Quantitative analysis of conditional gene inactivation using rationally designed, tetracycline-controlled miRNAs

The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo

Reelin Controls Progenitor Cell Migration in the Healthy and Pathological Adult Mouse Brain

Understanding the signals that control migration of neural progenitor cells in the adult brain may provide new therapeutic opportunities. Reelin is best known for its role in regulating cell migration during brain development, but we now demonstrate a novel function for reelin in the injured adult brain. First, we show that Reelin is upregulated around lesions. Second, experimentally increasing Reelin expression levels in healthy mouse brain leads to a change in the migratory behavior of subventricular zone-derived progenitors, triggering them to leave the rostral migratory stream (RMS) to which they are normally restricted during their migration to the olfactory bulb. Third, we reveal that Reelin increases endogenous progenitor cell dispersal in periventricular structures independently of any chemoattraction but via cell detachment and chemokinetic action, and thereby potentiates spontaneous cell recruitment to demyelination lesions in the corpus callosum. Conversely, animals lacking Reelin signaling exhibit reduced endogenous progenitor recruitment at the lesion site. Altogether, these results demonstrate that beyond its known role during brain development, Reelin is a key player in post-lesional cell migration in the adult brain. Finally our findings provide proof of concept that allowing progenitors to escape from the RMS is a potential therapeutic approach to promote myelin repair

Dendritic Spine Hypoplasticity and Downregulation of Reelin and GABAergic Tone in Schizophrenia Vulnerability

In this review, we will first present a brief overview of the current understanding of: (a) the biology of reelin; (b) the putative reelin signaling pathways via integrin receptor stimulation; (c) the cytosolic adapter protein DAB1, which appears to be operative in the transduction of reelin's pleiotropic actions in embryonic, adolescent, and adult brain; (d) the regulation of GABAergic function, including some aspects of GABAergic system development; and (e) dendritic spine function and its role in the regulation of synaptic plasticity. We argue that a downregulation of reelin expression occurring in prefrontal cortex and in every brain structure of schizophrenia patients so far studied may be associated with a decrease in dendritic spine expression that in turn may provide an important reduction of cortical function as documented by the downregulation of glutamic acid decarboxylase67 (GAD67) expression, which might be secondary to a reduction of GABAergic axon terminals. This hypothesis is supported by a genetic mouse model of reelin haploinsufficiency that replicates the above-described dendritic and presynaptic GABAergic defects documented in schizophrenia brains

Stress differentially regulates synaptophysin and synaptotagmin expression in hippocampus

BACKGROUND: In view of the effects of stress on synaptic plasticity, the regulation of synaptophysin and synaptotagmin expression by immobilization was analyzed by in situ hybridization. METHODS: Rats were exposed to immobilization stress, which induced typical behavioral alterations, such as reduced locomotor activity after stress exposure. Determination of mRNA levels of the integral synaptic vesicle proteins was performed immediately after acute or chronic immobilization. RESULTS: The results demonstrate that stress exposure leads to reduced expression of synaptophysin but increased expression of synaptotagmin in the hippocampus. CONCLUSIONS: This rapid and differential regulation of synaptic vesicle proteins could be responsible for some of the morphological, biochemical, and behavioral changes observed after stress exposure. These changes may be relevant to such clinical disorders as psychoses, depression, and posttraumatic stress disorder that are sensitive to stress and involve changes in neural and synaptic plasticity

Reelin and glutamic acid decarboxylase(67) promoter remodeling in an epigenetic methionine-induced mouse model of schizophrenia

Reduction of prefrontal cortex glutamic acid decarboxylase (GAD(67)) and reelin (mRNAs and proteins) expression is the most consistent finding reported by several studies of postmortem schizophrenia (SZ) brains. Converging evidence suggests that the reduced GAD(67) and reelin expression in cortical GABAergic interneurons of SZ brains is the consequence of an epigenetic hypermethylation of RELN and GAD(67) promoters very likely mediated by the overexpression of DNA methyltransferase 1 in cortical GABAergic interneurons. Studies of the molecular mechanisms (DNA methylation plus related chromatin remodeling factors) that cause the down-regulation of reelin and GAD(67) in SZ brains have important implications not only to understand the disease pathogenesis but also to improve present pharmacological interventions to treat SZ. The mouse treated with l-methionine models some of the molecular neuropathologies detected in SZ, including the hypermethylation of RELN promoter CpG islands and the down-regulation of reelin and GAD(67) expression. We now report that in these mice, RELN and GAD(67) promoters express an increased recruitment of methyl-CpG binding domain proteins. In these mice the histone deacetylase inhibitor valproate, which increases acetylated histone content in cortical GABAergic interneurons, also prevents MET-induced RELN promoter hypermethylation and reduces the methyl-CpG binding domain protein binding to RELN and GAD(67) promoters. These findings suggest that DNA hypermethylation and the associated chromatin remodeling may be critically important in mediating the epigenetic down-regulation of reelin and GAD(67) expression detected in cortical GABAergic interneurons of SZ patients