Figure 5.

Sirt6 overexpression in PrL neurons.

(A) Schematic diagram of the viral vector construction used to target the cells in the PrL. The Sirt6 transgene was placed under the control of the CMV promoter (top panel). Schematic of the area injected with AAV2/9-Sirt6 or AAV2/9-eGFP (below panel, left). Bilateral double injection of AAV2/9-Sirt6 or AAV2/9-eGFP in the PrL, verified by eGFP expression (green; right, below panel). (B) Overexpression of the Sirt6 protein according to a 3-fold change in vectors. Sirt6 expression (red, Cy3) in the PrL cells was transduced with empty vectors (left) and with the Sirt6 transgene (right). Data are presented as mean ± SEM, and were analyzed by Student’s t-test. (C) eGFP (green) was co-stained with the neuronal nuclear marker NeuN (red, Cy3). White arrows indicate the body of eGFP⁺ cells. (D, E) The specificity of eGFP in astrocytes and microglia. The astrocytes and microglia were seldom infected with the virus. (F) Analysis of the tropism of AAV9 for different kinds of cells, such as neurons (NeuN), astrocytes (GFAP), and microglia (IBA1). AAV2/9: Adeno-associated virus serotype 9; CMV: cytomegalovirus; DAPI: 4′,6-diamidino-2-phenylindole; eGFP: enhanced green fluorescent protein; GFAP: glial fibrillary acidic protein; IBA1: ionized calcium-binding adapter molecule 1; PrL: prelimbic cortex; Sirt6: Sirtuins 6.