Characterization of the AD7C-NTP cDNA expression in Alzheimer's disease and measurement of a 41-kD protein in cerebrospinal fluid.

Abstract

We have isolated a novel Alu sequence-containing cDNA, designated AD7c-NTP, that is expressed in neurons, and overexpressed in brains with Alzheimer's disease (AD). The 1,442-nucleotide AD7c-NTP cDNA encodes an approximately 41-kD protein. Expression of AD7c-NTP was confirmed by nucleic acid sequencing of reverse transcriptase PCR products isolated from brain. AD7c-NTP cDNA probes hybridized with 1. 4 kB mRNA transcripts by Northern blot analysis, and monoclonal antibodies generated with the recombinant protein were immunoreactive with approximately 41-45-kD and approximately 18-21-kD molecules by Western blot analysis. In situ hybridization and immunostaining studies localized AD7c-NTP gene expression in neurons. Using a quantitative enzyme-linked sandwich immunoassay (Ghanbari, K., I. Beheshti, and H. Ghanbari, manuscript submitted for publication) constructed with antibodies to the recombinant protein, AD7c-NTP levels were measured under code in 323 clinical and postmortem cerebrospinal fluid (CSF) samples from AD, age-matched control, Parkinson's disease, and neurological disease control patients. The molecular mass of the AD7c-NTP detected in CSF was approximately 41 kD. In postmortem CSF, the mean concentration of AD7c-NTP in cases of definite AD (9.2+/-8.2 ng/ml) was higher than in the aged control group (1.6+/-0.9; P < 0.0001). In CSF samples from individuals with early possible or probable AD, the mean concentration of AD7c-NTP (4.6+/-3.4) was also elevated relative to the levels in CSF from age-matched (1.2+/-0.7) and neurological disease (1.0+/-0.9) controls, and ambulatory patients with Parkinson's disease (1.8+/-1.1) (all P < 0.001). CSF levels of AD7c-NTP were correlated with Blessed dementia scale scores (r = 0. 66; P = 0.0001) rather than age (r = -0.06; P > 0.1). In vitro studies demonstrated that overexpression of AD7c-NTP in transfected neuronal cells promotes neuritic sprouting and cell death, the two principal neuroanatomical lesions correlated with dementia in AD. The results suggest that abnormal AD7c-NTP expression is associated with AD neurodegeneration, and during the early stages of disease, CSF levels correlate with the severity of dementia.

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

“…Can novel genes arise de novo from previously gene-free neutrally evolving genome regions? Despite some false positives (Monte et al 1997;Kriegs et al 2005), recruitment of entire protein-coding genes out of neutrally evolving sequences does occur (Long et al 2003Heinen et al 2009;Kaessmann 2010;Carvunis et al 2012;Murphy and McLysaght 2012;Neme and Tautz 2013). Recently, it has been emphasized that many long non-protein-coding RNAs originated from TEs (Kapusta et al 2013).…”

Cerebrospinal fluid protein biomarkers for Alzheimer’s disease

“…147 Using a new ELISA method based on antibodies generated against recombinant AD7c-NTP protein, a marked increase in CSF-AD7c-NTP was found in AD compared with controls and other neurological disorders including PD and multiple sclerosis. 147 Although initial studies reported a good separation between AD and control groups, 147,148 later studies showed a less pronounced increase, with a marked overlap between AD and both controls and patients with PD. 88 Furthermore, an evidence-based evaluation of this biomarker is rendered because, to my knowledge, the AD7c-NTP ELISA method is not commercially available; CSF samples have to be sent for analyses to the company that developed the method.…”

“…Screening a human brain cDNA library, again with the same antibodies against PTP, 144,145 a cDNA named AD7c was isolated. 147 Despite the nucleotide sequence showing no homology to PTP, this cDNA was named AD7c-NTP. 147 Using a new ELISA method based on antibodies generated against recombinant AD7c-NTP protein, a marked increase in CSF-AD7c-NTP was found in AD compared with controls and other neurological disorders including PD and multiple sclerosis.…”

“…147 Despite the nucleotide sequence showing no homology to PTP, this cDNA was named AD7c-NTP. 147 Using a new ELISA method based on antibodies generated against recombinant AD7c-NTP protein, a marked increase in CSF-AD7c-NTP was found in AD compared with controls and other neurological disorders including PD and multiple sclerosis. 147 Although initial studies reported a good separation between AD and control groups, 147,148 later studies showed a less pronounced increase, with a marked overlap between AD and both controls and patients with PD.…”

Oxidative stress and hypoxia-like injury cause Alzheimer-type molecular abnormalities in central nervous system neurons

“…It was used to measure cellular expression of CD95, mitochondrial protein, cytochrome oxidase, GAP-43, synaptophysin, and NTP. NTP was detected using the N2J1 monoclonal antibody [30] that binds to the 21-kDa form of NTP which increases in expression during experimental neuritic sprouting [31,32] and AD neurodegeneration [30,33]. The cells were fixed in Histochoice (Amresco, Solon, Ohio), permeabilized with 0.05% saponin in Trisbuffered saline (TBS; 50 mM Tris, pH 7.5, 0.9% NaCl), and blocked with Superblock-TBS (Pierce, Rockford, Ill.).…”