Neuronal expression of the transcription factor Gli1 using the Talpha1 alpha-tubulin promoter is neuroprotective in an experimental model of Parkinson's disease.

Nigrostriatal neurons degenerate during Parkinson's disease. Experimentally, neurotoxins such as 6-hydroxydopamine (6-OHDA) in rodents, and MPTP in mice and non-human primates, are used to model the disease-induced degeneration of midbrain dopaminergic neurons. Glial-cell-derived neurotrophic factor (GDNF) is a very powerful neuroprotector of dopaminergic neurons in all species examined. However, recent reports have indicated the possibility that GDNF may, in the long term and if expressed in an unregulated manner, exert untoward effects on midbrain dopaminergic neuronal structure and function. Although GDNF remains a powerful neurotrophin, the search for alternative therapies based on alternative and complementary mechanisms of action to GDNF is warranted. Recently, recombinant adenovirus-derived vectors encoding the differentiation factor Sonic Hedgehog (Shh) and its downstream transcriptional activator (Gli1) were shown to protect dopaminergic neurons in the substantia nigra pars compacta from 6-OHDA-induced neurotoxicity in rats in vivo. A pancellular human CMV (hCMV) promoter was used to drive the expression of both Shh and Gli1. Since Gli1 is a transcription factor and therefore exerts its actions intracellularly, we decided to test whether expression of Gli1 within neurons would be effective for neuroprotection. We demonstrate that neuronal-specific expression of Gli1 using the neuron-specific Talpha1 alpha-tubulin (Talpha1) promoter was neuroprotective, and its efficiency was comparable to the pancellular strong viral hCMV promoter. These results suggest that expression of the transcription factor Gli1 solely within neurons is neuroprotective for dopaminergic neurons in vivo and, furthermore, that neuronal-specific promoters are effective within the context of adenovirus-mediated gene therapy-induced neuroprotection of dopaminergic midbrain neurons. Since cell-type specific promoters are known to be weaker than the viral hCMV promoter, our data demonstrate that neuronal-specific expression of transcription factors is an effective, specific, and sufficient targeted approach for neurological gene therapy applications, potentially minimizing side effects due to unrestricted promiscuous gene expression within target tissues.

Differentiation and transcription factor gene therapy in experimental parkinson's disease: sonic hedgehog and Gli-1, but not Nurr-1, protect nigrostriatal cell bodies from 6-OHDA-induced neurodegeneration.

We tested the activity of the dopaminergic neuron differentiation factor sonic hedgehog, its downstream transcription factor target Gli-1, and an orphan nuclear receptor, Nurr-1, necessary for the induction of the dopaminergic phenotype of nigrostriatal neurons, in an in vivo model of nigrostriatal neurodegeneration. Our preliminary experiments demonstrated that all three constructs expressed the proper molecules and that these had the predicted biological activities in vitro. We expressed the N-terminal of sonic hedgehog (ShhN) and the Gli-1 and Nurr-1 entire coding regions from highly purified, and quality controlled, replication-defective adenoviral vectors injected into the brains of rats and used the dopaminergic growth factor GDNF as a positive control. The neurotoxin 6-hydroxydopamine was used to lesion the nigrostriatal dopaminergic innervation; RAd-ShhN and RAd-Gli-1 protected dopaminergic neuronal cell bodies in the substantia nigra, but not axonal terminals in the striatum, from 6-OHDA-induced cell death, while RAd-Nurr-1 was ineffective in protecting either cell bodies or axons. RAd-GDNF was able to protect both the dopaminergic cell bodies and the striatal axon terminals. Our results establish for the first time, to the best of our knowledge, that gene transfer of ShhN and one of its target transcription factors can selectively protect dopaminergic nigrostriatal neuronal cell bodies from a specific neurotoxic insult. Selective protection of nigrostriatal dopaminergic cell bodies by the differentiation factor ShhN and the transcription factor Gli-1 was achieved in a neurotoxic model that eliminates more than 70% of the nigral neurons under consideration. Differentiation and transcription factors can thus be used for the treatment of neurodegeneration by gene therapy.

Gene therapy strategies for intracranial tumours: glioma and pituitary adenomas.

Intracranial tumours such as brain gliomas and pituitary adenomas pose a challenging area of research for the development of gene therapy strategies, both from the point of view of the severity of the diseases, to the physiological implication of gene delivery into the central nervous system and pituitary gland. On the one hand, brain gliomas are very malignant tumours, with a life expectancy of six months to a year at the most after the time of diagnosis, in spite of advances in treatment modalities which involve chemotherapy, surgery and radiotherapy. Gene therapy for these tumours is therefore a very attractive therapeutic modality which due to the severity of the disease is already in clinical trials. On the other hand, pituitary tumours are usually benign, and in most cases, treatment is successful. Nevertheless, there are some instances, especially with the macroadenomas and some invasive tumours in which treatment fails. Gene therapy strategies for these adenomas therefore needs to progress substantially in terms of safety, adverse side effects and physiological impact on the normal pituitary gland before clinical implementation. In this paper, we will review gene delivery systems both viral and non-viral and several therapeutic strategies which could be implemented for the treatment of these diseases. These include cytotoxic approaches both conditional and direct, immune-stimulatory strategies, anti-angiogenic strategies and approaches which harness pro-apoptotic and tumour suppressor gene targets. We will also review the models which are currently available in which these gene therapy strategies can be tested experimentally. This new therapeutic modality holds enormous promise, but we still need substantial improvements both from the delivery, efficacy and safety stand points before it can become a clinical reality.

Fas ligand-transfected myoblasts and islet cell transplantation.

BACKGROUND: Expression of Fas ligand (FasL, CD95L) within the local environment of an allograft may protect from rejection by inducing apoptosis of infiltrating T cells. However, there is mounting evidence that ectopic expression of FasL stimulates an inflammatory response and targets the FasL-expressing tissue for destruction. Given the potential therapeutic applicability of FasL-based immune protection, we sought to determine whether ectopic FasL expression was detrimental and to analyze the inflammatory response induced by ectopic FasL expression in the absence of any confounding allo-immune responses. METHODS AND RESULTS: Two myoblast cell lines expressing different levels of functional FasL were produced. Co-implantation of FasL-expressing myoblasts with syngeneic islets allowed examination of the inflammatory response induced by ectopic FasL expression. In contrast to the suggested benefits of localized FasL expression, islets co-implanted with FasL-expressing myoblasts were destroyed in a vigorous inflammatory response predominated by neutrophils. Interestingly, FasL expression also had a marked anti-tumor effect. CONCLUSIONS: Unless FasL-dependent neutrophil-mediated inflammation can be prevented, it is unlikely that this strategy will be useful for preventing allograft rejection.

Neuropeptide tyrosine-like immunoreactivity (NPY-LI) in ganglion neurons in the adrenal gland of the flat snake (Waglerophis merremii)

In previous morphological and histochemical studies on the adrenal gland of the flat snake, no data demonstrating the existence of ganglion neurons has been reported. The aim of this paper was therefore to establish the presence of ganglion neurons in the adrenal gland of the flat snake Waglerophis merremii and, further to study their chemical phenotype using immunohistochemistry. Our results showed the presence of cells which were immunolabelled with the neuronal marker neurofilament 10 and were thus identified as large ganglion neurons. These cells were localized in the dorsal ribbon of the gland, suggesting a noradrenergic phenotype, exhibited long processes with a longitudinal direction and co-expressed neuropeptide tyrosine- (NPY) and tyrosine hydroxylase-like immunoreactivities (-LI).In addition, NPY-immunoreactive (-IR) fibers were recognized with a wide distribution throughout the gland whereas vasoactive intestinal polypeptide (VIP)-IR fibers were only observed between clusters of cortical and adrenergic chromaffin cells. No cells containing VIP-LI were detected within the gland. Based on their histochemical phenotype, ganglion cells containing NPY and TH could correspond to ganglion neurons type I of the rat. The possible absence of type II ganglion neurons in the adrenal gland of the snake is discussed

Neuropeptide tyrosine-like immunoreactivity (NPY-LI) in ganglion neurons in the adrenal gland of the flat snake (Waglerophis merremii).

In previous morphological and histochemical studies on the adrenal gland of the flat snake, no data demonstrating the existence of ganglion neurons has been reported. The aim of this paper was therefore to establish the presence of ganglion neurons in the adrenal gland of the flat snake Waglerophis merremii and, further to study their chemical phenotype using immunohistochemistry. Our results showed the presence of cells which were immunolabelled with the neuronal marker neurofilament 10 and were thus identified as large ganglion neurons. These cells were localized in the dorsal ribbon of the gland, suggesting a noradrenergic phenotype, exhibited long processes with a longitudinal direction and co-expressed neuropeptide tyrosine- (NPY) and tyrosine hydroxylase-like immunoreactivities (-LI). In addition, NPY-immunoreactive (-IR) fibers were recognized with a wide distribution throughout the gland whereas vasoactive intestinal polypeptide (VIP)-IR fibers were only observed between clusters of cortical and adrenergic chromaffin cells. No cells containing VIP-LI were detected within the gland. Based on their histochemical phenotype, ganglion cells containing NPY and TH could correspond to ganglion neurons type I of the rat. The possible absence of type II ganglion neurons in the adrenal gland of the snake is discussed.