The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research.

The inherently low regenerative capacity of the CNS demands effective strategies to inhibit neurodegeneration in acute lesions but also in slowly progressive neurological disorders. Therefore, therapeutic targets that can interact with the degeneration cascade to block, not just postpone, neuronal degeneration need to be defined. Bcl-X(L), a protein protecting the integrity of the mitochondrial membrane potential, was investigated for its neuroprotective properties in a long-term in vivo model of neuronal cell death. An AAV-2-based vector was used to express both Bcl-X(L) and EGFP in retinal ganglion cells (RGCs) of the adult rat retina. Transection of the optic nerve results in degeneration of RGCs in control retinae, while Bcl-X(L)-overexpressing ganglion cells were protected from degeneration. At 2 weeks after axotomy, 94% of the transduced RGCs survived the lesion (15% in controls). For the first time, we investigated RGC survival up to 8 weeks after axotomy and detected that 46% of the Bcl-X(L)-overexpressing RGCs still survived, representing significantly increased neuroprotection compared to neurotrophin-based approaches. We could also show that the axons of AAV-Bcl-X(L)-transduced RGCs remained morphologically intact after the lesion, thus providing the basis for regeneration-inducing attempts.

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Functional characterization of disease-related proteins, their splice variants and dominant negative mutants in the context of complex CNS tissues such as brain and retina is frequently assessed by in vivo gene transfer. For correct interpretation of results it is imperative that the protein under investigation is unambiguously detected in the transduced cell types and can be distinguished from any endogenously expressed physiological variants. Therefore the first systematic evaluation of epitope tags used to trace ectopically expressed proteins in the central nervous system is presented here. Substantial differences in the performances of various epitope tag-antibody combinations with respect to sensitivity, specificity and influence of the epitope tag on the fusion protein are elucidated. Epitope tags already established for protein detection in vitro and to some extent in vivo (c-Myc, HA and FLAG tags) were immunohistochemically detected with high sensitivity. However, detection of these tags revealed problems with background staining and we also document structural and functional influence of the tags on the fusion protein. In order to prevent such unwanted side-effects, epitope tags which have not yet been used for in vivo applications (IRS, EE and AU1 tags) were characterized in brain, retina and cultured neurons. While use of the IRS and EE tags was hindered by low sensitivity or specificity, optimal results were obtained with the AU1 epitope, which may develop into a standard tool for detection of ectopic protein expression in the central nervous system.