Succination is increased on select proteins in the brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome

We thank Ms. Jennifer Bethard, Medical University of South Carolina for the mass spectrometric analysis of the 2D gel spots. We would like to thank Professor John Baynes, USC for helpful discussion. We thank Dr. Boris Kantor, USC Viral Vector Core for production of the lentiviral vectors. This work was supported by grants from the National Institutes of Health (R03 HD077187, R01 NS092938, R37 DK19971 and P20 GM109091), the American Diabetes Association (1-11-JF-13), and a University of South Carolina Research Foundation ASPIRE-I award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-Translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltagedependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys77 and Cys48 were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model

Neuropeptide Y is an essential in vivo developmental regulator of cardiac I-Ca,I-L

Cell culture studies demonstrate an increase in cardiac L-type Ca2+ current (I-Ca,I-L) density on sympathetic innervation in vitro and suggest the effect depends on neurally released neuropeptide Y (NPY). To determine if a similar mechanism contributes to the postnatal increase in I-Ca,I-L in vivo, we prepared isolated ventricular myocytes from neonatal and adult mice with targeted deletion of the NPY gene (Npy(-/-)) and matched controls (Npy(+/+)). Whole-cell voltage clamp demonstrates I-Ca,I-L density increases postnatally in Npy(+/+) (by 56%), but is unchanged in Npy(-/-). Both I-Ca,I-L density and action potential duration are significantly greater in adult Npy(+/+) than Npy(-/-) myocytes, whereas I-Ca,I-L density is equivalent in neonatal Npy(+/+) and Npy(-/-) myocytes. These data indicate NPY does not influence ICa,L prenatally, but the postnatal increase in I-Ca,I-L density is entirely NPY-dependent. In contrast, there is a similar postnatal negative voltage shift in the I-V relation in Npy(+/+) and Npy(-/-), indicating NPY does not influence the developmental change in I-Ca,I-L voltage-dependence. Immunoblot analyses and measurements of maximally activated I-Ca,I-L (in presence of forskolin or BayK 8644) show that the differences in current density between Npy(+/+) and Npy(-/-) cannot be attributed to altered Ca2+ channel alpha(1C) subunit protein expression. Rather, these results suggest that the in vivo NPY-dependent postnatal increase in I-Ca,I-L density in cardiac myocytes results from regulation I-Ca,I-L properties by NPY

The neuropeptide Y Y1 receptor regulates leptin-mediated control of energy homeostasis and reproductive functions.

The orexigenic neurotransmitter neuropeptide Y (NPY) plays a central role in the hypothalamic control of food intake and energy balance. NPY also exerts an inhibition of the gonadotrope axis that could be important in the response to poor metabolic conditions. In contrast, leptin provides an anorexigenic signal to centrally control the body needs in energy. Moreover, leptin contributes to preserve adequate reproductive functions by stimulating the activity of the gonadotrope axis. It is of interest that hypothalamic NPY represents a primary target of leptin actions. To evaluate the importance of the NPY Y1 and Y5 receptors in the downstream pathways modulated by leptin and controlling energy metabolism as well as the activity of the gonadotrope axis, we studied the effects of leptin administration on food intake and reproductive functions in mice deficient for the expression of either the Y1 or the Y5 receptor. Furthermore, the role of the Y1 receptor in leptin resistance was determined in leptin-deficient ob/ob mice bearing a null mutation in the NPY Y1 locus. Results point to a crucial role for the NPY Y1 receptor in mediating the NPY pathways situated downstream of leptin actions and controlling food intake, the onset of puberty, and the maintenance of reproductive functions