Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter

Abstract

Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an approximately 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported approximately 90% glutamatergic neuron-specific expression. The GAD67 promoter supported approximately 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic neuron.

Novel strategy to selectively label excitatory and inhibitory neurons in the cerebral cortex of mice

“…The method we outline in this article enables to easily label many neurons differentially based on their neurochemical subtype (excitatory or inhibitory). While other vectors have been introduced to express various proteins in different subtypes of neurons (Rasmussen et al, 2007) our strategy has the advantage that it requires a single vector instead of two. This paper describes a method which has a great potential to investigate inhibitory and excitatory axons.…”

Transport of l-Glutamine, l-Alanine, l-Arginine and l-Histidine by the Neuron-Specific Slc38a8 (SNAT8) in CNS

“…SNAT8 localized to the cell body, in the cytosol close to the membrane and in the axon of neurons, in hypothalamus, cerebral cortex and in other areas of the brain. The microtubule-associated protein 2 (MAP2) 36 and 37 , was used to investigate if SNAT8 was expressed in excitatory glutamatergic neurons.…”

Progress and prospects: Biological properties and technological advances of herpes simplex virus type 1-based amplicon vectors

“…8 When delivered into the post-rhinal cortex, the phosphate-activated glutaminase and vesicular glutamate transporter 1 promoters supported B90% glutamatergic neuron-specific expression, whereas the glutamic acid decarboxylase 67 promoter supported B90% GABAergic neuron expression. Long-term expression, at least 2 months after injection into the cortex, was observed using these promoters.…”

“…Long-term expression, at least 2 months after injection into the cortex, was observed using these promoters. 8 In a second study, an amplicon containing a 13.5 kbp genomic fragment encoding the gonadotropin-releasing hormone (GnRH) gene, together with its cognate promoter and regulatory elements, was administrated into the hypothalamic preoptic area of hypogonadal mice, which are deficient in GnRH production because of a deletion in the GnRH gene. After vector injection, an improvement in reproductive parameters of adult female homozygous hypogonadal mice was observed.…”