Effects of Targeted Suppression of Glutaryl-CoA Dehydrogenase by Lentivirus-Mediated shRNA and Excessive Intake of Lysine on Apoptosis in Rat Striatal Neurons

Abstract

In glutaric aciduria type 1 (GA1), glutaryl-CoA dehydrogenase (GCDH) deficiency has been shown to be responsible for the accumulation of glutaric acid and striatal degeneration. However, the mechanisms by which GA1 induces striatal degeneration remain unclear. In this study, we aimed to establish a novel neuronal model of GA1 and to investigate the effects of GCDH deficiency and lysine-related metabolites on the viability of rat striatal neurons. Thus we constructed a lentiviral vector containing short hairpin RNA targeted against the GCDH gene expression (lentivirus-shRNA) in neurons. A virus containing a scrambled short hairpin RNA construct served as a control. Addition of lysine (5 mmol/L) was used to mimic hypermetabolism. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis was assessed using Hoechst33342 staining and Annexin V-PE/7-AAD staining. The mitochondrial membrane potential (MPP) was monitored using tetramethylrhodamine methyl ester. The expression levels of caspases 3, 8, and 9 were determined by Western blotting. We found that lentivirus-shRNA induced apoptosis and decreased MMP levels in neurons, and addition of 5 mmol/L lysine enhanced this effect markedly. Lentivirus-shRNA upregulated the protein levels of caspases 3 and 9 regardless of the presence of 5 mmol/L lysine. The expression level of caspase 8 was higher in neurons co-treated with lentivirus-shRNA and 5 mmol/L lysine than in control. Benzyloxy-carbonyl-Val-Ala-Asp(OMe)-fluoromethylketone, a pan-caspase inhibitor, blocked the apoptosis induced by lentivirus-shRNA and 5 mmol/L lysine to a great extent. These results indicate that the targeted suppression of GCDH by lentivirus-mediated shRNA and excessive intake of lysine may be a useful cell model of GA1. These also suggest that GA1-induced striatal degeneration is partially caspase-dependent.

Mechanistic Effects of Amino Acids and Glucose in a Novel Glutaric Aciduria Type 1 Cell Model

“…To assess the purity of the neurons, the stained cells were washed, subjected to fluorescence microscopy (Olympus BX51 AX-70, Tokyo, Japan), and analyzed using Image-Pro plus 6.0 (Media Cybernetics, Bethesda, MD, USA). A lentivirus-mediated short hairpin RNA (shRNA) targeting the rat GCDH gene (lentivirus-shRNA, Genechem, Shanghai, China) was successfully constructed in a previous study [30] . The primary striatal neurons were maintained in maintenance medium for 5 days before transduction.…”

“…The primary striatal neurons were maintained in maintenance medium for 5 days before transduction. Transduction was conducted as described by Gao et al [30] The transduction efficiency was assessed using fluorescence microscopy at 72 h after the transduction of the primary cells with a negative control (NC) lentivirus (Genechem) containing an empty vector that expresses enhanced green fluorescent protein (eGFP) at a multiplicity of infection (MOI) = 10 ( Figure S1 ). To generate GA1 model cells, the primary striatal neurons were transduced using lentivirus-shRNA (MOI = 10).…”

“…Total RNA was extracted from the striatal neurons plated in 6-well plates using TRIzol (Invitrogen, Life Technologies, Carlsbad, CA, USA). The procedures for cDNA synthesis and real-time PCR amplification were performed according to the descriptions of Gao et al [30] . The primer sequences designed using Primer 5.0 (synthesized by Invitrogen, China) are shown in Table S1 in File S1 .…”

“…Protein was extracted from the cells seeded in 6-well plates using cell lysis buffer (Beyotime, China) and quantified using a BCA protein assay kit (Beyotime). After denaturation, the protein samples (30 µg/lane) were prepared for subsequent electrophoresis, transfer, blocking and incubation of antibodies, as described by Gao et al [30] . Antibodies against caspase-8 (1∶1000, Cell Signaling Technology, Danvers, MA, USA), caspase-9 (1∶1,000, Cell Signaling Technology), fumarase (FH) (1∶1000, Cell Signaling Technology), jun (1∶1500, Abcam), GADPH (1∶1500, Cell Signaling Technology), fos (1∶500, Abcam), the α-ketoglutarate dehydrogenase complex (OGDC) (1∶500, Santa Cruz Biotechnology, Paso Robles, CA, USA), citrate synthase (CS) (1∶500, Santa Cruz Biotechnology), caspase-3 (1∶150, Abcam), bcl2 (1∶150, Santa Cruz Biotechnology), and bax (1∶150, Santa Cruz Biotechnology) were diluted in 5% skim milk powder with 0.2% PBS-Tween 20.…”

“…Lentivirus vectors, which are one of the most widely used types of vectors for exploring genetic diseases involving neurodegeneration, can maintain stable and durable target gene suppression in non-dividing cells, including neurons [26] , [27] , [29] . We recently established novel GA1 model cells in which we used lentivirus-mediated shRNA to knock down the GCDH gene in rat striatal neurons and subsequently cultured them in lysine-rich medium to simulate striatal degeneration similar to that observed in human GA1 encephalopathic crisis [30] .…”