The design and synthesis of a novel nuclear factor erythroid 2-related factor 2 (Nrf2) enhancer is reported. Using a structure-based virtual screening approach, several commercially available compounds were identified as having high probability to interact with the Nrf2-binding pocket in the Kelch-like ECH-associated protein 1 (Keap1). Keap1 is an adaptor protein that recruits Nrf2 to a cullin-3-dependent ubiquitin ligase complex. The identified compounds were tested against rat pheochromocytoma PC-12 cells for their cytoprotective activity, and one compound (SKT359126) demonstrated an Nrf2-mediated cell-protective effect. Based on the structure of SKT359126, 23 novel derivatives were synthesized and evaluated. Of the screened derivatives, 1-{4-[(3,4-dihydroxybenzylidene)amino]phenyl}-5-oxopyrrolidine-3-carboxylic acid demonstrated better activity than the parent molecules in activating the Nrf2 transduction pathway in a dose- and time-dependent manner. This compound represents a promising starting point for the development of therapeutics for the treatment of oxidative-stress-related diseases.
The role of the proapototic Bax gene in ischemia-reperfusion (I/R) injury was studied in three groups of mice: homozygotic knockout mice lacking the Bax gene (Bax(-/-)), heterozygotic mice (Bax(+/-)), and wild-type mice (Bax(+/+)). Isolated hearts were subjected to ischemia (30 min, 37 degrees C) and then to 120 min of reperfusion. The left ventricular developed force of Bax-deficient vs. Bax(+/+) hearts at stabilization and at 120 min of reperfusion was 1,411 +/- 177 vs. 1,161 +/- 137 mg and 485 +/- 69 vs. 306 +/- 68 mg, respectively. Superior cardiac function of Bax(-/-) hearts after I/R was accompanied by a decrease in creatine kinase release, caspase 3 activity, irreversible ischemic injury, and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cardiomyocytes. Electron microscopic evaluation revealed reduced damage to mitochondria and the nuclear chromatin structure in Bax-deficient mice. In the Bax(+/-) hearts, the damage markers were moderate. The superior tolerance of Bax knockout hearts to I/R injury recommends this gene as a potential target for therapeutic intervention in patients with severe and intractable myocardial ischemia.
Achieving safe and readily accessible sources for cell replacement therapy in Parkinson's disease (PD) is still a challenging unresolved issue. Recently, a primitive neural crest stem cell population (hOMSC) was isolated from the adult human oral mucosa and characterized in vitro and in vivo. In this study we assessed hOMSC ability to differentiate into dopamine-secreting cells with a neuronal-dopaminergic phenotype in vitro in response to dopaminergic developmental cues and tested their therapeutic potential in the hemi-Parkinsonian rat model. We found that hOMSC express constitutively a repertoire of neuronal and dopaminergic markers and pivotal transcription factors. Soluble developmental factors induced a reproducible neuronal-like morphology in the majority of hOMSC, downregulated stem cells markers, upregulated the expression of the neuronal and dopaminergic markers that resulted in dopamine release capabilities. Transplantation of these dopaminergic-induced hOMSC into the striatum of hemi-Parkinsonian rats improved their behavioral deficits as determined by amphetamine-induced rotational behavior, motor asymmetry and motor coordination tests. Human TH expressing cells and increased levels of dopamine in the transplanted hemispheres were observed 10 weeks after transplantation. These results demonstrate for the first time that soluble factors involved in the development of DA neurons, induced a DA phenotype in hOMSC in vitro that significantly improved the motor function of hemiparkinsonian rats. Based on their neural-related origin, their niche accessibility by minimal-invasive procedures and their propensity for DA differentiation, hOMSC emerge as an attractive tool for autologous cell replacement therapy in PD.
Cell-to-cell communication system involves Exosomes, small, membrane-enveloped nanovesicles. Exosomes are evolving as effective therapeutic tools for different pathologies. These extracellular vesicles can bypass biological barriers such as the blood-brain barrier, and can function as powerful nanocarriers for drugs, proteins and gene therapeutics. However, to promote exosomes' therapy development, especially for brain pathologies, a better understanding of their mechanism of action, trafficking, pharmacokinetics and bio-distribution is needed. In this research, we established a new method for non-invasive in-vivo neuroimaging of mesenchymal stem cell (MSC)-derived exosomes, based on computed tomography (CT) imaging with glucose-coated gold nanoparticle (GNP) labeling. We demonstrated that the exosomes were efficiently and directly labeled with GNPs, via an energy-dependent mechanism. Additionally, we found the optimal parameters for exosome labeling and neuroimaging, wherein 5 nm GNPs enhanced labeling, and intranasal administration produced superior brain accumulation. We applied our technique in a mouse model of focal ischemia. Imaging and tracking of intranasally-administered GNP-labeled exosomes revealed specific accumulation and prolonged presence at the lesion area, up to 24 hrs. We propose that this novel exosome labeling and in-vivo neuroimaging technique can serve as a general platform for brain theranostics.
