BDNF, NT-3 and Trk receptor agonist monoclonal antibodies promote neuron survival, neurite extension, and synapse restoration in rat cochlea ex vivo models relevant for hidden hearing loss

Abstract

Neurotrophins and their mimetics are potential treatments for hearing disorders because of their trophic effects on spiral ganglion neurons (SGNs) whose connections to hair cells may be compromised in many forms of hearing loss. Studies in noise or ototoxin-exposed animals have shown that local delivery of NT-3 or BDNF has beneficial effects on SGNs and hearing. We evaluated several TrkB or TrkC monoclonal antibody agonists and small molecules, along with BDNF and NT-3, in rat cochlea ex vivo models. The TrkB agonists BDNF and a monoclonal antibody, M3, had the greatest effects on SGN survival, neurite outgrowth and branching. In organotypic cochlear explants, BDNF and M3 enhanced synapse formation between SGNs and inner hair cells and restored these connections after excitotoxin-induced synaptopathy. Loss of these synapses has recently been implicated in hidden hearing loss, a condition characterized by difficulty hearing speech in the presence of background noise. The unique profile of M3 revealed here warrants further investigation, and the broad activity profile of BDNF observed underpins its continued development as a hearing loss therapeutic.

Fig 1. M-antibodies selectively activate Trk receptors as detected by ERK phosphorylation.

(A-B) In human TrkB-expressing cells, ERK phosphorylation increased dose-dependently in response to M3, M4, M5, M6, BDNF, and NT-3, but not in response to 7,8-DHF, LM22A4, M1, M2, or M7. (C-D) The potency (C) of M3, M4, M5, and M6 in TrkB-expressing cells was comparable to BDNF, although the maximal effect (D) was reduced with M4 and M5. (E-F) In human TrkC-expressing cells, phospho-ERK increased dose-dependently in response to M1, M2, M7, NT-3 and BDNF but did not respond to treatment with M3, M4, M5, or M6. (G-H) The potency (G) of M1, M2, and M7 in TrkC-expressing cells was similar to NT-3, though M7 had a significantly lower EC₅₀. Maximal activation (H) was significantly reduced with M1, M2, and M7 compared to NT-3. Dose-response curves and max effect bar graphs are normalized to either 10nM BDNF (A-D) or 10nM NT-3 (E-H). *p<0.01, **p<0.001, ***p<0.0001. Statistical tests were not performed on panels A, B, E, and F.

Fig 2. M-antibodies and neurotrophins increase SGN survival.

(A) Protocol for SGN dissection and culture. NT = neurotrophic agent (NT-3, BDNF, M-antibodies, etc.) (B) SGNs were fixed, immunostained for neurofilament (green) and DAPI (blue), and imaged. Surviving neurons were identified as cells with intact soma and at least one neurite. (C) The number of surviving SGNs increased when cultured with neurotrophins or M-antibodies, compared to treatment with mouse IgG1 and human IgG4 isotype controls. Survival of SGNs was not improved with LM22B10 compared to a vehicle-only control, 0.01% DMSO. Results are normalized to the number of surviving SGNs in wells treated with 1nM NT-3. Blue bars = TrkB agonist, green bars = TrkC agonist, white bar = non-selective TrkB/TrkC agonist. (D) The effect on SGN survival was evaluated across a range of doses, and normalized to 1nM NT-3, as in (C). For panel (C), N = 3 to 70 wells per treatment condition; for panel (D), the numbers of treated wells are provided in the figure key.

Fig 3. BDNF, NT-3, and M3 increase SGN complexity.

(A-D) SGNs were immunostained, imaged and batch analyzed using PerkinElmer Harmony 4.6. (A) SGNs were immunostained for neurofilament (green) and DAPI (blue) and imaged at 20x. All of the images in a single well were stitched together and analyzed. The analysis detects SGN somas (B, highlighted in yellow) and neurites (C, magenta). (D) Each neurite tree is assigned to a single cell for analysis. Depending on the stitching, occasionally neurites will not meet the criteria due to gaps that exceed a user-defined threshold (D, white arrowhead). (E) The number of roots (asterisk), extremities (circles), nodes (arrows), segments (discrete sections of neurite between nodes or extremities), and total neurite length were measured for each neuron. (F) Treatment with 10nM BDNF, 1nM NT-3, or 1nM M3 increased the number of roots compared to the IgG-treated control. (G-H) The total neurite length, as well as number of extremities, segments, and nodes per cell increased with BDNF, NT-3, and M3 treatments compared to hIgG4, but the differences were not statistically significant. *p< 0.05, **p<0.005, ***p<0.001. N = 67 to 834 neurons per treatment condition.

Fig 4. BDNF, NT-3 and M3 promote neurite extension in SGN explants.

(A) Protocol for explanted SGN dissection and culture. (B) Example of SGN explants under different treatment conditions after 48-72h in culture. Neurotrophic treatments increased the number of neurites that extend from the tissue. Tissues were fixed and immunostained for neurofilament (green). (C) BDNF, NT-3 and M3 had a dose-dependent effect on the number of neurites per explant. The isotype control, human IgG4, also appeared to promote neurite outgrowth at the highest dose (100nM). (D) Explants were imaged and analyzed individually using the Harmony neuron analysis to examine neurite length, nodes (arrows), segments (arrowheads), and the number of extremities (asterisks). (E-G) BDNF, NT-3, and M3 had a strong effect on neurite complexity of SGN explants compared to IgG-treated or untreated controls. *p<0.05, **p<0.005, ***p<0.0005. P-values for BDNF, NT-3, and hIgG4 were determined by comparison to untreated SGN explants in the same experiment, and values obtained for M3 were compared to SGN explants treated with 10nM hIgG4 in the same experiment. For BDNF, N = 18 to 50; for NT-3, N = 17 to 50; for M3, N = 19 to 46; for IgG, N = 2 to 37, representing the number of explanted tissues that were combined across multiple independent experiments. For neurite counts, data was combined from nine independent experiments, and for neurite morphology, data was combined from four independent experiments.

Fig 5. NT-3, BDNF and M3 promote formation of synapses and fiber growth in cochlear explants.

(A) Protocol for cochlea explantation and culture. (B) Explants were immunostained with Myo7A (blue) to identify hair cells and neurofilament (yellow) to label SGNs. The number of type I SGNs was calculated by counting all of the fibers approaching the hair cells and then subtracting those that continue to the outer hair cell layer. Green dots and magenta arrowheads represent a few examples of the fibers that were counted. Neurotrophins and M3 increased the number of type I SGN fibers contacting inner hair cells. (C) The pre-synaptic puncta were identified by an antibody that labels CtBP2 (green). Neurotrophins and possibly M3 increased the number of presynaptic puncta. (D) The postsynaptic puncta were identified by an antibody that labels PSD-95 (red). Neurotrophins and M3 increased the number of postsynaptic puncta. Bar graphs are normalized to control explants that were maintained in culture media but not treated with neurotrophic agents. The number of explanted tissues is indicated in parentheses and are combined from two (for PSD-95) or three (for SGNs and CtBP2) independent experiments. *p<0.05, **p<0.005, ***p<0.0005 (vs. CTL).

Fig 6. NT-3, BDNF and M3 restore SGN fiber density in organotypic cochlear explants after excitotoxic trauma.

(A) Protocol for cochlea explantation and culture. (B,D) Cochlear explants cultured without excitotoxin (B) and with excitotoxin (D). Explants were immunostained with Myo7A (blue) to identify hair cells and neurofilament (yellow) to label SGNs. White arrows in (D) highlight SGN fibers that are retracting or degenerating. (C) Quantification of type I SGNs, calculated as in Fig 5. The number of type I SGNs was reduced with excitotoxin (NK) treatment, but rescued when followed with 1nM NT-3, BDNF, or M3. NK = excitotoxin (NMDA + kainate). The number of explanted tissues is indicated in parentheses and were combined from three independent experiments. *p<0.05, **p<0.005 (vs. NK).

Fig 7. NT-3, BDNF and M3 restore synaptic puncta in organotypic cochlear explants after excitotoxic trauma.

(A) Protocol for cochlea explantation and culture. Some samples were fixed and immunostained 18h after excitotoxin to evaluate short-term synapse damage. Others were evaluated for restorative effects at 72h post-excitotoxin. (B) The number of synapses was measured by counting immunostained PSD-95 puncta (red). Hair cells are labeled with Myo7A (blue). (C-F) Example images of puncta counts after excitotoxin: (C) without trophic support, (D) with BDNF, (E) with NT-3, or (F) with M3. (G) The number of PSD-95 puncta was significantly reduced 18h after excitotoxin (NK) treatment but rescued (H) when followed with NT-3 (10nM), BDNF (10nM), or M3 (1nM). The number of explanted tissues is indicated in parentheses and were combined from two (18h timepoint) or five (72h timepoint) independent experiments. *p<0.05, **p<0.005, ***p<0.0005 (vs. CTL in panel G and vs. NK in panel H).