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, 6 (11), e1966

The Point Mutation UCH-L1 C152A Protects Primary Neurons Against Cyclopentenone Prostaglandin-Induced Cytotoxicity: Implications for Post-Ischemic Neuronal Injury

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The Point Mutation UCH-L1 C152A Protects Primary Neurons Against Cyclopentenone Prostaglandin-Induced Cytotoxicity: Implications for Post-Ischemic Neuronal Injury

H Liu et al. Cell Death Dis.

Abstract

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ(12,14)-prostaglandin J2 (15dPGJ2), are reactive prostaglandin metabolites exerting a variety of biological effects. CyPGs are produced in ischemic brain and disrupt the ubiquitin-proteasome system (UPS). Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain-specific deubiquitinating enzyme that has been linked to neurodegenerative diseases. Using tandem mass spectrometry (MS) analyses, we found that the C152 site of UCH-L1 is adducted by CyPGs. Mutation of C152 to alanine (C152A) inhibited CyPG modification and conserved recombinant UCH-L1 protein hydrolase activity after 15dPGJ2 treatment. A knock-in (KI) mouse expressing the UCH-L1 C152A mutation was constructed with the bacterial artificial chromosome (BAC) technique. Brain expression and distribution of UCH-L1 in the KI mouse was similar to that of wild type (WT) as determined by western blotting. Primary cortical neurons derived from KI mice were resistant to 15dPGJ2 cytotoxicity compared with neurons from WT mice as detected by the WST-1 cell viability assay and caspase-3 and poly ADP ribose polymerase (PARP) cleavage. This protective effect was accompanied with significantly less ubiquitinated protein accumulation and aggregation as well as less UCH-L1 aggregation in C152A KI primary neurons after 15dPGJ2 treatment. Additionally, 15dPGJ2-induced axonal injury was also significantly attenuated in KI neurons as compared with WT. Taken together, these studies indicate that UCH-L1 function is important in hypoxic neuronal death, and the C152 site of UCH-L1 has a significant role in neuronal survival after hypoxic/ischemic injury.

Figures

Figure 1
Figure 1
Adduct formation by 15dPGJ2 with cysteine152 is associated with decreased UCH-L1 hydrolase activity. (a) MS/MS spectrum of a tryptic fragment derived from Flag-tagged UCH-L1 expressed in primary neuronal cells following incubation with 15dPGJ2. Schematic representation of the amino-acid sequence, fragment ions, and the corresponding m/z values for the cysteine-modified tryptic peptide NEAIIQAAHDSVAQEGQC*R. 15dPGJ2 adduction (*) is shown to occur at C152. The spectrum was from an average of six tandem mass spectra from the doubly charged peptide ion at m/z 748.36 observed as eluted from C18 nanoLC separation with a retention range of 56.87–57.07 min. (b) Hydrolase activity in recombinant wild-type (WT) and mutant UCH-L1 C152A (C152A) proteins after incubation with 12.5 μM 15dPGJ2 for 2 h measured at 0–12 min post substrate addition. Data are in arbitrary fluorescence units (AFUs) normalized to their respective time 0 and are expressed as means±S.E. n=2 per group. *P<0.05 between recombinant UCH-L1 C152A and WT 15dPGJ2-treated groups using repeated measures ANOVA with Bonferroni post hoc testing
Figure 2
Figure 2
Overexpression of UCH-L1 C152A in rat primary neurons protects cells against 15dPGJ2-induced protein aggregation and cell death. Rat primary neurons were infected with flag-tagged lentivirus (LV)-UCH-L1 wild-type (WT) or LV-UCH-L1 C152A (C152A) at DIV2, then treated with 5 μM 15dPGJ2 (15d) or vehicle (Veh) for 48 h at DIV10. (a) Cell death as measured by LDH release, normalized to respective vehicle control. N=6–12 per group. *P>0.05; #P<0.01 versus UN; Student's t-test. (b) Representative photos of LV-infected rat primary neurons after anti-flag immunocytochemistry (red, UCH-L1). Green is EGFP (indicating lentiviral infection) and blue is DAPI nuclear stain. Bar=10 μm. Photos taken with an Olympus confocal microscope at 240 × . (c) UCH-L1 particle counts per cell: UCH-L1 particle sizes were measured and counted using NIH ImageJ software (National Institutes of Health). n=23–25 per group. *P<0.05 using repeated measures ANOVA with Bonferroni post hoc testing. (a and c) Data are means±S.E.
Figure 3
Figure 3
Generation of the UCH-L1 C152A knock-in (KI) mouse. (a) Schematic representation of homologous recombination of DNA fragments producing a point mutation in UCH-L1 converting the 152 cysteine to alanine. (b) UCH-L1 protein expression in UCH-L1 C152A KI and wild-type (WT) mouse brain cortex, hippocampus, and striatum. Brain regions (n=3 per group) were lysed and immunoblotted using anti-UCH-L1 and anti-GAPDH antibodies. Left: immunoblots; right: Graphical densitometric immunoblot analysis. (c) UCH-L1 protein expression in mouse UCH-L1 WT and KI primary neurons produced from UCH-L1 C152A KI and WT mice (n=4 per group). Left: immunoblots; right: Graphical densitometric immunoblot analysis. (b and c) Data are means±S.E. and normalized to their respective WT groups. GAPDH was used as a loading control
Figure 4
Figure 4
The UCH-L1 C152A mutation confers protection against 15dPGJ2-induced apoptotic cell death. (a and b) UCH-L1 C152A knock-in (KI) and wild-type (WT) primary neurons were treated with 15dPGJ2 or vehicle (DMSO, Veh) for 24 h. (a) Cell viability (WST-1 assay) after treatment with 5–15 μM 15dPGJ2. N=6 per group. (b) Immunoblot detection of cleaved caspase 3 (Casp 3), pro Casp 3 (upper group), and PARP (full length: full, cleaved: clvd, lower group) using anti-caspase 3 and anti-PARP antibodies, respectively, after treatment with 2.5 or 5 μM 15dPGJ2. GAPDH was used as a loading control. Left: Graphical densitometric analysis of immunoblots (right). N=3 per group. All: Data are means±S.E. and are normalized to their respective vehicle controls. **P<0.01 versus WT; NS, not significant. Black bar: WT; gray bar: UCH-L1 C152A
Figure 5
Figure 5
The UCH-L1 C152A mutation attenuates 15dPGJ2-induced protein aggregation in primary neurons. (a and b) Wild type (WT) and UCH-L1 C152A knock-in (KI) primary neurons were treated with 2.5 μM 15dPGJ2 or vehicle (DMSO, Veh) for 24 h then immunostained with anti-ubiquitin (green) and anti-UCH-L1 (red) antibodies. (a) Ubiquitin (left) and UCH-L1 (right) particles were counted using ImageJ software (National Institutes of Health, n=23–25 cells per group). *P<0.05; **P<0.01 using one-way ANOVA with Bonferroni post hoc testing. (b) Representative fluoromicrographs of cells measured in (a). Blue is DAPI nuclear stain. Bar=10 μm. Arrows indicate aggregates. (c) WT and KI cell lysates were prepared from WT and KI primary neurons treated with 15dPGJ2 or Vehicle (V) for 24 h and fractioned into RIPA-soluble and -insoluble fractions. (c) Representative immunoblots detecting poly-ubiquitin (left group) and Ubiquitin K48 (right group) in each fraction. (d) Immunoblot of ubiquitin K63 level in RIPA-soluble fraction. Corresponding densitometric immunoblot analysis is shown below. Data are normalized to their respective vehicle-treated groups. β-actin was used as a loading control. N=4 per group. *P<0.05; **P<0.01 versus WT. (a and c) Data are means±S.E.
Figure 6
Figure 6
The UCH-L1 C152A mutation protects 15dPGJ2-induced injury to neurites. Wild type (WT) and UCH-L1 C152A knock-in (KI) primary neurons were incubated with 1.25–5 μM 15dPGJ2 or vehicle (Veh) for 24 h then immunostained with anti-NeuN (green) and anti-neurofilament L (red) antibodies. Blue is DAPI nuclear stain. (a) Representative confocal fluoromicrographs taken at 240 × . Bar=20 μm. (b and c) Intact neurites (outlined arrows) and neurite fragments (solid arrows) were counted in eight fields per group and are normalized to the number of cells examined. Data are means±S.E. **P<0.01 versus WT; NS, not significant

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