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 a 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 dimensional mesencephalic reaggregates similarly are efficiently transduced within 3 days of HF HSV-TH9 vector exposure to express either ß-galactosidase or bcl-2 protein as confirmed by both immunohistochemistry and Western blots. With optimal transduction protocols identified, combined with confirmation of protein expression at the time of grafting, we were appropriately poised to definitively determine whether bcl-2 overexpression is capable of increasing grafted DA neuron survival. Our results indicate that gene transfer of bcl-2 does not prevent the cell death experienced by grafted cells after implantation and therefore does not represent an effective approach to increase grafted DA neuron survival.
The assessment of the potential toxicity of overexpression of native or exogenous proteins is critical to successful gene therapy. Previous studies in which primary cortical neurons were transduced with either helper or helper-free HSV packaged amplicon virus stocks expressing different reporter genes indicated that the toxicity observed in these cultures was related to the type of reporter gene expressed, not the presence of helper virus, with lacZ expression the least toxic of the reporter genes examined [12
]. In our studies using primary mesencephalic cells we found similar results in that the presence of helper virus did not impact the toxicity observed in the mesencephalic cultures in general. However, the presence of helper virus was significantly more toxic to the THir neurons in these same cultures.
Transduction of mesencephalic cultures with the HF TH9lac vector construct yielded significantly lower THir neurons at PID 4 compared to cultures transduced with HF HSVlac. However, by PID 7 the number of THir neurons in cultures transduced with TH9lac (MOIs 1.0, 2.0) had returned to the numbers observed with HF HSVlac transduction. This transient loss of TH phenotype is not indicative of toxicity of the TH9lac vector. In fact, analysis of LDH levels reveal that the TH9lac vector construct is significantly less toxic than both the HC and HF HSV vectors. Based on these results, as well as our results demonstrating superior transduction rate of THir neurons, we selected the HF TH9lac vector construct for our transplantation studies.
We have previously reported that apoptotic cell death of grafted cells peaks immediately after implantation [38
]. This underscores the concept that the fate of grafted DA neurons is determined during the immediate post-grafting interval, with survival after 4 days being equivalent to survival rates observed months later [4
]. Therefore, interventions aimed at augmenting grafted dopamine survival must target these critical first few days after implantation. Our in vitro results utilizing both monolayer cultures and aggregates suggests significant expression of the bcl-2 within THir neurons at the time of implantation (≈ 50%). However, by ten weeks after implantation the transduction rate of THir neurons had decreased markedly to approximately 2%. This down-regulation of transgene expression after only a few days is a prevailing characteristic of HSV amplicon-based vectors [12
] although slightly longer expression stability has been reported by using the TH promoter [48
]. For the purposes of improving graft survival, down-regulation of bcl-2 after a few days should prove adequate and in fact, may be optimal. With HSV-TH9bcl-2-mediated transduction bcl-2 expression is in essence “regulatable” with bcl-2 expression maximal when newly grafted cells are bombarded by numerous triggers of cell death [37
] and subsequently down-regulated when these challenges have past.
Bcl-2 is a member of a gene family that can be functionally subdivided into two groups with one group promoting apoptotic cell death (Bax, Bak) and the other group suppressing it (Bcl-2, Bcl-Xl) [51
]. Our present findings rule out bcl-2 overexpression as a method to increase grafted DA neuron survival. However, bcl-2 overexpression does protect nigral DA neurons from MPP+
toxicity in culture [33
] and programmed cell death during development [23
]. Given these findings it appears that the ability of bcl-2 overexpression to provide neuroprotection for nigral DA neurons depends upon the type of insult. Newly grafted DA neurons experience very specific kinds of insults that are unique to the grafting situation, i.e. anoikis, hypoxia/ischemia, trophic factor withdrawal [37
This study is not the first attempt to utilize bcl-2 overexpression to protect grafted DA neurons after transplantation. Initially, an immortalized cell line derived from rat mesencephalon was transduced with a retrovirus encoding bcl-2 and subsequently grafted [2
]. Two different groups have also examined the effect on graft survival of grafting mesencephalic suspensions from mice genetically engineered to overexpress bcl-2 [1
]. None of these three studies reported differences in cell survival between the bcl-2 overexpressing treatment groups and controls; however, enhanced THir neurite extension with bcl-2 overexpression was reported [21
]. This finding is not surprising given that the bcl-2 molecule has been demonstrated to have the capacity to induce and maintain axonal growth [10
]. The authors concluded from their findings that cell death in grafted cells can circumvent regulation by bcl-2 [36
]. While our present findings support this conclusion, these studies could not rule out the possibility that inadequate levels of bcl-2 were expressed at the time of implantation to provide protection. Given that grafted cells die immediately following implantation [38
], bcl-2 must be overexpressed during this interval in order to promote graft survival. While the aforementioned studies confirmed bcl-2 overexpression, this confirmation was not conducted immediately prior to implantation. Instead, verification of bcl-2 protein expression was conducted via Western blots of mesencephalic tissue pieces, post mortem examination of transgenic tissue or in vitro
at 48 hours after plating [21
]. Bcl-2 overexpression was not demonstrated after tissue dissociation, a disruptive process that can temporarily interrupt protein expression and render cells quiescent. It is possible that the stress of dissociation into cell suspension downregulated bcl-2 to physiologically inactive levels during the immediate post-grafting interval. We propose that our mesencephalic reaggregate system has eliminated this potential confound thereby allowing us to definitively determine that bcl-2 overexpression, at least at the present levels, does not elicit survival promoting effects.
One advantage of gene transfer to cells prior to implantation (ex vivo gene therapy) is the ability to methodically evaluate the extent and cell-specificity of transduction prior to implantation. In the case of primary mesencephalic cell suspensions, this opportunity is critical to predicting success. Mesencephalic cells are a heterogeneous mixture of neurons (GABAergic, serotonergic, dopaminergic) as well as glia. The THir cell population within this diverse cell mixture varies (dependent on dissection size) from around 1-20%. If the gene of interest to be used to promote DA neuron survival must be expressed by the DA neurons themselves in order to elicit survival effects then the ability of the vector to infect a significant percentage of THir neurons must be established. Our experimental design has allowed us to verify that THir neurons are indeed transduced with the HF HSVTH9 vector (≈50%). Conversely, if THir neurons do not require expression of the gene and protein to be investigated in order to elicit trophic effects then transduction of the nondopaminergic cell population may suffice. This latter situation may be most applicable to ex vivo transduction of mesencephalic cells with trophic factors such as glial cell line derived neurotrophic factor (GDNF) or brain derived neurotrophic factor (BDNF).
Significant agreement has emerged from the work of researchers worldwide that specific conditions associated with the transplant procedure render grafted cells susceptible to apoptotic death. The potential triggers of apoptosis during the transplantation procedure include: hypoxia, anoikis, oxidative stress and neurotrophic factor withdrawal. Treatment strategies that aim to reduce or eliminate the triggers of grafted cell death appear to be more successful than approaches that target the downstream intracellular apoptotic cascade [37
]. In particular, treatment of mesencephalic cell suspensions with isolated trophic factors (GDNF, BDNF, NT 4/5) as well as glial-derived factors, and antioxidant therapies have demonstrated consistent survival promoting effects. Nevertheless, despite significant improvements in grafted DA neuron survival using these treatments, the overwhelming majority of grafted embryonic cells (60-70%) still do not survive the grafting procedure. Perhaps the application of gene therapy delivery of trophic factors via carefully optimized ex vivo
transduction could further improve upon the survival rates observed with trophic factor protein incubation. Future studies are underway to investigate the effectiveness of such an approach.