Fig 2: Neurite arborisation of cortical neurons isolated from adult Mth1/Ogg1-DKO but not wild-type mice was significantly impaired in the absence of antioxidants.(a) Adult cortical neurons isolated from Mth1/Ogg1-DKO (TO-DKO) and wild-type mice were cultured for 5 days in the absence (-AO) or presence (+AO) of antioxidants and were subjected to MAP2-immunofluorescence microscopy. Representative images are shown, indicating a simpler neuritic branching pattern in neurons from TO-DKO mice cultured in the absence of antioxidants. Scale bar = 20 µm. (b) Sholl’s concentric sphere analysis shows severely impaired neurite arborisation in TO-DKO neurons in the absence of antioxidants. More than 16 neurons were examined in each culture condition. Combined data from two independent experiments are shown. Error bar = SEM. MANOVA, between subjects: F(3, 171) = 19.671, p < 0.0001; within subjects: interaction, p < 0.0001 (Univar G-G, e = 0.311). TO-DKO (-AO) vs. other three groups, *p < 0.0001.

Fig 3: Requirement for copper is increased in the secretory pathway upon neuronal differentiation.(a) Immunostaining of PAM (green) in a chicken spinal cord (HH stage 20 embryo). Traverse sections of chick embryos were analysed. Differentiated cells in marginal zones were identified by postmitotic marker isl1/2 (red). Scale bar, 50 µm. (b) Intensity profile along the line indicated by the arrow in a shows overlap between PAM and Isl1/2 expression. Medial and lateral borders are represented as M and L, respevtively. (c) Schematic of differentiation of SH-SY5Y cells by sequential treatments with retinoic acid (DIF RA) and BDNF (DIF BDNF). (d) Differentiation is associated with upregulation of genes involved in copper fluxes to mitochondria and, especially, the secretory pathway. The mRNA levels were determined by ??Ct analysis using GAPDH as a reference gene and normalized to non-differentiated cells (red line). Each value is presented as mean±s.d. (n=3). (e) Protein levels for ATP7A and Atox1 increase upon differentiation. Equal amount of protein was loaded to each lane. Neuronal differentiation was verified by upregulation of MAP2. (f) Cellular copper content in differentiated SH-SY5Y cells (BDNF) is higher than in non-differentiated (ND) cells. Copper amounts were determined by atomic absorption and normalized to protein amounts. Data from three independent measurements. (g,h) ATP7A is localized within TGN and vesicular structures which are distinct from endosomal or lysosomal compartments. Coimmunostaining with TGN was shown as a representative image (see Supplementary Fig. 2a for other images). Nucleus was stained with DAPI (blue). Scale bar, 10 µm. Colocalization of ATP7A and various markers were evaluated using Pearson's product-moment coefficients. Three replicate samples were prepared and analysed. Each value is presented as mean±s.d. (n=3).

Fig 4: (A–C) Silencing of fructose 1,6-bisphosphatase 2 (Fbp2) expression in cultured hippocampal neurons. (A) Expression of Fbp in hippocampal neurons: untreated (left image), treated with antisense shRNA (middle image) and shRNA against Fbp2 (right image). Bar = 20 µm, N = 30 regions of interest (ROIs). (B) Decrease in Fbp2-associated fluorescence (expressed as the ratio of Fbp2 to Map2 fluorescence). (C) Western blot analysis of Fbp2-silenced hippocampal neurons and densitometric analysis of the protein level. Tubulin ß3 was used as a reference protein. (D,E) Fbp2 is the main isoform of Fbp in hippocampal neurons. (D) In situ hybridization demonstrates that fluorescent signal associated with mRNA for Fbp2 is much stronger than for Fbp1. (E) Quantification of the mRNA for Fbp2 and Fbp1. Bar = 20 µm, N = 30 ROIs. (F) Fbp2 protects neuronal mitochondria against calcium-induced swelling. The amount of swollen mitochondria is inversely proportional to the decrease of relative absorbance at 540 nm. The absorbance measured before Ca2+ addition was assumed to be 100%. The values are given as a mean and SD. *** indicates p < 0.001.

Fig 5: Lack of improvement in neuropathology in minocycline treated animals 5 days post-TBI. A) Quantification of ventricular size 5 days post-TBI and B) quantification of the volume of tissue loss in the traumatized hemisphere 5 days post-TBI based on MAP-2 immunoreactivity, D) representative photomicrographs of MAP2-immunolabeled sections, scale bar 50 µm. C) and E) quantification and representative images of the volume of white matter loss in the traumatized hemisphere based on MBP immunoreactivity, scale bar 200 µm. Data are indicated by means ± SEM (n = 12–18 animals/group). PBS- and minocycline-treated groups were compared via a Student’s t-test.