Short Promoters in Viral Vectors Drive Selective Expression in Mammalian Inhibitory Neurons, but do not Restrict Activity to Specific Inhibitory Cell-Types

Short cell-type specific promoter sequences are important for targeted gene therapy and studies of brain circuitry. We report on the ability of short promoter sequences to drive fluorescent protein expression in specific types of mammalian cortical inhibitory neurons using adeno-associated virus (AAV) and lentivirus (LV) vectors. We tested many gene regulatory sequences derived from fugu (Takifugu rubripes), mouse, human, and synthetic composite regulatory elements. All fugu compact promoters expressed in mouse cortex, with only the somatostatin (SST) and the neuropeptide Y (NPY) promoters largely restricting expression to GABAergic neurons. However these promoters did not control expression in inhibitory cells in a subtype specific manner. We also tested mammalian promoter sequences derived from genes putatively coexpressed or coregulated within three major inhibitory interneuron classes (PV, SST, VIP). In contrast to the fugu promoters, many of the mammalian sequences failed to express, and only the promoter from gene A930038C07Rik conferred restricted expression, although as in the case of the fugu sequences, this too was not inhibitory neuron subtype specific. Lastly and more promisingly, a synthetic sequence consisting of a composite regulatory element assembled with PAX6 E1.1 binding sites, NRSE and a minimal CMV promoter showed markedly restricted expression to a small subset of mostly inhibitory neurons, but whose commonalities are unknown

Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins.

Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains. The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown. Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions. In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains. In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin. The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain. One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold

In vitro myelin formation using embryonic stem cells

Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation.National Institutes of Health: P30CA014195 National Institutes of Health: P30NS072031National Institutes of Health: P30NS072031National Multiple Sclerosis SocietySanofi and Salk Institute Discovery AwardChristopher & Dana Reeve FoundationLeona M. and Harry B. Helmsley Charitable TrustAnnette Merle-SmithCrick-Jacobs Junior FellowshipCalifornia Institute for Regenerative MedicineWaitt Advanced Biophotonics CenterW.M. Keck FoundationNational Cancer Institute (NCI) P30 Cancer Center Support GrantNational Institute of Neurological Disorders and Stroke (NINDS) P30 Neuroscience Center Core Gran

Expression and Functional Characterization of Membrane-Integrated Mammalian Corticotropin Releasing Factor Receptors 1 and 2 in <i>Escherichia coli</i>

<div><p>Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in <i>E. coli</i>. The characteristics of different CRFR2β -PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of <i>E. coli</i> bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our <i>E. coli</i> expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.</p></div

PD-sauvagine binding.

<p>Displacement by PD-sauvagine (♦) or antalarmin (●) of labeled PD-sauvagine bound to hCRFR1α expressed in <i>E. coli</i> membranes.</p

Comparison of the expression level of recombinant CRFRs.

<p>Expression of PelB-hCRFR1α (R1) and PelB-mCRFR2β (R2) was carried out either in LB (A) or in TB (B) medium in Rosetta2(DE3) strain. TB derived cultures were diluted twenty times before electrophoresis, while LB derived fractions were not diluted. With either medium, equivalent volumes of both the bacterial inclusion bodies and membrane fractions were analyzed by Western blot with His₆-tag antibody. IPTG induced bacteria Rosetta2(DE3) transformed with the parental vector pET-26b (-) were used as a negative control.</p

Influence of various <i>E. coli</i> host strain on the expression of CRFRs.

<p>Expressions of PelB-hCRFR1α (<b>A</b>) and PelB-mCRFR2β (<b>B</b>) were carried out in LB medium in four different strains. Equivalent volumes of the bacterial inclusion bodies and membrane fractions were analyzed by Western blot with His₆-tag antibody. Parental strain BL21(DE3) transformed with the same constructs but treated without IPTG inducer was used as a negative control.</p