My research focuses on third generation lentiviral vectors to identify potentially therapeutic transgenes, to study the regulation of their expression and their safe, direct delivery by viral vectors or after stable in vitro transduction of dividing stem cells prior to transplantation. The summary includes my own research and the status of each collaboration listed for each transgene. 1. Nrf2: neuroprotective during oxidative stress. 4 vector DNAs encoding Nrf2 and a constitutively active deletion mutant of Nrf2, under control of either the CMV IE or the GFAP promoter, were cloned and vectors were produced. This collaboration (Yarowsky/Jaiswal) was terminated, because the Nrf2 DNA provided by one of the collaborator unexpectedly encoded for 3 amino acid changes plus several silent mutations as compared to the gene bank sequence. 2. ND2: a protein, suggested to promote neuronal development. After infection of human neural progenitor cells, lentiviral vector expressing ND2 appears to prevent the development of astrocytes or causes selective astrocytic cell death. This collaboration (Fishman) was temporarily put on hold, because of the poor quality of the progenitor cells cultures. 3. PGC1a, Sirt1, TFAM: These regulator proteins of mitochondrial replication and for the establishment of metabolic homeostasis and neuroprotection were cloned and 3 vectors were isolated. 3 SH-SY5Y cell lines encoding these genes were generated. The proteins are expressed at their expected cellular locations: PGC1a and Sirt1 (nucleus), TFAM (mitochondria). As requested, these cells were provided to the collaborator (Russell) to be used for studies on diabetic peripheral neuropathy (with focus on Sirt1). The collaborator used these cell lines to apply for research funding, initially without proper acknowledgment. For this reason, I consider this collaboration terminated. In addition, due to the lack of new research data generated by my collaborator for almost 1 year, these vectors will now be used separately for my own studies (see below, 8.). 4. EGFPTTC/Lentiviral vector pseudotyped with rabies virus or VSV glycoproteins: Routes of gene delivery to the nervous system. 4 in vivo-grade vectors expression EGFP and EGFP-tetanus toxin C fragment fusion protein (EGFPTTC) were injected in the tongue and gastrocnemius muscles of mice or instilled into the nasal cavity. The purpose was to examine retrograde transport of the vector genome (via rabies glycoprotein) and potential subsequent retrograde transneuronal transport of EGFPTTC to higher order neurons. Unexpectedly, the detectability of EGFP expression in tissue slices has become a serious problem. For this study to be completed, the plan is to apply real-time PCR for the detection of integrated vector in tissue and antigen-retrieval from fixed tissue for more sensitive and specific protein detection. 5. Syn-DsRed, GFAP-ECYP, MBP-EYFP: cell-type specific promoters for neurons, astrocytes and oligodendrocytes expressing 3 different colored reporter genes: 3 lentiviral vectors were isolated and used to infect human neural progenitor cells. Removal of EGF and FGF induced cell differentiation. After 2 weeks, Syn-DsRed was expressed in a few neurons as a marker for more mature cells. ECFP expression mostly overlapped with GFAP expression, expression of ECFP and EYFP promoters was prevalent and was unexpectedly also found coexpressed in some precursor cells. 6. These vectors were used to transduce mouse CD34 stem cells provided by a collaborator (Trisler). As previously reported, CD34 cells express oligodendrocyte-specific proteins. Consistently, about 10-20% of CD34 cells expressed EYFP from the MBP promoter. 1 million transduced CD34 cells, either singly or triple infected with one or 3 vectors (see 5.), were stereotactically injected unilaterally into the hippocampus of normal mice or MBP-deficient Shiverer mice. The purpose was to examine the migration of these cells to the site of a lesion made on the opposite side, and to examine their differentiation to microglial cells. In addition, the different promoters and the colored reporter genes might confirm trans-differentiation of CD34 cells to the 3 neural cell types, as was reported earlier by Trisler. Unfortunately, severe autofluorescence of the tissue prevented a clear identification of the transplanted cells. Different techniques are currently considered to retrieve integrated vector genomic DNA and to retrieve the antigen for better detection. 7. ICP10PK: a neuroprotective, anti-apoptotic gene of HSV-2, which signals via crosstalk with microglia and creates an important neuroprotective bystander effect. The collaborator (Aurelian) provided the gene. A novel bicistronic ICP10PKmyc2AEYFP DNA was assembled and cloned into the lentiviral vector. A ribosome skipping mechanism caused by the 2A peptide allowed efficient coexpression of ICP10PK and EYFP from the same mRNA. Lentiviral vector was isolated and expressed in hippocampal neurons. In a pilot experiment expression of ICP10PK was neuroprotective against NMDA-induced excitotoxicity. This vector stably transduced human neural progenitor cells and the mouse embryonic stem cell line ES-D3. ES-D3 cells are currently examined for safety, cell differentiation, neuroprotection as well as cell migration in vivo. Infection of human neuron progenitor cells demonstrates ICP10PK expression in GFAP-positive cells and in b-tubulin positive neuronal cells. Apparently ICP10PK does not seem to interfere with the neural differentiation even though it is an anti-apoptotic protein. Apoptosis plays a normal role during neural differentiation. It also demonstrates that single expression of ICP10PK by the lentiviral vector remains neuroprotective in the absence of the other 75 HSV-2 genes. The future goal is to examine neuroprotection by ICP10PK in a kainic acid model of epilepsy and in an animal model of neurodegenerative disease. The neuroprotective bystander effect was important for initiating this collaboration. Delivery of this gene by migratory cells will be tested, which may be able to protect larger brain areas. 8. PGC1a: a key co-transcriptional regulator protein for downstream genes, affecting cell metabolism, mitochondrial replication, oxidative stress and others. Its involvement in HD has been proposed. My studies focus on the regulation of PGC1a expression in different cell types. We have interesting findings, which shed light on potential new transcriptional regulatory mechanism for this highly controlled protein. It is likely that this mechanism plays a role in some forms of neurodegeneration in human. These findings will be verified and should soon be ready for publication. Publications of several of the research findings and accomplishments depend on results obtained by collaborators, for which there is limited control on my part. For all projects, I provided functional, state of the art lentiviral vectors as requested by my collaborators. However, detailed studies using my vectors are either missing or were not shared by some collaborators. In some cases, there was no effort or intention to discuss hypotheses or experimental strategies in detail. Several collaborators only used my expertise and my materials as their pilot data to apply for their own research funding. This unfortunate situation appears to occur frequently. It was the reason for the termination of some collaboration. On the other hand, my expertise was beneficial and secured significant non-NIH research funding to one PI (Fishman), which pays for my technical support and supplies. A summary of my research was presented at the REAP meeting of VA Research Services and UMaryland.
| Choi, Joungil; Ravipati, Avinash; Nimmagadda, Vamshi et al. (2014) Potential roles of PINK1 for increased PGC-1?-mediated mitochondrial fatty acid oxidation and their associations with Alzheimer disease and diabetes. Mitochondrion 18:41-8 |
| Choi, Joungil; Batchu, Vera Venkatanaresh Kumar; Schubert, Manfred et al. (2013) A novel PGC-1? isoform in brain localizes to mitochondria and associates with PINK1 and VDAC. Biochem Biophys Res Commun 435:671-7 |
| Standley, Steve; Petralia, Ronald S; Gravell, Manneth et al. (2012) Trafficking of the NMDAR2B receptor subunit distal cytoplasmic tail from endoplasmic reticulum to the synapse. PLoS One 7:e39585 |
| Schubert, Manfred; Breakefield, Xandra; Federoff, Howard et al. (2008) Gene delivery to the nervous system. Mol Ther 16:640-6 |
