The Transdifferentiation of brain derived neurotrophic factor secreting mesenchymal stem cells for neuroprotection

The ultimate purpose of this project was to create a modified stem cell line which could enhance nerve regeneration following peripheral nerve trauma. Specifically, this work was focused upon answering two questions. First, could we chemically transdifferentiate genetically modified mesenchymal stem cells to resemble a Schwann cell-like state? A protocol for the chemical transdifferentiation of MSCs was validated and well tested in the Sakaguchi lab, but no one had ever attempted to chemically transdifferentiate BDNF hyper-secreting MSCs. Second, if we succeeded in creating BDNF hyper-secreting transdifferentiated MSCs (BDNF tMSCs), would levels of BDNF secretion be affected, and, more importantly, would the secreted BDNF still be biologically active? We hypothesized that BDNF tMSCs would still resemble a Schwann cell like phenotype and be able to produce the same or lower amounts of biologically active BDNF when compared to their undifferentiated and GFP control counterparts. Generated data relied largely on the use of immunocytochemistry to quantify the percentage of cells expressing Schwann cell markers. BDNF secretion was quantified by ELISA and bioactivity was tested using the PC12-trkB assay. This study was an important first step in characterizing these BDNF tMSCs by in vitro assays and was essentially a proof of concept study to show that genetically modified MSCs could still be chemically transdifferentiated. As a next step, we hope to seed these BDNF tMSCs within a polymeric conduit transplant used in a rat sciatic nerve transection model to test the ability of these cells to aid in nerve regeneration in vivo

An Interaction between the Walker A and D-loop Motifs Is Critical to ATP Hydrolysis and Cooperativity in Bacteriophage T4 Rad50

The ATP binding cassette (ABC) proteins make up a large superfamily with members coming from all kingdoms. The functional form of the ABC protein nucleotide binding domain (NBD) is dimeric with ATP binding sites shared between subunits. The NBD is defined by six motifs: the Walker A, Q-loop, Signature, Walker-B, D-loop, and H-loop. The D-loop contains a conserved aspartate whose function is not clear but has been proposed to be involved in cross-talk between ATP binding sites. Structures of various ABC proteins suggest an interaction between the D-loop aspartate and an asparagine residue located in Walker A loop of the opposing subunit. Here, we evaluate the functional role of the D-loop using a bacteriophage T4 ABC protein, Rad50 (gp46). Mutation of either the D-loop aspartate or the Walker A asparagine results in dramatic reductions in ATP affinity, hydrolysis rate, and cooperativity. The mutant proteins bind Mre11 (gp47) and DNA normally, but no longer support the ATP-dependent nuclease activities of Mre11. We propose that the D-loop aspartate functions to stabilize the Walker A asparagine in a position favorable for catalysis. We find that the asparagine is crucially important to the mechanism of ATP hydrolysis by increasing the affinity for ATP and positioning the γ-phosphate of ATP for catalysis. Additionally, we propose that the asparagine acts as a γ-phosphate sensor and, through its interaction with the conserved D-loop aspartate, transmits conformational changes across the dimer interface to the second ATP binding site

The Transdifferentiation of brain derived neurotrophic factor secreting mesenchymal stem cells for neuroprotection

The ultimate purpose of this project was to create a modified stem cell line which could enhance nerve regeneration following peripheral nerve trauma. Specifically, this work was focused upon answering two questions. First, could we chemically transdifferentiate genetically modified mesenchymal stem cells to resemble a Schwann cell-like state? A protocol for the chemical transdifferentiation of MSCs was validated and well tested in the Sakaguchi lab, but no one had ever attempted to chemically transdifferentiate BDNF hyper-secreting MSCs. Second, if we succeeded in creating BDNF hyper-secreting transdifferentiated MSCs (BDNF tMSCs), would levels of BDNF secretion be affected, and, more importantly, would the secreted BDNF still be biologically active? We hypothesized that BDNF tMSCs would still resemble a Schwann cell like phenotype and be able to produce the same or lower amounts of biologically active BDNF when compared to their undifferentiated and GFP control counterparts. Generated data relied largely on the use of immunocytochemistry to quantify the percentage of cells expressing Schwann cell markers. BDNF secretion was quantified by ELISA and bioactivity was tested using the PC12-trkB assay. This study was an important first step in characterizing these BDNF tMSCs by in vitro assays and was essentially a proof of concept study to show that genetically modified MSCs could still be chemically transdifferentiated. As a next step, we hope to seed these BDNF tMSCs within a polymeric conduit transplant used in a rat sciatic nerve transection model to test the ability of these cells to aid in nerve regeneration in vivo.</p