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.

Impulse oscillometry identifies peripheral airway dysfunction in children with adenosine deaminase deficiency.

Adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) is characterized by impaired T-, B- and NK-cell function. Affected children, in addition to early onset of infections, manifest non-immunologic symptoms including pulmonary dysfunction likely attributable to elevated systemic adenosine levels. Lung disease assessment has primarily employed repetitive radiography and effort-dependent functional studies. Through impulse oscillometry (IOS), which is effort-independent, we prospectively obtained objective measures of lung dysfunction in 10 children with ADA-SCID. These results support the use of IOS in the identification and monitoring of lung function abnormalities in children with primary immunodeficiencies.

The EXS Domain of PHO1 Participates in the Response of Shoots to Phosphate Deficiency via a Root-to-Shoot Signal.

The response of shoots to phosphate (Pi) deficiency implicates long-distance communication between roots and shoots, but the participating components are poorly understood. We have studied the topology of the Arabidopsis (Arabidopsis thaliana) PHOSPHATE1 (PHO1) Pi exporter and defined the functions of its different domains in Pi homeostasis and signaling. The results indicate that the amino and carboxyl termini of PHO1 are both oriented toward the cytosol and that the protein spans the membrane twice in the EXS domain, resulting in a total of six transmembrane α-helices. Using transient expression in Nicotiana benthamiana leaf, we demonstrated that the EXS domain of PHO1 is essential for Pi export activity and proper localization to the Golgi and trans-Golgi network, although the EXS domain by itself cannot mediate Pi export. In contrast, removal of the amino-terminal hydrophilic SPX domain does not affect the Pi export capacity of the truncated PHO1 in N. benthamiana. While the Arabidopsis pho1 mutant has low shoot Pi and shows all the hallmarks associated with Pi deficiency, including poor shoot growth and overexpression of numerous Pi deficiency-responsive genes, expression of only the EXS domain of PHO1 in the roots of the pho1 mutant results in a remarkable improvement of shoot growth despite low shoot Pi. Transcriptomic analysis of pho1 expressing the EXS domain indicates an attenuation of the Pi signaling cascade and the up-regulation of genes involved in cell wall synthesis and the synthesis or response to several phytohormones in leaves as well as an altered expression of genes responsive to abscisic acid in roots.

Genetically-defined deficiency of mannose-binding lectin is associated with protection after experimental stroke in mice and outcome in human stroke

The complement system is a major effector of innate immunity that has been involved in stroke brain damage. Complement activation occurs through the classical, alternative and lectin pathways. The latter is initiated by mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs). Here we investigated whether the lectin pathway contributes to stroke outcome in mice and humans.

Long term dietary deficiency in steers: vital functions and T3 and IGF-1 relationships

To evaluate the influence of diets with different degrees of energy deficiency on the hormonal profile and vital functions, 12 steers were randomly distributed into 3 groups of 4 animals. For 140 days, each group received (G1) a diet to promote a weight gain of 900gr/day (17.7 Mcal/d DE and 13% CP), (G2) 80% of the maintenance requirements (5.8 Mcal/d DE and 7% CP), or (G3) 60% of the maintenance requirements (4.7 Mcal/d DE and 5% CP). In G2 and G3, the energy deficit caused a marked decrease in the heart rate and respiratory rate and a reduction in the blood levels of Insulin like growth factor-1 (IGF-1) and triiodothyronine (T3). The decrease in heart rate, respiratory movement and, to a lesser extent, reduction of the rectal temperature, reflected the low status of energy and was negatively impacted by the low levels of T3. There was a strong correlation between the hormones T3 and IGF-1 (r=0.833). There were also strong correlations between T3 and HR (r=0.701), T3 and RR (r=0.632), IGF-1 and HR (r=0.731), and IGF-1 and RR (r=0.679). There were intermediate correlations between T3 and TºC (r=0.484), T3 and insulin (r=0.506), IGF-1 and insulin (r=0.517), and IGF-1 and TºC (r=0.548). This study showed the influence of a long period of providing an energy-deficient diet on animal performance, correlating hormonal status and vital functions in growing cattle. The results indicated that the evaluated parameters represent an important tool for the early detection of dietary deficiency.

Disaccharidase levels in normal epithelium of the small intestine of rats with iron-deficiency anemia

Iron-deficiency anemia is the nutritional deficiency most frequently occurring throughout the world, which manifests as a complex systemic disease involving all cells, affecting enzyme activities and modifying protein synthesis. In view of these considerations, the objective of the present study was to determine the effects of iron-deficiency anemia on disaccharidases and on the epithelial morphokinetics of the jejunal mucosa. Newly weaned male Wistar rats were divided into 4 groups of 10 animals each: C6w received a standard ration containing 36 mg elemental iron per kg ration for 6 weeks; E6w received an iron-poor ration (5-8 mg/kg ration) for 6 weeks; C10w received an iron-rich ration (36 mg/kg ration) for 10 weeks; E10w received an iron-poor ration for 6 weeks and then an iron-rich ration (36 mg/kg) for an additional 4 weeks. Jejunal fragments were used to measure disaccharidase content and to study cell proliferation. The following results were obtained: 1) a significant reduction (P<0.001) of animal weight, hemoglobin (Hb), serum iron and total iron-binding capacity (TIBC) in group E6w as compared to C6w; reversal of the alterations in Hb, serum iron and TIBC with iron repletion (E10w = C10w); animal weights continued to be significantly different in groups E10w and C10w. 2) Sucrase and maltase levels were unchanged; total and specific lactase levels were significantly lower in group E6w and this reduction was reversed by iron repletion (E10w = C10w). 3) The cell proliferation parameters did not differ between groups. On the basis of these results, we conclude that lactase production was influenced by iron deficiency and that this fact was not related to changes in cell population and proliferation in the intestinal mucosa

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens

Low and high molecular weight kininogens (LK and HK), containing 409 and 626 amino acids with masses of ~65 and 120 kDa after glycosylation, respectively, are coded by a single gene mapped to the human chromosome 3 by alternative splicing of the transcribed mRNA. The NH2-termini Glu1-Thr383 region, identical in LK and HK, contains bradykinin (BK) moieties Arg363-Arg371. LK, HK and their kinin products Lys-BK and BK are involved in several biologic processes. They are evolutionarily conserved and only 7 patients, all apparently normal, have been reported to lack them. In one of these patients (Williams' trait), a codon mutation (Arg178 ® stop) has been blamed for the absence of LK and HK. However, using Western blots with 2 monoclonal anti-HK antibodies, one that recognizes the region common to LK and HK and the other that recognizes only HK, I detected ~110-kDa bands in the plasma of this LK/HK-deficient patient vs ~120-kDa bands in normal human and ape plasmas. With polyclonal anti-Lys-BK antibody, which strongly detects BK cleaved at its COOH-terminus in purified HK, I detected ~110-kDa bands in the normal and the deficient plasmas. Western blots with a monoclonal anti-prekallikrein (PK) antibody showed that surface activation of PK and distribution of PK activation products, both dependent on HK, were similar in these plasmas. These findings suggest that a mutant gene yielded a kininogen-like species possibly involving aberrant mRNA splicing - structurally different from normal HK, but apparently with the capacity to carry out seemingly vital HK functions.

Relation of the disaccharidases in the small intestine of the rat to the degree of experimentally induced iron-deficiency anemia

Hypolactasia associated with severe iron-deficiency anemia has been reported in several studies. The objective of the present study was to determine whether hypolactasia is associated with the degree and duration of iron-deficiency anemia. Newly weaned male Wistar rats were divided into a control group receiving a diet supplemented with iron (C) and an experimental group (E) receiving a diet not supplemented with iron (iron-deficiency diet). The animals were studied on the 3rd, 5th, 7th, 14th, 21st, 28th and 35th days of the experiment, when overall and iron nutritional status and disaccharidase activity in the small intestine were determined by the Dahlqvist method. A reduction in weight occurred in the anemic animals starting on the 5th day of the study. Anemia was present in the experimental animals, with a progressive worsening up to the 14th day (hemoglobin: C = 13.27 and E = 5.37) and stabilizing thereafter. Saccharase and maltase activities did not differ significantly between groups, whereas lactase showed a significant reduction in total (TA) and specific activity (SA) in the anemic animals starting on the 21st day of the study. Median lactase TA for the C and E groups was 2.27 and 1.25 U on the 21st day, 2.87 and 1.88 U on the 28th day, and 4.20 and 1.59 U on the 35th day, respectively. Median lactase SA was 0.31 and 0.20 U/g wet weight on the 21st day, 0.39 and 0.24 U/g wet weight on the 28th day, and 0.42 and 0.23 U/g wet weight on the 35th day, respectively. These findings suggest a relationship between the enzymatic alterations observed and both the degree and duration of the anemic process. Analysis of other studies on intestinal disaccharidases in anemia suggests that the mechanism of these changes may be functional, i.e., that the enterocytes may suffer a reduction in their ability to synthesize these enzymes.

Dietary cellulose has no effect on the regeneration of hemoglobin in growing rats with iron deficiency anemia

The objective of the present study was to determine the effect of cellulose on intestinal iron absorption in rats during recovery from iron deficiency anemia. Twenty-one-day-old male Wistar-EPM rats were fed an iron-free ration for two weeks to induce anemia. At 5 weeks of age, the rats were divided into two groups (both groups receiving 35 mg of elemental iron per kg diet): cellulose group (N = 12), receiving a diet containing 100 g of cellulose/kg and control (N = 12), receiving a diet containing no cellulose. The fresh weight of the feces collected over a 3-day period between the 15th and 18th day of dietary treatment was 10.7 ± 3.5 g in the group receiving cellulose and 1.9 ± 1.2 g in the control group (P<0.001). Total food intake was higher in the cellulose group (343.4 ± 22.0 g) than in the control (322.1 ± 13.1 g, P = 0.009) during the 3 weeks of dietary treatment. No significant difference was observed in weight gain (cellulose group = 132.8 ± 19.2, control = 128.0 ± 16.3 g), hemoglobin increment (cellulose group = 8.0 ± 0.8, control = 8.0 ± 1.0 g/dl), hemoglobin level (cellulose group = 12.3 ± 1.2, control = 12.1 ± 1.3 g/dl) or in hepatic iron levels (cellulose group = 333.6 ± 112.4, control = 398.4 ± 168.0 µg/g dry tissue). We conclude that cellulose does not adversely affect the regeneration of hemoglobin, hepatic iron level or the growth of rats during recovery from iron deficiency anemia.

Antioxidant dietary deficiency induces caspase activation in chick skeletal muscle cells

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambdaexc = 320 nm and lambdaem = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.

Impact of congenital calcitonin deficiency due to dysgenetic hypothyroidism on bone mineral density

The objective of the present study was to determine the effect of chronic calcitonin deficiency on bone mass development. The results of 11 patients with thyroid dysgenesis (TD) were compared to those of 17 normal individuals (C) and of 9 patients with other forms of hypothyroidism (OH): 4 with hypothyroidism due to inborn errors of thyroid hormone synthesis and 5 with Hashimoto's thyroiditis. The subjects received an intravenous calcium stimulus and blood was collected for the determination of ionized calcium (Ca2+), calcitonin, and intact parathyroid hormone. Bone mineral density (BMD) was determined by dual-energy X-ray absorptiometry. After calcium administration the levels of Ca2+ in the two groups of hypothyroidism were significantly higher than in the normal control group (10 min after starting calcium infusion: C = 1.29 ± 0.08 vs TD = 1.34 ± 0.03 vs OH = 1.34 ± 0.02 mmol/l; P < 0.05), and only the TD group showed no calcitonin response (5 min after starting calcium infusion: C = 27.9 ± 5.8 vs TD = 6.6 ± 0.3 vs OH = 43.0 ± 13.4 ng/l). BMD values did not differ significantly between groups (L2-L4: C = 1.116 ± 0.02 vs TD = 1.109 ± 0.03 vs OH = 1.050 ± 0.04 g/cm²). These results indicate that early deficiency of calcitonin secretion has no detrimental effect on bone mass development. Furthermore, the increased calcitonin secretion observed in patients with inborn errors of thyroid hormone biosynthesis does not confer any advantage in terms of BMD.

Newborn screening for biotinidase deficiency in Brazil: biochemical and molecular characterizations

Biotinidase deficiency is an inherited metabolic disorder characterized by neurological and cutaneous symptoms. Fortunately, it can be treated and the symptoms prevented by oral administration of the vitamin biotin. Using dried blood-soaked filter paper cards, biotinidase activity was determined in the sera of 225,136 newborns in Brazil. Mutation analysis performed on DNA from 21 babies with low serum biotinidase activity confirmed that 3 had profound biotinidase deficiency (less than 10% of mean normal sera biotinidase activity), 10 had partial biotinidase deficiency (10 to 30% of mean normal serum activity), 1 was homozygous for partial biotinidase deficiency, 4 were heterozygous for either profound or partial deficiency, and 3 were normal. Variability in serum enzyme activities and discrepancies with mutation analyses were probably due to inappropriate handling and storage of samples sent to the laboratory. Obtaining an appropriate control serum at the same time as that of the suspected child will undoubtedly decrease the false-positive rate (0.09%). Mutation analysis can be used to confirm the genotype of these children. The estimated incidence of biotinidase deficiency in Brazil is about 1 in 9,000, higher than in most other countries. Screening and treatment of biotinidase deficiency are effective and warranted. These results strongly suggest that biotinidase deficiency should be included in the newborn mass screening program of Brazil.