Silencing Status Epilepticus-Induced BDNF Expression with Herpes Simplex Virus Type-1 Based Amplicon Vectors

Abstract

Brain-derived neurotrophic factor (BDNF) has been found to produce pro- but also anti-epileptic effects. Thus, its validity as a therapeutic target must be verified using advanced tools designed to block or to enhance its signal. The aim of this study was to develop tools to silence the BDNF signal. We generated Herpes simplex virus type 1 (HSV-1) derived amplicon vectors, i.e. viral particles containing a genome of 152 kb constituted of concatameric repetitions of an expression cassette, enabling the expression of the gene of interest in multiple copies. HSV-1 based amplicon vectors are non-pathogenic and have been successfully employed in the past for gene delivery into the brain of living animals. Therefore, amplicon vectors should represent a logical choice for expressing a silencing cassette, which, in multiple copies, is expected to lead to an efficient knock-down of the target gene expression. Here, we employed two amplicon-based BDNF silencing strategies. The first, antisense, has been chosen to target and degrade the cytoplasmic mRNA pool of BDNF, whereas the second, based on the convergent transcription technology, has been chosen to repress transcription at the BDNF gene. Both these amplicon vectors proved to be effective in down-regulating BDNF expression in vitro, in BDNF-expressing mesoangioblast cells. However, only the antisense strategy was effective in vivo, after inoculation in the hippocampus in a model of status epilepticus in which BDNF mRNA levels are strongly increased. Interestingly, the knocking down of BDNF levels induced with BDNF-antisense was sufficient to produce significant behavioral effects, in spite of the fact that it was produced only in a part of a single hippocampus. In conclusion, this study demonstrates a reliable effect of amplicon vectors in knocking down gene expression in vitro and in vivo. Therefore, this approach may find broad applications in neurobiological studies.

Fig 1. Structure of the amplicon plasmids.

(A) The pAM2-BDNF-antisense-GFP plasmid (6.84 Kb) results by insertion in antisense orientation of a fragment (1.1 Kb) containing the BDNF sequence and a poly-A tail. (B) In the pAM-CT-BDNF-GFP plasmid (7.07 Kb), the BDNF sequence (1.1 Kb) is inserted in convergent transcription, between two CMV promoters. (C) The control plasmid, pAM2-GFP plasmid (5.70 Kb). These 3 plasmids were used to produce stocks of amplicon vectors at high purities (see text).

Fig 2. In vitro validation of the amplicon vectors.

(A to D) Infection of mesoangioblast cells (MABs) constitutively expressing BDNF with the BDNF-antisense-GFP amplicon vector at MOI 5. Infection of the cells with amplicon vectors was confirmed by GFP fluorescence (A) and GFP detection on western blot (C). Pro-BDNF expression was analyzed by western blot in the 4 days following infection and pro-BDNF signal was normalized to α-actin for quantification (D). (E to H) Infection of MABs with the BDNF-CT-GFP amplicon vector at MOI 5. Infection of the cells was confirmed by GFP fluorescence (E) and GFP detection on western blot (G). Pro-BDNF expression was analyzed by western blot in the 4 days following infection and pro-BDNF signal was normalized to α-actin for quantification (H). Data in D and H are the mean±SEM of 6 experiments. * p<0.05, **p<0.01, ***p<0.001: ANOVA and post-hoc Dunnett test. Horizontal bars in panels A, B, E and F = 25 μm.

Fig 3. Absence of overt amplicon vector-induced toxicity after injection in the dorsal hippocampus.

Dentate gyrus (DG) of the dorsal hippocampus injected (ipsilateral) and non-injected (controlateral) with BDNF-antisense-GFP or with BDNF-CT-GFP amplicon vector. Nuclei are marked by DAPI in blue, GFAP-positive astrocytes in red, IBA-1-positive microglia in green and neuronal nuclei are labeled by NeuroTrace in magenta. Horizontal bars = 100 μm.

Fig 4. Transgene expression following injection of amplicon vectors in the right and left hippocampus at different time points after pilocarpine-induced status epilepticus.

(A) Representative GFP immunofluorescence in the dorsal hippocampus of a rat at 5 days post injection with the BDNF-antisense-GFP amplicon vector. (B) Quantification of the pro-BDNF signal, normalized to α-actin, 3, 6 and 24 h after pilocarpine status epilepticus induced 5 days after injection of the amplicon vectors in the right dorsal hippocampus. Data in B are the mean±SEM of 4–5 rats per group. * p<0.05, ANOVA and post-hoc Dunnett test. Horizontal bar in A = 250 μm.

Fig 5. Behavioral effects.

(A) Time to enter convulsive status epilepticus following administration of the different doses of BDNF-antisense-GFP vector. * p<0.05, ANOVA and post-hoc Dunnett test. (B) Mortality of pilocarpine-treated animals injected with the different doses of BDNF-antisense-GFP amplicon vector. Data in are the mean±SEM of 10–14 animals.