1. In last years report, we reported for the first time on the effects of the nuclear transcriptional co-activator PGC-1a on human neural progenitor cells (hNPC). We showed that PGC-1a expression inhibited cell proliferation, dramatically altering the shape and the cytoskeletal structure (microtubules) of hNPCs. Many hNPCs are bi- and multipolar cells with long projections, most of which were retracted upon PGC-1a transgene expression. Simultaneously, the cells showed increased b-tubulin III expression, which is known can de-polymerize microtubules, suggesting increased b-tubulin III expression might be responsible for destabilizing microtubules, potentially causing a catastrophe (a term used for microtubule de-polymerization). Paclitaxel, which preferentially interacts with b-tubulin III, stabilized the microtubules in hNPCs and appeared to partially reverse the microtubule-destabilizing effects of PGC-1a in hNPCs. PGC-1a transgene expression inhibited hNPC proliferation under growth conditions, suggesting it may also interfere with mitotic spindle formation during cell proliferation, which contain b-tubulin III. These effects were specific for hNPCs. They were not observed in embryonic kidney cells (Hek293) or in human neuroblastoma cells (SH-SY5Y), except possibly at very high PGC-1a expression levels in a few cells. Interestingly, in SH-SY5Y cells, the expression of the integrated PGC-1a transgene was epigenetically silenced after approximately 2 weeks. However, even after 3 months of continuous cell passages, the PGC-1a transgene could be reactivated within 24 hrs by the selective addition of the histone deacetylase inhibitors trichostatin A and valproic acid. Silencing of the PGC-1a transgene was also observed in N18 neuroblastoma cells but not in Hek293 cells, suggesting a a tight regulation of PGC-1a expression in these b-tubulin III+ neural cell types. A manuscript was submitted for publication and the data were presented at the American Society for Gene and Cell Therapy and will also be presented at the NIH Research Festival this year. PGC-1a is widely perceived as a neuroprotective protein, which serves the high energy needs of neurons;it is not considered to be a potential foe. The regulatory functions of the transcriptional co-activator PGC-1a are well established for mitochondrial replication and mitochondrial oxidative phosphorylation (Wu et al., 1999), as well as for the protection against reactive oxygen species (ROS) (St-Pierre et al., 2006). Without functional PGC-1a protein expression, the progression of neurodegenerative diseases such as Parkinsons (Pacelli et al., 2011;Shin et al., 2011), Huntingtons (Cui et al., 2006), Alzheimers (Qin et al., 2009) disease and amyotrophic lateral sclerosis (Liang et al., 2011) are accelerated. In contrast, Ciron et al., 2012 recently reported that, while targeted injection of an AAV vector encoding PGC-1a in vivo increased neuronal respiration as expected, prolonged over-expression of PGC-1a unexpectedly caused mitochondrial depolarization, inhibited retrograde transport within the nigrostriatal system and selectively killed dopaminergic neurons. We suggest that these observations may be explained in part by the retraction of cellular projections, which we observed with PGC-1a expressing hNPCs. A retraction of cellular processes (axons and dendrites) occurs during many neurodegenerative diseases. On the other hand, the disconnection and reestablishing new connections partly define neural plasticity and are important for nervous system function. Based on our observations, we hypothesize that PGC-1a plays an integrating role in regulating and balancing microtubule dynamics, besides its established roles in regulating energy metabolism and protecting against ROS. By regulating microtubule dynamics, PGC-1a affects intracellular transport over long distances, thereby facilitating the regeneration and the transport of mitochondria to active synapses within the cell, which require high levels of oxidative phosphorylation. This regulation complements the well-established PGC-1a functions by adding the ability to alter cell shape, its polarity and branching in order to maintain metabolic homeostasis throughout the cell. Our initial observations suggested that increased b-tubulin III expression may in part be responsible for this dynamic change. We therefore generated a lentiviral vector that encodes b-tubulin III. Vector-encoded b-tubulin III protein efficiently polymerized and integrated into Hek293 cell microtubules, even though these cells normally do not express this primarily neuron-specific protein. An increase in spherical as compared to polygonal Hek293 cell shapes was noted, as well as slightly reduced cell proliferation, suggesting b-tubulin III may also be responsible for the retraction of cellular processes in hNPCs. The expression of b-tubulin III in hNPCs, however, did not cause the dramatic retraction of cellular processes that was observed after PGC-1a expression, although it may still contribute to the microtubule dynamics in hNPCs. In addition, we compared the expression of several other hNPC cytoskeleton-related proteins with and without PGC-1a transgene expression. Most proteins showed no statistically significant differences in proteins levels, except for b-tubulin III. Two additional proteins were also significantly increased by PGC-1a. We believe one of these proteins is a strong candidate for being involved in the retraction of cellular projections and the depolarization of hNPCs, which include many radial glia and multipolar cells. The other protein is a multifunctional protein, which according to several earlier reports has surprisingly diverse properties and functions. This protein could potentially provide a link between PGC-1a over-expression and apoptosis as observed by Ciron et al., 2012 in vivo. At elevated expression levels together with nuclear translocation, this protein may also increase the oncogenic potential of affected cells depending on their stress levels. The identity of these two proteins will be revealed in a new and refocused manuscript. The known functions of these proteins, together with b-tubulin III, point to a novel role for PGC-1a in microtubule dynamics, and it indicates a potentiating function of PGC-1a for apoptosis and/or oncogenesis. Interestingly, high levels of b-tubulin III expression in many cancer cells correlate with high invasiveness and paclitaxel-resistance. In summary, PGC-1a appears to be a key transcriptional co-activator, which is controlled by several metabolic and environmental stress sensors reporting on the metabolic status of the cell. Our studies suggest, for the first time, that PGC-1a may also control microtubule dynamics in neural cells. We hypothesize that depending on the strength and duration of a variety of stress inputs, the cellular response through PGC-1a varies from basic cell maintenance, to cell adaptation and plasticity, to apoptosis and oncogenesis. We propose that understanding the role of the cytoskeleton and its potential regulation by PGC-1a during neurodevelopment and neurodegenerative diseases is critically important, as it may potentially reveal shared elements necessary to combat neurodegenerative diseases. 2. A collaboration with Dr. Steve Standley et al. at NIDCD and the OTT, OD, NIH involved the use of lentiviral vector-encoded, conditionally targeted, chimeric NMDA receptor C-terminal regions that contain PDZ-binding domains, which bind to PSD-95 family proteins. This approach allowed the study of NMDA-receptor/PSD-95 protein interactions during the trafficking of these proteins from the ER through the trans-Golgi network to the synapse of hippocampal and cortical neurons in culture.
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