Figure 1.
MARK2 is expressed in the developing brain, and its levels can be effectively reduced in vitro and in vivo. A, B, B′, In situ hybridization of E14 brain sections shows high expression of MARK2 mRNA in the CP and VZ/SVZ, where NeuN staining is excluded (brown), and lower expression levels in the IZ (A, coronal view; B, sagittal view). B′, No signal was detected using the sense probe. C, PAK5 mRNA is more abundant in the CP and IZ than in the VZ/SVZ of E14 mouse embryos brains. D, In E16 brain sections, MARK2 antibodies recognize the protein in locations similar to those at E14; note lower expression levels in the IZ. E–H, Exogenous MARK2 expressed as a GFP fusion protein in HEK293 cells was effectively knocked down by two different shRNA sequences, MARK2 shRNA A (G) and MARK2 shRNA C (H), compared with the GFP-MARK2 expression levels in the presence of a control shRNA plasmid (E, F). I–L, Resistance to shRNA of MARK2 WT (I, K) and MARK2-KD (J, L) after introduction of silent mutations in the shRNA target site. No reduction in the expression of GFP-MARK2R (K) or GFP-MARK2R-KD (L) was recorded when MARK2 shRNA A was introduced compared with cells expressing control shRNA (I, J). M, Real-time PCR demonstrates reduction in endogenous MARK2 mRNA levels after treatment of differentiated CAD cells with either MARK2 shRNA A or MARK2 shRNA C, whereas cells treated with control shRNA (CTR) continue to exhibit high levels of MARK2 mRNA expression. N–P, In situ hybridization of E16 brain slices electroporated in utero with MARK2 shRNA A at E14. A cluster of electroporated cells expressing GFP (N) correlates with reduction in MARK2 mRNA signal (O). P, Merge of N and O. Q, R, Single-cell images taken from electroporated brain slices 2 d after in utero electroporation of MARK2 shRNA (green cells) show lower MARK2 protein levels by immunostaining with anti-MARK2 antibodies (red) than the untreated cells in the surrounding area (cell nuclei are stained with DAPI, in blue). S, T, Cells electroporated with control shRNA (GFP-positive cells in S, cell nuclei in blue) show levels of MARK2 expression (red) similar to those of neighboring cells (note uniform staining in T). U–Z, An area in the SVZ with a cluster of neurons expressing a high concentration of MARK2 shRNA A (shRNA:GFP, 8:1). V, The area in U was immunostained with anti-phosphorylated tau (pTau pS262) antibodies (red). The reduction of pTau in the fibers is readily visible. A higher magnification is shown in the boxed inset. W, Merge of V and U. X, Area comparable with that in I from a brain section electroporated in utero with control shRNA and GFP; multiple green neurons are shown. Y, The section in X was immunostained with anti-phosphorylated tau (pTau pS262) antibodies (red); immunostaining is obvious mainly in the cell processes and is indicated by the white arrowheads. A higher magnification is shown in the boxed inset. Z, Merge of X and Y. Scale bar sizes are indicated in micrometers.