1. PGC-1α/ β-tubulin III/microtubule dynamics: The studies revealed a novel role of the transcriptional co-activator PGC-1αin regulating the dynamics of microtubule structures in neural and cancer cells through the induction of β-tubulin III expression. We show that β-tubulin III expression can, counter-intuitively, destabilize microtubules with dramatic effects on mitosis and microtubule stability, affecting cell structure and function. Increased microtubule dynamics may potentially affect neural differentiation and degeneration, and increase the resistance of cancer cells against drugs, such as taxol, that affect microtubule stability. These findings have important implications for the development and progression of neurodegenerative disease and for neuronal injury. They expand PGC-1αs multiple functions beyond its established role in metabolic homeostasis and the protection against reactive oxygen species (ROS). Our study links, for the first time, microtubule dynamics and cellular structure with metabolic homeostasis and ROS, affecting mitosis, potentially cell differentiation, neuronal plasticity and neuronal function. In addition, our results provide new insight in the transcriptional regulation of PGC-1αitself, with evidence for histone deacetylation as an epigenetic control mechanism for PGC-1αtransgene expression. Microtubule dynamics have long been studied in great detail. Our findings may also link ATP production and microtubule dynamics, which is especially critical for metabolically active neuronal cells, to PGC-1αexpression that is also neuroprotective. We propose that these functions are enhanced by increased microtubule dynamics. Whether this increase also leads to increased intracellular transport necessary to maintain metabolic homeostasis, while retaining neuronal connectivity and function, is an important question requiring further evaluation. It could, independent of other culprits that may induce neurodegeneration, provide a new disease mechanism that could potentially be self-inflicted, causing fibrosis, astrogliosis and axonal atrophy and/or promote neural plasticity. Future studies will be expanded to include less restricted promoters to also see the effect of PGC-1αexpression on microtubule dynamics in neuronal cells. A less restricted promoter willl be required to examine the neutrality of several transgenes during development of neurons from a mixed human neural progenitor cell population. Currently, transgene expression from the CMV promoter was restricted to radial glia and glial cells. For future transgene delivery via stably-transduced stem cells, it will be essential that vector-encoded transgene expression is neutral. In addition, it should neither interfere with cell differentiation nor induce cell transformation. 2. Transplantation of vector-transduced stem cells: Transgene delivery by lentiviral vector-transduced bone marrow stem cells following intracranial transplantation in one mouse gave rise to a solid tumor outside the brain, above the injection site. With 99% probability, this tumor had developed from as single bone marrow stem cell that had been transduced by a single lentiviral vector particle. This conclusion is based on observations that showed the selection of small cell subpopulation with an increased growth rate. Identification of the vector integration site could provide important clues about the mechanism of cell transformation and tumor development, which is important for the safety of future cell transplantations. It could identify a potential oncogenic integration site for stem cells. Interestingly, tumor cells, but not the transformed tumor-precursor cells, co-expressed high levels PGC-1αand β-tubulin III. It is well established that β-tubulin III expression is linked to cancer cell invasiveness and resistance to microtubule-targeting drugs. The intent is to continue this study, potentially in collaboration with colleagues at NINDS. The study has important clinical implications for cancer, but also for the transplantation of vector-transduced stem cells to the nervous system. 3. Neuroprotection by ICP10PK: Studies of the neuroprotective, anti-apoptotic properties of lentiviral vector-encoded HSV-2 protein, ICP10PK, and its important neuroprotective bystander effect will require additional NINDS support. New lentiviral vector DNAs will be generated for vector isolation that express several transgenes, including ICP10PK, from the ubiquitin C promoter. These vectors would be used to evaluate ICP10PK to see whether: 1. It is neutral with respect to cell differentiation, 2. It is neuroprotective, and 3. It continues to exert its protective bystander effect. Depending on NINDS support, novel ICP10PK transgene delivery routes would then be evaluated to provide least invasive, neuroprotective efficacy.
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|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|