Targeting Dyrk1A with AAVshRNA attenuates motor alterations in TgDyrk1A, a mouse model of Down syndrome

Genetic-dissection studies carried out with Down syndrome (DS) murine models point to the critical contribution of Dyrk1A overexpression to the motor abnormalities and cognitive deficits displayed in DS individuals. In the present study we have used a murine model overexpressing Dyrk1A (TgDyrk1A mice) to evaluate whether functional CNS defects could be corrected with an inhibitory RNA against Dyrk1A, delivered by bilateral intrastriatal injections of adeno-associated virus type 2 (AAVshDyrk1A). We report that AAVshDyrk1A efficiently transduced HEK293 cells and primary neuronal cultures, triggering the specific inhibition of Dyrk1A expression. Injecting the vector into the striata of TgDyrk1A mice resulted in a restricted, long-term transduction of the striatum. This gene therapy was found to be devoid of toxicity and succeeded in normalizing Dyrk1A protein levels in TgDyrk1A mice. Importantly, the behavioral studies of the adult TgDyrk1A mice treated showed a reversal of corticostriatal-dependent phenotypes, as revealed by the attenuation of their hyperactive behavior, the restoration of motor-coordination defects, and an improvement in sensorimotor gating. Taken together, the data demonstrate that normalizing Dyrk1A gene expression in the striatum of adult TgDyrk1A mice, by means of AAVshRNA, clearly reverses motor impairment. Furthermore, these results identify Dyrk1A as a potential target for therapy in DS.     

The nutritive value of concentrate feedstuffs for ruminant animals: Part III. Small intestinal digestibility as measured by in vitro or mobile bag techniques

The aim of the study was to generate a database of small intestinal digestibility (SID) of different sources of concentrate ingredients commonly offered to ruminants in European countries. Test protein feeds were sunflower meal (SUN), rapeseed meal (RAP), soyabean meal (SBM) and cottonseed meal (CSM). Test energy feeds were palm kernel meal (PK), pollard (PO), barley (BA) and beet pulp (BP). Test protein+energy feeds were maize distillers grains (MDG), maize gluten feed (MGF), copra meal (CO) and malt combings (MC). The ruminal undegradable protein (RUP) portion of the test feedstuffs was obtained by ruminal incubation in four Friesian steers offered grass silage and concentrate. The RUP fraction was digested with pepsin and pancreatin enzymes (PPD) or placed into the duodenal cannula of two Friesian cows using the mobile bag technique, which were recovered in the faeces. The average in situ SID (g/kg) of CSM, RSM, SBM, SUN, BA, BP, PK, CO, MDG, MGF and MC was 834 (S.D. 61), 711 (S.D. 47), 978 (S.D. 9), 586 (S.D. 170), 427 (S.D. 80), 712 (S.D. 24), 767 (S.D. 53), 816 (S.D. 38), 860 (S.D. 73), 656 (S.D. 65) and 510 (S.D. 41), respectively. Corresponding in vitro SID values (g/kg) were 641 (S.D. 65), 620 (S.D. 45), 840 (S.D. 27), 606 (S.D. 155), 445 (S.D. 36), 601 (S.D. 17), 640 (S.D. 63), 686 (S.D. 69), 756 (S.D. 63), 634 (S.D. 84) and 504 (S.D. 16), respectively. These results show that the SID of feeds can vary substantially between different sources and indicates that different feeds should be screened for their nutritive value as a result. As the relationship between in situ (Y) and in vitro (X) SID is best described by the linear regression equation Y=−91.9+12.7X (r=0.91), indicating a close relationship between results obtained using both techniques, the in vitro PPD technique offers a quick and reliable method for SID of feeds to be screened on a regular basis.     

Intestinal-epithelial LSD1 controls goblet cell maturation and effector responses required for gut immunity to bacterial and helminth infection

Infectious and inflammatory diseases in the intestine remain a serious threat for patients world-wide. Reprogramming of the intestinal epithelium towards a protective effector state is important to manage inflammation and immunity and can be therapeutically targeted. The role of epigenetic regulatory enzymes within these processes is not yet defined. Here, we use a mouse model that has an intestinal-epithelial specific deletion of the histone demethylase Lsd1 (cKO mice), which maintains the epithelium in a fixed reparative state. Challenge of cKO mice with bacteria-induced colitis or a helminth infection model both resulted in increased pathogenesis. Mechanistically, we discovered that LSD1 is important for goblet cell maturation and goblet-cell effector molecules such as RELMß. We propose that this may be in part mediated by directly controlling genes that facilitate cytoskeletal organization, which is important in goblet cell biology. This study therefore identifies intestinal-epithelial epigenetic regulation by LSD1 as a critical element in host protection from infection.     

A novel spirocyclic tropanyl-Δ2-isoxazoline derivative enhances citalopram and paroxetine binding to serotonin transporters as well as serotonin uptake

A group of spirocyclic tropanyl-Δ²-isoxazolines was synthesized exploiting the 1,3-dipolar cycloaddition of nitrile oxides to olefins. Their interaction with the dopamine and serotonin transporters (DAT and SERT, respectively) was evaluated through binding experiments. The majority of the compounds had no inhibitory effects (IC₅₀ >> 10 μM), while some had an IC₅₀ value in the range 5–10 μM (8a–c, 10b and 11c on DAT, 12b on SERT). Unexpectedly, one of the tertiary amines under investigation, that is 3′-methoxy-8-methyl-spiro{8-azabicyclo[3.2.1]octane-3,5′(4′H)-isoxazole 7a, was able to enhance at a concentration of 10 μM both [³H]citalopram and [³H]paroxetine binding to SERT in rat brain homogenate (up to 25%, due to an increase of B_max) and [³H]serotonin uptake (up to 30%) in cortical synaptosomes. This peculiar pharmacological profile of 7a suggests it binds to an allosteric site on SERT, and positions derivative 7a as a very useful tool to investigate SERT machinery.     

Antagonism of the Prokineticin System Prevents and Reverses Allodynia and Inflammation in a Mouse Model of Diabetes

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease.