Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors.

The transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein-labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter-transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

The effect of alpha-synuclein knockdown on MPP+ toxicity in models of human neurons.

The protein alpha-synuclein is central to the pathophysiology of Parkinson's disease (PD) but its role in the development of neurodegeneration remains unclear. alpha-Synuclein-knockout mice develop without gross abnormality and are resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial inhibitor widely used to model parkinsonism. Here we show that differentiated human dopaminergic neuron-like cells also have increased resistance to 1-methyl-4-phenylpyridine (MPP+), the active metabolite of MPTP, when alpha-synuclein is knocked down using RNA interference. In attempting to understand how this occurred we found that lowering alpha-synuclein levels caused changes to intracellular vesicles, dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2), each of which is known to be an important component of the early events leading to MPP+ toxicity. Knockdown of alpha-synuclein reduced the availability of DAT on the neuronal surface by 50%, decreased the total number of intracellular vesicles by 37% but increased the density of VMAT2 molecules per vesicle by 2.8-fold. However, these changes were not associated with any reduction in MPP+ -induced superoxide production, suggesting that alpha-synuclein knockdown may have other downstream effects which are important. We then showed that alpha-synuclein knockdown prevented MPP+ -induced activation of nitric oxide synthase (NOS). Activation of NOS is an essential step in MPTP toxicity and increasing evidence points to nitrosative stress as being important in neurodegeneration. Overall, these results show that as well as having a number of effects on cellular events upstream of mitochondrial dysfunction alpha-synuclein affects pathways downstream of superoxide production, possibly involving regulation of NOS activity.

Delivering RNA interference to the mammalian brain.

RNA interference (RNAi) is a new modality in gene therapy which can elicit down-regulation of gene expression and has enormous potential in the treatment of neurological diseases. RNAi is a conserved system through which double stranded RNA (dsRNA) guides sequence specific mRNA degradation. The RNAi apparatus may be artificially triggered by delivery of naked siRNA molecules or by plasmid-based expression of dsRNA. Before these techniques can be used as effective treatments in the brain, efficient methods of in vivo delivery must be devised. This review first describes the mechanism of RNAi, and then critically examines both viral and non-viral methods for delivery of RNAi to the mammalian brain. There have been a number of important recent publications in this field and the progress towards effective in vivo delivery of RNAi to the central nervous system is discussed. Finally, potential problems that must be considered before applying this technology to the human brain are outlined.

RNA interference-mediated knockdown of alpha-synuclein protects human dopaminergic neuroblastoma cells from MPP(+) toxicity and reduces dopamine transport.

The critical observation in the pathology of Parkinson's disease (PD) is that neurodegeneration is largely restricted to dopaminergic neurons that develop cytoplasmic inclusions called Lewy bodies. These aggregations contain the protein alpha-synuclein. Furthermore, it is becoming apparent that alpha-synuclein expression levels are a major factor in PD pathogenesis. Patients with additional copies of the alpha-synuclein gene develop PD with a severity proportional to levels of alpha-synuclein overexpression. Similarly, overexpression of alpha-synuclein in in vitro and in vivo models has been shown to be toxic. However, little is known about the effects of reducing alpha-synuclein expression in human neurons. To investigate this, we have developed a system in which levels of alpha-synuclein can be acutely suppressed by using RNA interference (RNAi) in a physiologically relevant human dopaminergic cellular model. By using small interfering RNA (siRNA) molecules targeted to endogenous alpha-synuclein, we achieved 80% protein knockdown. We show that alpha-synuclein knockdown has no effect on cellular survival either under normal growth conditions over 5 days or in the presence of the mitochondrial inhibitor rotenone. Knockdown does, however, confer resistance to the dopamine transporter (DAT)-dependent neurotoxin N-methyl-4-phenylpyridinium (MPP(+)). We then demonstrate for the first time that alpha-synuclein suppression decreases dopamine transport in human cells, reducing the maximal uptake velocity (V(max)) of dopamine and the surface density of its transporter by up to 50%. These results show that RNAi-mediated alpha-synuclein knockdown alters cellular dopamine homeostasis in human cells and may suggest a mechanism for the increased survival in the presence of MPP(+), a toxin used extensively to model Parkinson's disease.