Sensory neuropathy in progressive motor neuronopathy (pmn) mice is associated with defects in microtubule polymerization and axonal transport

Abstract

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) are now recognized as multi-system disorders also involving various non-motor neuronal cell types. The precise extent and mechanistic basis of non-motor neuron damage in human ALS and ALS animal models remain however unclear. To address this, we here studied progressive motor neuronopathy (pmn) mice carrying a missense loss-of-function mutation in tubulin binding cofactor E (TBCE). These mice manifest a particularly aggressive form of motor axon dying back and display a microtubule loss, similar to that induced by human ALS-linked TUBA4A mutations. Using whole nerve confocal imaging of pmn × thy1.2-YFP16 fluorescent reporter mice and electron microscopy, we demonstrate axonal discontinuities, bead-like spheroids and ovoids in pmn suralis nerves indicating prominent sensory neuropathy. The axonal alterations qualitatively resemble those in phrenic motor nerves but do not culminate in the loss of myelinated fibers. We further show that the pmn mutation decreases the level of TBCE, impedes microtubule polymerization in dorsal root ganglion (DRG) neurons and causes progressive loss of microtubules in large and small caliber suralis axons. Live imaging of axonal transport using GFP-tagged tetanus toxin C-fragment (GFP-TTC) demonstrates defects in microtubule-based transport in pmn DRG neurons, providing a potential explanation for the axonal alterations in sensory nerves. This study unravels sensory neuropathy as a pathological feature of mouse pmn, and discusses the potential contribution of cytoskeletal defects to sensory neuropathy in human motor neuron disease.

Keywords: axon degeneration; microtubule; motor neuron disease; sensory neuropathy; spheroid.

Figure 1

Sensory axon degeneration in suralis nerves of pmn mice revealed by axonal YFP imaging and electron microscopy. A. Images of suralis nerves from 25‐day old wildtype (wt) thy1‐YFP‐16 mice (A1) and pmn thy1‐YFP‐16 mice (A2) expressing YFP in axons. A pmn phrenic motor nerve with its distal branches (A3) is shown for comparison. Scale: 100 µm. B. Images of confocal z‐stacks showing axon caliber irregularities and axonal spheroids in peripheral nerves of pmn YFP mice (B2) as compared to wt (B1). In suralis nerves of pmn YFP mice, axonal spheroids occur in both large caliber axons (arrows) and small caliber axons (arrowheads). Signs of axon degeneration in suralis sensory axons are qualitatively similar to those in phrenic motor axons (B3) of pmn YFP mice but quantitatively less pronounced. Scale: 100 μm. C. High power microscopic images showing axonal spheroids in axons of pmn YFP suralis nerves (C2, arrows) but not in corresponding wt YFP axons (C1). Also note the presence of multiple small axonal YFP foci (asterisks) in the same axon. Scale: 30 μm. D. Quantitation of YFP spheroids in pmn suralis nerves. The number of spheroids per axon segment is about 100 fold higher in pmn YFP axons (4.69 ± 0.47) than in wt YFP axons (0.047 ± 0.013). Axons were monitored on single confocal sections and traced along > 200 μm length in z‐stacks of 20–30 μm depth. Number of axon blebs per mm axon segment is represented as mean of means per nerve ± sd. A total of 2701 axons (wt YFP: 203 to 427 per nerve, pmn YFP: 112 to 278 per nerve) was analyzed in five nerves per genotype. Statistical significance ****, P < 1.7 × 10⁻²³ by Student's t‐test, unpaired, unequal variance. E. Electron microscopy images of longitudinal nerve sections. In comparison to the wt suralis nerve (E1), the pmn suralis nerve (E2) presents some damaged myelinated axons. These myelinated fibers are now a row of several aligned ovoids, consisting of myelin debris and disrupted axons (arrow). N: normal axon. Similar lesions are seen in the pmn phrenic motor nerve where two Schwann cells contain numerous myelin and axonal debris (ovoids, arrows) (E3). Scale bars see images. F. Electron microscopy of transverse sections. The density of myelinated axons is similar in wt (F1) and pmn (F2) suralis nerves. A macrophage (M) in the pmn suralis nerve is loaded with some myelin debris. Macrophages have only a plasma membrane but no basal membrane and present elongated cytoplasmic expansions. Schwann cells have a plasma and a basal membrane, and no elongated cytoplasmic expansions. Scale bars see images. G. High power electron microscopy images of transverse sections. In the pmn suralis nerve (G2) note myelin and axonal debris inside a Schwann cell (SC) cytoplasm (ovoid, arrow) and disappearance of unmyelinated fibers in the other Schwann cell. Ovoids are also seen in the pmn phrenic nerve (G3, arrow). Scale: 2 μm. H. Entire nerve cross sections (semi‐thin sections) show normal number of myelinated fibers in pmn (H2) as compared to wt suralis nerves (H1). Note severe degeneration of myelinated fibers in the pmn phrenic motor nerve (H3). Scale: 50 μm.

Figure 2

TBCE expression in DRG neurons of wildtype and pmn mice. A. Immunoblot showing expression of TBCE (∼59 kD) at similar level in cortex, hippocampus, cerebellum, brainstem, spinal cord and DRGs of 35‐day‐old wt mice. Note loss of TBCE expression in DRGs of pmn mice as compared to wt. Loading control: GAPDH. B. Immunofluorescence labeling of a lumbar DRG from a wt mouse shows strong TBCE expression in neuronal cell bodies but not in axons. Scale: 50 µm. C. Immunofluorescence labeling shows cytosolic and perinuclear (arrows) distribution of TBCE. Scale: 10 µm. D, E. Confocal imaging showing TBCE protein expression at the p115‐labeled Golgi membrane in wt DRG neurons (D) and the loss of TBCE from the Golgi membrane in pmn DRG neurons (E). Scale: 10 µm.

Figure 3

Progressive microtubule loss and defective microtubule polymerization in pmn sensory neurons. A. Electron micrographs showing microtubules in suralis nerve axons from wildtype and pmn mice aged 35 days. B. High power magnifications showing individual microtubules in suralis nerve axons. C. Kinetics of microtubule loss. Microtubule densities (mean ± sem) in myelinated pmn suralis axons are significantly reduced at day 15 (19.3 ± 1.2 vs. 23.9 ± 1.2 MT/μm², *P = 0.0056) and at day 35 (16.1 ± 0.9 vs. 24.8 ± 2 MT/μm², ***P = 0.0001) with a significant difference between both time points (**P = 0.0072). Cross‐sectional area of axons does not significantly differ between wt and pmn (day 15: wt 3.59 ± 1.1 μm², pmn: 3.22 ± 1.2 μm²). Analyses at day 15 were done on a total of 50 axons (wt) and 51 axons (pmn) from n = 4 nerves per genotype. Analyses at day 35 were done on a total of 50 axons (wt) and 64 axons (pmn) from n = 4 wt and n = 5 pmn nerves. Statistical significance was determined by Mann–Whitney test. D. Axonal microtubule loss. In comparison to wt suralis nerves at day 15, microtubule densities (mean ± sem) in pmn suralis nerves are reduced by 20% in both large caliber axons (> 2 μm diameter) and small caliber axons (≤ 2 μm diameter). Note the higher density of microtubules in small caliber axons. **P < 0.001 by Student's t‐test, *P < 0.05 by Student's t‐test. Microtubule densities were calculated from total numbers of microtubules and cross sectional areas of axons. A total of n = 30 large diameter axons and n = 20 small diameter axons from 5 suralis nerves were analyzed per genotype. E. Images showing microtubule regrowth in DRG neurons purified from wt or pmn E15 embryos. Neurons were treated with nocodazole to depolymerize microtubules (0 minutes) and allowed to re‐grow microtubules for 30 minutes after nocodazole washout. Microtubules and centrosomes were labeled with β_III‐tubulin‐ and γ‐tubulin‐specific antibodies, respectively. Scale: 5 µm. F. Histogram showing reduced microtubule length in pmn DRG neurons 30 minutes after nocodazole washout (mean ± sd, n > 70 microtubules per genotype pooled from two independent experiments, P = 0.007 by Mann–Whitney test). G. Cumulative plot of microtubule length 30 minutes after nocodazole washout.

Figure 4

Reduced axonal transport in DRG neurons of pmn mice. A. Images of DRG neurons showing embryonic wt and pmn mice in culture after microtubule labeling with antibodies against detyrosinated tubulin (Detyr‐Tub): Scale: 50 µm. B. Confocal images show neuronal axons after internalization of GFP‐TTC into particles and labeling for detyrosinated tubulin (Detyr‐Tub): Scale: 1 µm. C. Mean velocity of GFP‐TTC axonal transport in DRG neurons [n = 74 (wt), n = 90 (pmn), *P = 0.033 by Mann–Whitney test]. D. Histogram analysis of GFP‐TTC axonal transport velocities reveals a global shift and a reduction of maximal particle velocities in pmn as compared to wt DRG neurons. E. Mean velocity of GFP‐TTC axonal transport in motor neurons [n = 151 (wt), n = 209 (pmn), **P < 0.001 by Mann–Whitney test]. The mean velocity is significantly lower in pmn motor neurons as compared to pmn DRG neurons (C), *P = 0.033 by Mann–Whitney test. F. Histogram analysis of GFP‐TTC axonal transport velocities in wt and pmn motor neurons.