1. The study on PGC1a expression in neural cells and its potential role in the regulation of cytoskeleton dynamics was temporarily put on hold. 2. For the study on the inhibition of Cdk5/p25 kinase hyperactivity during neurodegeneration, the following observations can be added: Cell supernatants from cells that express a transgene, which encodes a new chimeric, inhibitory peptide against Cdk5/p25 activity showed high peptide levels, clearly demonstrating efficient secretion of the peptide. In vitro assays using histone 1 phosphorylation confirmed that the peptide was functional, able to inhibit Cdk5/p25 kinase activity in vitro. As a control, cell supernatants containing a secreted, but scrambled peptide were only weakly inhibitory. Medium alone did not inhibit. Neural progenitor cells that were infected by a lentiviral vector, which encodes the inhibitory peptide, appeared unaffected by the expression, secretion and reuptake of this chimeric cell-penetrating peptide. Infected cells included neurons, glial cells and their precursor cells. These results confirmed the key functional properties of the vector-encoded peptide. Instead of infecting hematopoietic stem cells directly with the LV vector prior to transplantation into transgenic mouse models of neurodegenerative diseases such as the 5xFAD mouse (Alzheimers disease), we refined our experimental strategy to initially focus on the safety of the peptide and then on its therapeutic efficacy. Transgenic mice or other transgenic animal species will be created using high-titer LV vector co-expressing the peptide and EGFP. Normal development and behavior of these mice will indicate peptide safety. We anticipate that the peptide will be expressed, secreted, and it will penetrate many cell types. Potential inhibition of normal Cdk5/p35 activity during development could lead to perinatal lethality. Healthy transgenic mice will then be bred with mouse models of neurodegenerative diseases, with preference for the 5xFAD disease model, to demonstrate therapeutic efficacy of the peptide. If the peptide shows efficacy, EGFP and peptide expressing hematopoietic stem cells will be isolated from the initial transgenic mice and transplanted into ablated and conditioned 5xFAD mice to evaluate whether the transmigration of peptide-secreting monocytes to sites of injury and amyloid plaques is sufficient to ameliorate the disease. If successful, this delivery approach is scalable to human patients. Different from direct intraperitoneal systemic delivery of high amounts of the neuroprotective peptide, targeted transgene delivery via stereotactic injection of AAV vectors encoding the new secreted and cell-penetrating peptide may be employed in the case of Parkinson's disease. Transgene delivery via migratory stem cells or hematopoietic cells such as lymphocytes or monocytes may further expand the utility of this peptide transgene approach systemically and include many neurodegenerative diseases like Huntington's disease and especially diseases with immunological components such as amyotrophic lateral sclerosis, pain, stroke and multiple sclerosis. It is now recognized that Cdk5 as well as GSK3b hyperactivities are interrelated, both kinases contributing to disease progression and apoptosis. Surprisingly, p25 may affect each kinase activity, leading to a neurotoxic avalanche of events towards apoptosis. Our goal is to prevent this avalanche while maintaining both Cdk5 and GSK3b activities within their normal range. This is critically important for many essential cellular functions, from glucose control to cognition. We postulate an apoptosis triggering mechanism that is thresholded. The neuroprotective peptide only targets hyperactivities without affecting normal activities in vivo. To assure peptide safety, being able to generate a transgenic mouse or even a marmoset using the already isolated lentiviral vector, which encodes the secreted cell-penetrating peptide will, therefore, be an important experiment.
| 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 |
