency

Mutations in the solute carrier family 6-member 8 (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with intellectual disability, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood, hindering the development of therapies. In this study, we generated an extensive transcriptomic profile of CTD showing that Cr deficiency causes perturbations of gene expression in excitatory neurons, inhibitory cells, and oligodendrocytes which result in remodeling of circuit excitability and synaptic wiring. We also identified specific alterations of parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional electrophysiological phenotype. Mice lacking Slc6a8 only in PV+ interneurons recapitulated numerous CTD features, including cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ interneurons is sufficient to determine the neurological phenotype of CTD. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses significantly improved cortical activity in Slc6a8 knock-out animals. Altogether, these data demonstrate that Slc6a8 is critical for the normal function of PV+ interneurons and that impairment of these cells is central in the disease pathogenesis, suggesting a novel therapeutic venue for CTD