Creatine deficiency syndomes : a new model of partial GAMT deficiency affecting brain cell development

Les syndromes de déficiences cérébrales en créatine (CCDS) sont dus à des mutations dans les gènes GATM et G AMT (codant pour les enzymes AGAT et G AMT de la voie de synthèse de créatine) ainsi que SLC6A8 (transporteur de créatine), et génèrent une absence ou une très forte baisse de créatine (Cr) dans le cerveau, mesurée par spectroscopic de résonance magnétique. Les patients CCDS développent des handicaps neurologiques sévères. Les patients AGAT et GAMT peuvent être traités avec des doses importantes de Cr, mais gardent dans la plupart des cas des séquelles neurologiques irréversibles. Aucun traitement efficace n'existe à ce jour pour la déficience en SLC6A8. Bien que de nombreux modèles aient été développés pour comprendre la Cr cérébrale en conditions physiologiques, les pathomécanismes des CCDS ne sont pas encore compris. Des souris transgéniques pour les gènes Gatm, Gamt et Slc6a8 ont été générées, mais elles ne miment que partiellement la pathologie humaine. Parmi les CCDS, la déficience en GAMT est la plus sévère, en raison de l'accumulation cérébrale de l'intermédiaire guanidinoacétate (GAA). Alors que la toxicité cérébrale du GAA a été étudiée par exposition directe au GAA d'animaux adultes sains, les mécanismes de la toxicité du GAA en condition de déficience en GAMT dans le cerveau en développement sont encore inconnus. Le but de ce projet était donc de développer un modèle de déficience en GAMT dans des cultures 3D primaires de cellules nerveuses de rat en agrégats par knock-down du gène GAMT, en utilisant un virus adéno-associé (AAV) induisant le mécanisme d'interférence à l'ARN (RNAi). Le virus scAAV2, à la multiplicité d'infection de 1000, s'est révélé le plus efficace pour transduire tous les types de cellules nerveuses des cultures (neurones, astrocytes, oligodendrocytes), et générer un knock-down maximal de la protéine GAMT de 85% (jour in vitro 18). Cette déficience partielle en GAMT s'est révélée insuffisante pour générer une déficience en Cr, mais a causé l'accumulation attendue de GAA, à des doses comparables aux niveaux observés dans le LCR des patients GAMT. Le GAA a induit une croissance axonale anarchique accompagnée d'une baisse de l'apoptose naturelle, suivis par une induction tardive de mort cellulaire non-apoptotique. Le co-traitement par la Cr a prévenu tous les effets toxiques du GAA. Ce travail montre que l'accumulation de GAA en absence de déficience en Cr est suffisante pour affecter le développement du tissu nerveux, et suggère que des formes de déficiences en GAMT supplémentaires, ne présentant pas de déficiences en Cr, pourraient être découvertes par mesure du GAA, en particulier à travers les programmes récemment proposés de dépistage néonatal de la déficience en GAMT. -- Cerebral creatine deficiency syndromes (CCDS) are caused by mutations in the genes GATM and GAMT (respectively coding for the two enzymes of the creatine synthetic pathway, AGAT and GAMT) as well as SLC6A8 (creatine transporter), and lead to the absence or very strong decrease of creatine (Cr) in the brain when measured by magnetic resonance spectroscopy. Affected patients show severe neurological impairments. While AGAT and GAMT deficient patients can be treated with high dosages of Cr, most remain with irreversible brain sequelae. No treatment has been successful so far for SLC6A8 deficiency. While many models have helped understanding the cerebral Cr pathways in physiological conditions, the pathomechanisms underlying CCDS are yet to be elucidated. Transgenic mice carrying mutations in the Gatm, Gamt and Slc6a8 genes have been developed, but only partially mimic the human pathology. Among CCDS, GAMT deficiency is the most severe, due to the CNS accumulation of the guanidinoacetate (GAA) intermediate. While brain toxicity of GAA has been explored through direct GAA exposure of adult healthy animals, the mechanisms underlying GAA toxicity in GAMT deficiency conditions on the developing CNS are yet unknown. The aim of this project was thus to develop and characterize a GAMT deficiency model in developing brain cells by gene knockdown, by adeno-associated virus (AAV)-driven RNA interference (RNAi) in rat 3D organotypic primary brain cell cultures in aggregates. scAAV2 with a multiplicity of infection of 1000 was shown as the most efficient serotype, was able to transduce all brain cell types (neurons, astrocytes, oligodendrocytes) and to induce a maximal GAMT protein knockdown of 85% (day in vitro 18). Metabolite analysis showed that partial GAMT knockdown was insufficient to induce Cr deficiency but generated the awaited GAA accumulation at concentrations comparable to the levels observed in cerebrospinal fluid of GAMT-deficient patients. Accumulated GAA induced axonal hypersprouting paralleled with inhibition of natural apoptosis, followed by a later induction in non-apoptotic cell death. Cr supplementation led to the prevention of all GAA-induced toxic effects. This work shows that GAA accumulation without Cr deficiency is sufficient to affect CNS development, and suggests that additional partial GAMT deficiencies, which may not show the classical brain Cr deficiency, may be discovered through GAA measurement including by recently proposed neonatal screening programs for GAMT deficiency.

Severity and burden of partial-onset seizures in a phase III trial of eslicarbazepine acetate.

OBJECTIVE: The objective of this study was to compare posttreatment seizure severity in a phase III clinical trial of eslicarbazepine acetate (ESL) as adjunctive treatment of refractory partial-onset seizures. METHODS: The Seizure Severity Questionnaire (SSQ) was administered at baseline and posttreatment. The SSQ total score (TS) and component scores (frequency and helpfulness of warning signs before seizures [BS]; severity and bothersomeness of ictal movement and altered consciousness during seizures [DS]; cognitive, emotional, and physical aspects of postictal recovery after seizures [AS]; and overall severity and bothersomeness [SB]) were calculated for the per-protocol population. Analysis of covariance, adjusted for baseline scores, estimated differences in posttreatment least square means between treatment arms. RESULTS: Out of 547 per-protocol patients, 441 had valid SSQ TS both at baseline and posttreatment. Mean posttreatment TS for ESL 1200mg/day was significantly lower than that for placebo (2.68 vs 3.20, p<0.001), exceeding the minimal clinically important difference (MCID: 0.48). Mean DS, AS, and SB were also significantly lower with ESL 1200mg/day; differences in AS and SB exceeded the MCIDs. The TS, DS, AS, and SB were lower for ESL 800mg/day than for placebo; only SB was significant (p=0.013). For both ESL arms combined versus placebo, mean scores differed significantly for TS (p=0.006), DS (p=0.031), and SB (p=0.001). CONCLUSIONS: Therapeutic ESL doses led to clinically meaningful, dose-dependent reductions in seizure severity, as measured by SSQ scores. CLASSIFICATION OF EVIDENCE: This study presents Class I evidence that adjunctive ESL (800 and 1200mg/day) led to clinically meaningful, dose-dependent seizure severity reductions, measured by the SSQ.

Effect of partial-thickness tear on loading capacities of the supraspinatus tendon: a finite element analysis.

Partial-thickness tears of the supraspinatus tendon frequently occur at its insertion on the greater tubercule of the humerus, causing pain and reduced strength and range of motion. The goal of this work was to quantify the loss of loading capacity due to tendon tears at the insertion area. A finite element model of the supraspinatus tendon was developed using in vivo magnetic resonance images data. The tendon was represented by an anisotropic hyperelastic constitutive law identified with experimental measurements. A failure criterion was proposed and calibrated with experimental data. A partial-thickness tear was gradually increased, starting from the deep articular-sided fibres. For different values of tendon tear thickness, the tendon was mechanically loaded up to failure. The numerical model predicted a loss in loading capacity of the tendon as the tear thickness progressed. Tendon failure was more likely when the tendon tear exceeded 20%. The predictions of the model were consistent with experimental studies. Partial-thickness tears below 40% tear are sufficiently stable to persist physiotherapeutic exercises. Above 60% tear surgery should be considered to restore shoulder strength.

The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma.

Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.