Retrograde nerve growth factor signaling abnormalities in familial dysautonomia
Lin Li, … , Katherine Gruner, Warren G. Tourtellotte
Lin Li, … , Katherine Gruner, Warren G. Tourtellotte
Published April 13, 2020
Citation Information: J Clin Invest. 2020;130(5):2478-2487. https://doi.org/10.1172/JCI130401.
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Retrograde nerve growth factor signaling abnormalities in familial dysautonomia

Abstract

Familial dysautonomia (FD) is the most prevalent form of hereditary sensory and autonomic neuropathy (HSAN). In FD, a germline mutation in the Elp1 gene leads to Elp1 protein decrease that causes sympathetic neuron death and sympathetic nervous system dysfunction (dysautonomia). Elp1 is best known as a scaffolding protein within the nuclear hetero-hexameric transcriptional Elongator protein complex, but how it functions in sympathetic neuron survival is very poorly understood. Here, we identified a cytoplasmic function for Elp1 in sympathetic neurons that was essential for retrograde nerve growth factor (NGF) signaling and neuron target tissue innervation and survival. Elp1 was found to bind to internalized TrkA receptors in an NGF-dependent manner, where it was essential for maintaining TrkA receptor phosphorylation (activation) by regulating PTPN6 (Shp1) phosphatase activity within the signaling complex. In the absence of Elp1, Shp1 was hyperactivated, leading to premature TrkA receptor dephosphorylation, which resulted in retrograde signaling failure and neuron death. Inhibiting Shp1 phosphatase activity in the absence of Elp1 rescued NGF-dependent retrograde signaling, and in an animal model of FD it rescued abnormal sympathetic target tissue innervation. These results suggest that regulation of retrograde NGF signaling in sympathetic neurons by Elp1 may explain sympathetic neuron loss and physiologic dysautonomia in patients with FD.

Authors

Lin Li, Katherine Gruner, Warren G. Tourtellotte

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Figure 2

Abnormal retrograde TrkA receptor phosphorylation (activation) in the absence of Elp1.

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Abnormal retrograde TrkA receptor phosphorylation (activation) in the ab...
(A) TCtl and TcKO neurons grown in bulk culture and differentiated for 5–7 days were deprived of NGF for 24 hours using NGF-free medium, anti-NGF antibody (0.05 μg/mL), and BAF (50 μM) to prevent neuronal apoptosis. Cell-surface proteins were biotinylated at 4°C and the neurons were either left unchallenged or they were challenged with NGF (10 ng/mL) for 20 minutes at 37°C. Excess cell-surface biotin was stripped from the live neurons and total cellular and membrane protein lysates were obtained. Internalized biotinylated proteins were isolated by streptavidin affinity purification (AP) and the proteins were subjected to Western blotting and film densitometry. There was no detectable abnormality in TrkA receptor internalization in response to NGF in TcKO neurons (Student’s t test; P = 0.89, n = 7–9), and internalized TrkA receptors showed no difference in phosphorylation at Y490 20 minutes after NGF treatment (Student’s t test; P = 0.38, n = 3–6). (B) Similarly, DA cell-surface biotinylation in partitioned neuron cultures showed that TCtl and TcKO neurons were similar in their ability to internalize cell-surface TrkA receptors (Student’s t test; NS, P = 0.19, n = 3) and phosphorylate the receptors at Y490 within axons (green arrowhead) 20 minutes after NGF treatment (Student’s t test; NS, P = 0.92, n = 3). (C) Five hours after NGF treatment and DA cell-surface biotinylation, internalization and retrograde transport of biotinylated TrkA receptors to the cell body (green arrowhead) was similar between TCtl and TcKO neurons (Student’s t test; NS, P = 0.19, n = 8). However, the retrogradely transported TrkA axon-surface receptors were not phosphorylated in TcKO neurons compared with TCtl neurons (Student’s t test; *P < 0.00002, n = 11).