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  • Localization and Expression of Sirtuins 1, 2, 6 and Plasticity-Related Proteins in the Recovery Period after a Photothrombotic Stroke in Mice.

Localization and Expression of Sirtuins 1, 2, 6 and Plasticity-Related Proteins in the Recovery Period after a Photothrombotic Stroke in Mice.

Journal of stroke and cerebrovascular diseases : the official journal of National Stroke Association (2020-09-12)
Svetlana Demyanenko, Elena Gantsgorn, Stanislav Rodkin, Svetlana Sharifulina
ABSTRACT

Sirtuins, class III histone deacetylases, are involved in the regulation of tissue repair processes and brain functions after a stroke. The ability of some isoforms of sirtuins to circulate between the nucleus and cytoplasm may have various pathophysiological effects on the cells. In present work, we focused on the role of non-mitochondrial sirtuins SIRT1, SIRT2, and SIRT6 in the restoration of brain cells following ischemic stroke. Here, using a photothrombotic stroke (PTS) model in mice, we studied whether local stroke affects the level and intracellular localization of SIRT1, SIRT2, and SIRT6 in neurons and astrocytes of the intact cerebral cortex adjacent to the ischemic ipsilateral hemisphere and in the analogous region of the contralateral hemisphere at different time points during the recovery period after a stroke. We evaluated the co-localization of sirtuins with growth-associated protein-43 (GAP-43), the presynaptic marker synaptophysin (SYN) and acetylated α-tubulin (Ac-α-Tub), that are associated with brain plasticity and are known to be involved in brain repair after a stroke. The results show that during the recovery period, an increase in SIRT1 and SIRT2 levels occurred. The increase of SIRT1 level was associated with an increase in synaptic plasticity proteins, whereas the increase of SIRT2 level was associated with an acetylated of α-tubulin, that can reduce the mobility of neurites. SIRT6 co-localized with GAP-43, but not with SYN. Moreover, we showed that SIRT1, SIRT2, and SIRT6 are not involved in the PTS-induced apoptosis of penumbra cells. Taken together, our results suggest that sirtuins functions differ depending on cell type, intracellular localization, specificity of sirtuins isoforms to different substrates and nature of post-translational modifications of enzymes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-Synaptophysin antibody produced in mouse, clone SVP-38, ascites fluid
Sigma-Aldrich
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-15, ascites fluid
Sigma-Aldrich
Anti-Sirt2 antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Anti-SIRT1 antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, pH 7, ≥98%
Sigma-Aldrich
Anti-Mouse IgG1 (γ1), CF555 antibody produced in goat, ~2 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Anti-Neuron-Specific Enolase (NSE), Mouse monoclonal, clone NSE-P1, purified from hybridoma cell culture
Sigma-Aldrich
Anti-Acetylated Tubulin antibody, Mouse monoclonal, clone 6-11B-1, purified from hybridoma cell culture
Sigma-Aldrich
Anti-Rabbit IgG (H+L), highly cross-adsorbed, CF 488A antibody produced in goat, ~2 mg/mL, affinity isolated antibody
Roche
In Situ Cell Death Detection Kit, TMR red, sufficient for ≤50 tests
Sigma-Aldrich
Anti-Tubulin γ, C-Terminal antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
NuCLEAR Extraction Kit, For mammalian tissue or cultured cells
Sigma-Aldrich
Anti-Sirt6 (N-terminal) antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution