Nucleofection of whole murine retinas

The mouse retina constitutes an important research model for studies aiming to unravel the cellular and molecular mechanisms underlying ocular diseases. The accessibility of this tissue and its feasibility to directly obtain neurons from it has increased the number of studies culturing mouse retina, mainly retinal cell suspensions. However, to address many questions concerning retinal diseases and protein function, the organotypic structure must be maintained, so it becomes important to devise methods to transfect and culture whole retinas without disturbing their cellular structure. Moreover, the postmitotic stage of retinal neurons makes them reluctant to commonly used transfection techniques. For this purpose some published methods employ in vivo virus-based transfection techniques or biolistics, methods that present some constraints. Here we report for the first time a method to transfect P15-P20 whole murine retinas via nucleofection, where nucleic acids are directly delivered to the cell nuclei, allowing in vitro transfection of postmitotic cells. A detailed protocol for successful retina extraction, organotypic culture, nucleofection, histological procedures and imaging is described. In our hands the A-33 nucleofector program shows the highest transfection efficiency. Whole flat-mount retinas and cryosections from transfected retinas were imaged by epifluorescence and confocal microscopy, showing that not only cells located in the outermost retinal layers, but also those in inner retinal layers are transfected. In conclusion, we present a novel method to successfully transfect postnatal whole murine retina via nucleofection, showing that retina can be successfully nucleofected after some optimization steps.     

Leucine-rich repeat kinase 2 induces α-synuclein expression via the extracellular signal-regulated kinase pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes α-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.     

Moderate Accumulation of Calcium and Phosphorus in the Splenic Artery with Aging and Low Accumulation of Those in the Pulmonary Artery with Aging

To elucidate compositional changes of the arteries with aging, the authors investigated age-related changes of elements in the splenic and pulmonary arteries, which supplied blood to contractile organs. After ordinary dissection by medical students at Nara Medical University was finished, the splenic and pulmonary arteries were resected from the subjects, ranging in age from 58 to 94 years. The element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that a moderate accumulation of Ca and P occurred in the splenic artery with aging, but it hardly occurred in the pulmonary artery with aging. Regarding the relationship among elements, the finding that there were significant direct correlations among the contents of Ca, P, Mg, and Na was commonly obtained in both the splenic and pulmonary arteries. The accumulation of Ca and P in the splenic artery with aging occurred independently of that in the pulmonary artery. Histologic observation indicated that a major part of Ca deposits was seen in the middle tunica, but not in the internal tunica. Therefore, the calcification occurring in the splenic artery belonged to middle sclerosis.     

G Protein-coupled receptor kinase 2 (GRK2): A novel modulator of insulin resistance

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key, integrative node in many signalling pathways. Besides its canonical role in the modulation of the signalling mediated by many G protein-coupled receptors (GPCR), this protein can display a very complex network of functional interactions with a variety of signal transduction partners, in a stimulus, cell type, or context-specific way. We review herein recent data showing that GRK2 can regulate insulin-triggered transduction cascades at different levels and that this protein plays a relevant role in insulin resistance and obesity in vivo, what uncovers GRK2 as a potential therapeutic target in the treatment of these disorders.     

The empowerment of translational research: lessons from laminopathies

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.     

Insulin resistance associated to obesity: the link TNF-alpha

Adipose tissue secretes proteins which may influence insulin sensitivity. Among them, tumour necrosis factor (TNF)-alpha has been proposed as a link between obesity and insulin resistance because TNF-alpha is overexpressed in adipose tissue from obese animals and humans, and obese mice lacking either TNF-alpha or its receptor show protection against developing insulin resistance. The activation of proinflammatory pathways after exposure to TNF-alpha induces a state of insulin resistance in terms of glucose uptake in myocytes and adipocytes that impair insulin signalling at the level of the insulin receptor substrate (IRS) proteins. The mechanism found in brown adipocytes involves Ser phosphorylation of IRS-2 mediated by TNF-alpha activation of MAPKs. The Ser307 residue in IRS-1 has been identified as a site for the inhibitory effects of TNF-alpha in myotubes, with p38 mitogen-activated protein kinase (MAPK) and inhibitor kB kinase being involved in the phosphorylation of this residue. Moreover, up-regulation of protein-tyrosine phosphatase (PTP)1B expression was recently found in cells and animals treated with TNF-alpha. PTP1B acts as a physiological negative regulator of insulin signalling by dephosphorylating the phosphotyrosine residues of the insulin receptor and IRS-1, and PTP1B expression is increased in peripheral tissues from obese and diabetic humans and rodents. Accordingly, down-regulation of PTP1B activity by treatment with pharmacological agonists of nuclear receptors restores insulin sensitivity in the presence of TNF-alpha. Furthermore, mice and cells deficient in PTP1B are protected against insulin resistance induced by this cytokine. In conclusion, the absence or inhibition of PTP1B in insulin-target tissues could confer protection against insulin resistance induced by cytokines.     

IC-tagged proteins are able to interact with each other and perform complex reactions when integrated into muNS-derived inclusions

We have recently developed a versatile tagging system (IC-tagging) that causes relocation of the tagged proteins to ARV muNS-derived intracellular globular inclusions. In the present study we demonstrate (i) that the IC-tag can be successfully fused either to the amino or carboxyl terminus of the protein to be tagged and (ii) that IC-tagged proteins are able to interact between them and perform complex reactions that require such interactions while integrated into muNS inclusions, increasing the versatility of the IC-tagging system. Also, our studies with the DsRed protein add some light on the structure/function relationship of the evolution of DsRed chromophore.     

Serum Levels of Vasoactive Intestinal Peptide as a Prognostic Marker in Early Arthritis

Objective

Suitable biomarkers are essential for the design of therapeutic strategies in personalized medicine. Vasoactive intestinal peptide (VIP) has demonstrated immunomodulatory properties in autoimmune murine and ex vivo human models. Our aim was to study serum levels of VIP during the follow-up of an early arthritis (EA) cohort and to analyze its value as a biomarker predicting severity and therapeutic requirements.

Methods

Data from 91 patients on an EA register were analyzed (76% rheumatoid arthritis (RA), 24% undifferentiated arthritis, 73% women, and median age 54 years; median disease duration at entry, 5.4 months). We collected per protocol sociodemographic, clinical, and therapeutic data. VIP levels were determined by enzyme immunoassay in sera harvested from the 91 patients (353 visits; 3.9 visit/patient) and from 100 healthy controls. VIP values below the 25^th percentile of those assessed in healthy population were considered low. To determine the effect of independent variables on VIP levels, we performed a longitudinal multivariate analysis nested by patient and visit. A multivariate ordered logistic regression was modeled to determine the effect of low VIP serum levels on disease activity at the end of follow-up.

Results

VIP concentrations varied considerably across EA patients. Those fulfilling the criteria for RA had the lowest values in the whole sample, although no significant differences were observed compared with healthy donors. Disease activity, which was assessed using DAS28, inversely correlated with VIP levels. After a two-year follow-up, those patients with low baseline levels of VIP displayed higher disease activity and received more intensive treatment.

Conclusion

Patients who are unable to up-regulate VIP seem to have a worse clinical course despite receiving more intense treatment. Therefore, measurement of VIP levels may be suitable as a prognostic biomarker.