Abstract


Y. Peng Loh*1, Lin Cong2, Yong Cheng2, Niamh X Cawley3 and Saravana Murthy4
1NICHD, National Institutes of Health, Bethesda, MD, 2NICHD, NIH, Bethesda, 3National Institutes of Health, Bethesda, MD, 4National Institutes of Health

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Y. Peng Loh*1, Lin Cong2, Yong Cheng2, Niamh X Cawley3 and Saravana Murthy4
1NICHD, National Institutes of Health, Bethesda, MD, 2NICHD, NIH, Bethesda, 3National Institutes of Health, Bethesda, MD, 4National Institutes of Health

Carboxypeptidase E (CPE) is a prohormone processing enzyme. It is an exopeptidase that cleaves C-terminal basic residues from endoproteolytically cleaved peptide hormones. The human CPE gene is on chromosome 4q32.3 and recently a truncating homozygous mutation was found in a woman who presented clinical features including obesity, type 2 diabetes, hypogonadotrophic hypogonadism and intellectual disability. Furthermore a human mutation with 3 adenosine insertions in the CPE gene identified in an Alzheimer Disease patient was found to lead to neurodegeneration, memory deficits and depressive-like behavior in a mouse model. Previous in vitro studies have indicated that CPE acts extracellularly as a signaling molecule to mediate neuroprotection against oxidative stress through activation of ERK/AKT signaling pathway, and up-regulation of pro-survival protein, BCL2. Here we investigated a carboxypeptidase E mutation consisting of a T to C SNP at bp 980 of exon 4, which results in Tryptophan (W) to Arginine (R) substitution at codon 235 (W235R) found in 12.5 % of the AGI_ASP population of patients made up of 40 African-Americans and Caucasians (dbSNPrs cluster id: rs34516004), to determine if the mutant protein has lost its neuroprotective activity. Through in vitro studies we show that the SNP caused loss of enzymatic activity in the TC-CPE protein. Overexpression of this TC-CPE mutant protein in Neuro2a cells showed that it was poorly secreted compared to CPE-WT, and was retained in the endoplasmic reticulum (ER), causing ER stress, as demonstrated by the increased expression of the marker, CHOP. Double labeling of CPE and calnexin (an ER marker) suggested the accumulation of TC-CPE in the ER. Moreover, the cells were rounded characteristic of degenerating cells. The accumulation the TG-CPE was enhanced by the treatment with the proteasome inhibitor, MG132, in the cells, indicating that the mutant protein was degraded in proteosomes. Furthermore, the CPE mutant was able to “hijack” the WT-CPE into the degradation pathway. While Neuro2A cells transfected with WT-CPE showed reduced cytotoxicity when challenged with H2O2 compared to cells expressing an empty vector, cells transfected with TC-CPE had no neuroprotective effect. Our present study identified a new SNP in the human CPE gene which leads to loss of its function in neuroprotection. Such a CPE mutation could lead to neurodegeneration and dementia in humans. Thus CPE is a potential therapeutic target for treatment of neurodegenerative diseases. To this end, we have found that the anti-diabetic drug, rosiglitazone, can up-regulate the expression of CPE and can therefore be a candidate drug for treatment of dementia.
 

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