Identification of novel GDNF isoforms and cis-antisense GDNFOS gene and their regulation in human middle temporal gyrus of Alzheimer disease

Abstract

Primate-specific genes and isoforms could provide insight into human brain diseases. Our bioinformatic analysis revealed that there are possibly five isoforms of human GDNF gene with different pre- and pro-regions by inter- and intra-exon splicing. By using TaqMan primer probe sets, designed between exons, we verified the expression of all isoforms. Furthermore, a novel GDNFOS gene was found to be transcribed from the opposite strand of GDNF gene. GDNFOS gene has four exons that are spliced into different isoforms. GDNFOS1 and GDNFOS2 are long noncoding RNAs, and GDNFOS3 encodes a protein of 105 amino acids. To study human GDNF and GDNFOS regulation in neurodegenerative diseases, the protein and mRNA levels were measured by Western blot and RT-quantitative PCR, respectively, in postmortem middle temporal gyrus (MTG) of Alzheimer disease (AD) and Huntington disease (HD) patients in comparison with those of normal controls. In the MTG of AD patients, the mature GDNF peptide was down-regulated; however, the transcript of GDNF isoform from human exon 2 was up-regulated, whereas that of the conserved isoform from exon 1 remained unchanged in comparison with those of normal controls. In contrast, the mature GDNF peptide and the isoform mRNA levels were not changed in the MTG of HD. The findings of novel GDNF and GDNFOS isoforms and differences in tissue expression patterns dysregulated in AD brains may further reveal the role of endogenous GDNF in human brain diseases.

FIGURE 1.

A, human GDNF/GDNFOS genomic locus, gene structures, and splicing patterns. The boxes represent exons, the lines represent introns, and the triangles represent splicing patterns. B, transcripts of GDNF and GDNFOS isoforms and TaqMan probe designs. The boxes represent spliced exons, and the TaqMan probes are marked between exon junctions.

FIGURE 2.

A, rat mature GDNF peptide expression in brain regions. First lane, precision molecular mass marker; second lane, recombinant GDNF under reduced conditions; third lane, medial prefrontal cortex; fourth lane, dorsal prefrontal cortex; fifth lane, ventral prefrontal cortex; sixth lane, temporal cortex; seventh lane, nucleus accumbens; eighth lane, dorsal striatum; ninth lane, hippocampus CA1; tenth lane, hippocampus CA2&3; eleventh lane, hippocampus dentate gyrus; twelfth lane, amygdala; thirteenth lane, thalamus; fourteenth lane, hypothalamus; fifteenth lane, cerebellum. The y axis of the bar graph represents normalized (tubulin) integrated intensity (I.I.) of Li-Cor Odyssey scan. B, lane 1, recombinant GDNF under nonreduced condition; lane 2, recombinant GDNF under reduced conditions; lane 3, precision molecular mass marker; lane 4, heart; lane 5, intestine; lane 6, kidney; lane 7, liver; lane 8, lung; lane 9, muscle; lane 10, spleen; lane 11, stomach; lane 12, testis; lane 13, spinal cord. The y axis of the bar graph represents normalized (tubulin) integrated intensity (I.I.) of Li-Cor Odyssey scan.

FIGURE 3.

GDNFOS3 nucleotide and peptide sequences. The nucleotide sequence of ORF is represented by bold uppercase letters, and the noncoding sequence is represented by lowercase letters. The nucleotide numbers are marked on the right side, and the amino acid numbers are on the left side. The signal peptide and the consensus glycosylation site sequences are underlined, and the alternative polyadenylation signals are in bold and underlined.

FIGURE 4.

A, GDNFOS1 mRNA levels in human brain region and peripheral tissue. The abbreviations are the same with the Western blot. B, GDNFOS3 peptide levels in human, hamster, and rat cell lines and tissues. Lane 1, molecular mass marker; lane 2, HEK293 cell; lane 3, SH-SY5Y cell; lane 4, CHO cell; lane 5, human MTG; lane 6, rat kidney; lane 7, rat prefrontal cortex. The left gel is the immunostaining with GDNFOS3 antibody, and the right gel is preabsorption with GDNFOS3 antigenic peptide before incubation with the primary antibody.

FIGURE 5.

Western blot analysis of GDNF mature peptide. A, GDNF mature peptide levels in control (C) and AD (A) MTG and scatter plot of AD MTG normalized by control. B, GDNF mature peptide levels in control (C) and HD (H) MTG and scatter plot of HD MTG normalized by control. The y axis is the percentage changes over control.

FIGURE 6.

GDNF and GDNFOS isoform mRNA levels in AD, HD, and control MTG. A, GDNF Ex1_4 in AD. B, GDNF-βS in AD. C, GDNFOS1 in AD. D, GDNF Ex2_4 in AD. E, GDNF Ex2_4 in HD. F, GDNF-αL in AD.