Heterozygous NTF4 mutations impairing neurotrophin-4 signaling in patients with primary open-angle glaucoma

Abstract

Glaucoma, a main cause of blindness in the developed world, is characterized by progressive degeneration of retinal ganglion cells (RGCs), resulting in irreversible loss of vision. Although members of the neurotrophin gene family in various species are known to support the survival of numerous neuronal populations, including RGCs, it is less clear whether they are also required for survival and maintenance of adult neurons in humans. Here, we report seven different heterozygous mutations in the Neurotrophin-4 (NTF4) gene accounting for about 1.7% of primary open-angle glaucoma patients of European origin. Molecular modeling predicted a decreased affinity of neurotrophin 4 protein (NT-4) mutants with its specific tyrosine kinase receptor B (TrkB). Expression of recombinant NT-4 carrying the most frequent mutation was demonstrated to lead to decreased activation of TrkB. These findings suggest a pathway in the pathophysiology of glaucoma through loss of neurotrophic function and may eventually open the possibility of using ligands activating TrkB to prevent the progression of the disease.

Figure 1

Location of NT-4 Amino Acid Changes on Protein Structure and Evolutionary Conservation (A) Schematic representation of the intron-exon organization of the NTF4 gene and of the derived protein. NT-4 mature protein (starting from G81) is highlighted with gray transverse lines. All six mutations identified in this study are shown above the protein. Five variants are located in the NGF domain, and one is located in the N terminus of the preproprotein. (B) DNA chromatograms of the mutation sites. (C) Multiple amino acid sequence alignments show evolutionary conservation of mutated residues (shown in blue) among mamalian species. Amino acid changes are highlighted in red.

Figure 2

Location of the Mutations in the Crystal Structure of the NT-4-TrkB Complex The two chains of the dimeric NT-4 are colored in cyan and blue, respectively. The sites of the mutations are highlighted by magenta balls (prime symbols denote residues of the second subunit), indicating that these residues are located within or close to the TrkB binding interface. TrkB domains bound by NT-4 are colored in red and orange. Residue R209, which is C-terminally adjacent to R206, is not resolved in this crystal structure. Therefore, the approximate position of this residue is indicated in accordance with another NT-4 structure, in which the respective region is better resolved. The latter structure also provided the basis for the detailed analysis of the R209G mutation shown in Figure 3E.

Figure 3

Predicted Effects of NT-4 Amino Acid Changes on NT-4 Structure and Interaction with TrkB Contacts formed by the WT residues (left) and effects of the mutations (right). Residues of TrkB are denoted in italics. (A) Effect of the E84K mutation: The side chain of E84 is oriented toward the solvent. K84 is expected to adopt a different orientation as a result of electrostatic attraction by E293 of TrkB (green dotted line). As a consequence, the contacts from NTF4 residues 81–84, representing the N terminus of mature NT-4 to TrkB, will be disrupted (indicated by the broken line for the protein backbone). (B) Effect of the A88V mutation. The larger size of the V88 side chain results in steric clashes with L94 (red arrows), most probably causing structural rearrangement of the binding region and thus a poorer fit to the receptor. (C) Effect of the R90H mutation: R90 stacks on the side chain of H343 from TrkB. The shorter side chain of H90 results in poorer stacking in the mutant, suggesting a weaker binding. (D) Effect of the R206W and R206Q mutations: R206 forms hydrogen bonds (green dotted lines) to the backbone carbonyl of R163, thus stabilizing the three-dimensional structure of NT-4 in a region close to the receptor binding site. In the R206Q mutant, only one hydrogen bond can be formed, whereas no hydrogen bonds can be formed in the R206W mutant because of the length and chemical properties of the W side chain. (E) Effect of the R209G mutation: R209 forms a hydrogen bond (green dotted line) to the backbone carbonyl of R164, thereby stabilizing the three-dimensional structure of NT-4 in a fashion similar to that of R206 (see panel D).

Figure 4

R206W-myc NT-4 Shows Decreased TrkB Activation and Neurite Outgrowth in Comparison to WT-myc NT-4 (A) Treatment of TrkB-nnr5 cells with BDNF and NT-4 for 10 min similarly induced TrkB phosphorylation, whereas NT-3 was less active and NGF was inactive (100 ng/ml, respectively). (B) Treatment with R206W-myc NT-4 for 10 min induced TrkB activation significantly less than WT and WT-myc NT-4 (10 ng/ml, respectively). (C) Densitometry analysis of “phospho-TrkB” was performed after immunoblotting in (B). Data represent mean ± SEM (n = 4). ^∗∗∗ indicates p < 0.001 versus untreated, and ### indicates p < 0.001 versus R206W-myc NT-4. (D) Representative phase contrast images of TrkB-nnr5 cells treated with WT-myc or R206W-myc NT-4 (10 ng/ml) for 2 days. Scale bar represents 10 mm. (E) Graph shows the percentage of cells with neurites. TrkB-nnr5 cells were treated with WT, WT-myc, or R206W-myc NT-4 at 1 or 10 ng/ml for 2 days. Data represent mean ± SEM (n = 3). ^∗ indicates p < 0.05, ^∗∗∗ indicates p < 0.001 versus untreated, and ### indicates p < 0.001 versus R206W-myc NT-4 (10 ng/ml).

Figure 5

NTF4 mRNA Is Expressed in Human Retinal Ganglion Cells (A) Ganglion cells in the ganglion cell layer (GCL) label for NTF4 transcript. (B) In control sections (sense probe), no specific signal is detectable. Scale bars represent 50 mm (A) and 20 mm (B). Abbreviations are as follows: PhRs, photoreceptor segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.