Skip to main content

Advertisement

Advancing the pathologic phenotype of giant axonal neuropathy: early involvement of the ocular lens

Article metrics

  • 571 Accesses

  • 1 Citations

Abstract

Giant axonal neuropathy (GAN; ORPHA: 643; OMIM# 256850) is a rare, hereditary, pediatric neurodegenerative disorder associated with intracellular accumulations of intermediate filaments (IFs). GAN knockout (KO) mouse models mirror the IF dysregulation and widespread nervous system pathology seen in human GAN. Validation of therapeutic efficacy and viral vector delivery systems with these GAN KO models has provided the springboard for the development of a viral vector being delivered intrathecally in an ongoing Phase I gene therapy clinical trial for the treatment of children with GAN (https://clinicaltrials.gov/ct2/show/NCT02362438). During the course of a comprehensive pathologic characterization of the GAN KO mouse, we discovered the very early and unexpected involvement of the ocular lens. Light microscopy revealed the presence of intracytoplasmic inclusion bodies within lens epithelial cells. The inclusion bodies showed strong immunohistochemical positivity for glial fibrillary acidic protein (GFAP). We confirmed that intracytoplasmic inclusion bodies are also present within lens epithelial cells in human GAN. These IF inclusion bodies in lens epithelial cells are unique to GAN. Similar IF inclusion bodies in lens epithelial cells have not been reported previously in experimental animal models or human diseases. Since current paradigms in drug discovery and drug repurposing for IF-associated disorders are often hindered by lack of validated targets, our findings suggest that lens epithelial cells in the GAN KO mouse may provide a potential target, in vivo and in vitro, for evaluating drug efficacy and alternative therapeutic approaches in promoting the clearance of IF inclusions in GAN and other diseases characterized by intracellular IF accumulations.

Giant axonal neuropathy (GAN, OMIM# 256850) is a rare, hereditary, pediatric neurodegenerative disorder associated with intracellular accumulations of intermediate filaments (IFs) [1]. The disease affects both the peripheral nervous system (PNS) and central nervous system (CNS), and patients nearly always succumb to disease by the third decade. The pathologic signature of GAN in the PNS and CNS is giant axonal swellings filled with dense accumulations of whorled, structurally normal neurofilaments. GAN is caused by autosomal recessive loss-of-function mutations in the GAN gene that encodes the protein gigaxonin. Gigaxonin plays a pivotal role in the cytoskeletal organization and degradation of IFs. Loss of gigaxonin leads to accumulation of different types of IFs within a variety of cells, including desmin in muscle cells, vimentin in fibroblasts, neurofilaments in neurons, and glial fibrillary acidic protein (GFAP) in astrocytes [2]. Most GAN patients also have characteristically tightly curled hair due to alterations of keratin IFs [3].

Three mouse models of GAN have been developed by knocking out part of the endogenous GAN gene [4,5,6]. All three mouse models mirror the IF dysregulation and widespread nervous system pathology seen in human GAN [7]. Validation of therapeutic efficacy and viral vector delivery systems with these GAN KO models [8] has provided the springboard for the development of a viral vector to be delivered intrathecally in a Phase I gene therapy clinical trial for the treatment of children with GAN [9].

During the course of a comprehensive study of the pathologic findings in the GAN KO mouse, we encountered the unexpected and very early involvement of the ocular lens (Fig. 1). Here, described for the first time, we document the early appearance of abundant IF accumulations in lens epithelial cells of the GAN KO mouse. Lens epithelial cells potentially provide an easily accessible target for accelerating complementary drug discovery and drug repurposing strategies for human GAN.

Fig. 1
figure1

Ocular Lens. Control. Lens cells exist in two distinct forms, lens epithelial cells and lens fiber cells. The anterior surface of the lens is covered by a single layer of epithelial cells that serve as a reservoir for continual lens fiber cell formation and lens growth throughout life. The lens is unique as reflected in almost continuous cell production with negligible cell loss. On their path to becoming mature lens fiber cells, lens epithelial cells undergo extraordinary structural differentiation [10]. GAN KO mouse. Oval intracytoplasmic eosinophilic inclusion bodies within lens epithelial cells

GAN KO mice with a deletion of GAN exons 3–5 (GAN/Y) [4] or a deletion of GAN exon 1 (GAN/J) [6] were maintained at the University of North Carolina at Chapel Hill (UNC–CH) as previously described [8]. Heterozygous GAN mice are phenotypically normal [4, 6] and were used as controls. Mixed sex and age-matched littermates from both GAN KO models were used in these studies (4-month-old cohort: 4 KO, 2 heterozygotes; 24-month-old cohort: 10 KO, 15 heterozygotes).

In 4-month-old GAN KO mice, light microscopic examination of H&E-stained sections revealed oval, intracytoplasmic eosinophilic inclusion bodies within lens epithelial cells (Fig. 2a). Histologically identical inclusion bodies were found in 24-month-old GAN KO mice (Fig. 2b). In both 4-month-old and 24-month-old cohorts, inclusion bodies were present in almost every epithelial cell. A panel of immunohistochemical stains for lens IF proteins (GFAP, vimentin, keratin 8/18, CP49 and filensin) [10] showed strong immunoreactivity of inclusion bodies for GFAP (Fig. 2c). The epithelial cell inclusion bodies were present in both GAN/J and GAN/Y KO mice. Age-matched control mice had no inclusion bodies (Fig. 2d). The inclusion bodies were not present in lens fiber cells in the GAN KO mice or age-matched controls. No lens fiber cell degeneration was identified histologically in 4-month-old GAN KO mice or age-matched controls. Lens fiber cell degeneration, morphologically consistent with age-related degeneration [11], was present to a similar degree in both 24-month-old GAN KO mice and age-matched controls.

Fig. 2
figure2

Lens epithelial cells in GAN KO mice, age-matched controls and human GAN. a. GAN KO (4-month-old) lens epithelial cells show intracytoplasmic inclusion bodies (H&E original magnification 100X). b. GAN/J KO (24-month-old) lens epithelial cell inclusion bodies (H&E original magnification 100X). c. GAN/J KO (24-month-old) lens epithelial cell inclusion bodies show strong GFAP immunoreactivity (GFAP IHC original magnification 100X). d. Control mouse (24-month-old) histologically unremarkable lens epithelial cells (H&E original magnification 100X). e. Human GAN lens epithelial cells reveal intracytoplasmic inclusion bodies (H&E original magnification 100X, formalin fixed, paraffin embedded tissue. Decedent was a young child with phenotypically typical GAN) (arrows point to one of the numerous intracytoplasmic inclusion bodies). Inset. Lens epithelial cells. Control mouse (24-month-old) (H&E); GAN/J KO (24 -month-old) lens epithelial cell inclusion body (H&E); GAN/J KO (24-month-old) lens epithelial cell inclusion body shows strong GFAP immunoreactivity (GFAP IHC). (N designates nucleus, arrow points to cytoplasmic inclusion body)

The neuropathological phenotype of the GAN KO mouse model shares many morphological features with the human disease [7]. Here, described for the first time in the GAN KO mouse, we document the presence of intracytoplasmic IF inclusion bodies in lens epithelial cells. The inclusion bodies were present in the young 4-month-old KO mice and served as a reliable, easily identifiable, early marker of GAN.

These IF inclusion bodies in lens epithelial cells appear to be unique to GAN, as similar IF inclusion bodies have not been reported previously in experimental animal models or human diseases. Although lens abnormalities have not been reported in clinical or postmortem studies of human GAN [12,13,14,15,16], we confirmed in a specimen obtained at autopsy that similar appearing, intracytoplasmic inclusion bodies are also present in lens epithelial cells in human GAN (Fig. 2e).

The presence of GFAP-positive inclusion bodies in lens epithelial cells and their absence in lens fiber cells is intriguing. One difference between lens epithelial cells and lens fiber cells is the large concentration of the chaperone protein complex alpha-crystallin in lens fiber cells [17]. The chaperone activity of alpha-crystallin modulates the assembly of IFs, including GFAP, and assists IFs in recovery from stress by preventing inappropriate filament-filament interactions that would otherwise lead to aggregation [18].

Current paradigms in drug discovery and drug repurposing for IF-associated disorders are often hindered by lack of validated targets [19]. One strategy to circumvent this constraint is to screen against a disease phenotype in cell culture or animal model that recapitulates the pathologic phenotype of the human disease [19, 20]. Our findings suggest that lens epithelial cells in the GAN KO mouse may provide a potential target cell, in vivo, for evaluating the efficacy of drugs and other therapeutic approaches in promoting clearance of IF inclusions. Additionally, lens epithelial cells can be grown on their native basement membrane or as dissociated cells [21] and serve as a simple in vitro model system of target cells.

Intracytoplasmic accumulations of IFs are a distinctive pathological feature shared by common neurodegenerative diseases of adulthood, such as Alzheimer’s disease and Parkinson’s disease, as well as rare neurodegenerative diseases of childhood, such as Alexander disease and GAN [2]. It is possible that lens epithelial cells from the GAN KO mouse, if used as a drug repurposing screen, could be extended to address multiple diseases that share an IF accumulation pathologic phenotype [20, 22].

In summary, the GAN KO mouse exhibits great fidelity to the characteristic pathologic features and selected functional deficits of human GAN [7]. Here, we present the novel finding of GAN pathology in both mouse and human lens epithelial cells. We suggest that lens epithelium may serve as a target tissue in which to study the effects of pharmacological interventions on GAN and potentially other disorders characterized by intracytoplasmic IF accumulations.

Abbreviations

CNS:

Central nervous system

GAN:

Giant axonal neuropathy

GFAP:

Glial fibrillary acidic protein

IF:

Intermediate filament

KO:

Knockout

PNS:

Peripheral nervous system

References

  1. 1.

    Johnson-Kerner BL, Roth L, Greene JP, Wichterle H, Sproule DM. Giant axonal neuropathy: an updated perspective on its pathology and pathogenesis. Muscle Nerve. 2014;50(4):467–76.

  2. 2.

    Lin NH, Huang YS, Opal P, Goldman RD, Messing A, Perng MD. The role of gigaxonin in the degradation of the glial-specific intermediate filament protein GFAP. Mol Biol Cell. 2016;27(25):3980–90.

  3. 3.

    Soomro A, Alsop RJ, Negishi A, Kreplak DF, Kuczmarski ER, Goldman RD, Rheinstadter MC. Giant axonal neuropathy alters the structure of keratin intermediate fialments in human hair. JR Soc Interface. 2017;14:1–9.

  4. 4.

    Ding J, Allen E, Wang W, Valle A, Wu C, Nardine T, et al. Gene targeting of GAN in mouse causes a toxic accumulation of microtubule-associated protein 8 and impaired retrograde axonal transport. Hum Mol Genet. 2006;15(9):1451–63.

  5. 5.

    Ganay T, Boizot A, Burrer R, Chauvin JP, Bomont P. Sensory-motor deficits and neurofilament disorganization in gigaxonin-null mice. Mol Neurodegener. 2011;6(1):25.

  6. 6.

    Dequen F, Bomont P, Gowing G, Cleveland DW, Julien J-P. Modest loss of peripheral axons, muscle atrophy and formation of brain inclusions in mice with targeted deletion of gigaxonin exon 1. J Neurochem. 2008;107(1):253–64.

  7. 7.

    Armao D, Bailey RM, Bouldin TW, Kim Y, Gray SJ. Autonomic nervous system involvement in the giant axonal neuropathy (GAN) KO mouse: implications for human disease. Clin Auton Res. 2016;26(4):307–13.

  8. 8.

    Bailey RM, Armao D, Nagabhushan Kalburgi S, Gray SJ. Development of intrathecal AAV9 gene therapy for Giant axonal neuropathy. Mol Ther Methods Clin Dev. 2018;9:160–71.

  9. 9.

    Intrathecal Administration of scAAV9/JeT-GAN for the Treatment of Giant Axonal Neuropathy ClinicalTrials.gov [updated July 16, 2018. Available from: https://clinicaltrials.gov/ct2/show/NCT02362438.

  10. 10.

    Song S, Landsbury A, Dahm R, Liu Y, Zhang Q, Quinlan RA. Functions of the intermediate filament cytoskeleton in the eye lens. J Clin Invest. 2009;119(7):1837–48.

  11. 11.

    Wolf NS, Li Y, Pendergrass W, Schmeider C, Turturro A. Normal mouse and rat strains as models for age-related cataract and the effect of caloric restriction on its development. Exp Eye Res. 2000;70(5):683–92.

  12. 12.

    Roth LA, Marra JD, LaMarca NH, Sproule DM. Measuring disease progression in giant axonal neuropathy: implications for clinical trial design. J Child Neurol. 2015;30(6):741–8.

  13. 13.

    Kumar K, Barre P, Nigro M, Jones MZ. Giant axonal neuropathy: clinical, electrophysiologic, and neuropathologic features in two siblings. J Child Neurol. 1990;5(3):229–34.

  14. 14.

    Kretzschmar HA, Berg BO, Davis RL. Giant axonal neuropathy. A neuropathological study. Acta Neuropathol. 1987;73(2):138–44.

  15. 15.

    Peiffer J, Schlote W, Bischoff A, Boltshauser E, Muller G. Generalized giant axonal neuropathy: a filament-forming disease of neuronal, endothelial, glial, and schwann cells in a patient without kinky hair. Acta Neuropathol. 1977;40(3):213–8.

  16. 16.

    Thomas C, Love S, Powell HC, Schultz P, Lampert PW. Giant axonal neuropathy: correlation of clinical findings with postmortem neuropathology. Ann Neurol. 1987;22(1):79–84.

  17. 17.

    Horwitz J. Alpha-crystallin. Exp Eye Res. 2003;76:145–53.

  18. 18.

    Hagemann TL, Boelens WC, Wawrousek EF, Messing A. Supression of GFAP toxicity by alpha B-crystallin in mouse models of Alexander disease. Hum Mol Genet. 2009;18(7):1190–9.

  19. 19.

    Varma H. Drug screening for Huntington's disease and other neurodegenerative disorders. Curr Mol Pharmacol. 2010;3(3):164–73.

  20. 20.

    Sun W, Zheng W, Simeonov A. Drug discovery and development for rare genetic disorders. Am J Med Genet A. 2017;173(9):2307–22.

  21. 21.

    Martinez G, de Iongh RU. The lens epithelium in ocular health and disease. Int J Biochem Cell Biol. 2010;42(12):1945–63.

  22. 22.

    Snider NT, Omary MB. Post-translational modifications of intermediate filament proteins: mechanisms and functions. Nat Rev Mol Cell Biol. 2014;15(3):163–77.

Download references

Acknowledgements

We thank Kimberlie A. Burns, Research Specialist at the Marsico Lung Institute at UNC-CH, for her anatomical and histological expertise. We acknowledge Yuhui Hu and Mary Keener, UNC-CH, and Jowaly Schneider, the Johns Hopkins University School of Medicine, for their histological technical skills and support. The UNC Histology Research Core Facility in the Department of Cell Biology and Physiology provided some histology services for this project. We acknowledge the Microscopy Services Laboratory, Pathology and Laboratory Medicine, UNC-CH. We are especially thankful for the Johns Hopkins Autopsy Service. Finally, we are grateful for C. Bönnemann, MD and his team at NIH/NINDS, for their collaboration and the initiation of the Phase I GAN clinical trial.

Funding

This study was supported by Hannah’s Hope Fund, the NIH National Institute of Neurological Disorders and Stroke (NS087175, S.J.G.; NS095515, R.M.B.), and the NIH National Institute of Child Health and Human Development (HD040127, R.M.B.; U54HD079124). Indirect administrative support for S.J.G. was provided by the Research to Prevent Blindness to the UNC-CH Department of Ophthalmology.

Availability of data and materials

All data generated or analyzed during this study are included in this published article [and its Additional files 1 and 2].

Author information

DA., TWB, RMB, and SJG, contributed to study concept and design. DA, TWB, RMB, JEH, SJG contributed to data acquisition and analysis. DA, TWB, RMB, JEH, DXB and SJG contributed to drafting and reviewing the text. DA, TWB, RMB, and SJG contributed to composing the figures. All authors read and approved the final manuscript.

Correspondence to Steven J. Gray.

Ethics declarations

Ethics approval and consent to participate

All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85–23] and approved by the UNC–CH Institutional Animal Care and Use Committee. Human specimens were obtained through a validated autopsy consent from the legal next of kin, which explicitly stated that tissues could be used for research purposes. The consent and diagnostic autopsy report were filed in the deceased patient’s medical record.

Consent for publication

Not applicable.

Competing interests

All authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Additional files

Additional file 1:

Details regarding experimental procedures and immunohistochemistry (IHC). (DOCX 15 kb)

Additional file 2:

Details regarding immunoreactivity (IR). (DOCX 16 kb)

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Armao, D., Bouldin, T.W., Bailey, R.M. et al. Advancing the pathologic phenotype of giant axonal neuropathy: early involvement of the ocular lens. Orphanet J Rare Dis 14, 27 (2019) doi:10.1186/s13023-018-0957-5

Download citation

Keywords

  • Giant axonal neuropathy (GAN)
  • Intermediate filaments (IF)
  • GAN KO mouse model
  • Gigaxonin
  • Human GAN
  • Lens epithelium
  • IF accumulations