Our results suggest that the role of β-synuclein as a putative modulator of neuropathology in aggregopathies like Parkinson disease and dementia with Lewy bodies needs to be revisited.

No abstract

Experimentally restricting transgene expression exclusively to astrocytes has proven difficult. Using adeno-associated-virus-mediated gene transfer, we assessed two commonly used glial fibrillary acidic protein promoters: the full-length version gfa2 (2,210-bp human glial fibrillary acidic protein [GFAP] promoter) and the truncated variant gfaABC1D (681-bp GFAP promoter). The capacity to drive efficient, but also cell-type specific, expression of the EGFP in astrocytes was tested both in vitro in rat primary cortical cultures as well as in vivo in the rat striatum. We observed an efficient, but not entirely astrocyte-specific, gfa2-driven reporter expression. gfaABC1D exhibited a weaker activity, and most importantly, off-target, neuronal expression of the transgene occurred in a larger fraction of cells. Therefore, we explored the potential of a microRNA (miR)-specific target-sequence-based approach for abolishing off-target expression. When miR124 target sequences were incorporated into the 3′ UTR, neuronal gene expression was effectively silenced. However, unexpectedly, the insertion of an additional sequence in the 3′ UTR clearly diminished transgene expression. In conclusion, the gfaABC1D promoter on its own is not sufficient to specifically target transgene expression to astrocytes and is not well suited for AAV-based gene targeting, even if short promoter sequences are required. The combination with a miR de-targeting sequence represents a promising experimental strategy that eliminates off-target, neuronal expression.

Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson's disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.