Biological membranes : biophysical properties and aquaporin function

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

The effect of acute alcohol exposure on hepatic oxidative stress and function: prevention by micronutrients

Alcohol binge drinking, especially in teenagers and young adults is a major public health issue in the UK, with the number of alcohol related liver disorders steadily increasing. Understanding the mechanisms behind liver disease arising from binge-drinking and finding ways to prevent such damage are currently important areas of research. In the present investigation the effect of acute ethanol administration on hepatic oxidative damage and apoptosis was examined using both an in vivo and in vitro approach; the effect of micronutrient supplementation prior and during ethanol exposure was also studied. The following studies were performed: (1) ethanol administration (75 mmol/kg body weight) and cyanamide pre-treatment followed by ethanol to study elevated acetaldehyde levels with liver tissue analysed 2.5, 6 and 24 hours post-alcohol; (2). Using juvenile animals, 2% betaine supplementation followed by acute ethanol with tissue analysed 24 hrs post ethanol; and (3). Micronutrient supplementation during concomitant ethanol exposure to hepG2 cells. It was found that a single dose of alcohol caused oxidative damage to the liver of rats at 2.5 hr post-alcohol as evidenced by decreased glutathione levels and increased malondialdehyde levels in both the cytosol and mitochondria. Liver function was also depressed but there were no findings of apoptosis as cytochrome c levels and caspase 3 activity was unchanged. At 6 hours, the effect of ethanol was reduced suggesting some degree of recovery, however, by 24 hours, increased mitochondrial oxidative stress was apparent. The effect of elevated acetaldehyde on hepatic damage was particularly evident at 24 hours, with some oxidative changes at earlier time points. At 24 hours, acetaldehyde caused a profound drop in glutathione levels in the cytosol and hepatic function was still deteriorating. Studies examining ethanol exposure to juvenile livers showed that glutathione levels were increased, suggesting an overtly protective response not seen in with older animals. It also showed that despite cytochrome c release into the cytosol, caspase-3 levels were not increased. This suggests that ATP depletion is preventing apoptosis initiation. Betaine supplementation prevented almost all of the alcohol-mediated changes, suggesting that the main mechanism behind alcohol-mediated liver damage is oxidative stress. Results using the hepG2 cell line model showed that micronutrients involved in glutathione synthesis can protect against hepatocyte damage caused by alcohol metabolism, with reduced reactive oxygen species and increased/maintained glutathione levels. In summary, these results demonstrate that both acute alcohol and acetaldehyde can have damaging effects to the liver, but that dietary intervention may be able to protect against ethanol induced oxidative stress.

The impact of oligofructose on stimulation of gut hormones, appetite regulation and adiposity

Objective: To investigate the effect of nutrient stimulation of gut hormones by oligofructose supplementation on appetite, energy intake (EI), body weight (BW) and adiposity in overweight and obese volunteers. Methods: In a parallel, single-blind and placebo-controlled study, 22 healthy overweight and obese volunteers were randomly allocated to receive 30 g day−1 oligofructose or cellulose for 6 weeks following a 2-week run-in. Subjective appetite and side effect scores, breath hydrogen, serum short chain fatty acids (SCFAs), plasma gut hormones, glucose and insulin concentrations, EI, BW and adiposity were quantified at baseline and post-supplementation. Results: Oligofructose increased breath hydrogen (P < 0.0001), late acetate concentrations (P = 0.024), tended to increase total area under the curve (tAUC)420mins peptide YY (PYY) (P = 0.056) and reduced tAUC450mins hunger (P = 0.034) and motivation to eat (P = 0.013) when compared with cellulose. However, there was no significant difference between the groups in other parameters although within group analyses showed an increase in glucagon-like peptide 1 (GLP-1) (P = 0.006) in the cellulose group and a decrease in EI during ad libitum meal in both groups. Conclusions: Oligofructose increased plasma PYY concentrations and suppressed appetite, while cellulose increased GLP-1 concentrations. EI decreased in both groups. However, these positive effects did not translate into changes in BW or adiposity.

Reduction of trimethylamine N-oxide to trimethylamine by the human gut microbiota: supporting evidence for ‘metabolic retroversion’

Dietary sources of methylamines such as choline, trimethylamine (TMA), trimethylamine N-oxide (TMAO), phosphatidylcholine (PC) and carnitine are present in a number of foodstuffs, including meat, fish, nuts and eggs. It is recognized that the gut microbiota is able to convert choline to TMA in a fermentation-like process. Similarly, PC and carnitine are converted to TMA by the gut microbiota. It has been suggested that TMAO is subject to ‘metabolic retroversion’ in the gut (i.e. it is reduced to TMA by the gut microbiota, with this TMA being oxidized to produce TMAO in the liver). Sixty-six strains of human faecal and caecal bacteria were screened on solid and liquid media for their ability to utilize trimethylamine N-oxide (TMAO), with metabolites in spent media profiled by Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy. Enterobacteriaceae produced mostly TMA from TMAO, with caecal/small intestinal isolates of Escherichia coli producing more TMA than their faecal counterparts. Lactic acid bacteria (enterococci, streptococci, bifidobacteria) produced increased amounts of lactate when grown in the presence of TMAO, but did not produce large amounts of TMA from TMAO. The presence of TMAO in media increased the growth rate of Enterobacteriaceae; while it did not affect the growth rate of lactic acid bacteria, TMAO increased the biomass of these bacteria. The positive influence of TMAO on Enterobacteriaceae was confirmed in anaerobic, stirred, pH-controlled batch culture fermentation systems inoculated with human faeces, where this was the only bacterial population whose growth was significantly stimulated by the presence of TMAO in the medium. We hypothesize that dietary TMAO is used as an alternative electron acceptor by the gut microbiota in the small intestine/proximal colon, and contributes to microbial population dynamics upon its utilization and retroversion to TMA, prior to absorption and secondary conversion to TMAO by hepatic flavin-containing monooxygenases. Our findings support the idea that oral TMAO supplementation is a physiologically-stable microbiota-mediated strategy to deliver TMA at the gut barrier.

Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults.

OBJECTIVE: The colonic microbiota ferment dietary fibres, producing short chain fatty acids. Recent evidence suggests that the short chain fatty acid propionate may play an important role in appetite regulation. We hypothesised that colonic delivery of propionate would increase peptide YY (PYY) and glucagon like peptide-1 (GLP-1) secretion in humans, and reduce energy intake and weight gain in overweight adults. DESIGN: To investigate whether propionate promotes PYY and GLP-1 secretion, a primary cultured human colonic cell model was developed. To deliver propionate specifically to the colon, we developed a novel inulin-propionate ester. An acute randomised, controlled cross-over study was used to assess the effects of this inulin-propionate ester on energy intake and plasma PYY and GLP-1 concentrations. The long-term effects of inulin-propionate ester on weight gain were subsequently assessed in a randomised, controlled 24-week study involving 60 overweight adults. RESULTS: Propionate significantly stimulated the release of PYY and GLP-1 from human colonic cells. Acute ingestion of 10 g inulin-propionate ester significantly increased postprandial plasma PYY and GLP-1 and reduced energy intake. Over 24 weeks, 10 g/day inulin-propionate ester supplementation significantly reduced weight gain, intra-abdominal adipose tissue distribution, intrahepatocellular lipid content and prevented the deterioration in insulin sensitivity observed in the inulin-control group. CONCLUSIONS: These data demonstrate for the first time that increasing colonic propionate prevents weight gain in overweight adult humans.

Effect of Short-Chain Fatty Acid Acetate on Colon Cancer

Acetate is a short chain fatty acid produced as a result of fermentation of ingested fibers by the gut microbiota. While it has been shown to reduce cell proliferation in some cancer cell lines1,2, more recent studies on liver3 and brain4 tumours suggest that acetate may actually promote tumour growth. Acetate in the cell is normally converted into acetyl-coA by two enzymes and metabolized; mitochondrial (ACSS1) and cytosolic (ACSS2) acetyl-coA synthetase. In the mitochondria acetyl-coA is utilized in the TCA cycle. In the cytosol it is utilized in lipid synthesis. In this study, the effect of acetate treatment on the growth of HT29 colon cancer cell line and its mechanism of action was assessed. HT29 human colorectal adenocarcinoma cells were treated with 10mM NaAc and cell viability, cellular bioenergetics and gene expression were investigated. Cell viability was assessed 24 hours after treatment using an MTT assay (Sigma, UK, n=8). Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was measured by XFe Analyzer (Seahorse Bioscience, USA). After a baseline reading cells were treated and OCR and ECAR measurements were observed for 18 hours (n=4). Total mRNA was isolated 24 hours after treatment using RNeasy kit (Qiagen, USA). Quantitative PCR reactions were performed using Taqman gene expression assays and Taqman Universal PCR Master Mix (ThermoFisher Scientific, UK) on Applied Biosystems 7500 Fast Real-Time PCR System (Life Technologies, USA) and analysed using ΔΔCt method (n=3). Acetate treatment led to a significant reduction in cell viability (15.9%, Figure 1). OCR, an indicator of oxidative phosphorylation, was significantly increased (p<0.0001) while ECAR, an indicator of glycolysis, was significantly reduced (p<0.0001, Figure 2). Gene expression of ACSS1 was increased by 1.7 fold of control (p=0.07) and ACSS2 expression was reduced to 0.6 fold of control (p=0.06, Figure 3). In conclusion, in colon cancer cells acetate supplementation induces cell death and increases oxidative capacity. These changes together with the trending decrease in ACSS2 expression suggest suppression of lipid synthesis pathways. We hypothesize that the reduced tumor growth by acetate is a consequence of the suppression of ACSS2 and lipid synthesis, both effects reported previously to reduce tumor growth3–5. These effects clearly warrant further investigation.

Anthropometric evaluation and micronutrients intake in patients submitted to laparoscopic Roux-en-Y gastric bypass with a postoperative period of ≥1 year

Backgroung - Bariatric surgery is indicated as the most effective treatment for morbid obesity; the Roux-en-Y gastric bypass (RYGB) is considered the procedure of choice. However, nutritional deficiency may occur in the postoperative period as a result of reduced gastric capacity and change in nutrients absorption in the gastrointestinal tract. The prescription of vitamin and mineral supplementation is a common practice after RYGB; however, it may not be sufficient to prevent micronutrient deficiencies. The aim of this study was to quantify the micronutrient intake in patients undergoing RYGB and verify if the intake of supplementation would be enough to prevent nutritional deficiencies. Methods - The study was conducted on 60 patients submitted to RYGB. Anthropometric, analytical, and nutritional intake data were assessed preoperatively and 1 and 2 years postoperatively. The dietary intake was assessed using 24-h food recall; the values of micronutrients evaluated (vitamin B12, folic acid, iron, and calcium) were compared to the dietary reference intakes (DRI). Results - There were significant differences (p < 0.05) between excess weight loss at the first and second year (69.9 ± 15.3 vs 9.6 ± 62.9 %). In the first and second year after surgery, 93.3 and 94.1 % of the patients, respectively, took the supplements as prescribed. Micronutrient deficiencies were detected in the three evaluation periods. At the first year, there was a significant reduction (p < 0.05) of B12, folic acid, and iron intake. Conclusions - Despite taking vitamin and mineral supplementation, micronutrient deficiencies are common after RYGB. In the second year after surgery, micronutrient intake remains below the DRI.

Serum trace elements in dysphagic gastrostomy candidates before endoscopic gastrostomy for long term enteral feeding

Background & aims - Patients who underwent endoscopic gastrostomy (PEG) present protein-energy malnutrition, but little is known about Trace Elements (TE), Zinc (Zn), Copper (Cu), Selenium (Se), Iron (Fe), Chromium (Cr). Our aim was the evaluation of serum TE in patients who underwent PEG and its relationship with serum proteins, BMI and nature of underlying disorder. Methods - A prospective observational study was performed collecting: patient's age, gender, underlying disorder, NRS-2002, BMI, serum albumin, transferrin and TE concentration. We used ferrozine colorimetric method for Fe; Inductively Coupled Plasma-Atomic Emission Spectroscopy for Zn/Cu; Furnace Atomic Absorption Spectroscopy for Se/Cr. The patients were divided into head and neck cancer (HNC) and neurological dysphagia (ND). Results - 146 patients (89 males), 21–95 years: HNC-56; ND-90. Low BMI in 78. Low values mostly for Zn (n = 122) and Fe (n = 69), but less for Se (n = 31), Cu (n = 16), Cr (n = 7); low albumin in 77, low transferrin in 94 and 66 with both proteins low. Significant differences between the groups of underlying disease only for Zn (t140.326 = −2,642, p < 0.01) and a correlation between proteins and TE respectively albumin and Zn (r = 0.197, p = 0.025), and albumin and Fe (r = 0.415, p = 0.000). Conclusions - When gastrostomy was performed, patients display low serum TE namely Zn, but also Fe, less striking regarding others TE. It was related with prolonged fasting, whatever the underlying disease. Low proteins were associated with low TE. Teams taking care of PEG-patients should use Zn supplementation and include other TE evaluation as part of the nutritional assessment of PEG candidates.

Targeting specific nutrient deficiencies in proteinrestricted diets: some practical facts in PKU dietary management

Among aminoacidopathies, phenylketonuria (PKU) is the most prevalent one. Early diagnosis in the neonatal period with a prompt nutritional therapy (low natural-protein and phenylalanine diet, supplemented with phenylalanine-free amino acid mixtures and special low-protein foods) remains the mainstay of the treatment. Data considering nutrient contents of cooked dishes is lacking. In this study, fourteen dishes specifically prepared for PKU individuals were analysed, regarding the lipid profile and iron and zinc contents. These dishes are poor sources of essential nutrients like Fe, Zn or n-3 fatty acids, reinforcing the need for adequate supplementation to cover individual patients’ needs. This study can contribute to a more accurate adjustment of PKU diets and supplementation in order to prevent eventual nutritional deficiencies. This study contributes to a better understanding of nutrient intake from PKU patients’ meals, showing the need for dietary supplementation.

Iron Overload Favors the Elimination of Leishmania

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The host’s iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the host’s oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.

Pseudohypoaldosteronisms, report on a 10-patient series.

BACKGROUND: Type 1 pseudohypoaldosteronism (PHA1) is a salt-wasting syndrome caused by mineralocorticoid resistance. Autosomal recessive and dominant hereditary forms are caused by Epithelial Na Channel and Mineralocorticoid Receptor mutation respectively, while secondary PHA1 is usually associated with urological problems. METHODS: Ten patients were studied in four French pediatric units in order to characterize PHA1 spectrum in infants. Patients were selected by chart review. Genetic, clinical and biochemistry data were collected and analyzed. RESULTS: Autosomal recessive PHA1 (n = 3) was diagnosed at 6 and 7 days of life in three patients presenting with severe hyperkalaemia and weight loss. After 8 months, 3 and 5 years on follow-up, neurological development and longitudinal growth was normal with high sodium supplementation. Autosomal dominant PHA1 (n = 4) was revealed at 15, 19, 22 and 30 days of life because of failure to thrive. At 8 months, 3 and 21 years of age, longitudinal growth was normal in three patients who were given salt supplementation; no significant catch-up growth was obtained in the last patient at 20 months of age. Secondary PHA1 (n = 3) was diagnosed at 11, 26 days and 5 months of life concomitantly with acute pyelonephritis in three children with either renal hypoplasia, urinary duplication or bilateral megaureter. The outcome was favourable and salt supplementation was discontinued after 3, 11 and 13 months. CONCLUSIONS: PHA1 should be suspected in case of severe hyperkalemia and weight loss in infants and need careful management. Pathogenesis of secondary PHA1 is still challenging and further studies are mandatory to highlight the link between infection, developing urinary tract and pseudohypoaldosteronism.

High prevalence of hypovitaminosis D in a Swiss rheumatology outpatient population.

Vitamin D is important for bone metabolism and neuromuscular function. While a routine dosage is often proposed in osteoporotic patients, it is not so evident in rheumatology outpatients where it has been shown that the prevalence of hypovitaminosis D is high. The aim of the current study was to systematically evaluate the vitamin D status in our outpatient rheumatology population to define the severity of the problem according to rheumatologic diseases. During November 2009, all patients were offered a screening test for 25-OH vitamin D levels and categorised as deficient (<10 µg/l [ng/ml] [25 nmol/l]), insufficient (10 µg/l to 30 µg/l [25 to 75 nmol/l]) or normal (>30 µg/l [75 nmol/l]). A total of 272 patients were included. The mean 25-OH vitamin D level was 21 µg/l (range 1.5 to 45.9). A total of 20 patients had vitamin D deficiency, 215 patients had an insufficiency and 37 patients had normal results. In the group of patients with osteoporosis mean level of 25-OH vitamin D was 25 µg/l and 31% had normal results. In patients with inflammatory rheumatic diseases (N = 219), the mean level of 25-OH vitamin D was 20.5 µg/l, and only 12% had normal 25-OH vitamin D levels. In the small group of patients with degenerative disease (N = 33), the mean level of 25-OH vitamin D was 21.8 µg/l, and 21% had normal results. Insufficiency and deficiency were even seen in 38% of the patients who were taking supplements. These results confirm that hypovitaminosis D is highly prevalent in an outpatient population of rheumatology patients, affecting 86% of subjects. Despite oral supplementation (taken in 38% of our population), only a quarter of those on oral supplementation attained normal values of 25-OH vitamin D.

Perioperative nutrition in abdominal surgery: recommendations and reality.

Introduction. Preoperative malnutrition is a major risk factor for increased postoperative morbidity and mortality. Definition and diagnosis of malnutrition and its treatment is still subject for controversy. Furthermore, practical implementation of nutrition-related guidelines is unknown. Methods. A review of the available literature and of current guidelines on perioperative nutrition was conducted. We focused on nutritional screening and perioperative nutrition in patients undergoing digestive surgery, and we assessed translation of recent guidelines in clinical practice. Results and Conclusions. Malnutrition is a well-recognized risk factor for poor postoperative outcome. The prevalence of malnutrition depends largely on its definition; about 40% of patients undergoing major surgery fulfil current diagnostic criteria of being at nutritional risk. The Nutritional Risk Score is a pragmatic and validated tool to identify patients who should benefit from nutritional support. Adequate nutritional intervention entails reduced (infectious) complications, hospital stay, and costs. Preoperative oral supplementation of a minimum of five days is preferable; depending on the patient and the type of surgery, immune-enhancing formulas are recommended. However, surgeons' compliance with evidence-based guidelines remains poor and efforts are necessary to implement routine nutritional screening and nutritional support.

Preoperative nutritional screening by the specialist instead of the nutritional risk score might prevent excess nutrition: a multivariate analysis of nutritional risk factors.

BACKGROUND: The aim of the current study was to assess whether widely used nutritional parameters are correlated with the nutritional risk score (NRS-2002) to identify postoperative morbidity and to evaluate the role of nutritionists in nutritional assessment. METHODS: A randomized trial on preoperative nutritional interventions (NCT00512213) provided the study cohort of 152 patients at nutritional risk (NRS-2002 ≥3) with a comprehensive phenotyping including diverse nutritional parameters (n=17), elaborated by nutritional specialists, and potential demographic and surgical (n=5) confounders. Risk factors for overall, severe (Dindo-Clavien 3-5) and infectious complications were identified by univariate analysis; parameters with P<0.20 were then entered in a multiple logistic regression model. RESULTS: Final analysis included 140 patients with complete datasets. Of these, 61 patients (43.6%) were overweight, and 72 patients (51.4%) experienced at least one complication of any degree of severity. Univariate analysis identified a correlation between few (≤3) active co-morbidities (OR=4.94; 95% CI: 1.47-16.56, p=0.01) and overall complications. Patients screened as being malnourished by nutritional specialists presented less overall complications compared to the not malnourished (OR=0.47; 95% CI: 0.22-0.97, p=0.043). Severe postoperative complications occurred more often in patients with low lean body mass (OR=1.06; 95% CI: 1-1.12, p=0.028). Few (≤3) active co-morbidities (OR=8.8; 95% CI: 1.12-68.99, p=0.008) were related with postoperative infections. Patients screened as being malnourished by nutritional specialists presented less infectious complications (OR=0.28; 95% CI: 0.1-0.78), p=0.014) as compared to the not malnourished. Multivariate analysis identified few co-morbidities (OR=6.33; 95% CI: 1.75-22.84, p=0.005), low weight loss (OR=1.08; 95% CI: 1.02-1.14, p=0.006) and low hemoglobin concentration (OR=2.84; 95% CI: 1.22-6.59, p=0.021) as independent risk factors for overall postoperative complications. Compliance with nutritional supplements (OR=0.37; 95% CI: 0.14-0.97, p=0.041) and supplementation of malnourished patients as assessed by nutritional specialists (OR=0.24; 95% CI: 0.08-0.69, p=0.009) were independently associated with decreased infectious complications. CONCLUSIONS: Nutritional support based upon NRS-2002 screening might result in overnutrition, with potentially deleterious clinical consequences. We emphasize the importance of detailed assessment of the nutritional status by a dedicated specialist before deciding on early nutritional intervention for patients with an initial NRS-2002 score of ≥3.

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.