Twist-1 regulates the miR-199a/214 cluster during development

MicroRNAs are known to regulate developmental processes but their mechanism of regulation remains largely uncharacterized. We show the transcription factor Twist-1 drives the expression of a 7.9-kb noncoding RNA transcript (from the Dynamin-3 gene intron) that encodes a miR-199a and miR-214 cluster. We also show that knocking down Twist-1 with shRNAs decreased miR-199a/214 levels and that Twist-1 bound an E-Box promoter motif to developmentally regulate the expression of these miRNAs. The expression of HIF-1 (known to mediate Twist-1 transcription), miR-199a and miR-214 was maximal at E12.5 and the miRNAs were expressed specifically in mouse cerebellum, midbrain, nasal process and fore- and hindlimb buds. This study shows the expression of the miR199a/214 cluster is controlled by Twist-1 via an E-Box promoter element and supports a role for these miRNAs as novel intermediates in the pathways controlling the development of specific neural cell populations

Cross-regulation of Connexin43 and β-catenin influences differentiation of human neural progenitor cells

Connexin43 (Cx43) is the most widely and abundantly expressed gap junction (GJ) protein and it is strongly associated with the regulation of cell cycle progression. Emerging roles for Cx43 in cell adhesion and migration during neural differentiation have also been recently recognized, and this has emphasized the involvement of Cx43 in different physiological process beyond its role as a GJ protein. In this study, we explore the function of Cx43 in the differentiation of human neural progenitor cells (hNPCs) using viral vectors that mediate the overexpression or knockdown of the protein. Results showed that in the absence of this protein fetal cortex-derived hNPCs differentiated toward a neuronal phenotype at expenses of a glial phenotype. Furthermore, the silencing of Cx43 did not affect hNPC proliferation rate or numbers of apoptotic cells. The increase in the number of neurons was not recapitulated when GJ intercellular communications were pharmacologically blocked, and this suggested that Cx43 was influencing hNPCs differentiation with a GJ-independent effect. In addition, Cx43 knockdown significantly increased β-catenin signaling, which has been shown to regulate the transcription of pro-neuronal genes during embryonic neural development. Our results add further support to the hypothesis that Cx43 protein itself regulates key signaling pathways during development and neurogenesis beyond its role as GJ protein

cAMP responsive element-binding protein phosphorylation is necessary for perirhinal long-term potentiation and recognition memory

We established the importance of phosphorylation of cAMP responsive element-binding protein (CREB) to both the familiarity discrimination component of long-term recognition memory and plasticity within the perirhinal cortex of the temporal lobe. Adenoviral transduction of perirhinal cortex (and adjacent visual association cortex) with a dominant-negative inhibitor of CREB impaired the preferential exploration of novel over familiar objects at a long (24 h) but not a short (15 min) delay, disrupted the normal reduced activation of perirhinal neurons to familiar compared with novel pictures, and impaired long-term potentiation of synaptic transmission in perirhinal slices. The consistency of these effects across the behavioral, systems, and cellular levels of analysis provides strong evidence for involvement of CREB phosphorylation in synaptic plastic processes within perirhinal cortex necessary for long-term recognition memory

Loss of MicroRNA-7 Regulation Leads to α-Synuclein Accumulation and Dopaminergic Neuronal Loss In Vivo

Abnormal alpha-synuclein (α-synuclein) expression and aggregation is a key characteristic of Parkinson's disease (PD). However, the exact mechanism(s) linking α-synuclein to the other central feature of PD, dopaminergic neuron loss, remains unclear. Therefore, improved cell and in vivo models are needed to investigate the role of α-synuclein in dopaminergic neuron loss. MicroRNA-7 (miR-7) regulates α-synuclein expression by binding to the 3' UTR of the Synuclein Alpha Non A4 Component of Amyloid Precursor (SNCA) gene and inhibiting its translation. We show that miR-7 is decreased in the substantia nigra of patients with PD and, therefore, may play an essential role in the regulation of α-synuclein expression. Furthermore, we have found that lentiviral-mediated expression of miR-7 complementary binding sites to stably induce a loss of miR-7 function results in an increase in α-synuclein expression in vitro and in vivo. We have also shown that depletion of miR-7 using a miR-decoy produces a loss of nigral dopaminergic neurons accompanied by a reduction of striatal dopamine content. These data suggest that miR-7 has an important role in the regulation of α-synuclein and dopamine physiology and may provide a new paradigm to study the pathology of PD

Enhanced Efficacy of the CDNF/MANF Family by Combined Intranigral Overexpression in the 6-OHDA Rat Model of Parkinson's Disease

Cerebral Dopamine Neurotrophic Factor (CDNF) and Mesencephalic Astrocyte-derived Neurotrophic factor (MANF) are members of a recently discovered family of neurotrophic factors (NTFs). Here, we used intranigral or intrastriatal lentiviral vector-mediated expression to evaluate their efficacy at protecting dopaminergic function in the 6-OHDA model of Parkinson's disease (PD). In contrast to the well-studied Glial-Derived Neurotrophic Factor (GDNF), no beneficial effects were demonstrated by striatal overexpression of either protein. Interestingly, nigral overexpression of CDNF decreased amphetamine-induced rotations and increased tyroxine hydroxylase (TH) striatal fiber density but had no effect on numbers of TH(+) cells in the SN. Nigral MANF overexpression had no effect on amphetamine-induced rotations or TH striatal fiber density but resulted in a significant preservation of TH(+) cells. Combined nigral overexpression of both factors led to a robust reduction in amphetamine-induced rotations, greater increase in striatal TH-fiber density and significant protection of TH(+) cells in the SN. We conclude that nigral CDNF and MANF delivery is more efficacious than striatal delivery. This is also the first study to demonstrate that combined NTF can have synergistic effects that result in enhanced neuroprotection, suggesting that multiple NTF delivery may be more efficacious for the treatment of PD than the single NTF approaches attempted so far.Molecular Therapy (2014); doi:10.1038/mt.2014.206

Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells)

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described