Mechanistic Insights on the Inhibition of C5 DNA Methyltransferases by Zebularine

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)—the enzymes responsible for DNA methylation—are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(β-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites

Transmission of Chronic Wasting Disease Identifies a Prion Strain Causing Cachexia and Heart Infection in Hamsters

Chronic wasting disease (CWD) is an emerging prion disease of free-ranging and captive cervids in North America. In this study we established a rodent model for CWD in Syrian golden hamsters that resemble key features of the disease in cervids including cachexia and infection of cardiac muscle. Following one to three serial passages of CWD from white-tailed deer into transgenic mice expressing the hamster prion protein gene, CWD was subsequently passaged into Syrian golden hamsters. In one passage line there were preclinical changes in locomotor activity and a loss of body mass prior to onset of subtle neurological symptoms around 340 days. The clinical symptoms included a prominent wasting disease, similar to cachexia, with a prolonged duration. Other features of CWD in hamsters that were similar to cervid CWD included the brain distribution of the disease-specific isoform of the prion protein, PrPSc, prion infection of the central and peripheral neuroendocrine system, and PrPSc deposition in cardiac muscle. There was also prominent PrPSc deposition in the nasal mucosa on the edge of the olfactory sensory epithelium with the lumen of the nasal airway that could have implications for CWD shedding into nasal secretions and disease transmission. Since the mechanism of wasting disease in prion diseases is unknown this hamster CWD model could provide a means to investigate the physiological basis of cachexia, which we propose is due to a prion-induced endocrinopathy. This prion disease phenotype has not been described in hamsters and we designate it as the ‘wasting’ or WST strain of hamster CWD

Variably Protease-sensitive Prionopathy in an Apparent Cognitively Normal 93-Year-Old

Variably protease-sensitive prionopathy (VPSPr) is a recently described novel prion disease biochemically characterized by abnormal prion protein (PrP) which has various degrees of sensitivity to proteases and generates a distinct profile on Western blots 1. VPSPr is the second most common sporadic prion protein disease after CJD 2. All reported cases to date have exhibited cognitive, behavioral, and psychiatric deficits with an age at onset range of 48-81 years and disease duration range of 7-72 months 1-4. Initial clinical diagnoses included normal pressure hydrocephalus (NPH), dementia with Lewy bodies (DLB), or frontotemporal dementia (FTD) 1,5, but Creutzfeldt-Jakob disease (CJD) often was considered clinically after rapid decline became apparent 1. All VPSPr cases evaluated have lacked mutations in the prion protein gene (PRNP) 1,2,5. Initial reports indicated all cases were homozygous for valine at codon 129 (VV) in PRNP 1. However, more recent reports have found VPSPr in all three codon 129 genotypes 2,5-7. Here we report a case of VPSPr in which there were no known clinical manifestations of prion disease during life and yet pathognomonic findings were revealed at autopsy