TMEM184b Promotes Axon Degeneration and Neuromuscular Junction Maintenance

Abstract

Complex nervous systems achieve proper connectivity during development and must maintain these connections throughout life. The processes of axon and synaptic maintenance and axon degeneration after injury are jointly controlled by a number of proteins within neurons, including ubiquitin ligases and mitogen activated protein kinases. However, our understanding of these molecular cascades is incomplete. Here we describe the phenotype resulting from mutation of TMEM184b, a protein identified in a screen for axon degeneration mediators. TMEM184b is highly expressed in the mouse nervous system and is found in recycling endosomes in neuronal cell bodies and axons. Disruption of TMEM184b expression results in prolonged maintenance of peripheral axons following nerve injury, demonstrating a role for TMEM184b in axon degeneration. In contrast to this protective phenotype in axons, uninjured mutant mice have anatomical and functional impairments in the peripheral nervous system. Loss of TMEM184b causes swellings at neuromuscular junctions that become more numerous with age, demonstrating that TMEM184b is critical for the maintenance of synaptic architecture. These swellings contain abnormal multivesicular structures similar to those seen in patients with neurodegenerative disorders. Mutant animals also show abnormal sensory terminal morphology. TMEM184b mutant animals are deficient on the inverted screen test, illustrating a role for TMEM184b in sensory-motor function. Overall, we have identified an important function for TMEM184b in peripheral nerve terminal structure, function, and the axon degeneration pathway.

Significance statement: Our work has identified both neuroprotective and neurodegenerative roles for a previously undescribed protein, TMEM184b. TMEM184b mutation causes delayed axon degeneration following peripheral nerve injury, indicating that it participates in the degeneration process. Simultaneously, TMEM184b mutation causes progressive structural abnormalities at neuromuscular synapses and swellings within sensory terminals, and animals with this mutation display profound weakness. Thus, TMEM184b is necessary for normal peripheral nerve terminal morphology and maintenance. Loss of TMEM184b results in accumulation of autophagosomal structures in vivo, fitting with emerging studies that have linked autophagy disruption and neurological disease. Our work recognizes TMEM184b as a new player in the maintenance of the nervous system.

Keywords: autophagy; axon degeneration; intraepidermal nerve fibers; mouse; neuromuscular junction.

Figure 1.

TMEM184b is expressed in central and peripheral neurons. A, Hydrophobicity plot of fly, mouse, and human TMEM184b showing seven transmembrane domains. B, Relative expression of TMEM184b mRNA in wild-type tissues by quantitative PCR (n = 3–4 animals per tissue). S.C., Spinal cord; aDRG, adult dorsal root ganglia. Error bars are SEM. C, Quantitative PCR of TMEM184 family members from brain tissue of wild-type and mutant animals. Data are normalized to wild-type TMEM184b expression. n.s., Not significant. Student's t test values for wild-type versus mutant: TMEM184a, p = 0.62; TMEM184b, p = 2.0 × 10⁻⁶; TMEM184c, p = 0.26. D, E, Sections of adult dorsal root ganglion (D) and spinal cord (E) from a TMEM184b heterozygous mouse containing one copy of the gene-trapped allele. Xgal stain is shown in blue. In D, an entire ganglion is shown to illustrate the extent of expression within sensory neurons. Red staining is β-III tubulin (tuj1). In E, red staining is ChAT, a marker of acetylcholine-producing motor neurons. Scale bars: D, 100 μm; inset, 20 μm; E, 10 μm.

Figure 2.

TMEM184b localizes to recycling endosomes within neuronal axons and soma. In all panels, TMEM184b-Myc has been virally expressed and identified with antibody to Myc (red). A, Cell bodies of embryonic dorsal root ganglia neurons expressing TMEM184b-Myc. Box in the upper portion of the merged image shows region from which inset (bottom right corner of each image) is taken. Yellow in the merged image indicates colocalization of Rab11 (green), a marker of recycling endosomes, and TMEM184b-Myc (red); 90 ± 7% (SD) of TMEM184b-Myc signal overlaps with Rab11 signal; 92 ± 5% (SD) of Rab11 signal overlaps with TMEM184b-Myc signal (Manders' correlation coefficient calculation, n = 53). Arrows within insets show clusters of recycling endosomes that contain TMEM184b. B, Axons of embryonic DRG neurons (7 d in vitro) stained for Tuj1 (green) and Myc (red). C, Co-occurrence of TMEM184b (red) and Rab11 (green) within axons. D, E, Double immunostaining of TMEM184b-Myc (red) with (D) early endosomes (EEA1, green) or with (E) Golgi (GM130, green). Little to no overlap is seen for either compartment. Representative images from four independent experiments are shown. Scale bars: A, D, E, 5 μm; C, 10 μm.

Figure 3.

TMEM184b participates in the axon degeneration cascade. A, Toluidine blue-stained sections through sciatic nerves (SN) of spared and injured (distal) sciatic nerves in wild-type and mutant animals. B, Quantification of the percentage of intact myelinated axons (showing cytoplasm and mitochondria) on the injured versus spared side at 3 and 5 d following sciatic nerve cut. N = 4 animals per genotype and treatment. **p = 0.0004 (two-tailed t test). C, Electron micrographs of representative myelinated axons in the sciatic nerves of wild-type and mutant animals. D, Representative sensory (Remak) axon bundles in each genotype and treatment. Scale bars: A, 20 μm; C, 1 μm; D, 500 nm.

Figure 4.

TMEM184b loss causes morphological defects at motor and sensory terminals. A, B, NMJs at the EHL muscle in heterozygous (A) or mutant (B) 8–10-week-old mice. All nerves are green (YFP); acetylcholine receptors in muscle are red (labeled with BTX). Arrows point to synaptic swellings at mutant synapses; yellow asterisk indicates a morphologically normal synapse. C, D, TMEM184b mutant NMJs showing individual nerves (YFP, green) and acetylcholine receptors (BTX, red) in 6.5-month-old mice. Dashed lines in D indicate locations of presynaptic swellings where little receptor apposition is found. E, F, Intraepidermal nerve fibers of wild-type and mutant animals, stained with PGP9.5 (red) and DAPI (blue). Dashed line shows the border between dermis (left) and epidermis (right). Mutants show abnormal swellings within epidermal fibers (arrows). G, Quantification of synaptic swellings in control (filled boxes) and mutant (open boxes) animals over time. p = 2.5 × 10⁻⁴ at 18 d; 1.6 × 10⁻⁴ at 8–10 weeks; 2.1 × 10⁻⁵ at 6.5 months. N = 3–4 animals per genotype (**p < 0.01). H, Quantification of the percentage of presynaptic terminal area opposed by acetylcholine receptors (BTX). Black bars, Wild type; gray bars, mutant. Graph shows the percentage of analyzed synapses (n = 40–60 synapses per mouse, 3 mice per genotype) that have the indicated levels of apposition. P values calculated using one-way ANOVA (p = 0.025, 0.16, and 0.0033 for lowest, middle, and highest levels of apposition, respectively). Scale bars: (in A) A, B, 20 μm; (in C) C, D, 10 μm; (in E) E, F, 20 μm.

Figure 5.

Electron and light microscopy reveal abnormal autophagosomal structures in TMEM184b mutant mice. A–F, Electron micrographs of TMEM184b mutant and control EHL muscles. A, B, NMJs on the EHL in wild-type (A) or mutant (B) animals. Mutant synaptic terminals are abnormally shaped (protrusion in upper right corner) and contain membranous inclusions (arrows). C, D, Enlargements of regions in B (mutant) shown by the white arrows. E, F, Examples of abnormal vesicular structures found near the muscle fiber surface in TMEM184b mutant mice. The inset in E shows a double membrane surrounding this structure (arrow). G, H, Sections of skeletal muscle (tibialis anterior) stained with the autophagosomal marker LC3 (G) or the lysosomal marker Lamp2 (H). In mutant animals, occasional accumulations of LC3 or Lamp2-positive structures are found within muscle fibers. Dotted lines show borders of a single myofiber. Scale bars: A, B, 1 μm; C, D, 500 nm; E, F, 250 nm; inset in E, 100 nm.

Figure 6.

TMEM184 loss causes sensory-motor impairment. Analysis of electrophysiological and functional parameters in TMEM184b mutant mice. A, Mutant mice hold on to an inverted screen for significantly less time than their littermate controls. Two-tailed t test, p = 0.0002. B–E, The compound motor action potential (CMAP) recorded from the plantar muscles of the foot (B), motor nerve conduction velocity of the sciatic nerve (C), mixed orthodromic compound nerve action potential amplitude (D), and conduction velocity of the tail nerve (E) were unchanged between littermate controls and TMEM184b mutants. For B–F, two-tailed t test, p > 0.05. F, The weight of TMEM184b mutant mice is the same as littermate controls. N = 8 mice/genotype/trial.