Restricted transgene expression in the brain with cell-type specific neuronal promoters

Abstract

Tissue-targeted expression is of major interest for studying the contribution of cellular subpopulations to neurodegenerative diseases. However, in vivo methods to investigate this issue are limited. Here, we report an analysis of the cell specificity of expression of fluorescent reporter genes driven by six neuronal promoters, with the ubiquitous phosphoglycerate kinase 1 (PGK) promoter used as a reference. Quantitative analysis of AcGFPnuc expression in the striatum and hippocampus of rodents showed that all lentiviral vectors (LV) exhibited a neuronal tropism; however, there was substantial diversity of transcriptional activity and cell-type specificity of expression. The promoters with the highest activity were those of the 67 kDa glutamic acid decarboxylase (GAD67), homeobox Dlx5/6, glutamate receptor 1 (GluR1), and preprotachykinin 1 (Tac1) genes. Neuron-specific enolase (NSE) and dopaminergic receptor 1 (Drd1a) promoters showed weak activity, but the integration of an amplification system into the LV overcame this limitation. In the striatum, the expression profiles of Tac1 and Drd1a were not limited to the striatonigral pathway, whereas in the hippocampus, Drd1a and Dlx5/6 showed the expected restricted pattern of expression. Regulation of the Dlx5/6 promoter was observed in a disease condition, whereas Tac1 activity was unaffected. These vectors provide safe tools that are more selective than others available, for the administration of therapeutic molecules in the central nervous system (CNS). Nevertheless, additional characterization of regulatory elements in neuronal promoters is still required.

FIG. 1.

Transduction efficiency and tropism in adult rats of lentiviral vectors containing the neuronal promoters. (A) Double immunofluorescence staining for the neuronal marker NeuN or (B) the astrocytic marker S100β, and AcGFPnuc after the injection of vesicular stomatitis virus G protein (VSV-G) pseudotyped lentiviral vectors (LVs) into the striatum. Acquisition parameters were optimized for each promoter. Quantification of AcGFPnuc-NeuN- and AcGFPnuc-S100β-positive cells are indicated and confirm the neuronal tropism of all vectors (n=5–6). Scale bar=20 μm.

FIG. 2.

Transcriptional activity of the neuronal promoters. (A) Quantification of the number of infected cells for each promoter. To avoid saturation, a low dose of lentiviral vector has been injected, explaining the limited number of transduced cells. (B) Graphs of mean fluorescence intensity per cell (MFI/cell) showing the distribution of the transcriptional activity of the different neuronal promoters in the striatum of adult rats (n=5–6).

FIG. 3.

Cell-type specificity and activity of PGK, GAD67, Dlx5/6, and Drd1a promoters in the hippocampus. (A) A weak transgene expression was observed with the SIN-W-Drd1a-AcGFPnuc LV. Scale bar=20 μm. (B) Representative picture of the transduced area, which was localized predominantly in the dentate gyrus. Scale bar=200 μm. (C) Representative images showing difference in fluorescence intensity between promoters. PGK image has been taken with a 50 ms exposition, whereas both GAD67 and Dlx5/6 have been taken with a 10 ms exposition. Scale bar=20 μm. (D) Double-fluorescence immunostaining with either NeuN (blue) or S100β (red), showing co-localization of AcGFPnuc with the neuronal marker. Acquisition parameters were optimized for each promoter. Scale bar=20 μm. (E) Quantitative analysis of MFI/cell in the hippocampus and comparison with that in the striatum (n=5 except for Dlx5/6, n=3).

FIG. 4.

The tetracycline amplification system improves the transcriptional activity of the weak Drd1a and neuron-specific enolase (NSE) promoters. (A) Scheme of the lentiviral vectors used for regulated expression of the reporter gene. (B) Quantitative analysis of MFI/cell for each promoter (n=6 except for NSE, n=4).

FIG. 5.

Expression in D1R- or D2R-positive striatal neurons. (A) LVs coding for the nuclear DsRed (DsRed2nuc) under the control of Drd1a, Dlx5/6, or Tac1 promoters were injected into the striatum of transgenic BAC-Drd1a-eGFP or Drd2-eGFP mice. Representative images of co-localization of the DsRednuc, with either the Drd1a-eGFP-positive cells or the Drd2-eGFP-positive cells. (B) Quantitative analysis of the co-localization of DsRed2nuc and GFP expression in BAC-Drd1a-eGFP or BAC-Drd2-eGFP mice (n=5–6, except for Drd1a promoter in BAC-Drd2-eGFP mice, n=3). Scale bar=20 μm.

FIG. 6.

Effect of a disease state on transcriptional activity. (A) LVs encoding a fluorescent reporter gene under the control of Dlx5/6 or Tac1 promoters were co-injected with LVs expressing the first 171 aa of either the wild-type huntingtin (Htt171-18Q) or the mutant huntingtin (Htt171-82Q) into the striatum of adult rats. (B) MFI/cell showing the weaker expression from the Dlx5/6 promoter in animals expressing the mutant than wild-type Htt and indicating that the Tac1 promoter was unaffected (n=5). Scale bar=20 μm.