Substantia nigra dopaminergic neurons and striatal interneurons are engaged in three parallel but interdependent postnatal neurotrophic circuits

The striatum integrates motor behavior using a well‐defined microcircuit whose individual components are independently affected in several neurological diseases. The glial cell line‐derived neurotrophic factor (GDNF), synthesized by striatal interneurons, and Sonic hedgehog (Shh), produced by the dopaminergic neurons of the substantia nigra (DA SNpc), are both involved in the nigrostriatal maintenance but the reciprocal neurotrophic relationships among these neurons are only partially understood. To define the postnatal neurotrophic connections among fast‐spiking GABAergic interneurons (FS), cholinergic interneurons (ACh), and DA SNpc, we used a genetically induced mouse model of postnatal DA SNpc neurodegeneration and separately eliminated Smoothened (Smo), the obligatory transducer of Shh signaling, in striatal interneurons. We show that FS postnatal survival relies on DA SNpc and is independent of Shh signaling. On the contrary, Shh signaling but not dopaminergic striatal innervation is required to maintain ACh in the postnatal striatum. ACh are required for DA SNpc survival in a GDNF‐independent manner. These data demonstrate the existence of three parallel but interdependent neurotrophic relationships between SN and striatal interneurons, partially defined by Shh and GDNF. The definition of these new neurotrophic interactions opens the search for new molecules involved in the striatal modulatory circuit maintenance with potential therapeutic value.     

Serological response against myxoma virus and rabbit hemorrhagic disease virus in European wild rabbits using commercial vaccines

We carried out an experimental study to determine the serological response against myxoma virus (MV) and rabbit hemorrhagic disease virus (RHDV) in wild rabbits using commercial vaccines. Seroconversion against MV ranged between 72.7% and 97.2% in animals vaccinated by subcutaneous and intradermal route, respectively, whereas between 75.0% and 77.8% of the animals presented antibodies against RHDV after inoculation with subcutaneous and intradermal vaccines, respectively. Regardless of the inoculation route, vaccination against MV resulted in a significant increase of seropositivity 5 days post-vaccination (dpv), which did not occur in animals vaccinated against RHDV. Furthermore, seroconversion against MV was significantly higher and faster in intradermally vaccinated rabbits as compared to those inoculated subcutaneously due to either the route of application and/or the type of vaccine used. The results indicated that vaccination significantly increased the prevalence of antibodies against MV and RHDV and suggested that the vaccines currently available induce a safe and effective immune response against both diseases in wild rabbits. Vaccination may be a useful management tool to control both viral diseases in field conditions, particularly in wild rabbits captured for translocations and restocking purposes in which a large number of animals are handled. © 2011 The Wildlife Society.     

Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling

Nucleocytoplasmic shuttling of Hxk2 induced by glucose levels has been reported recently. Here we present evidence that indicates that Hxk2 nucleocytoplasmic traffic is regulated by phosphorylation and dephosphorylation at serine 14. Moreover, we identified the protein kinase Snf1 and the protein phosphatase Glc7-Reg1 as novel regulatory partners for the nucleocytoplasmic shuttling of Hxk2. Functional studies revealed that, in contrast to the wild-type protein, the dephosphorylation-mimicking mutant of Hxk2 retains its nuclear localization in low glucose conditions, and the phosphomimetic mutant of Hxk2 retains its cytoplasmic localization in high glucose conditions. Interaction experiments of Hxk2 with Kap60 and Xpo1 indicated that nuclear import of the S14D mutant of Hxk2 is severely decreased but that the export is significantly enhanced. Conversely, nuclear import of the S14A mutant of Hxk2 was significantly enhanced, although the export was severely decreased. The interaction of Hxk2 with Kap60 and Xpo1 was found to occur in the dephosphorylated and phosphorylated states of the protein, respectively. In addition, we found that Hxk2 is a substrate for Snf1. Mutational analysis indicated that serine 14 is a major in vitro and in vivo phosphorylation site for Snf1. We also provide evidence that dephosphorylation of Hxk2 at serine 14 is a protein phosphatase Glc7-Reg1-dependent process. Taken together, this study establishes a functional link between Hxk2, Reg1, and Snf1 signaling, which involves the regulation of Hxk2 nucleocytoplasmic shuttling by phosphorylation-dephosphorylation of serine 14.     

Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv. ‘Grande Naine’ (Musa AAA). To achieve this, the embryo specific REG-2 promoter was isolated from rice and its expression pattern in banana cell clumps, somatic embryos and regenerated plantlets was characterized by using a pREG2::uidA fusion construct. Subsequently, the REG-2 promoter was placed upstream of the cre gene, conferring Cre functionality in somatic embryos and recombination of lox sites resulting in excision of the selectable marker and cre genes. PCR analysis revealed that 41.7 % of the analysed transgenic plants were completely marker free, results that were thereafter confirmed by Southern blot hybridization. These results demonstrate the feasibility of using developmentally controlled promoters to mediate marker excision in banana. This system does not require any extra handling compared to the conventional transformation procedure and might be useful in other species regenerating through somatic embryogenesis.     

Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism

Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.     

Evaluating mortality rates and causalities in a critically endangered felid across its whole distribution range

The conservation of endangered species requires accurate data, and knowledge of cause-specific mortality rates is one of the most important issues. In recent years, conservation programs for the critically endangered Iberian lynx Lynx pardinus have been developed on the basis of mortality data derived 30 years ago from the small Doñana population. Thus, there is an urgent need for an update of mortality rates and causes in both populations (Sierra Morena and Doñana). Here we use radio-tracking information from the whole range of the Iberian lynx to quantify mortality rates and identify their causes. Between 2006 and 2011, we radio-tagged 78 Iberian lynxes from its two remaining populations (39 from Sierra Morena and 39 from Doñana). Mortality events were evaluated to identify causes, and cause-specific annual mortality rates (AMR) were obtained using the nonparametric cumulative incidence function estimator. Overall, AMR was estimated at 0.16?±?0.05 (0.19?±?0.09 in Sierra Morena and 0.12?±?0.07 in Doñana). Disease was the main cause of mortality both for the whole population and the Doñana population. Poaching was the main cause of mortality in Sierra Morena. Our results suggest that the best strategy for conserving this species is to focus action on decreasing the fatal effect of disease and poaching. Given the possible existence of an underlying inbreeding-mediated immunosuppression, genetic management aimed at increasing the genetic diversity of this population is also recommended.     

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.