Uncoupling Proteins Prevent Glucose-Induced Neuronal Oxidative Stress and Programmed Cell Death

Abstract

The central role of mitochondria in most pathways leading to programmed cell death (PCD) has focused our investigations into the mechanisms of glucose-induced neuronal degeneration. It has been postulated that hyperglycemic neuronal injury results from mitochondria membrane hyperpolarization and reactive oxygen species formation. The present study not only provides further evidence to support our model of glucose-induced PCD but also demonstrates a potent ability for uncoupling proteins (UCPs) to prevent this process. Dorsal root ganglion (DRG) neurons were screened for UCP expression by Western blotting and immunocytochemistry. The abilities of individual UCPs to prevent hyperglycemic PCD were assessed by adenovirus-mediated overexpression of UCP1 and UCP3. Interestingly, UCP3 is expressed not only in muscle, but also in DRG neurons under control conditions. UCP3 expression is rapidly downregulated by hyperglycemia in diabetic rats and by high glucose in cultured neurons. Overexpression of UCPs prevents glucose-induced transient mitochondrial membrane hyperpolarization, reactive oxygen species formation, and induction of PCD. The loss of UCP3 in DRG neurons may represent a significant contributing factor in glucose-induced injury. Furthermore, the ability to prevent UCP3 downregulation or to reproduce the uncoupling response in DRG neurons constitutes promising novel approaches to avert diabetic complications such as neuropathy. Diabetes 53: 726 -734, 2004

The effects of the overexpression of recombinant uncoupling protein 2 on proliferation, migration and plasminogen activator inhibitor 1 expression in human vascular smooth muscle cells

“…In cultured neuronal cells, high glucose induces initial hyperpolarisation followed by depolarisation of the inner mitochondrial membrane potential, which is associated with increased generation of ROS [27,29]. Consistent with these studies, we found that high glucose and Ang II caused mitochondrial membrane hyperpolarisation followed by depolarisation.…”

“…Consistent with these studies, we found that high glucose and Ang II caused mitochondrial membrane hyperpolarisation followed by depolarisation. As expected, UCP-2 overexpression prevented these processes as well as ROS production [27]. Cell membrane NAD(P)H oxidase is another major source of ROS generation in VSMCs.…”

“…The degree of polarisation of the mitochondria was determined by loading with tetramethylrhodamine (TMRM; Molecular Probes, Eugene, OR, USA) as described previously [27]. Forty-eight hours after infecting cells with Ad-UCP-2 or Ad-Null, cells were seeded to 96-well culture plates and exposed to either control, Ang II (100 nmol/l) or high glucose (22 mmol/l D-glucose) conditions for the given time periods.…”

SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy

“…Adenovirus (Ad) constructs containing cDNA for SOD1, SOD2, oxomaleate carrier protein (OMC) or green fluorescent protein (GFP) were prepared as previously described (Hong et al, 2001;Du et al, 2000;Vincent et al, 2004a;Vincent et al, 2004b) and purified at the University of Iowa Gene Transfer Vector Core. At 24 h, 95-100% of DRG neurons were infected using 1,000 plaque forming units (pfu) per cell, determined by counting GFP positive neurons.…”

“…DRG were collected from C57BL/6J control and C57BL/6J SOD2 +/− mice when they were 6-8 weeks of age. Coverslips were coated with 0.01% poly-L-ornithine (0.1mg/ml) (Sigma) overnight, washed, dried and then further coated with rat tail collagen as previously described (Vincent et al, 2004b). Mouse DRG were extracted from adult mice and placed in L-15 media during dissection, dissociated in papain (2 mg/ml) and 2.5% collagenase for 30 min, and then quenched in calf serum.…”