Elsevier

Brain Research

Volume 1134, 23 February 2007, Pages 33-44
Brain Research

Research Report
Effects of ex vivo transduction of mesencephalic reaggregates with bcl-2 on grafted dopamine neuron survival

https://doi.org/10.1016/j.brainres.2006.11.079Get rights and content

Abstract

Survival rates of dopamine (DA) neurons grafted to the denervated striatum are extremely poor (5–20%). Gene transfer of survival promoting factors, such as the anti-apoptotic protein bcl-2, to mesencephalic DA neurons prior to transplantation (ex vivo transduction) offers a novel approach to increase graft survival. However, specific criteria to assess the efficacy of various vectors must be adhered to in order to reasonably predict successful gene transfer with appropriate timing and levels of protein expression. Cell culture results utilizing three different herpes simplex virus (HSV) vectors to deliver the reporter β-galactosidase gene (lacZ) indicate that transduction of mesencephalic cells with a helper virus-free HSV amplicon (HF HSV-TH9lac) that harbors the 9-kb tyrosine hydroxylase (TH) promoter to drive lacZ gene expression elicits the transduction of the highest percentage (≈ 50%) of TH-immunoreactive (THir) neurons without significant cytotoxic effects. This transduction efficiency and limited cytotoxicity was superior to that observed following transduction with helper virus-containing HSV (HC HSVlac) and helper virus-free HSV amplicons (HF HSVlac) expressing lacZ under the transcriptional control of the HSV immediate-early 4/5 gene promoter. Subsequently, we assessed the ability of HSV-TH9lac and the bcl-2 expressing HSV-TH9bcl-2 amplicon to transduce mesencephalic reaggregates. Although an increase in bcl-2 and β-galactosidase protein was induced by transduction, amplicon-mediated overexpression of bcl-2 did not lead to an increase in grafted THir neuron number. Even with highly efficient viral vector-mediated transduction, our results demonstrate that ex vivo gene transfer of bcl-2 to mesencephalic reaggregates is ineffective in increasing grafted DA neuron survival.

Introduction

Parkinson's disease (PD) is a chronic, neurodegenerative disorder that affects approximately 1–2% of the population over the age of 65 (Mouradian, 2002). The disease is caused by the specific degeneration of the dopaminergic neurons of the substantia nigra pars compacta, which is likely due to cumulative effects of genetic and environmental factors. Replacement strategies for PD, such as transplantation of primary embryonic dopamine (DA) neurons, are directed at restoring lost DA neurochemistry, returning brain function to the state that existed prior to the onset of symptoms of PD. Many years of successful research on neural grafting in animal models of PD (Brundin et al., 1987, Yurek and Sladek, 1990) have led to several clinical trials worldwide (Olanow et al., 1996, Piccini et al., 1999).

While the field of transplantation continues to advance at a great pace, several challenges remain. In contrast to what might have been anticipated, double-blind clinical trials with grafting of DA neurons failed to provide clinical benefits for all patient groups and yielded dyskinetic behaviors (Freed et al., 2001, Olanow et al., 2003). It was originally hypothesized that postoperative exacerbation of dyskinesias was due to graft overgrowth in the striatum and a generalized hyperdopaminergic effect. However, data from both clinical trials (Ma et al., 2002) and animal studies (Maries et al., 2006, Steece-Collier et al., 2003) suggest that non-homogeneous DA fiber reinnervation is more likely causative. The percentage of grafted fetal DA neurons that survive transplantation is extremely low (5–20%), further limiting the ability of these grafted cells to provide innervation. The reported negative impact of grafting on dyskinesias does not imply that neural grafting has failed to fulfil its clinical promise, but rather that it remains critical to the field of cell replacement, whether the source is embryonic, stem or other, to understand the means by which enhanced survival, and therefore enhanced and homogeneous DA fiber reinnervation, can be attained.

Gene therapy for the treatment of PD has mainly been directed at the delivery of trophic factors and dopaminergic enzymes to cells of the striatum (in vivo gene therapy) (Sortwell and Kordower, 2006) or to non-neuronal cells that are subsequently grafted (ex vivo) (Bankiewicz et al., 1997, Fisher et al., 1991, Freed et al., 1990, Horellou et al., 1990, Lundberg et al., 1996, Tseng et al., 1997, Wolff et al., 1989). Very little research has investigated the feasibility of ex vivo gene delivery to mesencephalic DA neurons in an effort to increase their survival rate after grafting. In our laboratory we have observed a peak of apoptotic nuclear profiles in mesencephalic grafts immediately after implantation (1–4 days) (Sortwell et al., 2000b, Sortwell et al., 2001). These studies, along with previous findings (Barker et al., 1996, Duan et al., 1995, Emgard et al., 1999), underscore the fact that the critical interval during which grafted DA neurons are dying in grafts to rats is during the first 4 days following implantation. Therefore, neuroprotection of DA neurons to be implanted will most effectively be accomplished via ex vivo transduction that generates optimal protein expression at the time of implantation.

In the present study, we examine the ex vivo transduction of primary mesencephalic DA neurons and reaggregate cultures with different herpes simplex viral (HSV) vectors to derive an optimal set of transduction conditions and to assess the effects of amplicon-mediated delivery of the gene encoding the anti-apoptotic factor bcl-2 on graft survival and efficacy in vivo.

Section snippets

Cytotoxicity of vectors

Three HSV amplicon vectors were assessed for cytotoxic effects on transduced mesencephalic cultures in general (lactate dehydrogenase, LDH, assay) and on THir neurons specifically (counts of THir neurons). On DIV 4, all vectors displayed significantly higher LDH levels at an MOI of 2.0 when compared to their respective LDH values at MOI levels of 0.5 and 1.0 [F(8,36) = 114.525; P  0.0001]. Across vectors, both the HC HSVlac and the HF HSVlac amplicons displayed significantly higher LDH levels than

Discussion

The present group of experiments systematically evaluates three different HSV vector constructs for their ability to efficiently transduce embryonic mesencephalic DA neurons without eliciting cytotoxicity. We demonstrate that a helper virus-free preparation of an HSV vector that uses the 9-kb TH promoter to drive expression is optimal for the purpose of transducing THir neurons within the heterogeneous mesencephalic monolayer culture population. We provide evidence to validate that three

Dissection and dissociation

Ventral mesencephalic brain regions were dissected using sterile techniques from embryonic day 14 F344 rat fetuses and pooled in a cold, sterile calcium–magnesium-free buffer (CMF) as described previously (Sortwell et al., 2000a, Sortwell et al., 2000b, Sortwell et al., 2001, Sortwell et al., 2004). Cell suspensions of embryonic mesencephalic tissue were then prepared through a series of CMF rinses, incubated in 0.125% trypsin for 10 min at 37°C, rinsed in CMF again and triturated in 0.004%

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      Citation Excerpt :

      A further study from the same group analyzed the effects of ex vivo transduction of mesencephalic reaggregates with the anti-apoptotic protein bcl-2 on grafted dopamine neuron survival. Using an amplicon expressing bcl-2 under the control of the TH promoter (HSV-TH9bcl-2) to transduce mesencephalic reaggregates, it was shown that, in spite of the efficiency of the infection, since many cells were effectively transduced, amplicon-mediated overexpression of bcl-2 did not lead to an increase in grafted TH-immune-reactive neuron number (Sortwell et al., 2007). Mitochondrial alterations are detected in most neurodegenerative disorders and may contribute to the dysfunction and demise of neurons.

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