Or cortex (Loizzo et al., 2010; Mattiazzi et al., 2002). In addition, mitochondrial
Or cortex (Loizzo et al., 2010; Mattiazzi et al., 2002). In addition, mitochondrial Ca2+ uptake capacity is affected in ALS mice before motor neuron dysfunction (Damiano et al., 2006). Even so, it remains unclear regardless of whether mitochondrial dysfunction is usually a trigger or a consequence of oxidative harm. Due to the proposed metabolic and oxidative damage elements in the disease, therapeutic approaches tested inside the ALS mouse models have usually broadly focused on bioenergetics and antioxidant agents, for example vitamin E (Gurney et al., 1996), creatine (Klivenyi et al., 1999), and catalase (Reinholz et al., 1999), with mixed outcomes (for a review see (Turner and Talbot, 2008)). Within the present study, we crossed a human UCP2 (hUCP2) transgenic mouse together with the G93A mutant SOD1 mouse, to test whether UCP2 overexpression could particularly lower mitochondrial ROS production, modulate bioenergetics and calcium uptake, and afford neuroprotection inside a familial ALS model. In addition, we expected that metabolic investigations inside the double transgenic mice would shed new light on the functions of UCP2 within the wholesome and diseased CNS.Mol Cell Neurosci. Author manuscript; readily available in PMC 2014 November 01.Peixoto et al.PageMaterials and MethodsGenetically modified miceNIH-PA Author BRDT Storage & Stability Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptG93A mutant human SOD1 mice inside a C57BL/6J genetic background have been obtained from Jackson Laboratories (strain B6.Cg-Tg(SOD1-G93A)1Gur/J). C57BL/6J mice overexpressing human UCP2 below the handle of its endogenous promoter have been generous gifts from Dr. Tamas L. Horvath (Yale University). Overexpression of human UCP2 inside the brain was assessed by real time PCR as previously described (Horvath et al., 2003). Double transgenic mice expressing SOD1 G93A and hUCP2 (hUCP2 G93A) had been generated by crossing female hUCP2+/+ with male SOD1 G93A+/- mice. Resulting Females hUCP2+/- SOD1 G93A-/- have been crossed with male SOD1 G93A+/- mice to yield hUCP2+/- SOD1 G93A+/-, SOD1 G93A+/-, hUCP2+/-, and non-transgenic handle mice (ntg). Mice have been genotyped by PCR of tail DNA at 21 days of age as previously described, (Horvath et al., 2003; Kim et al., 2012). Central nervous system UCP2 and SOD1 mRNA overexpression was confirmed by quantitative actual time PCR. All animal experiments had been carried out in sibling- and gender-matched pairs just after approval by the Institutional Animal Care and Use Committee (IACUC). Mouse phenotypes Survival, body weight, and motor efficiency on an accelerating rod have been determined as previously described (Kim et al., 2012). When mice became unable to right themselves within 20 s of being placed on their side they have been euthanized and age at time of death was recorded. Physique weight and physical efficiency on an accelerating rod (Rotarod, Columbus HDAC9 Source Instruments) had been assessed every single 2 weeks beginning at 80 days of age. Oxygen consumption and carbon dioxide production prices (VO2 and VCO2, respectively) have been determined at resting circumstances (absence of physical exercise, no dietary restrictions) for five minutes by putting animals inside a 2 L sealed chamber with dual gas sensors (Vernier Soft. Tech. LLC). The prices had been plotted as mL gas/min/kg at 120, 130, and 140 days of age. Isolation of brain mitochondria and measurement of mitochondrial ATP synthesis, ROS emission, Ca2+ uptake, and membrane potential Isolation and purification of mouse brain mitochondria was performed by differential centrifugation of homogenates on a discontinuous p.