The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury

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.Ubiquitin C-terminal hydrolase L1 is a multifunctional protein that is highly expressed in neurons throughout brain.¹ UCH-L1 closely interacts with proteins of the neuronal cytoskeleton and may have an important role in axonal transport and maintaining axonal integrity.^{2, 3} UCH-L1 regulates synaptic function and long-term potentiation (LTP) under normal and pathological conditions and may be involved in memory function.⁴ Mutations and altered function of UCH-L1 have been associated with neurological diseases including Parkinson''s (PD) and Alzheimer''s (AD) diseases and early onset neurodegeneration involving white matter.^{2, 3, 4, 5, 6, 7} However, the role of UCH-L1 function in cerebral ischemic injury and recovery has not been thoroughly investigated.Cyclopentenone prostaglandins (CyPGs) are the reactive metabolites of prostaglandins containing a carbonyl moiety that may covalently modify cysteine in a variety of proteins.^{8, 9, 10} CyPG concentration is dramatically increased in ischemic brain.¹¹ CyPGs such as 15dPGJ2 disrupt the ubiquitin-proteasome system (UPS), resulting in accumulation and aggregation of ubiquitinated (Ub) proteins and neuronal cell death.^{12, 13}UCH-L1 is a target of CyPG modification.^{13, 14, 15} In the current study, mass spectrometry (MS)/MS was used to determine that cysteine152 is the binding site of the CyPG 15dPGJ2 to UCH-L1. We then constructed a knock-in (KI) mouse using the bacterial artificial chromosome (BAC) technique with a cysteine to alanine mutation at this 15dPGJ2 binding site on UCH-L1. Primary neurons derived from KI and wild-type (WT) mice were used to determine the effect of CyPG binding to UCH-L1 on cell death and disruption of the UPS. These studies address a potential role for modification of UCH-L1 by CyPGs and other reactive lipid species in stroke and neurodegenerative diseases.