RESUMO
The anterior piriform cortex (APC) is activated by, and is the brain area most sensitive to, essential (indispensable) amino acid (IAA) deficiency. The APC is required for the rapid (20 min) behavioral rejection of IAA deficient diets and increased foraging, both crucial adaptive functions supporting IAA homeostasis in omnivores. The biochemical mechanisms signaling IAA deficiency in the APC block initiation of translation in protein synthesis via uncharged tRNA and the general amino acid control kinase, general control nonderepressing kinase 2. Yet, how inhibition of protein synthesis activates the APC is unknown. The neuronal K(+) Cl(-) cotransporter, neural potassium chloride co-transporter (KCC2), and GABAA receptors are essential inhibitory elements in the APC with short plasmalemmal half-lives that maintain control in this highly excitable circuitry. After a single IAA deficient meal both proteins were reduced (vs. basal diet controls) in western blots of APC (but not neocortex or cerebellum) and in immunohistochemistry of APC. Furthermore, electrophysiological analyses support loss of inhibitory elements such as the GABAA receptor in this model. As the crucial inhibitory function of the GABAA receptor depends on KCC2 and the Cl(-) transmembrane gradient it establishes, these results suggest that loss of such inhibitory elements contributes to disinhibition of the APC in IAA deficiency. The circuitry of the anterior piriform cortex (APC) is finely balanced between excitatory (glutamate, +) and inhibitory (GABA, -) transmission. GABAA receptors use Cl(-), requiring the neural potassium chloride co-transporter (KCC2). Both are rapidly turning-over proteins, dependent on protein synthesis for repletion. In IAA (indispensable amino acid) deficiency, within 20 min, blockade of protein synthesis prevents restoration of these inhibitors; they are diminished; disinhibition ensues. GCN2 = general control non-derepressing kinase 2, eIF2α = α-subunit of the eukaryotic initiation factor 2.
Assuntos
Aminoácidos Essenciais/deficiência , Condutos Olfatórios/metabolismo , Receptores de GABA-A/biossíntese , Simportadores/biossíntese , Animais , Regulação para Baixo , Potenciais Pós-Sinápticos Excitadores , Masculino , Ratos , Cotransportadores de K e Cl-RESUMO
Protein synthesis requires a continuous supply of all of the indispensable (essential) amino acids (IAAs). If any IAA is deficient, animals must obtain the limiting amino acid by diet selection. Sensing of IAA deficiency requires an intact anterior piriform cortex (APC), but does it act alone? Shortly after rats begin eating an IAA-deficient diet, the meal ends and EPSPs are activated in the APC; from there, neurons project to feeding circuits; the meal ends within 20 min. Within the APC in vivo, uncharged tRNA activates the general amino acid control non-derepressing 2 (GCN2) enzyme system increasing phosphorylation of eukaryotic initiation factor (P-eIF2α), which blocks general protein synthesis. If this paleocortex is sufficient for sensing IAA depletion, both neuronal activation and P-eIF2α should occur in an isolated APC slice. We used standard techniques for electrophysiology and immunohistochemistry. After rats ate IAA-devoid or -imbalanced diets, their depleted slices responded to different stimuli with increased EPSP amplitudes. Slices from rats fed a control diet were bathed in artificial CSF replete with all amino acids with or without the IAA, threonine, or a tRNA synthetase blocker, l-threoninol, or its inactive isomer, d-threoninol. Thr depletion in vitro increased both EPSP amplitudes and P-eIF2α. l (but not d)-threoninol also increased EPSP amplitudes relative to control. Thus, we show independent excitation of the APC with responses parallel to those known in vivo. These data suggest a novel idea: in addition to classical processing of peripheral sensory input, direct primary sensing may occur in mammalian cortex.
Assuntos
Aminoácidos Essenciais/deficiência , Córtex Cerebral/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Neurônios/metabolismo , Biossíntese de Proteínas , Proteínas Quinases/metabolismo , Aminoácidos Essenciais/biossíntese , Aminoácidos Essenciais/metabolismo , Animais , Córtex Cerebral/fisiologia , Dieta , Eletrofisiologia , Imuno-Histoquímica , Masculino , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Fosforilação , Ratos , Ratos Sprague-Dawley , Treonina/deficiênciaRESUMO
Citrullinemia type I (CTLN1, OMIM# 215700) is an inherited urea cycle disorder that is caused by an argininosuccinate synthetase (ASS) enzyme deficiency. In this report, we describe two spontaneous hypomorphic alleles of the mouse Ass1 gene that serve as an animal model of CTLN1. These two independent mouse mutant alleles, also described in patients affected with CTLN1, interact to produce a range of phenotypes. While some mutant mice died within the first week after birth, others survived but showed severe retardation during postnatal development as well as alopecia, lethargy, and ataxia. Notable pathological findings were similar to findings in human CTLN1 patients and included citrullinemia and hyperammonemia along with delayed cerebellar development, epidermal hyperkeratosis, and follicular dystrophy. Standard treatments for CTLN1 were effective in rescuing the phenotype of these mutant mice. Based on our studies, we propose that defective cerebellar granule cell migration secondary to disorganization of Bergmann glial cell fibers cause cerebellar developmental delay in the hyperammonemic and citrullinemic brain, pointing to a possible role for nitric oxide in these processes. These mouse mutations constitute a suitable model for both mechanistic and preclinical studies of CTLN1 and other hyperammonemic encephalopathies and, at the same time, underscore the importance of complementing knockout mutations with hypomorphic mutations for the generation of animal models of human genetic diseases.
Assuntos
Argininossuccinato Sintase/fisiologia , Citrulinemia/etiologia , Modelos Animais de Doenças , Hiperamonemia/etiologia , Mutação de Sentido Incorreto/genética , Alelos , Animais , Arginina/farmacologia , Western Blotting , Movimento Celular , Cerebelo/anormalidades , Citrulinemia/tratamento farmacológico , Deficiências do Desenvolvimento/tratamento farmacológico , Deficiências do Desenvolvimento/etiologia , Feminino , Transtornos do Crescimento/tratamento farmacológico , Transtornos do Crescimento/etiologia , Humanos , Hiperamonemia/tratamento farmacológico , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Óxido Nítrico/metabolismo , Fenótipo , Benzoato de Sódio/farmacologia , SíndromeRESUMO
The anterior piriform cortex (APC) has been shown to be an essential brain structure for the detection of dietary indispensable amino acid (IAA) deficiency, but little has been known about possible molecular detection mechanisms. Increased phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2alpha (eIF2alpha) has been directly linked to amino acid deficiency in yeast. Recently, we have shown increased phosphorylation of eIF2alpha (p-eIF2alpha) in the rat APC 20 minutes after ingestion of an IAA-deficient meal. We suggest that if phosphorylation of eIF2alpha is an important mechanism in detection of IAA deficiency, then APC neurons that show p-eIF2alpha should also show molecular evidence of potentiation. The present research demonstrates increased expression and co-localization of p-eIF2alpha and phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in APC neurons, but not in the primary motor or agranular insular cortices in response to an IAA-deficient diet. ERK1/2 is an element of the mitogen-activated protein kinase cascade, an intraneuronal signaling mechanism associated with neuronal activation. The region of the APC that responds to IAA deficiency with increased p-eIF2alpha and p-ERK1/2 labeling ranges from 3.1 to 2.5 mm rostral of bregma. Within this region, only a few neurons respond to IAA deficiency with co-localization of abundant p-eIF2alpha and p-ERK1/2. These chemosensory neurons probably detect IAA deficiency and generate neuronal signaling to other portions of the brain, changing feeding behavior.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Giro Para-Hipocampal/enzimologia , Treonina/deficiência , Ração Animal , Animais , Células Quimiorreceptoras/enzimologia , Imuno-Histoquímica , Masculino , Neurônios/enzimologia , Giro Para-Hipocampal/citologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Estatísticas não Paramétricas , Treonina/metabolismoRESUMO
Diets deficient in an indispensable amino acid are known to suppress food intake in rats. Few studies were focused at understanding how amino acid-deficient diets may elicit biochemical changes at the mitochondrial level. The goal of this study was to evaluate mitochondrial function in rats fed diets with 0.00, 0.18, 0.36, and 0.88% threonine (Thr) (set at 0, 30, 60, and 140% of Thr requirement for growth). Here, it is described for the first time that Thr-deficient diets induce a specific uncoupling of mitochondria in liver, especially with NADH-linked substrates, not observed in heart (except for Thr-devoid diet). The advantage of this situation would be to provide ATP to support growth and maintenance when high-quality protein food (or wealth of high-quality food in general) is available, whereas Thr-deficient diets (or deficient-quality protein food) promote the opposite, increasing mitochondrial uncoupling in liver. The uncoupling with NADH substrates would favor the use of nutrients as energy sources with higher FADH-to-NADH ratios, such as fat, minimizing the first irreversible NADH-dependent catabolism of many amino acids, including Thr, thus enhancing the use of the limiting amino acid for protein synthesis when a low quality protein source is available.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Proteínas Alimentares/metabolismo , Metabolismo Energético , Fígado/metabolismo , Mitocôndrias Hepáticas/metabolismo , Deficiência de Proteína/metabolismo , Treonina/deficiência , Trifosfato de Adenosina/metabolismo , Animais , Peso Corporal , Proteínas Alimentares/administração & dosagem , Modelos Animais de Doenças , Ingestão de Alimentos , Flavina-Adenina Dinucleotídeo/metabolismo , Masculino , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , NAD/metabolismo , Fosforilação Oxidativa , Deficiência de Proteína/fisiopatologia , Ratos , Ratos Sprague-Dawley , Treonina/administração & dosagem , Fatores de TempoRESUMO
Recognizing a deficiency of indispensable amino acids (IAAs) for protein synthesis is vital for dietary selection in metazoans, including humans. Cells in the brain's anterior piriform cortex (APC) are sensitive to IAA deficiency, signaling diet rejection and foraging for complementary IAA sources, but the mechanism is unknown. Here we report that the mechanism for recognizing IAA-deficient foods follows the conserved general control (GC) system, wherein uncharged transfer RNA induces phosphorylation of eukaryotic initiation factor 2 (eIF2) via the GC nonderepressing 2 (GCN2) kinase. Thus, a basic mechanism of nutritional stress management functions in mammalian brain to guide food selection for survival.