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. 2011 Feb 2;31(5):1624-34.
doi: 10.1523/JNEUROSCI.3347-10.2011.

Sensory neuropathy attributable to loss of Bcl-w

Stephanie L Courchesne  1 , Christoph Karch, Maria F Pazyra-Murphy, Rosalind A Segal
Affiliations

Sensory neuropathy attributable to loss of Bcl-w

Stephanie L Courchesne et al. J Neurosci. .

Abstract

Small fiber sensory neuropathy is a common disorder in which progressive degeneration of small-diameter nociceptors causes decreased sensitivity to thermal stimuli and painful sensations in the extremities. In the majority of patients, the cause of small fiber sensory neuropathy is unknown, and treatment options are limited. Here, we show that Bcl-w (Bcl-2l2) is required for the viability of small fiber nociceptive sensory neurons. Bcl-w(-/-) mice demonstrate an adult-onset progressive decline in thermosensation and a decrease in nociceptor innervation of the epidermis. This denervation occurs without cell body loss, indicating that lack of Bcl-w results in a primary axonopathy. Consistent with this phenotype, we show that Bcl-w, in contrast to the closely related Bcl-2 and Bcl-xL, is enriched in axons of sensory neurons and that Bcl-w prevents the dying back of axons. Bcl-w(-/-) sensory neurons exhibit mitochondrial abnormalities, including alterations in axonal mitochondrial size, axonal mitochondrial membrane potential, and cellular ATP levels. Collectively, these data establish bcl-w(-/-) mice as an animal model of small fiber sensory neuropathy and provide new insight regarding the role of Bcl-w and of mitochondria in preventing axonal degeneration.

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Figures

Figure 1.
Figure 1.
Bcl-w−/− mice show progressive deficits in behavioral response to thermal stimulation. A, Behavioral response to thermal and mechanical stimulation was measured in bcl-w+/+ and bcl-w−/− mice using the hotplate (50.0°C) and von Frey assays, respectively. bcl-w−/− mice show no impairment in response to thermal stimulation at 2–3 months of age (n = 16 bcl-w+/+; n = 17 bcl-w−/−). By 4–8 months of age, response to thermal stimulation was impaired in bcl-w−/− mice. (**p < 0.01; n = 21 bcl-w+/+; n = 13 bcl-w−/−). There is no difference in response to mechanical stimulation as measured by force to elicit hindpaw withdrawal (n = 16 bcl-w+/+; n = 17 bcl-w−/− for 2–3 months of age; n = 21 bcl-w+/+; n = 12 bcl-w−/− at 4–8 months of age; one statistically defined outlier removed from analysis in bcl-w−/− 4–8 month group). B, Grip strength was tested in bcl-w+/+ and bcl-w−/− mice using a digital grip strength meter. bcl-w−/− mice show no impairment in forelimb or hindlimb grip strength through 12 months (n = 11–34 bcl-w+/+; n = 2–9 bcl-w−/−).
Figure 2.
Figure 2.
bcl-w−/− mice show progressive loss of small fiber nociceptor innervation. A, B, bcl-w−/− mice show normal epidermal nociceptor innervation in both thin and thick skin as revealed by PGP9.5 staining (white arrowheads) at 2 months old. By 6 months old, bcl-w−/− mice show decreased nociceptor innervation in both thin and thick skin. Scale bars, 50 μm. C, D, Quantification of fiber number per 225 μm of epidermal length shows a significant decrease in fiber innervation in bcl-w−/− mice at 6 months old (*p < 0.05; **p < 0.01; n = 3 animals per genotype, 3–5 sections examined per animal at each age, for both thick and thin skin). E, F, A significant decrease in fibers innervating both the superficial and inner layers of thin and thick skin seen in bcl-w−/− mice at 6 months old (*p < 0.05; **p < 0.01).
Figure 3.
Figure 3.
Dorsal root ganglia from bcl-w−/− mice do not show compensatory alterations in mRNA levels of bcl-2 family members. Quantitative RT-PCR of bcl-2 family genes shows that bcl-w is the only mRNA significantly altered in DRGs of bcl-w−/− mice. Data represent mean ± SEM for at least three animals, samples run in triplicate. Values are normalized to bcl-w+/+ value for each gene. ND, Not detected.
Figure 4.
Figure 4.
Stereological counting of neurons in 6-month-old bcl-w−/− mice demonstrates no cell body loss. Neuronal cell counts were obtained using an Optical Fractionator Probe (StereoInvestigator) in 30-μm-thick sections labeled with the neuron-specific marker Tuj1. A, Representative images of L4 and L5 DRGs from bcl-w+/+ and bcl-w−/− mice labeled with Tuj1 to identify neurons used for counting. Scale bar, 20 μm. B, Stereological neuronal cell counts reveal no difference between bcl-w+/+ and bcl-w−/− DRGs (p > 0.05; n = 8 bcl-w+/+; n = 7 bcl-w−/−). C, Immunostaining for CGRP and IB4 reveals no morphologic changes or loss of peptidergic or nonpeptidergic nociceptors in bcl-w−/− DRGs. Scale bar, 20 μm. D, Immunostaining for CGRP (green) and IB4 (red) reveals no difference in the central process that innervates the spinal cord in bcl-w+/+ and bcl-w−/− mice. Scale bar, 20 μm.
Figure 5.
Figure 5.
Bcl-w shows unique regulation and localization in DRG sensory neurons. A, Bcl-w (red) is expressed in the majority of Tuj1-positive (green) DRG neurons. Scale bar, 20 μm. Specificity of the Bcl-w antibody is demonstrated by absence of staining in bcl-w−/− DRG. B, bcl-w mRNA is upregulated by distal axon stimulation with neurotrophins (100 ng/ml NGF + BDNF for 2 h vs vehicle treatment) but not by global stimulation. In contrast, bcl-2 mRNA is upregulated by global stimulation, and bcl-xL mRNA is not affected by neurotrophin stimulation. Data represent mean ± SEM for five experiments (data analyzed by z test; *p < 0.05, for a difference from 1). C, In vivo, bcl-w mRNA is upregulated at late embryonic stages and expression continues through adulthood. Data represent mean ± SEM for three to six animals per age, normalized to E14 values for each gene (data analyzed by z test; *p < 0.05, for a difference from 1), bcl-2 mRNA levels are stable over time, and bcl-xL levels increased in late embryonic and postnatal stages. D, Immunoblotting with antibodies specific to Bcl-w, Bcl-2, or Bcl-xL reveal that Bcl-w is expressed at high levels in axons, whereas Bcl-2 and Bcl-xL are predominantly localized to cell bodies. Normalized relative band density of Bcl-2 family member/actin show a distal axon to cell body ratio of 1.18 ± 0.03 for Bcl-w, 0.34 ± 0.07 for Bcl-2, and 0.78 ± 0.08 for Bcl-xL (n = 3). E, Immunostaining of DRGs grown in mass cultures with antibodies specific for Bcl-w, Bcl-2, or Bcl-xL reveal that Bcl-w is expressed in both axons and cell bodies, whereas Bcl-2 and Bcl-xL are predominantly localized to cell bodies. Scale bar, 10 μm.
Figure 6.
Figure 6.
bcl-w−/− DRG sensory neurons undergo axonal degeneration in vitro. A, C, Representative images of Tuj1-labeled axons from bcl-w+/+ and bcl-w−/− DRG explant and compartmented chamber cultures, respectively. Degenerated axon pictures show fragmented axons defined by the Analyze Particle function in NIH ImageJ software. B, D, Quantification of axonal degeneration as measured by the degeneration index (ratio of area of fragmented axons to total axon area) shows significantly more axonal degeneration in bcl-w−/− axons after 24 h of starvation (for explant cultures, *p < 0.005, n = 50 axonal fields from 4 bcl-w+/+ animals, n = 18 axonal fields from 5 bcl-w−/− animals; for compartmented chamber cultures, **p < 0.001, n = 36 axonal fields from 5 bcl-w+/+ animals, n = 39 axonal fields from 4 bcl-w−/− animals).
Figure 7.
Figure 7.
Mitochondria from bcl-w−/− DRGs have abnormal morphology and impaired function. A, Representative images of mitochondria stained with MitoTracker dye in Tuj1-labeled axons of cultured DRG neurons. Scale bars: top, 1 μm; bottom, 10 μm. Mitochondrial length in DRG axons from bcl-w−/− mice is increased compared with bcl-w+/+ (*p < 0.001; n = 1350 mitochondria from 5 bcl-w+/+ animals; n = 1198 mitochondria from 6 bcl-w−/− animals). B, Distribution plots of mitochondrial sizes show that the population of mitochondria in bcl-w−/− DRG axons is shifted toward longer lengths. C, Cumulative percentage plot of mitochondria distribution shows there is no difference in axonal mitochondria distributions in bcl-w+/+ and bcl-w−/− DRG neurons (p > 0.05 by Kolmogorov–Smirnov test; n = 82 axons from 2 bcl-w+/+ animals; n = 89 axons from 2 bcl-w−/− animals). D, Measurements of fluorescence intensity of the voltage-sensitive dye TMRE demonstrates increased membrane potential in axonal mitochondria of bcl-w−/− DRG neurons (*p < 0.001; n = 609 cell body mitochondria and 822 axonal mitochondria from 2 bcl-w+/+ animals; n = 1688 cell body mitochondria and 1608 axonal mitochondria from 3 bcl-w−/− animals). E, Mitochondrial fission and fusion proteins are expressed at normal levels in bcl-w−/− DRGs. Immunoblotting of mitochondrial proteins in bcl-w+/+ and bcl-w−/− DRGs shows no change in the levels of VDAC, MFN2, OPA1, DRP1, or FIS1. Loading control for VDAC levels is actin; VDAC is used as loading control for mitochondrial content for other proteins (n = 3).
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