Proof-of-concept: neonatal intravenous injection of adeno-associated virus vectors results in successful transduction of myenteric and submucosal neurons in the mouse small and large intestine.

BACKGROUND: Despite the success of viral vector technology in the transduction of the central nervous system in both preclinical research and gene therapy, its potential in neurogastroenterological research remains largely unexploited. This study asked whether and to what extent myenteric and submucosal neurons in the ileum and distal colon of the mouse were transduced after neonatal systemic delivery of recombinant adeno-associated viral vectors (AAVs). METHODS: Mice were intravenously injected at postnatal day one with AAV pseudotypes AAV8 or AAV9 carrying a cassette encoding enhanced green fluorescent protein (eGFP) as a reporter under the control of a cytomegalovirus promoter. At postnatal day 35, transduction of the myenteric and submucosal plexuses of the ileum and distal colon was evaluated in whole-mount preparations, using immunohistochemistry to neurochemically identify transduced enteric neurons. KEY RESULTS: The pseudotypes AAV8 and AAV9 showed equal potential in transducing the enteric nervous system (ENS), with 25-30% of the neurons expressing eGFP. However, the percentage of eGFP-expressing colonic submucosal neurons was significantly lower. Neurochemical analysis showed that all enteric neuron subtypes, but not glia, expressed the reporter protein. Intrinsic sensory neurons were most efficiently transduced as nearly 80% of calcitonin gene-related peptide-positive neurons expressed the transgene. CONCLUSIONS & INFERENCES: The pseudotypes AAV8 and AAV9 can be employed for gene delivery to both the myenteric and the submucosal plexus, although the transduction efficiency in the latter is region-dependent. These findings open perspectives for novel preclinical applications aimed at manipulating and imaging the ENS in the short term, and in gene therapy in the longer term.

Impairing MLL-fusion gene-mediated transformation by dissecting critical interactions with the lens epithelium-derived growth factor (LEDGF/p75).

The lens epithelium-derived growth factor (LEDGF/p75) tethers the mixed-lineage leukemia (MLL1) protein complex to chromatin. Likewise, LEDGF/p75 tethers the HIV-1 pre-integration complex to chromatin. We previously demonstrated that expression of the C-terminal fragment fused to enhanced green fluorescent protein (eGFP) (eGFP-LEDGF(325-530)) impaired HIV-1 replication. Here, we explored this strategy to selectively interfere with the leukemogenic activity of MLL-fusion proteins. We found that expression of LEDGF(325-530) impaired the clonogenic growth of MLL-fusion gene transformed human and mouse hematopoietic cells, without affecting the growth of control cells immortalized by the FLT3-ITD mutant or normal lineage-marker-depleted murine bone marrow cells. Expression of LEDGF(325-530) was associated with downregulation of the MLL target Hoxa9 and impaired cell cycle progression. Structure-function analysis revealed two small eGFP-fused LEDGF/p75 peptides, LEDGF(424-435) and LEDGF(375-386) phenocopying these effects. Both LEDGF(325-530) and the smaller active peptides were able to disrupt the LEDGF/p75-MLL interaction. Expression of LEDGF(325-530) or LEDGF(375-386) fragments increased the latency period to disease development in vivo in a mouse bone marrow transplant model of MLL-AF9-induced AML. We conclude that small peptides disrupting the LEDGF/p75-MLL interface have selective anti-leukemic activity providing a direct rationale for the design of small molecule inhibitors targeting this interaction.

Polycystin-1 and polycystin-2 are both required to amplify inositol-trisphosphate-induced Ca2+ release.

Autosomal dominant polycystic kidney disease is caused by loss-of-function mutations in the PKD1 or PKD2 genes encoding respectively polycystin-1 and polycystin-2. Polycystin-2 stimulates the inositol trisphosphate (IP(3)) receptor (IP(3)R), a Ca(2+)-release channel in the endoplasmic reticulum (ER). The effect of ER-located polycystin-1 is less clear. Polycystin-1 has been reported both to stimulate and to inhibit the IP(3)R. We now studied the effect of polycystin-1 and of polycystin-2 on the IP(3)R activity under conditions where the cytosolic Ca(2+) concentration was kept constant and the reuptake of released Ca(2+) was prevented. We also studied the interdependence of the interaction of polycystin-1 and polycystin-2 with the IP(3)R. The experiments were done in conditionally immortalized human proximal-tubule epithelial cells in which one or both polycystins were knocked down using lentiviral vectors containing miRNA-based short hairpins. The Ca(2+) release was induced in plasma membrane-permeabilized cells by various IP(3) concentrations at a fixed Ca(2+) concentration under unidirectional (45)Ca(2+)-efflux conditions. We now report that knock down of polycystin-1 or of polycystin-2 inhibited the IP(3)-induced Ca(2+) release. The simultaneous presence of the two polycystins was required to fully amplify the IP(3)-induced Ca(2+) release, since the presence of polycystin-1 alone or of polycystin-2 alone did not result in an increased Ca(2+) release. These novel findings indicate that ER-located polycystin-1 and polycystin-2 operate as a functional complex. They are compatible with the view that loss-of-function mutations in PKD1 and in PKD2 both cause autosomal dominant polycystic kidney disease.

The fetal respiratory system as target for antenatal therapy.

The widespread use of prenatal ultrasound has made the fetus a patient. A number of conditions diagnosed as such may require therapy prior to birth. Herein we describe past, current and potential future procedures designed to treat pulmonary conditions in the antenatal period. When congenital cystic adenomatoid malformation (CCAM) is -associated with fetal hydrops, treatment is required. Prior to viability this may be in utero resection of the pathologic lung lobe or shunting of cystic lesions. More recently, fetuses with isolated congenital diaphragmatic hernia (CDH) with lethal lung hypoplasia have been offered percutaneous fetal tracheal occlusion to provoke lung growth. A very rare condition is laryngeal atresia, which requires peripartum re-establishment of the airways. As we get more -experience with access to the fetal airways, this may open the doors for novel therapies. One of these is gene delivery to treat fetuses with serious monogenic disorders or to induce transient overexpression of certain proteins. We review the individual hurdles that are being met by researchers when designing fetal gene therapeutic strategies, in particular for the fetal lung. Also the use of stem cells for pulmonary disorders is currently explored.

In vivo bioluminescence imaging in an experimental mouse model for dendritic cell based immunotherapy against malignant glioma.

The value of bioluminescence imaging (BLI) for experimental cancer models has become firmly established. We applied BLI to the GL261 glioma model in the context of dendritic cell (DC) immunotherapy. Initial validation revealed robust linear correlations between in vivo, ex vivo and in vitro luciferase activity measurements. Ex vivo BLI demonstrated midline crossing and leakage of tumor cells. Orthotopically challenged mice followed with BLI showed an initial adaptation phase, after which imaging data correlated linearly with stereologically determined tumor dimensions. Transition from healthy to moribund state corresponded with an increasing in vivo flux but the onset of neurological deficit was clearly delayed compared to the onset of in vivo flux increase. BLI was implemented in prophylactic immunotherapy and imaging data were prognostic for therapy outcome. Three distinct response patterns were detected. Our data underscore the feasibility of in vivo BLI in an experimental immunotherapeutic setting in the GL261 glioma model.

Structural and catalytic similarities between nucleotide pyrophosphatases/phosphodiesterases and alkaline phosphatases.

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) generate nucleoside 5'-monophosphates from a variety of nucleotides and their derivatives. Here we show by data base analysis that these enzymes are conserved from eubacteria to higher eukaryotes. We also provide evidence for the existence of two additional members of the mammalian family of ecto-NPPs. Homology searches and alignment-assisted mutagenesis revealed that the catalytic core of NPPs assumes a fold similar to that of a superfamily of phospho-/sulfo-coordinating metalloenzymes comprising alkaline phosphatases, phosphoglycerate mutases, and arysulfatases. Mutation of mouse NPP1 in some of its predicted metal-coordinating residues (D358N or H362Q) or in the catalytic site threonine (T238S) resulted in an enzyme that could still form the nucleotidylated catalytic intermediate but was hampered in the second step of catalysis. We also obtained data indicating that the ability of some mammalian NPPs to auto(de)phosphorylate is due to an intrinsic phosphatase activity, whereby the enzyme phosphorylated on Thr-238 represents the covalent intermediate of the phosphatase reaction. The results of site-directed mutagenesis suggested that the nucleotide pyrophosphatase/phosphodiesterase and the phosphatase activities of NPPs are mediated by a single catalytic site.

Nucleotide pyrophosphatases/phosphodiesterases on the move.

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) release nucleoside 5'-monophosphates from nucleotides and their derivatives. They exist both as membrane proteins, with an extracellular active site, and as soluble proteins in body fluids. The only well-characterized NPPs are the mammalian ecto-enzymes NPP1 (PC-1), NPP2 (autotaxin) and NPP3 (B10; gp130(RB13-6)). These are modular proteins consisting of a short N-terminal intracellular domain, a single transmembrane domain, two somatomedin-B-like domains, a catalytic domain, and a C-terminal nuclease-like domain. The catalytic domain of NPPs is conserved from prokaryotes to mammals and shows remarkable structural and catalytic similarities with the catalytic domain of other phospho-/sulfo-coordinating enzymes such as alkaline phosphatases. Hydrolysis of pyrophosphate/phosphodiester bonds by NPPs occurs via a nucleotidylated threonine. NPPs are also known to auto(de)phosphorylate this active-site threonine, a process accounted for by an intrinsic phosphatase activity, with the phosphorylated enzyme representing the catalytic intermediate of the phosphatase reaction. NPP1-3 have been implicated in various processes, including bone mineralization, signaling by insulin and by nucleotides, and the differentiation and motility of cells. While it has been established that most of these biological effects of NPPs require a functional catalytic site, their physiological substrates remain to be identified.

Differential regulation of the expression of nucleotide pyrophosphatases/phosphodiesterases in rat liver.

We propose the name nucleotide pyrophosphatases/phosphodiesterases (NPP) for the enzymes that release nucleoside-5'-monophosphates from various pyrophosphate and phosphodiester bonds. Three structurally related mammalian NPPs are known, i.e. NPPalpha (autotaxin), NPPbeta (B10/gp130RB13-6) and NPPgamma (PC-1). We report here that these isozymes have a distinct tissue distribution in the rat but that they are all three expressed in hepatocytes. In FAO rat hepatoma cells only the level of NPPgamma was stimulated by TGF-beta1. In rat liver, the concentration of the transcripts of all three isozymes was found to increase manyfold during the first weeks after birth, but the increased expression of the NPPalpha mRNA was transient. The level of the NPP transcripts transiently decreased after hepatectomy, but NPPalpha mRNA was also lost after sham operation, which suggests that it may belong to the negative acute-phase proteins. The loss of the beta- and gamma-transcripts after hepatectomy was not due to a decreased NPP gene transcription or an increased turnover of the mature transcripts. However, hepatectomy also caused a similar loss of the nuclear pool of the NPPbeta and NPPgamma mRNAs. We conclude that a deficient processing and/or an increased turnover of the NPP pre-mRNAs underlies the hepatectomy-induced decrease of the beta- and gamma-transcripts. A similar loss of nuclear NPPgamma mRNA was also noted after treatment with cycloheximide, indicating that protein(s) with a high turnover control the stability and/or processing of the immature NPPgamma transcript.