CNS SIRT3 expression is altered by reactive oxygen species and in Alzheimer's disease

Abstract

Progressive mitochondrial dysfunction contributes to neuronal degeneration in age-mediated disease. An essential regulator of mitochondrial function is the deacetylase, sirtuin 3 (SIRT3). Here we investigate a role for CNS Sirt3 in mitochondrial responses to reactive oxygen species (ROS)- and Alzheimer's disease (AD)-mediated stress. Pharmacological augmentation of mitochondrial ROS increases Sirt3 expression in primary hippocampal culture with SIRT3 over-expression being neuroprotective. Furthermore, Sirt3 expression mirrors spatiotemporal deposition of β-amyloid in an AD mouse model and is also upregulated in AD patient temporal neocortex. Thus, our data suggest a role for SIRT3 in mechanisms sensing and tackling ROS- and AD-mediated mitochondrial stress.

Figure 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–C = 10 µm 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 µm. 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).

Figure 2. CNS Sirt3 mRNA expression is regulated by mitochondrial ROS and Sirt3 over-expression increases neuronal longevity.

A. Primary hippocampal cultures were loaded with MitoSOX, treated with antimycin A (AA, 250 nM, 12 hr) and/or pre-treated with N-acetyl-L-cysteine (NAC, 100 µM, o/n) and phase and fluorescent still images taken at 12 hrs. B. Mitochondrial ROS is significantly increased following AA treatment, which is partially blunted by NAC (n ≥50 neurons per treatment, one-way ANOVA ***P<0.001, **P<0.01, *P<0.05). C. Sirt3, but not Sirt5 mRNA expression is upregulated in response to AA treatment in primary hippocampal cultures, which is blocked by NAC ROS scavenging (n = 10). Sirt3/5 mRNA was measured and normalized to 18S rRNA using TaqMan multiplex QPCR. D. Sirt3 over-expression significantly increases neuronal lifespan. Hippocampal primary cultures were transduced with neuronal-specific lenti-GFP (control) or lenti-mSIRT3iGFP lentivirus and treated with AA (250 nM). Time until fluorescent neuronal death was recorded and is expressed as %control (n>150, ***P>0.0001).

Figure 3. Sirt3 mRNA is upregulated in a specific spatio-temporal pattern in a mouse model of AD.

A. Sirt3 mRNA expression is significantly upregulated in hippocampus samples of 6 months-old PDAPP mice (n = 9−10, ***P<0.001) with no alteration in cortex (n = 10) and cerebellum (n = 10). Sirt3 mRNA was measured and normalized to 18S rRNA using TaqMan multiplex QPCR. B. Sirt3 mRNA expression is significantly upregulated in cortex samples of 26 months-old PDAPP mice (n = 6−7, ***P<0.001) with no alteration in hippocampus (n = 8) and cerebellum (n = 6−7). C. Sirt5 mRNA is unaltered in 6 months-old hippocampus and 26 months-old cortex PDAPP samples.

Figure 4. Sirt3 is upregulated in human Alzheimer’s disease (AD).

A. Sirt3 mRNA expression is significantly increased in AD temporal cortex samples compared to matched controls (n = 14, **P<0.01). Protein and RNA were extracted from neuropathologically confirmed sporadic AD and matched control temporal cortex dissected samples. Sirt3 and Eno2 mRNA were measured and normalized to 18S rRNA using TaqMan multiplex QPCR. B. Cleaved (active) SIRT3 protein is increased in AD temporal cortex samples (n = 14−15). Protein levels were determined by Western Blot and normalized to α-tubulin and synaptophysin.