Proneurotrophin-3 may induce Sortilin-dependent death in inner ear neurons

Abstract

The precursor of the neurotrophin (NT) nerve growth factor (NGF) (proNGF) serves physiological functions distinct from its mature counterpart as it induces neuronal apoptosis through activation of a p75 NT receptor (p75(NTR) ) and Sortilin death-signalling complex. The NTs brain-derived nerve growth factor (BDNF) and NT3 provide essential trophic support to auditory neurons. Injury to the NT-secreting cells in the inner ear is followed by irreversible degeneration of spiral ganglion neurons with consequences such as impaired hearing or deafness. Lack of mature NTs may explain the degeneration of spiral ganglion neurons, but another mechanism is possible as unprocessed proNTs released from the injured cells may contribute to the degeneration by induction of apoptosis. Recent studies demonstrate that proBDNF, like proNGF, is a potent inducer of Sortilin:p75(NTR) -mediated apoptosis. In addition, a coincident upregulation of proBDNF and p75(NTR) has been observed in degenerating spiral ganglion neurons, but the Sortilin expression in the inner ear is unresolved. Here we demonstrate that Sortilin and p75(NTR) are coexpressed in neurons of the neonatal inner ear. Furthermore, we establish that proNT3 exhibits high-affinity binding to Sortilin and has the capacity to enhance cell surface Sortilin:p75(NTR) complex formation as well as to mediate apoptosis in neurons coexpressing p75(NTR) and Sortilin. Based on the examination of wildtype and Sortilin-deficient mouse embryos, Sortilin does not significantly influence the developmental selection of spiral ganglion neurons. However, our results suggest that proNT3 and proBDNF may play important roles in the response to noise-induced injuries or ototoxic damage via the Sortilin:p75(NTR) death-signalling complex.

Figure 1. SPR analysis of ligand binding to immobilised Sortilin

SPR analysis of receptor-ligand interactions between the NT3 and immobilised Sortilin demonstrated that mature NT3 binds to Sortilin with a Kd ~20 μM (A). This affinity is significantly lower than the affinity found for the interaction between proNT3 and Sortilin (Kd ~20 nM) (B), and for the interaction between the receptor and the prodomain sequence of proNT3 fused to GST (Kd~20 nM) (D). The interaction between Sortilin and proNT3 can be inhibited by the well-known Sortilin ligand neurotensin (C). NT3 neurotrophin-3, proNT3 proform of NT3, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase NTS neurotensin, Kd dissociation constant, SPR Surface plasmon resonance

Figure 2. Sortilin-mediated binding and internalisation of NT3 and proNT3

(A–J) HEK293 cells transfected with Sortilin, p75^NTR or proSortilin as indicated were incubated with 50nM of the given ligand (NT3 (A–C), proNT3 (D–F), or GST-NT3pro (G–J)) for 40 minutes at 37° C. Internalised ligand was visualised by immunofluorescence labelling with specific antibodies. Mature NT3 and proNT3 were detected with anti-NT3, whereas GST-NT3pro was detected with an anti-GST antibody. Cells transfected with Sortilin showed no binding and uptake of NT3 (A), but internalisation was detected of both proNT3 (D) and GST-NT3pro (G). The uptake of proNT3 could be inhibited by addition of NTS (E). Cells transfected with p75^NTR displayed strong surface labelling after incubation with NT3 (C) and proNT3 (F), but no binding of GST-NT3pro (I). HEK293 human embryonic kidney 293 cells, NT3 neurotrophin-3, proNT3 proform of NT3, p75^NTR p75 neurotrophin receptor, NTS neurotensin, GST-NT3pro prodomain of NT3 coupled to glutathione-s-transferase.

Figure 3. Crosslinking and precipitation of p75^NTR:Sortilin:proNT3 complexes

HEK293 cells transfected with an endocytosis-impaired variant of Sortilin, Sortilin-mut, and p75^NTR as indicated were incubated with or without 50 nM ligand (proNT3 or proNGF) before treatment with crosslinker. Receptor:ligand complexes were immunoprecipitated with anti-p75^NTR, and components were visualised after reducing SDS-PAGE by immunoblotting with anti-Sortilin, anti-proNT3, and anti-p75^NTR. The expression of Sortilin and p75^NTR in input cell lysates were confirmed by immunoblotting. HEK293 human embryonic kidney 293 cells, p75^NTR p75 neurotrophin receptor, Sortilin-mut endocytosis-impaired variant of Sortilin, proNGF proform of nerve growth factor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3

Figure 4. Immunoblotting of inner ear tissue for Sortilin, p75^NTR, proNT3 and mature NT3

Subfractions of membranous labyrinth from dissected newborn rats (P3–P5) were pooled, homogenised and centrifuged. The supernatants were subjected to SDS-PAGE followed by immunoblotting for Sortilin, p75^NTR, proNT3 and mature NT3. Beta-actin (~42 kDa) blots are included for evaluation of total protein concentration. (A) Sortilin (~98 kDa) was detected in whole-organ cochleae (Co) and in all in the subfractions i.e. the stria vascularis (I), the organ of Corti with Reissner’s membrane attached (II), the modiolus (not including the spiral ganglion) (III), and the spiral ganglion (IV). (B) P75^NTR (~64 kDa) was detected in the modiolus (III) and the SG (IV). (C) ProNT3 and NT3 were detected in all subfractions. (D) The specificity of proNT3 and NT3 antibodies was tested by immunoblotting of recombinant human proNGF (lane 1 (5 ng) and 2 (1 ng)), proNT3 (lane 3 (5 ng) and 4 (1 ng)), and NT3 (lane 5 (5 ng) and 6 (1 ng)). The proNT3 antibody recognises only proNT3, while the NT3 antibody detects both the proform and the mature form of NT3. kDa kilodalton, Co whole-organ membranous cochlea, p75^NTR p75 neurotrophin receptor, proNT3 proform of neurotrophin-3, NT3 mature form of neurotrophin-3, αSortilin anti-Sortilin, αp75^NTR anti- p75^NTR, αproNT3 anti-proNT3, αNT3 anti-NT3, αbeta-actin anti-beta actin, P3–P5 postnatal day 3–5

Figure 5. Immunolabelling of Sortilin and p75^NTR in the inner ear of neonatal rats

(A–D) Expression of Sortilin (A–C) and p75^NTR (D) in the cochlea. (E–H) High magnification of the organ of Corti showing Sortilin (E) and p75^NTR (F) expression, as well as double labelling of these receptors with the hair cell marker myosin VIIa (G+H). Note the absence of Sortilin in hair cells and the co-localisation of p75^NTR with the neuronal marker (Tuj1) in nerve terminals (E–I). (J–M) Coexpression of Sortilin (J+L) and p75^NTR (K+M) in SGN. (N–O) IF detection of Sortilin in marginal cells of the stria vascularis (N) and epithelial cells of Reissner’s membrane (O) facing the cochlear duct. (P–S) Sortilin (P+R+S) and p75^NTR (Q) expression in the saccule and semicircular ducts of the vestibular apparatus. Note the differential distribution of Sortilin and p75^NTR in the sensory epithelium of the vestibular apparatus in analogy to the hair cell region of the cochlea. RM Reissner’s membrane, SV stria vascularis, MC strial marginal cells, HC hair cells, SGN spiral ganglion neurons, SG spiral ganglion, SGNF spiral ganglion nerve fibers, *) staining artifact, DE Deiters cells, HE Hensen cells, IHC inner hair cells, OHC outer hair cells, SGNT spiral ganglion nerve terminals, Tuj1class III β-tubulin, HC-I type-1 hair cells HC-II type-2 hair cells, VGNT vestibular ganglion nerve terminals, SC supporting cells, CA crista ampullaris.

Figure 6. ProNT3 induced apoptosis in SCG neurons

The apoptotic potential of proNT3 was tested in SCG neurons. Here is shown the number of apoptotic neurons in percent of the total number counted. SCG neurons were incubated in growth media containing NGF (20ng/ml), NT3 (2ng/ml), proNGF (2ng/ml) or proNT3 (4ng/ml). After 36 hours, SCG cultures were fixed, immunostained with α-Tuj1, α-Sortilin and DAPI and subjected to apoptosis analysis by morphological evaluation. The results demonstrate that NGF (as opposed to NT3) is imperative for SCG neuron survival. In contrast, both proNGF and proNT3 significantly increased the proportion of apoptotic neurons compared to NGF withdrawal alone. SCG superior cervical ganglion, proNT proneurotrophins, NGF nerve growth factor, proNGF proform of NGF, None no (pro)neurotrophins added, NT3 neurotrophin-3, proNT3 proform of NT3.

Figure 7. ProNT3 induced apoptosis in SG neurons

ProNT3-induced apoptosis was also investigated in p75^NTR- and Sortilin-coexpressing SG neurons. Cultured SGs were rinsed in NT-free medium prior to incubation in modified media containing proNT3 (4ng/ml), proNT3 (8ng/ml) or no NTs. After 36 hours, SG cultures were fixed, stained with DAPI and a neuronal marker, and subjected to apoptosis analysis by morphological evaluation. Here is shown the number of apoptotic cells in percent of total number of neurons counted. The results demonstrate that proNT3 significantly enhanced apoptosis in SG neurons compared to the effect of NT withdrawal alone. The data represents a single experiment and bars indicate standard deviation between apoptosis-ratios on the individual slides (9–12) counted for each condition (~1500 neurons per additive). SG spiral ganglion, NT neurotrophins, proNT3 proform of neurotrophin-3, No add no neurotrophins added.