RESUMO
BACKGROUND: Exposure of the developing brain to propofol results in cognitive deficits. Recent data suggest that inhibition of neuronal apoptosis does not prevent cognitive defects, suggesting mechanisms other than neuronal apoptosis play a role in anaesthetic neurotoxicity. Proper neuronal growth during development is dependent upon growth cone morphology and axonal transport. Propofol modulates actin dynamics in developing neurones, causes RhoA-dependent depolymerisation of actin, and reduces dendritic spines and synapses. We hypothesised that RhoA inhibition prevents synaptic loss and subsequent cognitive deficits. The present study tested whether RhoA inhibition with the botulinum toxin C3 (TAT-C3) prevents propofol-induced synapse and neurite loss, and preserves cognitive function. METHODS: RhoA activation, growth cone morphology, and axonal transport were measured in neonatal rat neurones (5-7 days in vitro) exposed to propofol. Synapse counts (electron microscopy), dendritic arborisation (Golgi-Cox), and network connectivity were measured in mice (age 28 days) previously exposed to propofol at postnatal day 5-7. Memory was assessed in adult mice (age 3 months) previously exposed to propofol at postnatal day 5-7. RESULTS: Propofol increased RhoA activation, collapsed growth cones, and impaired retrograde axonal transport of quantum dot-labelled brain-derived neurotrophic factor, all of which were prevented with TAT-C3. Adult mice previously treated with propofol had decreased numbers of total hippocampal synapses and presynaptic vesicles, reduced hippocampal dendritic arborisation, and infrapyramidal mossy fibres. These mice also exhibited decreased hippocampal-dependent contextual fear memory recall. All anatomical and behavioural changes were prevented with TAT-C3 pre-treatment. CONCLUSION: Inhibition of RhoA prevents propofol-mediated hippocampal neurotoxicity and associated cognitive deficits.
Assuntos
Transporte Axonal/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Cones de Crescimento/efeitos dos fármacos , Propofol , Sinapses/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Animais , Toxinas Botulínicas , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Hipnóticos e Sedativos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas , Ratos , Ratos Sprague-Dawley , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 5'-diphosphate dissociation from Rho GTPases, keeping these small GTPases in an inactive state. The GDIs are made up of two domains: a flexible N-terminal domain of about 70 amino acid residues and a folded 134-residue C-terminal domain. Here, we characterize the conformation of the N-terminal regions of both RhoGDI and D4GDI using a series of NMR experiments which include (15)N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to form helices are identified: residues 36 to 58 and 9 to 20 in RhoGDI, and residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles of the N-terminal domain of RhoGDI, in vitro and in vivo functional assays have been carried out with N-terminally truncated proteins. These studies show that the first 30 amino acid residues are not required for inhibition of GDP dissociation but appear to be important for GTP hydrolysis, whilst removal of the first 41 residues completely abolish the ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI and D4GDI are able to interact in similar ways with the guanosine 5'-diphosphate-bound GTPase, but differ in their ability to regulate GTP-bound forms; these functional differences are attributed to the conformational differences of the N-terminal domains of the guanosine 5'-diphosphate dissociation inhibitors. Therefore, the two transient helices, appear to be associated with different biological effects of RhoGDI, providing a clear example of structure-activity relationships in a flexible protein domain.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/química , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Amidas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , NADPH Oxidases/metabolismo , Ressonância Magnética Nuclear Biomolecular , Maleabilidade , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Deleção de Sequência/genética , Solventes , Relação Estrutura-Atividade , Transfecção , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/genética , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Small GTPases of the Rho family regulate a wide variety of cell functions. In this review, we briefly describe the biological activities of Rho GTPases. Using the Rac-regulated NADPH oxidase as an example, we discuss possible regulatory points that might be exploited for drug development. Finally, we explore strategies for specific targeting of Rho GTPase-regulated signaling pathways.