Efficient gene expression from integration-deficient lentiviral vectors in the spinal cord.

Gene transfer to spinal cord cells may be crucial for therapy in spinal muscular atrophy, amyotrophic lateral sclerosis and spinal cord injury. Lentiviral vectors are efficient for transduction of a variety of cells, but like all integrating vectors they pose a risk of insertional mutagenesis. Integration-deficient lentiviral vectors (IDLVs) remain episomal but retain the transduction efficiency of standard integrating lentiviral vectors, particularly when the episomes are not diluted out through repeated cell division. We have now applied IDLVs for transduction of spinal cord in vitro, in explants and in vivo. Our results demonstrate similar efficiency of eGFP expression from integrating lentiviral vectors and IDLVs in most cell types analyzed, including motor neurons, interneurons, dorsal root ganglia (DRG) neurons and astroglia. IDLV-mediated expression of pro-glial-cell-derived neurotrophic factor (Gdnf) rescues motor neuron cultures from death caused by removal of exogenous trophic support. IDLVs also mediate efficient RNA interference in DRG neuron cultures. After intraparenchymal injection in the rat and mouse cervical and lumbar regions in vivo, transduction is mainly neuronal, with both motor neurons and interneurons being efficiently targeted. These results suggest that IDLVs could be efficient and safer tools for spinal cord transduction in future therapeutic strategies.

AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na+ transport across human airway epithelial cells.

BACKGROUND AND PURPOSE: Pulmonary transepithelial Na(+) transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na(+) channels and basolateral Na(+) K(+) ATPase activity. EXPERIMENTAL APPROACH: H441 human airway epithelial cells were used to examine the effects of hypoxia on Na(+) transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS. KEY RESULTS: AMPK was activated by exposure to 3% or 0.2% O(2) for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm(-2) ) was added to the apical surface of cells grown at the air-liquid interface. Only 0.2% O(2) activated AMPK in cells grown at the air-liquid interface. AMPK activation was associated with elevation of cellular AMP:ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I(sc) (I(ouabain) ) and apical amiloride-sensitive Na(+) conductance (G(Na+) ). Modification of AMPK activity prevented the effect of hypoxia on I(ouabain) (Na(+) K(+) ATPase) but not apical G(Na+) . Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G(Na+) (epithelial Na(+) channels). CONCLUSIONS AND IMPLICATIONS: Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na(+) channels and basolateral Na(+) K(+) ATPase activity to decrease transepithelial Na(+) transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.

Impaired cannabinoid receptor type 1 signaling interferes with stress-coping behavior in mice.

Dysregulation of the endocannabinoid system is known to interfere with emotional processing of stressful events. Here, we studied the role of cannabinoid receptor type 1 (CB1) signaling in stress-coping behaviors using the forced swim test (FST) with repeated exposures. We compared effects of genetic inactivation with pharmacological blockade of CB1 receptors both in male and female mice. In addition, we investigated potential interactions of the endocannabinoid system with monoaminergic and neurotrophin systems of the brain. Naive CB1 receptor-deficient mice (CB1-/-) showed increased passive stress-coping behaviors as compared to wild-type littermates (CB1+/+) in the FST, independent of sex. These findings were partially reproduced in C57BL/6N animals and fully reproduced in female CB1+/+ mice by pharmacological blockade of CB1 receptors with the CB1 receptor antagonist SR141716. The specificity of SR141716 was confirmed in female CB1-/- mice, where it failed to affect behavioral performance. Sensitivity to the antidepressants desipramine and paroxetine was preserved, but slightly altered in female CB1-/- mice. There were no genotype differences between CB1+/+ and CB1-/- mice in monoamine oxidase A and B activities under basal conditions, nor in monoamine content of hippocampal tissue after FST exposure. mRNA expression of vesicular glutamate transporter type 1 was unaffected in CB1-/- mice, but mRNA expression of brain-derived neurotrophic factor (BDNF) was reduced in the hippocampus. Our results suggest that impaired CB1 receptor function promotes passive stress-coping behavior, which, at least in part, might relate to alterations in BDNF function.