S Expressed in Neurons and Glia with KN-93 (phosphate) site long-form SIRT3 being Exclusively MitochondrialInitially we investigated SIRT3 protein JNJ-7706621 web expression in the mouse CNS by immunohistochemistry. SIRT3 was expressed throughout the CNS, although not in every cell, with labeling appearing strongest in nuclei (Fig. S1). For further analysis of CNS SIRT3 subcellular localization, we generated hippocampal primary cultures; co-staining of SIRT3 with NeuN revealed that SIRT3 was expressed in neurons as well as glia (Fig. 1A). Co-localization of SIRT3 with the mitochondrial protein HSP60 demonstrated that SIRT3 was present in somatic, as well as dendritic and axonalCNS SIRT3 in AD Mitochondrial StressFigure 1. Subcellular localization of CNS SIRT3. Confocal analysis of SIRT3 subcellular localization: A. SIRT3 is expressed in neurons and glia. Left: SIRT3 immunohistochemistry in primary hippocampal cultures. Middle: NeuN, Right: red and green channels merged. B, C. SIRT3 expression localizes to the nucleus and mitochondria of the cell body, axons and dendrites. Left: SIRT3 immunohistochemistry. Middle: mitochondrial HSP60, Right: red and green channels merged. Scale bar A = 10 mm D and E. Over-expression of long-form SIRT3eGFP results in exclusively mitochondrial localization. D. HeLa cells were co-transfected with Mito-DsRed and `long-form’ SIRT3eGFP plasmids (left: green SIRT3eGFP, middle: red Mito-DsRed, right: merge of channels). E. Primary hippocampal cultures were transfected with a `long-form’ SIRT3eGFP plasmid, fixed and immunohistochemistry performed for HSP60 (middle: red) and eGFP (left: green, right: merge of channels). Scale bar D, E = 5 mm. F. Over-expression of `short-form’ Sirt3eGFP results in cytoplasmic and nuclear localization. HEK293T cells were transfected with `short-form’ SIRT3eGFP, fixed and labeled with DAPI (left panel) and anti-GFP (middle panel, right: merge of channels). doi:10.1371/journal.pone.0048225.gmitochondria (Fig. 1B, 1C). Labeling for SIRT3 was also detected in the nucleus. In mouse, two main splice variants of Sirt3 generate either `longform’ SIRT3 containing an N-terminal mitochondrial localization signal (MLS) or `short-form’ SIRT3 starting at a methionine 78 amino acids downstream [18]. The SIRT3 antibody used for immunohistochemistry recognizes an epitope at the C-terminus of SIRT3; images shown in Fig. 1A are thus likely to reflect the localization of both long- and short-forms of SIRT3. We cloned both forms of Sirt3 from mouse brain cDNA and to analyze localization of both forms of SIRT3 we generated `long-form’ and `short-form’ Sirt3 C-terminal-eGFP fusion constructs. Transfection of `long-form’ SIRT3eGFP into HeLa cells and primary hippocampal cultures resulted in exclusively mitochondrial staining for SIRT3, as indicated by co-localization with mitochondrialtargeted dsRed (Fig. 1D) or with the anti-HSP60 antibody (Fig. 1E) respectively. In contrast, transfection of `short-form’ SIRT3eGFP into HEK293T cells resulted in cytoplasmic and nuclear SIRT3 localization, with no apparent mitochondrial SIRT3eGFP (Fig. 1F).In vitro Pharmacological Interference with the Mitochondrial ETC Upregulates Sirt3 mRNA ExpressionSince mitochondrial oxidative stress is a hallmark of several neuropathological diseases, including AD, we aimed to investigate whether interference with the ETC and induction of ROS could trigger changes in Sirt3 expression. Treatment of primary hippocampal cultures with antimycin A (AA, an ETC complexIII inhi.S Expressed in Neurons and Glia with Long-form SIRT3 being Exclusively MitochondrialInitially we investigated SIRT3 protein expression in the mouse CNS by immunohistochemistry. SIRT3 was expressed throughout the CNS, although not in every cell, with labeling appearing strongest in nuclei (Fig. S1). For further analysis of CNS SIRT3 subcellular localization, we generated hippocampal primary cultures; co-staining of SIRT3 with NeuN revealed that SIRT3 was expressed in neurons as well as glia (Fig. 1A). Co-localization of SIRT3 with the mitochondrial protein HSP60 demonstrated that SIRT3 was present in somatic, as well as dendritic and axonalCNS SIRT3 in AD Mitochondrial StressFigure 1. Subcellular localization of CNS SIRT3. Confocal analysis of SIRT3 subcellular localization: A. SIRT3 is expressed in neurons and glia. Left: SIRT3 immunohistochemistry in primary hippocampal cultures. Middle: NeuN, Right: red and green channels merged. B, C. SIRT3 expression localizes to the nucleus and mitochondria of the cell body, axons and dendrites. Left: SIRT3 immunohistochemistry. Middle: mitochondrial HSP60, Right: red and green channels merged. Scale bar A = 10 mm D and E. Over-expression of long-form SIRT3eGFP results in exclusively mitochondrial localization. D. HeLa cells were co-transfected with Mito-DsRed and `long-form’ SIRT3eGFP plasmids (left: green SIRT3eGFP, middle: red Mito-DsRed, right: merge of channels). E. Primary hippocampal cultures were transfected with a `long-form’ SIRT3eGFP plasmid, fixed and immunohistochemistry performed for HSP60 (middle: red) and eGFP (left: green, right: merge of channels). Scale bar D, E = 5 mm. F. Over-expression of `short-form’ Sirt3eGFP results in cytoplasmic and nuclear localization. HEK293T cells were transfected with `short-form’ SIRT3eGFP, fixed and labeled with DAPI (left panel) and anti-GFP (middle panel, right: merge of channels). doi:10.1371/journal.pone.0048225.gmitochondria (Fig. 1B, 1C). Labeling for SIRT3 was also detected in the nucleus. In mouse, two main splice variants of Sirt3 generate either `longform’ SIRT3 containing an N-terminal mitochondrial localization signal (MLS) or `short-form’ SIRT3 starting at a methionine 78 amino acids downstream [18]. The SIRT3 antibody used for immunohistochemistry recognizes an epitope at the C-terminus of SIRT3; images shown in Fig. 1A are thus likely to reflect the localization of both long- and short-forms of SIRT3. We cloned both forms of Sirt3 from mouse brain cDNA and to analyze localization of both forms of SIRT3 we generated `long-form’ and `short-form’ Sirt3 C-terminal-eGFP fusion constructs. Transfection of `long-form’ SIRT3eGFP into HeLa cells and primary hippocampal cultures resulted in exclusively mitochondrial staining for SIRT3, as indicated by co-localization with mitochondrialtargeted dsRed (Fig. 1D) or with the anti-HSP60 antibody (Fig. 1E) respectively. In contrast, transfection of `short-form’ SIRT3eGFP into HEK293T cells resulted in cytoplasmic and nuclear SIRT3 localization, with no apparent mitochondrial SIRT3eGFP (Fig. 1F).In vitro Pharmacological Interference with the Mitochondrial ETC Upregulates Sirt3 mRNA ExpressionSince mitochondrial oxidative stress is a hallmark of several neuropathological diseases, including AD, we aimed to investigate whether interference with the ETC and induction of ROS could trigger changes in Sirt3 expression. Treatment of primary hippocampal cultures with antimycin A (AA, an ETC complexIII inhi.