The Shortest Isoform of Dystrophin (Dp40) Interacts with a Group of Presynaptic Proteins to Form a Presumptive Novel Complex in the Mouse Brain

Duchenne muscular dystrophy (DMD) causes cognitive impairment in one third of the patients, although the underlying mechanisms remain to be elucidated. Recent studies showed that mutations in the distal part of the dystrophin gene correlate well with the cognitive impairment in DMD patients, which is attributed to Dp71. The study on the expression of the shortest isoform, Dp40, has not been possible due to the lack of an isoform specific antibody. Dp40 has the same promoter as that found in Dp71 and lacks the normal C-terminal end of Dp427. In the present study, we have raised polyclonal antibody against the N-terminal sequence common to short isoforms of dystrophin, including Dp40, and investigated the expression pattern of Dp40 in the mouse brain. Affinity chromatography with this antibody and the consecutive LC-MS/MS analysis on the interacting proteins revealed that Dp40 was abundantly expressed in synaptic vesicles and interacted with a group of presynaptic proteins, including syntaxin1A and SNAP25, which are involved in exocytosis of synaptic vesicles in neurons. We thus suggest that Dp40 may form a novel protein complex and play a crucial role in presynaptic function. Further studies on these aspects of Dp40 function might provide more insight into the molecular mechanisms of cognitive impairment found in patients with DMD

Assembly of the Dystrophin-Associated Protein Complex Does Not Require the Dystrophin Cooh-Terminal Domain

Dystrophin is a multidomain protein that links the actin cytoskeleton to laminin in the extracellular matrix through the dystrophin associated protein (DAP) complex. The COOH-terminal domain of dystrophin binds to two components of the DAP complex, syntrophin and dystrobrevin. To understand the role of syntrophin and dystrobrevin, we previously generated a series of transgenic mouse lines expressing dystrophins with deletions throughout the COOH-terminal domain. Each of these mice had normal muscle function and displayed normal localization of syntrophin and dystrobrevin. Since syntrophin and dystrobrevin bind to each other as well as to dystrophin, we have now generated a transgenic mouse deleted for the entire dystrophin COOH-terminal domain. Unexpectedly, this truncated dystrophin supported normal muscle function and assembly of the DAP complex. These results demonstrate that syntrophin and dystrobrevin functionally associate with the DAP complex in the absence of a direct link to dystrophin. We also observed that the DAP complexes in these different transgenic mouse strains were not identical. Instead, the DAP complexes contained varying ratios of syntrophin and dystrobrevin isoforms. These results suggest that alternative splicing of the dystrophin gene, which naturally generates COOH-terminal deletions in dystrophin, may function to regulate the isoform composition of the DAP complex

Tissue Plasminogen Activator Contributes to the Late Phase of LTP and to Synaptic Growth in the Hippocampal Mossy Fiber Pathway

AbstractThe expression of tissue plasminogen activator (tPA) is increased during activity-dependent forms of synaptic plasticity. We have found that inhibitors of tPA inhibit the late phase of long-term potentiation (L-LTP) induced by either forskolin or tetanic stimulation in the hippocampal mossy fiber and Schaffer collateral pathways. Moreover, application of tPA enhances L-LTP induced by a single tetanus. Exposure of granule cells in culture to forskolin results in secretion of tPA, elongation of mossy fiber axons, and formation of new, active presynaptic varicosities contiguous to dendritic clusters of the glutamate receptor R1. These structural changes are blocked by tPA inhibitors and induced by application of tPA. Thus, tPA may be critically involved in the production of L-LTP and specifically in synaptic growth

Increased activity of cell membrane-associated prothrombinase, fibrinogen-like protein 2, in peripheral blood mononuclear cells of B-cell lymphoma patients.

Fibrinogen-like protein 2, FGL-2, was reported to be overexpressed in various cancer tissues, where it acts as a transmembrane prothrombinase. This study aims to determine the prothrombinase activity of FGL-2 in peripheral blood mononuclear cells (PBMC) of patients with B-cell lymphoma. FGL-2 activity was determined in patients with B-cell lymphoma (n = 53), and healthy controls (n = 145). FGL-2 activity in patients at diagnosis increased 3 ± 0.3 fold (p < 0.001). Sensitivity and specificity of the test was established at 73.6% and 80.7%, respectively, using a cutoff of 150% activity over control. Moreover, FGL-2 activity in 10 of 11 patients in remission decreased by 76%. In contrast, no significant difference was observed in expression levels of fgl-2 gene in patients and controls. Taken together, our study indicates that FGL-2 prothrombinase activity in PBMC of lymphoma patients is increased in active disease and normalizes during remission, thus being a potential marker for follow up of lymphoma patients

Increased Activity of Prothrombinase Fgl-2 in Peripheral Blood Mononuclear Cells of Patients with B-Cell Lymphoma.

Abstract Abstract 2665 Background: Hematologic and solid tumors are associated with hypercoagulability the reason for which has not been delineated. Prothrombinase named fibrinogen-like protein 2 (FGL-2) is a 70 kD transmembrane protein that was found to have a quality of a serine protease capable of directly cleaving prothrombin to thrombin. FGL-2 is synthesized by monocytes, T-lymphocytes and endothelial cells. FGL-2 protein and its mRNA have been previously found within different tumor cells. Aim: To study the role of FGL-2 in patients with lymphoproliferative disorders. Our hypothesis is that upregulation of FGL-2 activity in patients with B-cell malignancies may contribute to tumorigenesis via generation of thrombin leading to increased angiogenesis and spread/metastasis of malignant cells. Methods: Thrombin generation reflecting FGL-2 activity was measured in homogenized peripheral blood mononuclear cells (PBMC) from 29 patients with active lymphoproliferative disorders and 107 normal controls. Informed consent was obtained from every participant. PBMC extracts were incubated with an equal volume of human prothrombin (final concentration10 μM) for 30 min at 37 °C. Thrombin generation was measured at 405 nm using an automated plate reader after addition of chromogen S-2238. The thrombin activity of each sample was calculated by comparison with absorbance curve generated by known concentrations of human thrombin. FGL-2 was immunoprecipitated (IP) from PBMC with an anti-FGL-2 antibody and EZview Red Protein A Affinity Gel (sigma # P6486). The activity of IP FGL-2 was measured by thrombin generation assay. The expression of FGL-2 was analyzed in HUVEC and PBMC in the presence or absence of IF-γ at 20 ng/ml. Total RNA was isolated using RNAqueous™ (Ambion #AM1912) and RT-PCR, analysis was performed using Rotor-gene RG-3000 (Corbett). The difference in cycle time (ΔCT) was measured by comparing FGL-2 gene with ABL-1 gene (house keeping gene). The relative quantification was calculated by the formula RQ= 2−ΔCT. HUVEC were transfected with 0.5 μM SiRNA synthesized complementary to FGL-2 (Target SiRNA) using Dharmacon ON-TARGET plus SMART pool reagent (Thermo Fisher Scientific) according to manufacturer instructions. FGL-2 expression following addition of target SiRNA was compared to that obtained following addition of non-specific (non-target) SiRNA. Student's t-test was used for all comparisons. Results: As shown in the table, almost 3-fold increase in FGL-2 activity in PBMC was observed among patients with active B-cell lymphoma, either aggressive or indolent, as compared to that of healthy controls (p&lt;0.0001 for all comparisons). No difference in FGL-2 activity in PBMC between patients with aggressive and indolent lymphoma was observed (p=0.68). Three-fold increase in thrombin generation was obtained in PBMC from the patients following IP, similarly to that observed in non-IP PBMC. A significant increase in mRNA of FGL-2 was found in either PBMC of lymphoma patient or HUVEC treated with IF-γ. In HUVEC the increase in FGL-2 mRNA was inhibited by 80% following treatment with specific SiRNA. Conclusions: Disclosures: No relevant conflicts of interest to declare. </jats:sec