Saturday, November 17, 2012

ABCA siRNA Transfection of Neurons & Astrocytes

Neuromics' i-FectTM Transfection Kit successfully meets the challenge of transfecting neurons and astrocytes in culture. The kit has been used for gene expression analysis studies for: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , RANK, TRPV1, Survivin, Flaviviruses, NOV, Troy β-arrestin, TRPV1 CAV1.2 TLR4 and ASIC. Related publication reference use of the kit for both in vitro and in vivo studies

In this study, Dr. Marina Guizzetti and his team use our i-Fect Kit and several other commercially available kits to knockdown ABCG1 and ABCG4: Jing Chen, Xiaolu Zhang, Handojo Kusumo, Lucio G. Costa, c, Marina Guizzettia. Cholesterol efflux is differentially regulated in neurons and astrocytes: Implications for brain cholesterol homeostasis. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1831 (2), p.263-275, Feb 2013. doi:10.1016/j.bbalip.2012.09.007.
Abstract: Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood–brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS.

siRNA transfection: ABCA1 SiRNA transfections: Neurons and astrocytes were transfected using the Nucleofector™ technology (Lonza/Amaxa; Walkersville, MD) as per the manufacturer's optimized protocol. In brief, primary neurons immediately after isolation, or astrocytes harvested after 7–10 days in vitro (DIV), were resuspended in Nucleofector solution. Aliquots of neurons or astrocytes were mixed with 200 pmol ABCA1 siRNA or non-targeting siRNA and were transfected using the Nucleofector programs O-007 and T-20 respectively. Exogenous cholesterol efflux was measured 96 h post transfection. ABCA1 down-regulation in ABCA1 siRNA-transfected cells was verified by Western blot. ABCG1 and ABCG4 Stealth RNAiTM siRNA transfections: on the day of transfection primary astrocytes were switched to a medium (DMEM with 10%FBS) without antibiotics and supplemented with 50 nM ABCG1 or ABCG4 siRNA, lipofectamine RNAiMAX Transfection Reagent, and Opti-MEM I according to the manufacturer's instruction for 24 h followed by the removal of the medium containing transfection reagents. Six days after preparation, primary cortical neurons were shifted to a medium (Neurobasal/B27) without antibiotic; transfection was carried out by adding to the cultures a solution containing 12 nM ABCG1 or ABCG4 SiRNA, i-Fect siRNA Transfection Reagent, and Opti-MEM I for 24 h. Exogenous cholesterol efflux was measured 48 h after the removal of the transfection reagents. The specific silencing of ABCG1 in astrocytes and neurons was confirmed by Western blot and by qRT-PCR; silencing of ABCG4 was confirmed only by qRT-PCR because no specific antibody to ABCG4 is available.

Figures: Effect of ABCG1 and ABCG4 silencing on cholesterol efflux from neurons and astrocytes. A: Primary rat cortical neurons were transfected with a non-target (NT siRNA), an ABCG1 specific siRNA (ABCG1 siRNA) or an ABCG4 siRNA using the i-Fect™ siRNA transfection reagent. B: Primary rat cortical astrocytes were transfected with an ABCG1 siRNA or an ABCG4 siRNA using lipofectamine RNAiMAX. Twenty-four hours after transfection, cells were labeled with 1 μCi/ml [3H]cholesterol for 24 h followed by a 6 h incubation with cholesterol acceptors. [3H]Cholesterol was quantified in the medium and in the cellular lipids (n=11–12). **pb0.01; ***pb0.001 compared to acceptor-matched controls by Student's t test. C: ABCG1 (left) and ABCG4 (right) mRNA levels were quantified by qPCR in neurons transfected with NT siRNA, ABCG1 siRNA or ABCG4 siRNA (n=4). D: the levels of ABCG1, ABCG4 and ABCA1 mRNA were determined by qPCR in ABCG1 siRNA-transfected (left) and ABCG4 siRNA transfected astrocytes (n=4). Representative immunoblots of ABCG1 levels (upper blots) and β-actin levels (lower blots) in neurons transfected with NT siRNA and ABCG1 siRNA (E) and astrocytes transfected with ABCG1 siRNA (F) and the densitometric quantification of ABCG1 levels normalized to β-actin from 4 independent determination (G) are shown.

The efficiency of transfection is higher in astrocytes than in neurons, it is possible that the lack of an effect of ABCG1 siRNA on cholesterol efflux from neurons may be due to the fact that ABCG1 is not sufficiently down-regulated in these cells. Further study on this phenomena is needed and are important for the discovery of druggable targets that could positively modulate cholesterol homeostasis.

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