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

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 beta-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 (approximately 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 beta-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.

Figure 1. Cytotoxicity of Three Different HSV Amplicon Vectors

HC HSVlac, HF HSVlac and HF HSV-TH9lac were assessed for cytotoxic effects on transduced mesencephalic monolayer cultures. A. Lactate Dehydrogenase (LDH) Assay absorbance level results indicated that the highest multiplicity of infection level (MOI) of 2.0 all three vectors displayed significantly higher LDH levels than lower levels of the same vector (* = P < 0.0001, comparisons within vector treatments). Additionally, both HC HSVlac and HF HSVlac amplicons displayed significantly higher LDH levels than HF TH9lac (+ = P < 0.003, comparisons between same MOI, different vector treatments). B. Counts of THir neurons revealed that only HC HSV transduction yielded significantly fewer THir neurons due to higher levels of vector exposure (* = P < 0.0001, comparisons within vector treatments). No significant differences were observed in the number of THir neurons within cultures transduced at MOI levels of 1.0 and 2.0 with HF HSVlac on post infection day 4 (PID4) and HF TH9lac on PID7 (P ≥ 0.5, + = P < 0.0001, comparisons between same MOI, different vector treatments). Values represent the mean ± S.E.M.

Figure 2. Transduction Efficiency of Three Different HSV Amplicon Vectors

HC HSVlac, HF HSVlac and HF HSV-TH9lac were assessed for their ability to transduce mesencephalic cells in general (A, B) and THir neurons in particular (C, D). Analysis was conducted at either 4 days (A, C) or 7 days (B, D) after infection. HC HSV displayed significantly higher overall transduction efficiency at MOI concentrations of 1.0 and 2.0 on post infection day 7 (B, P < 0.0001) however 7 days after infection exposure to HF TH9-lac at an MOI of 2.0 generated significantly greater transduction efficiency of dopamine neurons within the cultures (D, P < 0.0001). * = P < 0.0001, comparisons within vector treatments; + = P < 0.003, comparisons between same MOI, different vector treatments. Values represent the mean ± S.E.M.

Figure 3. Transduction of Mesencephalic Reaggregates

Cross sections were taken through mesencephalic reaggregates 4 days after infection with either HF HSV-TH9lac (A, C) or HF HSV-TH9bcl-2 amplicon vectors. X-gal histochemistry and bcl-2 immunohistochemistry (A and B respectively) reveal efficient transduction of mesencephalic reaggregates. These same reagreggates possess numerous THir neurons (C). Scale bars in A, B, C = 100 μm.

Figure 4. Effects of Bcl-2 Transduction on Graft-Induced Recovery from Rotational Asymmetry and Grafted THir Neuron Survival

A. Unilaterally lesioned rats received grafts of equivalent numbers of mesencephalic cells that were either freshly put into suspension (Susp), aggregated in rotary culture for 3 days (Cont Agg), aggregated and transduced with HF HS TH9lac (Lac Agg) or aggregated and transduced with HF HSV TH9bcl-2 (Bcl-2 Agg). Six weeks after transplantation all graft treatments displayed statistically similar behavioral recovery profiles (* = P < 0.05, compared to baseline rotational scores). B. No significant difference was observed between treatment groups in survival of grafted THir neurons 10 weeks after implantation. Values represent the mean ± S.E.M. C. Representative photomicrographs of THir neurons within grafts of mesencephalic cell suspension (Susp), control mesencephalic reaggregates (Control), aggregates transduced with HF HSV TH9lac (Lac) and aggregrates transduced with HF HSV TH9bcl-2 (Bcl-2). Scale bar in A = 250 μm.

Figure 5. Coexpression of TH and β-gal in Mesencephalic Grafts

Mesencephalic reaggregates were transduced with HF HSV-TH9lac, implanted to the denervated striatum of rats and analyzed using double label immunofluorescence. A. 10 weeks after transplantation β-gal-ir cells (green), THir neurons (red) and neurons coexpressing both β-gal and TH (yellow) are observed. B. An individual grafted neuron (arrow) expressing TH, B. Expressing β-gal and C. Merged images. Scale bar in A = 15 μm.