Behavioral Resilience and Sensitivity to Locally Restricted Cortical Migration Deficits Induced by In Utero Knockdown of Disabled-1 in the Adult Rat.

Irregular neuronal migration plays a causal role in mental illnesses such as schizophrenia and autism, but the very nature of the migration deficits necessary to evoke adult behavioral changes is unknown. Here, we used in utero electroporation (IUE) in rats to induce a locally restricted, cortical migration deficit by knockdown of disabled-1 (Dab1), an intracellular converging point of the reelin pathway. After birth, selection of successfully electroporated rats by detection of in vivo bioluminescence of a simultaneously electroporated luciferase gene correlated to and was thus predictive to the number of electroporated neurons in postmortem histochemistry at 6 months of age. Rat neurons silenced for Dab1 did not migrate properly and their number surprisingly decreased after E22. Behavioral tests at adult ages (P180) revealed increased sensitivity to amphetamine as well as decreased habituation, but no deficits in memory tasks or motor functions. The data suggest that even subtle migration deficits involving only ten-thousands of cortical neurons during neurodevelopment can lead to lasting behavioral and neuronal changes into adulthood in some very specific behavioral domains. On the other hand, the lack of effects on various memory-related tasks may indicate resilience and plasticity of cognitive functions critical for survival under these specific conditions.

Nrf2, the Master Regulator of Anti-Oxidative Responses.

Tight regulation of inflammation is very important to guarantee a balanced immune response without developing chronic inflammation. One of the major mediators of the resolution of inflammation is the transcription factor: the nuclear factor erythroid 2-like 2 (Nrf2). Stabilized following oxidative stress, Nrf2 induces the expression of antioxidants as well as cytoprotective genes, which provoke an anti-inflammatory expression profile, and is crucial for the initiation of healing. In view of this fundamental modulatory role, it is clear that both hyper- or hypoactivation of Nrf2 contribute to the onset of chronic diseases. Understanding the tight regulation of Nrf2 expression/activation and its interaction with signaling pathways, known to affect inflammatory processes, will facilitate development of therapeutic approaches to prevent Nrf2 dysregulation and ameliorate chronic inflammatory diseases. We discuss in this review the principle mechanisms of Nrf2 regulation with a focus on inflammation and autophagy, extending the role of dysregulated Nrf2 to chronic diseases and tumor development.

Rational design of a minimal and highly enriched CYP102A1 mutant library with improved regio-, stereo- and chemoselectivity.

A minimal CYP102A1 mutant library of only 24 variants plus wild type was constructed by combining five hydrophobic amino acids (alanine, valine, phenylalanine, leucine and isoleucine) in two positions. Both positions are located close to the centre of the haem group. The first, position 87, has been shown to mediate substrate specificity and regioselectivity in CYP102A1. The second hotspot, position 328, was predicted to interact with all substrates during oxidation and has previously been identified by systematic analysis of 31 crystal structures and 6300 sequences of cytochrome P450 monooxygenases. By systematically altering the size of the side chains, a broad range of binding site shapes was generated. All variants were functionally expressed in E. coli. The library was screened with four terpene substrates geranylacetone, nerylacetone, (4R)-limonene and (+)-valencene. Only three variants showed no activity towards all four terpenes, while eleven variants demonstrated either a strong shift or improved regio- or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type.

Generation of topically transgenic rats by in utero electroporation and in vivo bioluminescence screening.

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at ≥6 months after birth. We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.