Research reportDifferential expression of neural cell adhesion molecule isoforms in normal and glaucomatous human optic nerve heads
Introduction
Primary open angle glaucoma (POAG) is one of the most common blinding diseases worldwide, and is characterized by chronic loss of retinal ganglion cells. The early changes that occur in POAG in humans and in experimental glaucoma in non-human primates are visible by ophthalmoscopy and by microscopy as loss of tissue from the optic nerve head (ONH) (cupping). In the glaucomatous ONH of many patients, cupping of the optic disc and compression, stretching, and remodeling of the cribriform plates of the lamina cribrosa occur in response to elevated intraocular pressure (IOP) 19, 38, 64. The mechanisms underlying loss of axons and ONH remodeling in glaucoma are under investigation. POAG is unique to humans. Small animal and in vitro models are still under development. Therefore, studies of human glaucomatous tissues may suggest novel end points for experimental systems.
Previous work in our laboratory indicated that astrocytes (type 1B) are involved in the remodeling of the connective tissues in the ONH. Changes in the quantity and composition of several extracellular matrix (ECM) macromolecules significantly affect the biomechanical properties of the tissue supporting the nerve fibers 17, 24, 27, 28, 29, 50, 56, 58, 57, 63. Changes in the ECM support component of the ONH in glaucoma are likely irreversible, perhaps setting the stage for further optic nerve damage from IOP during the progression of the disease [for a comprehensive review, see Ref. [25]].
The ONH is a specialized region of the optic nerve. In humans and most mammals, the lamina cribrosa of the ONH represents a barrier that prevents myelination of the ONH and the retina 16, 59, 78. The lamina cribrosa is a sieve-like meshwork through which the axons pass on their way to the brain, and is composed of collagen and elastic tissue and lined by astrocytes 2, 15, 24. Type 1B astrocytes in the lamina cribrosa express neural cell adhesion molecule (NCAM) constitutively in vivo and in vitro 37, 78. Astrocytes of the ONH exhibit unique morphological and biochemical characteristics that can have an impact on axonal survival, degeneration, and regeneration when the astrocytes acquire a reactive phenotype 18, 37, 55, 66. The reactive astrocyte phenotype consists of changes in morphology and increased expression of glial fibrillary acidic protein (GFAP) and NCAM 66, 75.
NCAM is an important multifunctional member of the immunoglobulin gene superfamily. NCAM belongs to a family of cell surface glycoproteins that accomplish Ca2+ independent adhesion, through either homophilic or heterophilic binding between molecules on neighboring cells or between cell and ECM proteins. NCAM is abundantly expressed in the CNS during development 14, 76, and many studies have demonstrated NCAM expression in a variety of adult neural and non-neural tissues 3, 33, 42, 46, 72. The expression of NCAM is dynamically regulated in different tissues and changes in the surface density of the molecule are known to produce important alterations in cell adhesion 12, 13. The carbohydrates of NCAM may be extensively polysialated resulting in different adhesive properties 69, 70, 72. NCAM expression may be regulated by hormones, growth factors, and the ECM 32, 43, 45, 68. A single gene encodes NCAM but alternative splicing of the pre-mRNA produces three major isoforms, NCAM 180, NCAM 140 and NCAM 120 9, 71. Due to the different exons they contain, these isoforms differ in the size of their cytoplasmic domains and in the mode of association to the membrane. NCAM 120 lacks a cytoplasmic domain and is anchored to the membrane by glycosylphosphatidylinositol 4, 20, 21, 22. NCAM 140 and NCAM 180 possess transmembrane and cytoplasmic domains [51], but NCAM 180 contains 272 additional amino acids in the cytoplasmic domain that are encoded by exon 18.
The present study was designed to investigate whether NCAM expression in the human ONH was altered in POAG. The evidence accumulated in our previous work strongly suggests that NCAM expression by astrocytes may correlate with a transition from quiescent to reactive phenotype in glaucomatous optic neuropathy. Because we hypothesize that reactive astrocytes are related to glaucomatous damage in the ONH, we examined NCAM gene expression in normal and glaucomatous tissues. By utilizing reverse transcriptase-polymerase chain reaction (RT-PCR) with exon-specific primers and tissues from donors of different ages, we analyzed the pattern of NCAM expression in human ONHs and compared NCAM expression in donors with glaucoma with age matched normals. We also localized NCAM mRNA to specific cell types in the ONH by in situ hybridization (ISH) using a probe that recognizes mRNA of all three major NCAM isoforms and an exon 18 specific probe that distinguishes NCAM 180 mRNA.
Section snippets
Human tissues
Four pairs of normal human fetal eyes (gestational age 18–20 weeks) were obtained from the Anatomic Gift Foundation (Laurel MD). Fifty-five pairs of normal human eyes (donor ages 4 days to 94 years) with no history of eye disease, diabetes, or neurodegenerative disease, and 31 pairs of eyes with a history of POAG were obtained from eye banks throughout the United States through NDRI, Mid-America Transplant Services, and the Glaucoma Research Foundation. The average age of the patients with
RT-PCR analysis of NCAM isoforms expressed in cells cultured from human ONHs
Total RNA from cultured cells was reverse-transcribed to first strand cDNA using random and oligo dT primers. Using primers spanning exons 16 through 19 (16A and 19B), a 443 bp PCR fragment would be expected from NCAM 140. Exon 18 is present only in NCAM 180, therefore, a 1259 bp product for this isoform would be expected from the same primer pair (primers 16A and 19B, Table 1; Fig. 1A). Under these conditions NCAM amplification did not produce PCR products visible by ethidium bromide staining.
Discussion
This study documents de novo expression of the 180-kDa isoform of NCAM in astrocytes of the ONH of patients with glaucomatous optic neuropathy. In contrast, ONH tissues from normal age-matched donors express the 140-kDa isoform and do not express NCAM 180. The ONH is the earliest site of degeneration of the axons of the retinal ganglion cells in glaucoma 47, 64, 65. This tissue is undergoing marked remodeling by the astrocyte population and affecting the ECM [for a comprehensive review, see
Acknowledgements
The authors would like to thank Ms. Belinda McMahan and Ms. Jacqueline Selvidge for excellent technical assistance. The National Disease Research Interchange (NDRI) and the Glaucoma Research Foundation (San Francisco) provided the human eyes used in this study. The authors wish to thank Dr. John J. Hemperly (Becton Dickinson, Research Triangle, NC) for the gift of the recombinant NCAM DNA probes. Supported by NIH grants EY-06416 and EY-02687, a grant from The Glaucoma Foundation, New York, and
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2020, Progress in Retinal and Eye ResearchCitation Excerpt :In glaucoma, activated astrocytes become hypertrophied and migrate to where the nerve fibers previously existed (Varela and Hernandez, 1997). Their location outside the original columns is apparent in various histologic studies as disorganization of the glial columns (Hernandez, 2000; Morrison, 2006; Pena et al., 1999; Ricard et al., 1999; Yan et al., 2000). Even eyes at early stages of glaucoma showed slight disorganization of the previously orderly arranged glial columns (Agapova et al., 2003; Minckler and Spaeth, 1981).
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2017, Experimental Eye ResearchCitation Excerpt :Expression analysis with glaucoma-affected human Schlemm canal endothelial cells suggests the contribution of cell adhesion, heparin binding, glycosaminoglycan binding, and extracellular matrix remodeling pathways in the pathogenesis of POAG (Cai et al., 2015; Overby et al., 2014). Microarray analyses in astrocytes obtained from optic nerve head tissue have demonstrated the involvement of a variety of biological processes in glaucoma, which include cell adhesion and proliferation, cell motility, extracellular matrix synthesis and degradation, neurosteroids, and signal transduction (Agapova et al., 2003; Hernandez et al., 2002; Lukas et al., 2008; Ricard et al., 1999). Expression analysis of primary lamina cribrosa cells has implicated involvement of TGF-β, extracellular matrix, and fibrosis in POAG (Kirwan et al., 2009).
Investigating the potential immune role of fish NCAMs: Molecular cloning and expression analysis in mandarin fish
2015, Fish and Shellfish ImmunologyCitation Excerpt :Mammalian NCAM family consists of two members, NCAM1 and NCAM2, which are paralogs and possibly originating from whole genome duplication [3]. Human NCAM1 has 3 isoforms, NCAM-120, NCAM-140 (also named CD56) and NCAM-180 [4,5]. CD56, as the only isoform expressed on immune cells, has been found distributed in a variety of cells including neurons, astrocytes, Schwann cells, natural killer (NK) cells, and a subset of T (natural killer T, NKT) cells [6].
State-dependent changes in the expression levels of NCAM-140 and L1 in the peripheral blood cells of bipolar disorders, but not in the major depressive disorders
2008, Progress in Neuro-Psychopharmacology and Biological PsychiatryPurification of astrocytes from adult human optic nerve heads by immunopanning
2003, Brain Research Protocols
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Current address: Department of Ophthalmology, Escola Paulista de Medicina, São Paulo, Brazil.