Low-Anxiety Rat Phenotypes Can Be Further Reduced through Genetic Intervention

Background

A previous study using an intercross between the inbred rat strains Lewis (LEW) and Spontaneously Hypertensive Rats (SHR) identified a locus on chromosome 4, named Anxrr16, influencing an experimental index of anxiety and showing a transgressive effect, with alleles from the LEW strain (more anxious) decreasing rather than increasing anxiety.

Objective

To confirm the location and isolate the effect of a rat genome region named Anxrr16 through a planned genomic recombination strategy, where the target locus in SHR rats was replaced with LEW genetic material.

Methods

A new congenic strain, named SHR.LEW-Anxrr16 (SLA16), was developed from a cross between LEW (donor) and SHR (receptor) rats and then evaluated in several anxiety-related tests. The activity and attention levels of the new strain were also evaluated, since hyperactivity was observed during its construction and because SHR is a model of attention deficit hyperactivity disorder.

Results

Significant effects of Anxrr16 were found for open field central locomotion, as well as for other indices of anxiety from the light/dark box, triple test and T-maze. In all cases, the low-anxiety levels of SHR rats were further reduced by the insertion of LEW alleles. Differences in locomotor activity were found only in unfamiliar (hence stressful) environments and no genetic effects were observed in indices of attention.

Conclusion

The SLA16 strain can help in the identification of the molecular pathways involved in experimental anxiety and it demonstrates how apparently extreme phenotypes sometimes hide major opposite-acting genes.     

Toxicologic and pharmacokinetic study of low doses of verapamil combined with doxorubicin

The effect of a chronic treatment with low oral doses of verapamil, a calcium channel blocker commonly employed in cardiovascular therapy, on doxorubicin toxicity, was evaluated in CD1 mice. Verapamil, administered at a dosage corresponding to a typical cardiovascular posology in humans, significantly increased doxorubicin toxicity. In particular the mortality was significantly higher and earlier and histological analysis revealed an increase in the severity of lesions in the liver, kidney and small bowel of verapamil pretreated animals. The pharmacokinetic profiles revealed that verapamil treated group had higher doxorubicin peak plasma and tissue levels and AUCs.This study shows that verapamil, administered at low doses, dramatically increases doxorubicin toxicity, probably through an interaction between the two drugs, both P-glycoprotein substrates, on the protein expressed in normal tissues, and suggests caution in the use of the calcium channel blocker for cardiovascular pathologies in patients who have to be treated with antineoplastic agents, substrates of P-glycoprotein.     

Mutations of the Huntington’s Disease Protein Impact on the ATM-Dependent Signaling and Repair Pathways of the Radiation-Induced DNA Double-Strand Breaks: Corrective Effect of Statins and Bisphosphonates

Huntington’s disease (HD) is a neurodegenerative syndrome caused by mutations of the IT15 gene encoding for the huntingtin protein. Some research groups have previously shown that HD is associated with cellular radiosensitivity in quiescent cells. However, there is still no mechanistic model explaining such specific clinical feature. Here, we examined the ATM-dependent signaling and repair pathways of the DNA double-strand breaks (DSB), the key damage induced by ionizing radiation, in human HD skin fibroblasts. Early after irradiation, quiescent HD fibroblasts showed an abnormally low rate of recognized DSB managed by non-homologous end-joining reflected by a low yield of nuclear foci formed by phosphorylated H2AX histones and by 53BP1 protein. Furthermore, HD cells elicited a significant but moderate yield of unrepaired DSB 24 h after irradiation. Irradiated HD cells also presented a delayed nucleo-shuttling of phosphorylated forms of the ATM kinase, potentially due to a specific binding of ATM to mutated huntingtin in the cytoplasm. Our results suggest that HD belongs to the group of syndromes associated with a low but significant defect of DSB signaling and repair defect associated with radiosensitivity. A combination of biphosphonates and statins complements these impairments by facilitating the nucleo-shuttling of ATM, increasing the yield of recognized and repaired DSB.     

Expression and function of P-glycoprotein and absence of multidrug resistance-related protein in rat and beige mouse peritoneal mast cells

To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein. This compound is also transported by Multidrug Resistance-related Protein (MRP), another membrane transport protein expressed in many tumour resistant cells as well as in normal cells. When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed. Pretreatment with modulators of P-glycoprotein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations. In addition, Bodipy-verapamil efflux from these cells was rapid and also inhibited by verapamil and vinblastine. In contrast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP. Methylamine and monensin, substances that modify the pH values in the granules, were able to lower the concentrations of Bodipy-verapamil. Microscopical observations, conducted in both rat and beige mouse mast cells, demonstrated that the fluorochrome accumulated in the cytoplasmic secretory granules. RT–PCR performed on rat peritoneal mast cells revealed the presence of MDR1a and MDR1b mRNAs; on the contrary, MRP mRNA was not expressed. Mast cells were further treated with the fluorescent probe LysoSensor Blue, a weak base that becomes fluorescent when inside acidic organelles. This substance accumulated in mast cell granular structures and its fluorescence was reduced either by treatment with P-glycoprotein modulators or with agents that disrupt pH gradients. In conclusion, these data further confirm the presence of an active P-glycoprotein, but not of MRP, in rat peritoneal mast cells. These findings, coupled with previous ultrastructural data, lend further support to the assumption that this protein is located on the mast cell perigranular membrane. The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized. Alternatively, this protein might be indirectly implicated in changes of pH values inside secretory granules.     

The evolutionary dynamics of the Helena retrotransposon revealed by sequenced Drosophila genomes

Background  

Several studies have shown that genomes contain a mixture of transposable elements, some of which are still active and others ancient relics that have degenerated. This is true for the non-LTR retrotransposon Helena, of which only degenerate sequences have been shown to be present in some species (Drosophila melanogaster), whereas putatively active sequences are present in others (D. simulans). Combining experimental and population analyses with the sequence analysis of the 12 Drosophila genomes, we have investigated the evolution of Helena, and propose a possible scenario for the evolution of this element.     

Physiological cholesterol concentration is a neuroprotective factor against β-amyloid and β-amyloid-metal complexes toxicity

Alzheimer's disease is one of the most common forms of dementia in the elderly. One of its hallmarks is the abnormal aggregation and deposition of β-amyloid (Aβ). Endogenous and exogenous metal ions seem to influence β-amyloid folding process, aggregation and deposition. Besides these variables other elements appear to affect β-amyloid behavior, such as cholesterol. The physiological concentration of cholesterol in the cerebrospinal fluid (CSF) was used in order to determine the extent in which Aβ and Aβ-metal complexes in vitro aggregation and their toxicity on human neuroblastoma cell cultures is affected. Cholesterol did not appear to influence Aβ and Aβ-metal complexes aggregation, but it was effective in protecting neuroblastoma cells against Aβ complexes' toxicity. The Aβ-Al complex seemed to be the most effective in disrupting and damaging membrane external layer, and simultaneously it appears to increase its toxicity on cell cultures; both of these effects are preventable by cholesterol. The presence in physiological concentrations of cholesterol seemed to compensate membrane damage that occurred to neuroblastoma cells. These findings appear to contradict some data reported in literature. We believe that our results might shed some light on the role played by cholesterol at physiological concentrations in both cellular balance and membrane protection.     

Resveratrol acts not through anti-aggregative pathways but mainly via its scavenging properties against Aβ and Aβ-metal complexes toxicity

It has been recently suggested that resveratrol can be effective in slowing down Alzheimer's disease (AD) development. As reported in many biochemical studies, resveratrol seems to exert its neuro-protective role through inhibition of β-amyloid aggregation (Aβ), by scavenging oxidants and exerting anti-inflammatory activities. In this paper, we demonstrate that resveratrol is cytoprotective in human neuroblastoma cells exposed to Aβ and or to Aβ-metal complex. Our findings suggest that resveratrol acts not through anti-aggregative pathways but mainly via its scavenging properties.