January 2003
Volume 44, Issue 1
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Glaucoma  |   January 2003
Increase in Dephosphorylation of the Heavy Neurofilament Subunit in the Monkey Chronic Glaucoma Model
Author Affiliations
  • Kenji Kashiwagi
    From the Department of Ophthalmology, University of Yamanashi Faculty of Medicine, Tamaho, Yamanashi, Japan; the
  • Bo Ou
    From the Department of Ophthalmology, University of Yamanashi Faculty of Medicine, Tamaho, Yamanashi, Japan; the
  • Shinichiro Nakamura
    Department of Veterinary Pathology, Nippon Veterinary and Animal Science University, Musashino, Tokyo, Japan; and the
  • Yuko Tanaka
    From the Department of Ophthalmology, University of Yamanashi Faculty of Medicine, Tamaho, Yamanashi, Japan; the
  • Michihiro Suzuki
    Corporation for Production and Research of Laboratory Primates, Tsukuba, Ibaraki, Japan.
  • Shigeo Tsukahara
    From the Department of Ophthalmology, University of Yamanashi Faculty of Medicine, Tamaho, Yamanashi, Japan; the
Investigative Ophthalmology & Visual Science January 2003, Vol.44, 154-159. doi:10.1167/iovs.02-0398
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      Kenji Kashiwagi, Bo Ou, Shinichiro Nakamura, Yuko Tanaka, Michihiro Suzuki, Shigeo Tsukahara; Increase in Dephosphorylation of the Heavy Neurofilament Subunit in the Monkey Chronic Glaucoma Model. Invest. Ophthalmol. Vis. Sci. 2003;44(1):154-159. doi: 10.1167/iovs.02-0398.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To investigate the phosphorylation of the heavy neurofilament subunit (NF-H), which could be deeply involved in axonal transport of retinal ganglion cells (RGCs), in an experimental glaucoma model of chronic elevation of intraocular pressure (IOP) in monkeys.

methods. One eye in adult monkeys was randomly selected for laser treatment, and IOP was maintained between 30 and 40 mm Hg throughout the experiment. The eyeballs with the optic nerve and optic chiasm were enucleated as one tissue and were subject to immunocytochemical observation, using two NF-H–specific antibodies, NF-200 and SMI31. NF-200 reacts with both phosphorylated and dephosphorylated NF-H, whereas SMI reacts only with phosphorylated NF-H. Ratios of SMI31-positive to NF-200-positive areas were calculated for quantitative evaluation of phosphorylation status. Specimens from the retina, lamina cribrosa (LC), post-LC, and optic chiasm were evaluated separately. Phosphorylation of NF-H at the retina and optic nerve head was compared between specimens from temporal retina and nasal retina, or between temporal and nasal regions of the optic disc. The status of phosphorylation was confirmed by Western blot analysis.

results. An enlargement of the disc cup was observed on the temporal side, and the superior and inferior poles were preferentially involved in the neuronal damage in laser-treated eyes. Most NF-Hs in the control eyes were phosphorylated in all investigated regions, whereas those in the glaucomatous eyes were significantly dephosphorylated, and NF-Hs in the temporal region were significantly dephosphorylated compared with those in the nasal region. At the optic chiasm, NF-Hs in axons traveling from laser-treated eyes were highly dephosphorylated, and the extent of NF-H dephosphorylation corresponded to the degree of glaucoma-induced axonal damage. Western blot analysis showed the change in the phosphorylation of NF-Hs.

conclusions. NF-Hs in RGC axons are dephosphorylated by elevated IOP, which may be deeply involved in glaucoma-induced damage to axonal transport.

Neurofilaments (NFs) are major neuronal intermediate filaments expressed in most neurons. These heteropolymers are composed of three subunits, NF-H, NF-M, and NF-L, in order of decreasing molecular weight. 1 Although these NF proteins are primarily dephosphorylated in the perikarya and dendrites of neurons, almost all NFs in neuronal axons are phosphorylated and form crossbridges that provide resistance to severe mechanical stresses. 2 Phosphorylation of NF-H appears to be a major mechanism of the formation of NF crossbridges, although the precise details of this mechanism remain unclear. It has recently been reported that phosphorylation of NF-H is deeply involved in axonal transport, axonal plasticity, and neuronal morphology in addition to maintaining the integrity of the neuronal cytoskeleton. 1 3 4 5  
Glaucoma-induced optic nerve damage has been hypothesized to be caused by apoptosis of retinal ganglion cells (RGCs), due to a disturbance of axonal transport. 6 7 8 Veckers et al. 9 have reported a loss of NF-immunoreactive optic nerve fibers in experimentally induced glaucoma in monkeys. However, to our best knowledge, there has been no study investigating changes in the status of NF-H phosphorylation in RGC axons in glaucoma. Thus, in the present study, we examined the status of phosphorylation of NF-H in RGC axons, using a model of experimental glaucoma in monkeys, with chronic elevation of intraocular pressure (IOP). 
Materials and Methods
All experiments were conducted and all laboratory animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Subjects
Four adult Macaca irus monkeys weighing from 4.5 to 4.8 kg were used. IOP was measured with a handheld electronic tonometer (TonoPen XL; Bio-Rad, Glendale, CA) in monkeys under general anesthesia induced by intramuscular injection of 9 mg/kg of ketamine hydrochloride (Sankyo, Tokyo, Japan). 
Laser Treatment
Laser treatment was performed according to a previously described method. 10 11 Under general anesthesia as described, the monkeys were set in front of the slit lamp of an argon laser delivery system (Novus 2000; Coherent, Santa Clara, CA). One eye was randomly selected for laser treatment and was treated with 0.4% oxybuprocaine hydrochloride. Approximately 80 to 120 burns were made with the laser being aimed at the middle of the trabecular meshwork with a beam diameter of 100 μm for 0.2 second at 600 to 800 mW. The laser treatment was repeated weekly for 6 to 8 weeks. Measurement of IOP and slit lamp examination were performed weekly. Fundus photography with a fundus camera (NF-505; Nikon, Tokyo, Japan) was performed every 2 to 3 weeks and fluorescent angiography was performed monthly. 
Tissue Preparation
When an increase in severity of optic nerve head cupping occurred in the temporal half while optic nerve head cupping in the nasal half remained static, the primates were killed by an overdose of ketamine hydrochloride and exsanguinated with approximately 2 L saline followed by 2 L 2% paraformaldehyde and 0.05% glutaraldehyde in 0.1 mM phosphate buffer (PB; pH 7.4). Bilateral eyeballs attached to the optic nerve and optic chiasm were enucleated as one tissue, and eyeballs were dissected at the equator for further fixation with 2% paraformaldehyde and 0.05% glutaraldehyde in 0.1 mM PB for 1 hour. The tissues were rinsed with 0.1 mM PB, dehydrated with graded ethanol, immersed in xylene, embedded in paraffin, and cut into 4-μm specimens. 
Two retinal regions approximately 10 mm away from the optic disc on the temporal and nasal sides were chosen to investigate phosphorylation in the retina, and specimens from the lamina cribrosa (LC) and post-LC were evaluated separately. The central region of the optic chiasm was cut vertically and was also examined. 
Light Microscopy
Specimens were deparaffinized in xylene, hydrated through graded ethanol, stained with hematoxylin and eosin, and mounted. 
Immunohistochemical Processing
Specimens were deparaffinized in xylene, rinsed with 0.1 mM phosphate-buffered saline (PBS; pH 7.4), incubated with 0.4% trypsin at 37°C for 20 minutes to provide better infiltration of antibodies according to the manufacturer’s recommendation, rinsed with PBS, and blocked with 2% bovine serum albumin-PBS at room temperature for 30 minutes. Specimens then were incubated sequentially with one of the primary antibodies: rabbit monoclonal anti-neurofilament 200 (NF200: Sigma Chemical Co., St. Louis, MO) diluted 1:250 or mouse monoclonal anti-human neurofilament (SMI31: Sternberger Monoclonals, Baltimore, MD) diluted 1:250. NF200 recognizes both phosphorylated and dephosphorylated forms of the 200-kDa neurofilaments, whereas SMI31 reacts only with a phosphorylated epitope in extensively phosphorylated forms of this polypeptide. After three washes in PBS, specimens were incubated with secondary antibodies, either Texas red–conjugated anti-rabbit IgG for NF200 or FITC-conjugated anti-mouse IgG for SMI31. Specimens then were washed and mounted. A confocal laser microscope (TSC4D; Leica Microsystems, Wetzlar, Germany) was used for observation. 
Semiquantitative Evaluation of NF-H Phosphorylation
Areas in each specimen reacting with NF200 or SMI31 were measured using the NIH image-analysis program (ver. 1.61; W. Rasband, National Institutes of Health; available by ftp from zippy.nimh.nih.gov or on floppy disc from NTIS, Springfield, VA, part number PB95-500195GEI). In brief, SMI31-positive areas are green and NF200-positive areas are red. These areas were separately measured on computer, and the ratio of SMI31-positve to NF200-positive areas in the same specimen reflected the extent of phosphorylation. Among the retinal specimens, 10 consecutive specimens each from both the temporal and nasal retina were analyzed. In the optic disc region, 10 consecutive specimens from LC and post-LC regions were chosen, and the ratio of phosphorylation on both the temporal and nasal sides was calculated separately, as described. Three monkeys were used for quantitative evaluation of NF-H phosphorylation in the retina and optic disc region. Specimens obtained from the middle of the optic chiasm were analyzed. Unlike the specimens of the retina and optic disc region, it was difficult to measure the colored areas separately in the optic chiasm, and therefore quantitative analysis in this region was not performed. Preparations from two monkeys were used independently for histologic observation in the optic chiasm. 
Western Blot Analysis
The status of NF-H in the experimental glaucoma model was confirmed by Western blot analysis. In the present study, SMI32, a dephosphorylated NF-H–specific antibody (Sternberger Monoclonals), was used in addition to antibodies NF200 and SMI31. Blocks of the optic nerve head approximately 5 mm in length were dissected from the glaucomatous and control eyes and washed with PBS. Samples were placed in the sample buffer containing 20% glycerol, 1% sodium dodecyl sulfate, and 1% β-mercaptoethanol in 0.5 M Tris-HCl buffer (pH 6.8) and were thoroughly homogenized. Samples were then boiled for 5 minutes and centrifuged at 14,000g for 15 minutes. Protein levels in supernatants were quantified by the Lowry method and were adjusted at 16 μg/lane with the sample buffer. Supernatants with 0.1% bromophenol blue and prestained molecular weight standards (Calbiochem, San Diego, CA) were separated on a 7.5% polyacrylamide gel at a constant 40 mA for 1 hour and were then transferred to a nitrocellulose membrane by Western blot analysis at a constant 2 mA/cm2 for 1 hour. The membranes were blocked by incubation with a blocking solution containing 3% bovine serum albumin (BSA) in 0.01% Tween-PBS for 2 hours at room temperature and were then incubated with SMI31 diluted at 1:1000, SMI32 diluted at 1:1000, or NF200 diluted at 1:250 overnight at 4°C. On the next day, after three washes in 0.01% Tween-PBS, the membranes were incubated with horseradish peroxidase–conjugated anti-mouse IgG diluted at 1:1000 for SMI31 and SMI32, or horseradish peroxidase–conjugated anti-rabbit IgG diluted at 1:500 for NF200 for 1 hour at room temperature. The labeling was then developed with diaminobenzidine. 
Chemicals used in the present study were purchased from Sigma Chemical Co. unless noted otherwise. Preparations from two monkeys were used independently to confirm the results of Western blot analysis. 
Statistical Analysis
The NF200-positive area and the extent of phosphorylation were compared with the Mann-Whitney test. A significant difference was defined as P < 0.05. All data are expressed as the mean ± SD. 
Results
Procedural Course of the Experimental Glaucoma Model in Monkeys
The IOP in laser-treated eyes decreased during the initial laser treatments and then elevated to 30 to 40 mm Hg after repeated treatments. When IOP returned to baseline, additional laser treatments were performed. An enlargement of the optic nerve head was observed more than 8 months after initiation of laser treatment. IOP in non–laser-treated eyes was maintained between 10 and 20 mm Hg throughout the experiment. Laser-treated eyes showing an obvious increase in disc cupping were enucleated 8 to 10 months after initiation of laser treatment. An enlargement of the disc cup was observed in the temporal side of the optic disc, and the superior and inferior poles were preferentially involved in the neuron damage in both laser-treated eyes, with the cup-to-disc ratios at the time of enucleation being approximately 0.7 to 0.8. No obvious abnormality was observed in the fundus, except for an increase in disc-to-cup ratio in all eyes studied. 
Phosphorylation of NF-H in the Retina
In the control, most axonal fibers were well phosphorylated in all investigated regions (Fig. 1) . In contrast, laser-treated eyes showed a significant reduction of phosphorylation compared with the control eyes. The reduction of phosphorylation in the temporal retina was significantly greater than that in the nasal retina, although the ratio of phosphorylation between the nasal and temporal retinas was quite similar in control eyes. 
Morphologic Changes in the Optic Disc
Laser treatment resulted in deformation of the optic disc similar to that observed in human optic discs with glaucoma-induced atrophy. This deformation included an increase in optic disc cupping in the temporal region, a loss of axonal bands, bowing of the cribriform fascia, an increasing amount of connective tissue, and preferential damage in the superior to inferior poles of the temporal side. These changes in laser-treated eyes were predominantly observed in the LC (Fig. 2) , whereas the morphologic appearance in the post-LC was less different between laser-treated and control eyes. 
Phosphorylation of NF-H in the Optic Disc
Axonal fibers in the optic disc were almost completely phosphorylated in the control eyes, whereas they were significantly dephosphorylated in the glaucomatous eyes. The region of greater damage, primarily the temporal region, showed a greater dephosphorylation of NF-Hs, whereas the less-damaged nasal region showed less dephosphorylation in the glaucomatous eyes, both in the LC (Fig. 3) and post-LC (Fig. 4) . Although the morphologic difference between the temporal and nasal regions of the post-LC was less significant than that in the LC, dephosphorylation in the temporal region was significantly greater than in the nasal region of the post-LC. 
Semiquantitative Analysis of Phosphorylation in the Retina, LC, and Post-LC
The ratios of phosphorylation in control eyes were very high, whereas those in experimental eyes were significantly reduced in all investigated specimens (Fig. 5) . The phosphorylation in the control eyes was 96.7% ± 5.4% (temporal retina), 84.7% ± 18.6% (nasal retina), 84.6% ± 11.4% (temporal LC), 94.3% ± 9.4% (nasal LC), 94.8% ± 7.4% (temporal post-LC), and 84.7% ± 4.8% (nasal post-LC), whereas the phosphorylation in the glaucomatous eyes was 21.5% ± 5.4% (temporal retina), 55.9% ± 18.8% (nasal retina), 22.4% ± 7.2% (temporal LC), 52.7% ± 10.3% (nasal LC), 22.9% ± 9.6% (temporal post-LC), and 49.9% ± 4.8% (nasal post-LC). Differences in NF-H phosphorylation between nasal and temporal areas were significant in all regions of the glaucomatous eyes, except for the nasal retina (P = 0.13). Otherwise, no significant differences in phosphorylation were observed in the glaucomatous eyes or in control eyes. All data are expressed as mean ± SD. 
Phosphorylation of NF-H in the Optic Chiasm
The extent of dephosphorylation of NF-Hs in the optic chiasm faithfully corresponded to the degree of glaucoma-induced damage to nerve fibers by laser treatment (Fig. 6) . The extent of phosphorylation was lowest in the left lateral region, where nerve fibers travel through the temporal disc of the laser-treated eye, followed by the region of the right inner side, where a large number of nerve fibers travel from the nasal disc of the laser-treated eye. Many nerve fibers in the left inner region of the optic chiasm, where a large number of nerve fibers traveling from the nasal disc of the control eye were phosphorylated, and nerve fibers in the right lateral area, where most nerve fibers traveling from the temporal disc of the control eyes were almost completely phosphorylated. Similar results were observed in preparations examined independently from two monkeys. 
Western Blot Analysis
Phosphorylated and dephosphorylated NF-H proteins were detected by Western blot analysis with three antibodies (Fig. 7) . The extract from both the glaucomatous and control eyes showed a band at approximately 200 kDa corresponding positively to NF200. The band intensity in the glaucomatous eyes was slightly lower than in the control eyes. Two positive bands were detected with SMI31, as previously reported, 12 13 and the glaucomatous eyes showed lower intensity of both bands compared with the control eyes. However, only the glaucomatous eyes showed a positive band when SMI32, a specific antibody against dephosphorylated NF-H, was used. These results were duplicated in two independent experiments. 
Discussion
In the present study, normal RGC axons were highly phosphorylated, which is consistent with other axons in the central nervous system, 2 but RGC axons were significantly dephosphorylated in eyes with chronically elevated IOP in the regions of the retina and optic nerve head and even in the optic chiasm. The extent of dephosphorylation of NF-H appeared to parallel the extent of glaucoma-induced damage to axons in these eyes. Significant dephosphorylation was observed, even in axons showing less damage, as shown by the histologic study of the glaucomatous eyes. In addition to these immunohistochemical observations, Western blot analysis confirmed the change in the status of phosphorylation. This is the first study to investigate the status of NF-H phosphorylation, although Vickers et al. 14 15 have reported that the number of NF-protein–containing RGCs is reduced and that the extent of NF-protein loss is variable among the RGC subpopulation in an experimental model of glaucoma in monkeys. 
It is well-known that disturbance of axonal transport results in glaucoma-induced degeneration of the optic nerve. Recent reports have shown that phosphorylation of NF-H is deeply involved in axonal transport, axonal plasticity, and neuronal morphology, and also in maintenance of the integrity of the neuronal cytoskeleton. 1 3 4 5 Phosphorylated NF-Hs extend carboxyl-terminal tails radially as side arms to facilitate binding with other types of NF proteins and cytoskeleton components, whereas dephosphorylation inhibits their normal contact. 3 16 Several reports have demonstrated that perturbations in phosphorylation or disorganized neurofilaments result in neurofilament-induced diseases, such as amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease. 16 17  
In the present study, we used an experimental model of glaucoma in the monkey, in which structures of the retina, optic nerve head, and optic chiasm are closest to those in humans among all experimental animals. IOP was maintained between 30 and 40 mm Hg, which is considered to cause minimal disturbance in local circulation. Indeed, we performed fluorescence angiography several times during the experiment and confirmed no disturbance in local circulation in the optic nerve head. Therefore, we believe that this glaucoma model has several advantages over other glaucoma models with acute elevation of IOP and/or the use of nonprimate animals, and that the elevation of IOP in this model primarily causes structural and neurochemical changes. 
The antibodies used in the present study have a high affinity for specific antigens. NF200 reacts with epitopes in the tail domain of the 200-kDa neurofilaments that are present in both the phosphorylated and dephosphorylated forms of this polypeptide, whereas SMI31 reacts only with a phosphorylated epitope in extensively phosphorylated 200-kDa NF. Therefore, these two antibodies allowed us to determine the amount of NF-H and the phosphorylation status. 
NF-H is present in neurons in a dephosphorylated form and is probably transported as subunits or small oligomers along microtubules, which are major routes for slow axonal transport. 18 NF-H is phosphorylated when transported into an axon, although the precise mechanism of phosphorylation is not fully understood. In neurons, the NF-H proteins have sidearms that limit packing density. Only NF-M and NF-H subunits contribute to the sidearms, which are thought to be formed by carboxyl-terminal tails. 19 20 The phosphorylation sites have been identified as KSP (Lys-Ser-Pro) repeats in the tail domain of NF-H, 4 and phosphorylation of NF-H appears to be catalyzed by cyclin-dependent kinase 5 (CDK5), 21 22 23 24 external signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), or stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). 5 25 26 However, it is not known how these kinases contribute to the constitutive phosphorylation of NF proteins in neurons. 
Tokuoka et al. 13 have reported that some neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 stimulate phosphorylation of NF-H. Quigley et al. 27 and Pease et al. 28 have reported that the obstruction of the transport of brain-derived neurotrophic factor (BDNF) in the area of the optic nerve head presumably results in ganglion loss in glaucoma. The absence of BDNF transport in the glaucomatous eye may be related to the dephosphorylation of NF-H observed in the present study. 
The phosphorylation states of NF-H and NF-M have been shown to change the spacing between NFs, probably by strengthening the repulsive forces between projections, 29 which may be related to a reduction of optic nerve diameter in glaucomatous eyes, as previously reported. 30 31 32  
It remains unclear whether glaucoma-induced damage causes dephosphorylation of axons or dephosphorylation causes the damage. In the present study we found that the degree of axon phosphorylation was highest in the control eyes, less in mildly damaged regions, and least in severely damaged regions. In addition, there were significant differences in the ratio of phosphorylation among these three conditions. Axons showing less morphologic damage, as confirmed by light microscopy, showed significantly greater dephosphorylation than those in the control. All evidence taken together, dephosphorylation, at least, could be a risk factor for further deterioration of the optic nerve in glaucoma. However, the present study is not sufficient to conclude that dephosphorylation of NF-H precedes axonal loss in glaucoma, because of the limited number of monkeys included in the study and the low level of accuracy of the evaluation of lost axonal fibers. Therefore, further investigation that could clarify this hypothesis is necessary. 
Sawaguchi et al. 33 have reported that distortion of the LC results in damaged axonal transport in glaucomatous eyes and that axonal transport is the most damaged in the LC portion. In the present study, we did not detect a significant difference in the dephosphorylation ratio between the LC portion and post-LC portion, although one glaucomatous eye (monkey 1128302052) showed a higher rate of dephosphorylated NF-H in the LC region than in the post-LC region. 
In axons, NF-H interacts with microtubules playing a primary role in axonal transport. We have reported that expression of microtubule-associated protein 1, which binds to and stabilizes microtubules, is reduced in guinea pig optic nerves in a model of acutely elevated IOP. 34 Further investigation should reveal the interactions among neurochemicals and their roles in axonal transport. 
The present study revealed the presence of significant dephosphorylation in the axons at post-LC, even in the optic chiasm, indicating that glaucoma-induced damage may influence the central visual nervous pathway. Yucel et al. 35 36 have reported the loss and atrophy of relay neurons in the region of the lateral geniculate nucleus in monkeys with experimental glaucoma. It is, therefore, necessary to investigate the changes in axonal neurochemistry, even in the central visual system. 
 
Figure 1.
 
Phosphorylation of retinal NF-H in monkey 1128302052. The control eyes showed that most NF-Hs in axonal fibers were phosphorylated either in the temporal (a) or nasal retina (b). In contrast, laser-treated eyes showed a significant reduction in phosphorylation. NF-Hs were moderately dephosphorylated in the nasal retina (c), whereas most NF-Hs were dephosphorylated in the temporal retina (d). Only very faint signals were observed in the negative control (e). Magnification, ×400.
Figure 1.
 
Phosphorylation of retinal NF-H in monkey 1128302052. The control eyes showed that most NF-Hs in axonal fibers were phosphorylated either in the temporal (a) or nasal retina (b). In contrast, laser-treated eyes showed a significant reduction in phosphorylation. NF-Hs were moderately dephosphorylated in the nasal retina (c), whereas most NF-Hs were dephosphorylated in the temporal retina (d). Only very faint signals were observed in the negative control (e). Magnification, ×400.
Figure 2.
 
Morphologic changes in the optic disc in the LC region in the monkey in Figure 1 . Compared with the control eyes (a), a loss of axonal bands and an increasing amount of connective tissue were observed in the temporal LC region of laser-treated eyes (b). Magnification, ×400.
Figure 2.
 
Morphologic changes in the optic disc in the LC region in the monkey in Figure 1 . Compared with the control eyes (a), a loss of axonal bands and an increasing amount of connective tissue were observed in the temporal LC region of laser-treated eyes (b). Magnification, ×400.
Figure 3.
 
Phosphorylation of NF-H in the LC region in the monkey in Figure 1 . NF-H in axons was almost completely phosphorylated in control eyes (a), whereas that in laser-treated eyes was significantly dephosphorylated (b, c, d). NF-H in the nasal region was moderately phosphorylated (c), whereas most NF-H in the temporal region was dephosphorylated (d). The loss of axonal bands in the temporal region was substantial, compared with that in the nasal region. Magnification: (a, b) ×200; (c, d) ×400.
Figure 3.
 
Phosphorylation of NF-H in the LC region in the monkey in Figure 1 . NF-H in axons was almost completely phosphorylated in control eyes (a), whereas that in laser-treated eyes was significantly dephosphorylated (b, c, d). NF-H in the nasal region was moderately phosphorylated (c), whereas most NF-H in the temporal region was dephosphorylated (d). The loss of axonal bands in the temporal region was substantial, compared with that in the nasal region. Magnification: (a, b) ×200; (c, d) ×400.
Figure 4.
 
Phosphorylation of NF-H in the post-LC region in the monkey in Figure 1 . NF-H in axons was almost completely phosphorylated in control eyes (a), whereas that in laser-treated eyes was significantly dephosphorylated (b, c, d). NF-H in the nasal region was moderately phosphorylated (b, c, arrow), whereas most NF-H in the temporal region was dephosphorylated (b, d, arrowhead). Magnification: (b) ×200; (a, c, d) ×400.
Figure 4.
 
Phosphorylation of NF-H in the post-LC region in the monkey in Figure 1 . NF-H in axons was almost completely phosphorylated in control eyes (a), whereas that in laser-treated eyes was significantly dephosphorylated (b, c, d). NF-H in the nasal region was moderately phosphorylated (b, c, arrow), whereas most NF-H in the temporal region was dephosphorylated (b, d, arrowhead). Magnification: (b) ×200; (a, c, d) ×400.
Figure 5.
 
Comparison of ratios of phosphorylation between glaucomatous eyes and control eyes. Ratios of phosphorylation in the glaucomatous eyes were significantly lower than those in the control eyes, and NF-H in the temporal area was more dephosphorylated than in nasal areas. Bars, SD. *P = 0.13, †P < 0.01; Mann-Whitney test; n = 3.
Figure 5.
 
Comparison of ratios of phosphorylation between glaucomatous eyes and control eyes. Ratios of phosphorylation in the glaucomatous eyes were significantly lower than those in the control eyes, and NF-H in the temporal area was more dephosphorylated than in nasal areas. Bars, SD. *P = 0.13, †P < 0.01; Mann-Whitney test; n = 3.
Figure 6.
 
Phosphorylation of NF-H in the optic chiasm in monkey 1127903018. The extent of dephosphorylation in laser-treated eyes was as follows (from greatest to least): (a) the left lateral region, where most nerve fibers travel through the temporal disc, and (c) the right inner region, where most nerve fibers travel from the nasal disc. In control eyes the extent of dephosphorylation was as follows: (b) the left inner region, where a large number of nerve fibers travel from the nasal disc, and (d) the right lateral region, where most nerve fibers travel from the temporal disc. Magnification, ×100.
Figure 6.
 
Phosphorylation of NF-H in the optic chiasm in monkey 1127903018. The extent of dephosphorylation in laser-treated eyes was as follows (from greatest to least): (a) the left lateral region, where most nerve fibers travel through the temporal disc, and (c) the right inner region, where most nerve fibers travel from the nasal disc. In control eyes the extent of dephosphorylation was as follows: (b) the left inner region, where a large number of nerve fibers travel from the nasal disc, and (d) the right lateral region, where most nerve fibers travel from the temporal disc. Magnification, ×100.
Figure 7.
 
Western blot analysis in monkey 1118505065. The control eye showed a slightly more increased band corresponding to NF-H compared with the glaucomatous eye (NF200). A greater degree of phosphorylation was detected in the control eye than in the glaucomatous eye (SMI31). However, only the glaucomatous eye showed a positive band corresponding to dephosphorylated NF-H (SMI32). NF200 recognized both the phosphorylated and dephosphorylated forms of the 200-kDa neurofilaments, whereas SMI31 and SMI32 reacted with the phosphorylated form and dephosphorylated form, respectively. CONT, control; MS, molecular standards; GL, glaucoma.
Figure 7.
 
Western blot analysis in monkey 1118505065. The control eye showed a slightly more increased band corresponding to NF-H compared with the glaucomatous eye (NF200). A greater degree of phosphorylation was detected in the control eye than in the glaucomatous eye (SMI31). However, only the glaucomatous eye showed a positive band corresponding to dephosphorylated NF-H (SMI32). NF200 recognized both the phosphorylated and dephosphorylated forms of the 200-kDa neurofilaments, whereas SMI31 and SMI32 reacted with the phosphorylated form and dephosphorylated form, respectively. CONT, control; MS, molecular standards; GL, glaucoma.
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Figure 1.
 
Phosphorylation of retinal NF-H in monkey 1128302052. The control eyes showed that most NF-Hs in axonal fibers were phosphorylated either in the temporal (a) or nasal retina (b). In contrast, laser-treated eyes showed a significant reduction in phosphorylation. NF-Hs were moderately dephosphorylated in the nasal retina (c), whereas most NF-Hs were dephosphorylated in the temporal retina (d). Only very faint signals were observed in the negative control (e). Magnification, ×400.
Figure 1.
 
Phosphorylation of retinal NF-H in monkey 1128302052. The control eyes showed that most NF-Hs in axonal fibers were phosphorylated either in the temporal (a) or nasal retina (b). In contrast, laser-treated eyes showed a significant reduction in phosphorylation. NF-Hs were moderately dephosphorylated in the nasal retina (c), whereas most NF-Hs were dephosphorylated in the temporal retina (d). Only very faint signals were observed in the negative control (e). Magnification, ×400.
Figure 2.
 
Morphologic changes in the optic disc in the LC region in the monkey in Figure 1 . Compared with the control eyes (a), a loss of axonal bands and an increasing amount of connective tissue were observed in the temporal LC region of laser-treated eyes (b). Magnification, ×400.
Figure 2.
 
Morphologic changes in the optic disc in the LC region in the monkey in Figure 1 . Compared with the control eyes (a), a loss of axonal bands and an increasing amount of connective tissue were observed in the temporal LC region of laser-treated eyes (b). Magnification, ×400.
Figure 3.
 
Phosphorylation of NF-H in the LC region in the monkey in Figure 1 . NF-H in axons was almost completely phosphorylated in control eyes (a), whereas that in laser-treated eyes was significantly dephosphorylated (b, c, d). NF-H in the nasal region was moderately phosphorylated (c), whereas most NF-H in the temporal region was dephosphorylated (d). The loss of axonal bands in the temporal region was substantial, compared with that in the nasal region. Magnification: (a, b) ×200; (c, d) ×400.
Figure 3.
 
Phosphorylation of NF-H in the LC region in the monkey in Figure 1 . NF-H in axons was almost completely phosphorylated in control eyes (a), whereas that in laser-treated eyes was significantly dephosphorylated (b, c, d). NF-H in the nasal region was moderately phosphorylated (c), whereas most NF-H in the temporal region was dephosphorylated (d). The loss of axonal bands in the temporal region was substantial, compared with that in the nasal region. Magnification: (a, b) ×200; (c, d) ×400.
Figure 4.
 
Phosphorylation of NF-H in the post-LC region in the monkey in Figure 1 . NF-H in axons was almost completely phosphorylated in control eyes (a), whereas that in laser-treated eyes was significantly dephosphorylated (b, c, d). NF-H in the nasal region was moderately phosphorylated (b, c, arrow), whereas most NF-H in the temporal region was dephosphorylated (b, d, arrowhead). Magnification: (b) ×200; (a, c, d) ×400.
Figure 4.
 
Phosphorylation of NF-H in the post-LC region in the monkey in Figure 1 . NF-H in axons was almost completely phosphorylated in control eyes (a), whereas that in laser-treated eyes was significantly dephosphorylated (b, c, d). NF-H in the nasal region was moderately phosphorylated (b, c, arrow), whereas most NF-H in the temporal region was dephosphorylated (b, d, arrowhead). Magnification: (b) ×200; (a, c, d) ×400.
Figure 5.
 
Comparison of ratios of phosphorylation between glaucomatous eyes and control eyes. Ratios of phosphorylation in the glaucomatous eyes were significantly lower than those in the control eyes, and NF-H in the temporal area was more dephosphorylated than in nasal areas. Bars, SD. *P = 0.13, †P < 0.01; Mann-Whitney test; n = 3.
Figure 5.
 
Comparison of ratios of phosphorylation between glaucomatous eyes and control eyes. Ratios of phosphorylation in the glaucomatous eyes were significantly lower than those in the control eyes, and NF-H in the temporal area was more dephosphorylated than in nasal areas. Bars, SD. *P = 0.13, †P < 0.01; Mann-Whitney test; n = 3.
Figure 6.
 
Phosphorylation of NF-H in the optic chiasm in monkey 1127903018. The extent of dephosphorylation in laser-treated eyes was as follows (from greatest to least): (a) the left lateral region, where most nerve fibers travel through the temporal disc, and (c) the right inner region, where most nerve fibers travel from the nasal disc. In control eyes the extent of dephosphorylation was as follows: (b) the left inner region, where a large number of nerve fibers travel from the nasal disc, and (d) the right lateral region, where most nerve fibers travel from the temporal disc. Magnification, ×100.
Figure 6.
 
Phosphorylation of NF-H in the optic chiasm in monkey 1127903018. The extent of dephosphorylation in laser-treated eyes was as follows (from greatest to least): (a) the left lateral region, where most nerve fibers travel through the temporal disc, and (c) the right inner region, where most nerve fibers travel from the nasal disc. In control eyes the extent of dephosphorylation was as follows: (b) the left inner region, where a large number of nerve fibers travel from the nasal disc, and (d) the right lateral region, where most nerve fibers travel from the temporal disc. Magnification, ×100.
Figure 7.
 
Western blot analysis in monkey 1118505065. The control eye showed a slightly more increased band corresponding to NF-H compared with the glaucomatous eye (NF200). A greater degree of phosphorylation was detected in the control eye than in the glaucomatous eye (SMI31). However, only the glaucomatous eye showed a positive band corresponding to dephosphorylated NF-H (SMI32). NF200 recognized both the phosphorylated and dephosphorylated forms of the 200-kDa neurofilaments, whereas SMI31 and SMI32 reacted with the phosphorylated form and dephosphorylated form, respectively. CONT, control; MS, molecular standards; GL, glaucoma.
Figure 7.
 
Western blot analysis in monkey 1118505065. The control eye showed a slightly more increased band corresponding to NF-H compared with the glaucomatous eye (NF200). A greater degree of phosphorylation was detected in the control eye than in the glaucomatous eye (SMI31). However, only the glaucomatous eye showed a positive band corresponding to dephosphorylated NF-H (SMI32). NF200 recognized both the phosphorylated and dephosphorylated forms of the 200-kDa neurofilaments, whereas SMI31 and SMI32 reacted with the phosphorylated form and dephosphorylated form, respectively. CONT, control; MS, molecular standards; GL, glaucoma.
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