Mitochondrial alterations in <i>Npc1</i>^-/- cerebellum.

<p>A) Cerebellar RNA was prepared from 3-, 5- or 7-week old WT and <i>Npc1</i>^-/- mice. Target gene mRNA levels were measured by qPCR using primers against PGC1α (<i>Ppargc1a</i>) standardized to <i>Ppia</i>. B) Immunoblots of cerebellar tissue homogenates from 3- or 5-week old wildtype (WT) and <i>Npc1</i>^-/- mice probed with antibodies against VDAC1 and actin as loading control. C) Genomic DNA was prepared from cerebella of 5-week old WT and <i>Npc1</i>^-/- mice. Mitochondrial DNA was determined by qPCR using primers against mitochondrial cytochrome c oxidase subunit 1 (<i>Mtco1</i>) DNA per nuclear <i>Ndufv1</i> DNA, and standardized to WT. D and E) Cerebellar RNA was prepared as in panel A. Target gene mRNA levels were measured by qPCR using primers against mitochondrially encoded subunit 2 of cytochrome c oxidase (<i>Mtco2</i>) or uncoupling protein 2 (<i>Ucp2</i>) standardized to <i>Ppia</i>. F to H) Mitochondria were isolated by differential centrifugation from combined homogenates of the cerebella, hippocampi and cerebral cortices of wildtype (WT) and <i>Npc1</i>^-/- mice at 3 and 7 weeks of age. F) Mitochondria were stained with nonyl acridine orange to verify the maintenance of their membrane potential. G and H) The rate of ATP generation was measured in the presence of 1 mM pyruvate, 1 mM malate and 300 µM ADP (Pyr/Mal) or in the presence of 5 mM succinate and 300 µM ADP as luminescence generated by the ATP-dependent D-luciferin/luciferase reaction as luminescence per second per protein. F) Mitochondria isolated from 3-week old mice. G) Mitochondria isolated from 7-week old mice. Data shown in panels F – H) are expressed as percent of WT of the same experiment, and are derived from three independent experiments each with two independent mitochondria preparations of each genotype and each age. I) Cerebellar tissue homogenates were prepared from 3-, 4- or 5-week old WT and <i>Npc1</i>^-/- mice and analyzed by immunoblotting using antibodies against phospho-AMPKα (Thr172), AMPKα, phospho-AMPKβ (Ser108), AMPKβ, and actin as loading control. Bar graphs show the ratio of band intensities of phosphorylated to total AMPK expressed as percent of the average of WT on the same membrane. All bar graphs show the mean ± SEM. * p<0.05, <i>Npc1</i>^-/- vs. WT.