- Email: [email protected]
Retinal tau pathology in human glaucomas
Retinal tau pathology in human glaucomas Neeru Gupta,*†‡§ MD, PhD, FRCSC; Jessica Fong;*†‡ Lee C. Ang,|| MD, FRCSC; Yeni H. Yücel*†‡¶ MD, PhD, FRCPC ABSTRACT • RÉSUMÉ
Background: Tau protein is a microtubule-associated protein critical to neuron structure and integrity. The abnormal hyperphosphorylated tau protein AT8 disrupts microtubules, interferes with axonal transport, and is associated with neuron injury in neurodegenerative diseases such as Alzheimer’s disease. The purpose of this study was to assess the presence of tau protein and abnormal tau protein AT8 in human glaucomas and to determine whether abnormal tau protein plays a role in glaucomatous neural degeneration. Methods: Sections from 11 surgical eye specimens with glaucoma from elevated intraocular pressure causes and 10 age-matched control eye specimens were immunostained for normal tau protein (BT2) and hyperphosphorylated tau protein (AT8). Postmortem specimens with incidental open-angle glaucoma (n = 6) were compared with controls (n = 3). Measurements of immunofluorescence intensity in glaucoma retinas were compared with those in control retinas. Abnormal tau AT8 and parvalbumin, a horizontal cellspecific marker, were studied with double-immunofluorescence techniques to determine colocalization. Results: In surgical glaucoma specimens, normal tau protein was decreased in both the optic nerve and retina compared with age-matched controls. Abnormal tau AT8 was evident within the posterior retina, predominantly at the outer border of the inner nuclear layer in surgical glaucoma specimens, and this was not observed in controls or incidental glaucoma cases. Quantitative immunofluorescence techniques demonstrated significantly increased abnormal tau AT8 in surgical glaucoma specimens compared with controls. Abnormal tau AT8 colocalized with parvalbumin in horizontal cells of the retina. Interpretation: Abnormal tau AT8, a marker of injury in various neurological diseases, is present in human glaucomas with uncontrolled intraocular pressure. The finding of abnormal tau protein in retinal horizontal cells may relate to elevated intraocular pressure and (or) neural degeneration in glaucoma. Tau protein abnormality in glaucoma underscores shared pathways with other neurodegenerative diseases. Contexte : La protéine tau, protéine associée aux microtubules, est cruciale pour la structure et l’intégrité des neurones. La protéine tau hyperphosphorylée AT8 dissocie les microtubules, gène le transport axonal et est associée au traumatisme des neurones dans les maladies neurodégénératives, telle la maladie d’Alzheimer. Cette étude a donc pour objet d’évaluer la présence de protéine tau et de la protéine tau anormale AT8 dans les glaucomes humains et d’établir si la protéine tau anormale joue un rôle dans la dégénérescence neurale glaucomateuse. Méthodes : Des coupes de 11 spécimens oculaires chirurgicaux atteints de glaucome à cause d’une pression intraoculaire élevée et 10 spécimens oculaires d’un même groupe d’âge ont été colorées par voie immunologique pour la protéine tau normale (BT2) et protéine tau hyperphosphorylée (AT8). Coupes oculaires post-mortem ayant un diagnostic de glaucome à angle ouvert incident (n = 6) ont été évaluées comparativement à témoins du même âge (n = 3). On a comparé les degrés d’intensité de l’immunofluorescence des rétines glaucomateuses avec des rétines témoins. On a enfin la protéine tau anormale AT8 et la parvalbumine, marqueur spécifique des cellules horizontales, en utilisant les techniques de double immunofluorescence pour en établir la colocalisation.
From *the Department of Ophthalmology & Vision Sciences, †the Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Ont.; ‡Keenan Research Centre at the Li Ka Shing Knowledge Institute, Toronto, Ont.; §Glaucoma & Nerve Protection Unit, St. Michael’s Hospital, University of Toronto, Toronto, Ont.; ||the Department of Pathology, Neuropathology, University of Western Ontario, London, Ont.; and ¶Ophthalmic Pathology Laboratory, Department of Ophthalmology & Vision Sciences, University of Toronto, Toronto, Ont.
Correspondence to: Neeru Gupta, MD, Glaucoma & Nerve Protection Unit, St. Michael’s Hospital, University of Toronto, 30 Bond Street, Cardinal Carter Wing, Suite 8-072, Toronto, ON M5B 1W8; [email protected] This article has been peer-reviewed. Cet article a été évalué par les pairs. Can J Ophthalmol 2008;43:53–60 doi:10.3129/i07-185
Presented at the annual meeting of the American Academy of Ophthalmology in New Orleans, La., November 2007 Originally received Mar. 23, 2007. Revised July 24, 2007 Accepted for publication Aug. 15, 2007 Published online Jan. 21, 2008 CAN J OPHTHALMOL—VOL. 43, NO. 1, 2008
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Retinal tau pathology in human glaucomas—Gupta et al. Résultats : Chez les spécimens chirurgicaux de glaucome, la protéine tau normale avait baissé dans le nerf optique et la rétine comparativement aux témoins du même âge. La protéine tau anormale AT8 s’est avérée évidente dans la partie postérieure de la rétine et prédominante dans la couche nucléaire interne des spécimens glaucomateux chirurgicaux, alors qu’on n’en a pas observé chez les groupes témoins ni les spécimens oculaires post-mortem. Les techniques quantitatives d’immunofluorescence ont démontré que la protéine tau anormale AT8 avait augmenté façon substantielle par rapport aux groupes témoins. La protéine tau anormale AT8 était colocalisée avec la parvalbumine dans les cellules horizontales de la rétine. Interprétation : La protéine tau anormale AT8, marqueur de lésions dans diverses maladies neurologiques, est présente dans le glaucome humain à cause d’une pression intraoculaire non maîtrisée. La présence de la protéine tau anormale dans les cellules horizontales de la rétine peut être reliée a une forte pression intraoculaire et (ou) à une dégénérescence neurale avancée dans le glaucome. L’anormalité de la protéine tau dans le glaucome met en évidence le partage de voies avec d’autres maladies dégénératives.
G
laucoma is a leading cause of irreversible blindness, with elevated intraocular pressure (IOP) as a major risk factor. Elevated IOP may lead to retinal ganglion cell (RGC) death, characteristic optic nerve head changes, and central visual pathway degeneration.1–8 Blocked axon transport following IOP elevation may contribute to neural injury by deprivation of essential nutrient and growth factor supplies.9,10 Tau protein is a microtubule-associated protein associated with axon transport in healthy nerve cells. The abnormal phosphorylation of tau may disrupt microtubules, interfere with axon transport mechanisms,11 and be toxic to neurons.12 Abnormal tau protein is a hallmark of a number of neurodegenerative diseases collectively known as tauopathies and these include Alzheimer’s disease, supranuclear palsy, corticobasal degeneration, Pick’s disease, argyrophilic grain disease, and frontotemporal dementia and parkinsonism linked to chromosome 17 with tau mutations, among others.13,14 Abnormal tau AT8 with the phospho-epitope at serine 202 is well characterized, and AT8 antibody is used routinely to stage human neurodegenerative diseases in neuropathology.15–18 It is not known whether abnormal tau protein AT8 plays a role in glaucomatous neural degeneration. Using immunocytochemical and quantitative methodologies, this study investigates normal tau protein and abnormal tau AT8 in the human retina in glaucomas with uncontrolled IOP, and incidental primary open-angle glaucoma, compared with age-matched controls. METHODS Specimens and processing
Following institutional research ethics board approval, normal and glaucoma human eye specimens were obtained from the Eye Bank of Canada, Ontario Division, and the Ophthalmic Pathology Laboratory, University of Toronto. Inclusion criteria for surgical glaucoma specimens were a history of glaucoma necessitating enucleation for uncontrolled IOP and histopathological demonstration of optic nerve head excavation, a patho-
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logical hallmark of glaucomatous damage. Incidental glaucoma cases were based on a history of glaucoma at the time of death. Exclusion criteria included chronic central nervous system disorder, diabetes, and intraocular tumor. Eleven glaucoma surgical eye specimens enucleated because of uncontrolled IOP from different subjects and 10 control eyes from different age-matched control subjects were studied (Table 1). The mean ages of surgical glaucoma patients and control patients were 42.7 (SD 20.0) years and 49.3 (SD 20.6) years, respectively. To assess whether these changes were related to uncontrolled IOP, 6 incidental open-angle glaucoma specimens and 3 age-matched control eyes were also studied, with mean ages of 77 (SD 5.55) years and 74 (SD 14.45) years, respectively (Table 2). The specimens were fixed in 10% neutral-buffered formalin, processed routinely, embedded in paraffin, and sectioned with a rotatory microtome at 7 μm. Sections were deparaffinized in Histoclear II (National Diagnostics, Atlanta, Ga.) and rehydrated in a Table 1—Details of surgical glaucoma and control eyes Sex
Eye
Pathological diagnosis/ cause of death
Time to fixation* (h)
19 19 24 29 37 38 49 52 57 66 80
M M M M M M M M M M M
OD OS OD OS OS OD OS OD OS OD OD
Angle closure Juvenile glaucoma Juvenile glaucoma Angle closure Angle closure Angle closure Angle closure Angle closure Angle recession Primary open-angle glaucoma Angle closure
<1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
18 26 27 35 58 59 59 68 69 74
M M M M F M F M M M
OD OD OS OS OD OS OD OD OD OD
Head trauma Heart transplant rejection Liver failure Myocardial fibrosis Intracerebral hemorrhage Ischemic bowel, sepsis Cardiac arrest, trauma Arteriosclerotic heart disease Pancreatic cancer Myocardial infarction
>48 9 >24 21 15 >48 21 8 >24 10
Patient no. Age (y) Glaucoma 1 2 3 4 5 6 7 8 9 10 11 Control 1 2 3 4 5 6 7 8 9 10
*Time from surgery or death to fixation. Note: y, years; h, hours; F, female; M, male.
Retinal tau pathology in human glaucomas—Gupta et al. graded alcohol series prior to immunocytochemical stainings. Experiments of all glaucoma and age-matched control retinas were performed simultaneously under similar conditions, and experiments were performed at least twice. Negative controls were obtained by omitting the primary antibodies. Brain sections from patients with Alzheimer’s disease with AT8-positive neurofibrillary tangles were used as positive controls. Normal tau and abnormal tau Immunoperoxidase labeling
Sections were washed in 0.05 mol/L tris-buffered saline and incubated in 3% hydrogen peroxide for 10 minutes to quench endogenous peroxide. Sections were incubated in citrate–ethylenediaminetetraacetic acid (EDTA) buffer (pH 6.2) and heated for 30 seconds in a microwave oven (Danby Products Ltd, Guelph, Ont.). After incubating in Power Block (BioGenex, San Ramon, Calif.) for 10 minutes at room temperature, they were washed in 0.01 mol/L phosphate-buffered saline (PBS). Primary antibody, BT2 diluted 1:400 (monoclonal antibody, Pierce Biotech Inc, Rockford, Ill.) or AT8 diluted 1:800 (monoclonal antibody, Pierce Biotech Inc) in blocking reagent, was applied for 1 hour at room temperature. BT2 antibody recognizes normal tau between residue 194 and 198, whereas AT8 antibody recognizes abnormally phosphorylated tau at the serine 202 residue (numbering according to human Tau40). The antibody–antigen complexes were detected with a supersensitive detection kit (BioGenex) that included horseradish peroxidase-conjugated streptavidin and 3,3-diaminobenzidine. Sections were then counterstained with Mayer’s hematoxylin and rinsed in water. Finally, they were dehydrated in a graded alcohol series and mounted before applying a coverslip using Histomount (National Diagnostics). Negative controls were obtained by omitting the primary antibodies.
heated in a pressure cooker (Biocare Medical DC2002, Concord, Calif.) for 15 seconds for antigen retrieval. After incubating the sections in PBS with 10% normal goat serum, 1% bovine serum albumin, and 0.1% Triton X-100 for 1 hour at room temperature, the primary antibody, either BT2 (1:100 dilution) or AT8 (1:200 dilution) in PBS with 3% normal goat serum, was applied for 1 hour at room temperature. The secondary antibody, Alexa 488conjugated goat anti-mouse immunoglobulin (Ig) G (Invitrogen Canada Inc, Burlington, Ont.) diluted 1:400 in PBS with 1% normal goat serum, was applied for 1 hour at room temperature. Sections were coverslipped with polyvinyl alcohol (PVA) (Sigma, Oakville, Ont.) and 1,4diazabicyclo[2,2,2]octane (DABCO) (Sigma) antifade mounting medium. Double labeling for abnormally phosphorylated tau and parvalbumin
Parvalbumin is a well-established marker for horizontal cells,19,20 and double labeling with anti-parvalbumin antibody was used to demonstrate that hyperphosphorylated tau is located in horizontal cells. Sections were prepared as above and incubated overnight at 4 oC with the first primary antibody, AT8 diluted 1:200 in PBS with 3% normal goat serum. The secondary antibody, Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc, West Grove, Penn.) diluted 1:200 in PBS with 1% normal goat serum, was applied for 1 hour at room temperature. Sections were then washed in PBS and incubated for 1 hour at room temperature with the second primary antibody against parvalbumin polyclonal antibody (Swant Inc, Bellinzona, Switzerland) diluted 1:1000 in PBS with 3% normal goat serum. This was followed by application of the second secondary antibody, Alexa 488conjugated goat anti-rabbit IgG (Invitrogen Canada Inc) with 1:400 dilution in PBS with 1% normal goat serum, for 1 hour at room temperature. Sections were coverslipped using PVA-DABCO antifade mounting medium.
Immunofluorescence labeling
After several washes in 0.05 mol/L tris-buffered saline, sections were incubated in citrate-EDTA buffer (pH 6.0) Table 2—Details of postmortem eyes with incidental glaucoma and control eyes Sex
Eye
Pathological diagnosis/ cause of death
Time to fixation* (h)
81 81 68 77 73 82
F F F F M F
OS OD OS OD OD OD
Primary open-angle glaucoma Primary open-angle glaucoma Primary open-angle glaucoma Primary open-angle glaucoma Primary open-angle glaucoma Primary open-angle glaucoma
7 17 21 21 6 9
57 83 81
F M M
OS OD OD
Cerebrovascular accident Natural causes Cerebrovascular accident
6 1 17
Patient no. Age (y) Glaucoma 1 2 3 4 5 6 Control 1 2 3
*Time from surgery or death to fixation. Note: y, years; h, hours; F, female; M, male.
Confocal laser scanning microscopy
Immunofluorescence-labeled sections were viewed using a confocal laser scanning microscope (Bio-Rad Radiance 2000, Hercules, Calif.). An acousto-optical tunable filter was used to maintain spectral separation and prevent bleedthrough. Pixel images (512–512) of the retina were obtained using LaserSharp 2000 software (Bio-Rad Cell Science Division, Hernel Hempstead, U.K.) and exported in *.TIF format. Immunofluorescence intensity measurements
Confocal images of the retina sections immunolabeled for BT2 or AT8 were obtained by a reader without knowledge of glaucoma or control status. Anterior and posterior retinal images were captured at one-third of the distance from the pars plana to the equator, and at one-third of the distance from the optic disc to the equator, respectively, for CAN J OPHTHALMOL—VOL. 43, NO. 1, 2008
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Retinal tau pathology in human glaucomas—Gupta et al. consistency. During this procedure, gain and exposure settings were adjusted and kept constant for all sections. Digital images were imported into ImageJ (v. 1.37, National Institutes of Health, Bethesda, Md.) for immunofluorescence intensity analysis. A constant threshold value was applied to all images. Using the “measure” tool on ImageJ, the number of pixels that matched or exceeded the set threshold value was calculated for each image. This value was divided by the total number of pixels in the image to determine the fluorescence intensity index, a measure of the level of immunofluorescence. Immunofluorescence intensity index values in glaucoma retinas were compared with controls using a two-tailed t test, using GraphPad Prism (v. 3.02, GraphPad Software
Inc, San Diego, Calif.). A p value of less than 0.05 was considered statistically significant. Colocalization study of dual-color confocal images
Confocal images of the retina were shown using ImageJ in their corresponding channel windows, channel 1 (red) and channel 2 (green). The background was subtracted from the images. Manders’ overlap coefficient and Pearson’s correlation coefficient were calculated. Manders’ coefficient ranges in value from 0 to 1, and is independent of pixel intensity. It represents the number of colocalized pixels expressed as a fraction of the number of pixels in the dualcolor image. Pearson’s coefficient ranges in value from –1 to 1, and is an index to determine pattern similarity.21,22 To determine whether colocalization occurred as a matter of chance, random images were generated as previously described.23 Blocks of pixels from channel 2 were rearranged at random to create a set of scrambled images. These were compared with channel 1, and Pearson’s correlation coefficient was calculated for each new random image. For each comparison, 100 such random images were generated, and the values of the random Pearson’s coefficient were compared with the actual value. RESULTS Normal tau protein in control and glaucoma
All control eyes showed normal tau immunoreactivity (BT2) in the inner nuclear and inner plexiform layers, with variable staining in RGC and nerve fiber layers (Fig. 1A).
Fig. 1—In the control retina (A), normal tau protein (brown) was observed in the inner nuclear and inner plexiform layers and the RGC layer (BT2 immunoreactivity with hematoxylin counterstain). In glaucoma (B), normal tau protein was absent. Immunofluorescence BT2 stain (bright green) confirms normal tau protein in control (C), and reduced tau protein in the glaucoma (D) inner retinas. Calibration bar indicates 25 μm.
Fig. 2—Normal tau protein immunofluorescence by mean intensity index in control (grey) and glaucoma (black). The difference in normal tau protein between control and glaucoma groups was statistically significant (*, p < 0.05).
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Fig. 3—In the normal optic nerve head, normal tau protein (BT2) (brown) was observed (A) and in glaucoma, it was mainly absent (B). Calibration bar indicates 250 μm.
Retinal tau pathology in human glaucomas—Gupta et al. In all surgical glaucoma retinas, normal tau profiles were either significantly reduced or absent (Fig. 1B). BT2 immunofluorescence labeling confirmed the presence of normal tau in the inner nuclear and inner plexiform layers in control retinas (Fig. 1C) and decreased normal tau in surgical glaucoma retinas (Fig. 1D). The mean immuno-
fluorescence intensity index for BT2 tau protein immunoreactivity in the surgical glaucoma group was significantly decreased compared with the control group (0.008 [SD 0.014] vs. 0.107 [SD 0.056]; p = 0.0002) (Fig. 2). Incidental postmortem glaucoma cases were not different from age-matched controls (0.156 [SD 0.081] vs. 0.154 [SD 0.078]; p > 0.05). Control optic nerves showed abundant normal tau (BT2) in the prelaminar and postlaminar portions of the optic nerve (Fig. 3A), and this was similar to most incidental postmortem glaucoma cases. In contrast, immunoreactivity for normal tau was absent in all surgical glaucomatous optic nerves studied (Fig. 3B). Abnormal tau (AT8) in control and glaucoma
Fig. 4—AT8 immunoreactivity was not observed in control posterior retinas. Calibration bar indicates 25 μm.
Control eyes showed very weak to absent immunoreactivity for abnormally phosphorylated tau AT8 in the posterior retina (Fig. 4), similar to incidental open-angle glaucoma eyes. All surgical glaucoma eyes were immunoreactive for tau AT8 at the outer border of the inner nuclear retinal layer (Fig. 5). AT8 immunoreactivity was also noted in the inner plexiform layer in the rare case. Optic nerve heads examined in control and glaucoma cases did not show evidence of AT8 immunoreactivity.
Fig. 5—Glaucomatous optic nerve head excavation was evident in glaucoma cases (left column). Abnormal tau AT8 (brown) in the retina was consistently observed in glaucoma (middle column). Boxed areas in middle column are seen at higher power in the right column. Red arrows indicate AT8 immunoreactivity observed at the outer border of the inner nuclear layer. Calibration bars indicate 250 μm (left column), and 25 μm (middle and right columns). (Row A, patient no. 10; B, no. 9; C, no. 8; D, no. 7.) CAN J OPHTHALMOL—VOL. 43, NO. 1, 2008
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Retinal tau pathology in human glaucomas—Gupta et al. Double labeling with AT8 and parvalbumin in the retina
AT8 immunofluorescence labeling in red confirmed the presence of abnormal tau at the outer border of the inner nuclear layer in surgical glaucoma cases (Fig. 6D). Minimal to no AT8 signal was observed in posterior control retinas (Fig. 6C). The same sections were labeled also for parvalbumin-immunofluorescence, a horizontal cell marker. Figures 6A and B show the presence of parvalbumin labeling in green in control and glaucoma retinas, respectively. AT8 immunoreactivity colocalized to parvalbumin-immunoreactive horizontal cells in glaucoma (Fig. 6F), and this was not seen in the control (Fig. 6E). Colocalization analysis of glaucoma retina images generated values for Manders’ overlap and Pearson’s correlation coefficients that were close to 1. Manders’ overlap coefficient ranged in value from 0.709 to 0.881, and Pearson’s correlation coefficient ranged from 0.514 to 0.716, indicating a high degree of colocalization for abnormal tau (AT8) and parvalbumin. Costes’
Fig. 6—Parvalbumin immunofluorescence in green labels horizontal cells in control (A) and glaucoma (B) retinas. Abnormal tau AT8 immunofluorescence in red is absent in controls (C), and present in horizontal cells in glaucoma (D). Double labeling with parvalbumin (green) and abnormal tau AT8 (red) shows only parvalbumin in control (E) and colocalization of parvalbumin and abnormal tau protein (yellow) in glaucoma (F).The calibration bar indicates 25 μm.
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analysis generated random values for Pearson’s correlation coefficient that were lower than the actual value in 100% of iterations, indicating that this colocalization is not due to chance alone. The immunofluorescence intensity index for abnormal tau AT8 in the posterior retina in glaucoma was significantly increased compared with controls (0.030 [SD 0.020] vs. 0.003 [SD 0.003]; p = 0.0012) (Fig. 7). In control and surgical glaucoma groups, mild AT8 staining adjacent to pars plana was observed in some cases, with no significant difference in immunofluorescence intensity index observed between the surgical glaucoma and control groups (0.016 [SD 0.014] vs. 0.018 [SD 0.015]; p = 0.79). INTERPRETATION
Normal tau protein has been described in normal human retina, and the present study confirms this in the inner nuclear retinal layers, including the RGCs and their nerve fibers.24 The decrease in normal tau in the inner retinal layers in glaucoma compared with controls is consistent with the advanced glaucoma seen in the surgical glaucoma enucleation specimens. The finding of abnormal tau protein was observed across a broad age range, from 19 years to 80 years, in cases of glaucoma from primary and secondary causes with uncontrolled elevated IOP. Abnormal tau protein is implicated in altered microtubule stability,11 axonal transport,25,26 and neurodegenerative diseases.14 Abnormal tau AT8 detected in these cases of glaucomas from causes of elevated IOP and (or) transneuronal degeneration is likely to be a posttranslational modification secondary to insult, as seen in various types of neuron injury.27,28 The absence of hyperphosphorylated AT8 in postmortem eye specimens with incidental open-angle glaucoma at the time of death further supports the theory that abnormal tau is related to uncontrolled elevated IOP and/or advanced glaucomatous damage. The specific localization of abnormal tau protein in
Fig. 7—Mean immunofluorescence intensity index in the retina for abnormal tau AT8 in control (grey) and glaucoma (black). The difference between glaucoma and control groups is statistically significant (*, p < 0.05).
Retinal tau pathology in human glaucomas—Gupta et al. human glaucoma to retinal horizontal cells was an unexpected finding. Despite known variation among human tissue specimens, the positive finding of abnormal tau in horizontal cells was consistent for each surgical glaucoma eye, across all ages. Horizontal cell pathology was noted in 2 cases of secondary glaucoma.29 Horizontal cells influence the spatial-chromatic organization of the receptive fields of RGCs.30 Recent experimental evidence in nonhuman primates suggests that the surrounds of parasol ganglion cells are generated mainly by horizontal cell negative feedback to the photoreceptors.31 One hypothesis for the horizontal cell abnormality in glaucoma takes into account the unique lateral arrangement of horizontal cell processes, possibly predisposing them to IOP-induced retinal stretch injury, inducing structural changes and tau protein alterations. It is also possible that the remodelling of horizontal cell processes observed following retinal degeneration and retinitis pigmentosa might also play a role following RGC loss in glaucoma.32–34 In experimental glaucoma, a number of molecules have been implicated in microtubule stability and intraneuronal transport, including kinesin and dynein,35 amyloid precursor protein,36 kinases,37–40 and phosphatases.41–44 Further studies of abnormal tau protein in experimental ocular hypertension models are needed to determine a possible relationship between tau changes and elevated IOP and (or) glaucomatous RGC injury. In addition, access to human cases with mild and moderate disease states and better characterized clinical history as it relates to visual field loss and IOP elevation would be helpful. The possible relationship of horizontal cell injury to glaucoma is intriguing, and whether horizontal cells contribute to visual dysfunction in glaucoma remains to be determined. Tau protein pathology in glaucoma underscores the presence of shared pathways with other neurodegenerative diseases, and may add to our understanding of the neuropathological processes leading to vision loss from glaucoma.45 Furthermore, treatment strategies directed at preliminary steps in the molecular cascades causing tauopathies may also be relevant to glaucomatous disease. Eye specimens were obtained from the Eye Bank of Canada, Ontario Division, and Ophthalmic Pathology Laboratory, University of Toronto. We thank Judy Trogadis of the St. Michael’s Hospital Bioimaging Centre. We are grateful to Eileen Girard, MLT, for her excellent technical assistance. This work was supported by the Glaucoma Research Society of Canada (Neeru Gupta, Yeni Yücel), the Thor Eaton Fund (Neeru Gupta) and Roy Foss fund (Neeru Gupta), and the Institute of Medical Science Summer Research Program, University of Toronto (Jessica Fong).
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Optic nerve dynein motor protein distribution changes with intraocular pressure elevation in a rat model of glaucoma. Exp Eye Res 2006;83:255–62. McKinnon SJ, Lehman DM, Kerrigan-Baumrind LA, et al. Caspase activation and amyloid precursor protein cleavage in rat ocular hypertension. Invest Ophthalmol Vis Sci 2002;43:1077–87. Lovell MA, Xiong S, Xie C, Davies P, Markesbery WR. Induction of hyperphosphorylated tau in primary rat cortical neuron cultures mediated by oxidative stress and glycogen synthase kinase-3. J Alzheimers Dis 2004;6:659–71. Ferrer I, Barrachina M, Puig B. Glycogen synthase kinase-3 is associated with neuronal and glial hyperphosphorylated tau deposits in Alzheimer’s disease, Pick’s disease, progressive supranuclear palsy and corticobasal degeneration. Acta Neuropathol 2002;104:583–91. Augustinack JC, Sanders JL, Tsai LH, Hyman BT. Colocalization and fluorescence resonance energy transfer between cdk5 and AT8 suggests a close association in pre-neurofibrillary tangles and neurofibrillary tangles. J Neuropathol Exp Neurol 2002;61:557–64. Plattner F, Angelo M, Giese KP. The roles of cyclin-dependent kinase 5 and glycogen synthase kinase 3 in tau hyperphosphorylation. J Biol Chem 2006;281(35):25457–65. Gong CX, Shaikh S, Grundke-Iqbal I, Iqbal K. Inhibition of protein phosphatase-2B (calcineurin) activity towards Alzheimer abnormally phosphorylated tau by neuroleptics. Brain Res 1996;741:95–102. Gong CX, Lidsky T, Wegiel J, Zuck L, Grundke-Iqbal I, Iqbal K. Phosphorylation of microtubule-associated protein tau is regulated by protein phosphatase 2A in mammalian brain. Implications for neurofibrillary degeneration in Alzheimer’s disease. J Biol Chem 2000;275:5535–44. Goedert M, Satumtira S, Jakes R, et al. Reduced binding of protein phosphatase 2A to tau protein with frontotemporal dementia and parkinsonism linked to chromosome 17 mutations. J Neurochem 2000;75:2155–62. Rahman A, Grundke-Iqbal I, Iqbal K. PP2B isolated from human brain preferentially dephosphorylates Ser-262 and Ser396 of the Alzheimer disease abnormally hyperphosphorylated tau. J Neural Transm 2006;113:219–30. Gupta N, Yücel YH. Glaucoma as a neurodegenerative disease. Curr Opin Ophthalmol 2007;18:110–4.
Key words: microtubule-associated protein, neurodegenerative diseases, Alzheimer’s, tauopathy, horizontal cells, optic nerve
Background: Tau protein is a microtubule-associated protein critical to neuron structure and integrity. The abnormal hyperphosphorylated tau protein AT8 disrupts microtubules, interferes with axonal transport, and is associated with neuron injury in neurodegenerative diseases such as Alzheimer’s disease. The purpose of this study was to assess the presence of tau protein and abnormal tau protein AT8 in human glaucomas and to determine whether abnormal tau protein plays a role in glaucomatous neural degeneration. Methods: Sections from 11 surgical eye specimens with glaucoma from elevated intraocular pressure causes and 10 age-matched control eye specimens were immunostained for normal tau protein (BT2) and hyperphosphorylated tau protein (AT8). Postmortem specimens with incidental open-angle glaucoma (n = 6) were compared with controls (n = 3). Measurements of immunofluorescence intensity in glaucoma retinas were compared with those in control retinas. Abnormal tau AT8 and parvalbumin, a horizontal cellspecific marker, were studied with double-immunofluorescence techniques to determine colocalization. Results: In surgical glaucoma specimens, normal tau protein was decreased in both the optic nerve and retina compared with age-matched controls. Abnormal tau AT8 was evident within the posterior retina, predominantly at the outer border of the inner nuclear layer in surgical glaucoma specimens, and this was not observed in controls or incidental glaucoma cases. Quantitative immunofluorescence techniques demonstrated significantly increased abnormal tau AT8 in surgical glaucoma specimens compared with controls. Abnormal tau AT8 colocalized with parvalbumin in horizontal cells of the retina. Interpretation: Abnormal tau AT8, a marker of injury in various neurological diseases, is present in human glaucomas with uncontrolled intraocular pressure. The finding of abnormal tau protein in retinal horizontal cells may relate to elevated intraocular pressure and (or) neural degeneration in glaucoma. Tau protein abnormality in glaucoma underscores shared pathways with other neurodegenerative diseases. Contexte : La protéine tau, protéine associée aux microtubules, est cruciale pour la structure et l’intégrité des neurones. La protéine tau hyperphosphorylée AT8 dissocie les microtubules, gène le transport axonal et est associée au traumatisme des neurones dans les maladies neurodégénératives, telle la maladie d’Alzheimer. Cette étude a donc pour objet d’évaluer la présence de protéine tau et de la protéine tau anormale AT8 dans les glaucomes humains et d’établir si la protéine tau anormale joue un rôle dans la dégénérescence neurale glaucomateuse. Méthodes : Des coupes de 11 spécimens oculaires chirurgicaux atteints de glaucome à cause d’une pression intraoculaire élevée et 10 spécimens oculaires d’un même groupe d’âge ont été colorées par voie immunologique pour la protéine tau normale (BT2) et protéine tau hyperphosphorylée (AT8). Coupes oculaires post-mortem ayant un diagnostic de glaucome à angle ouvert incident (n = 6) ont été évaluées comparativement à témoins du même âge (n = 3). On a comparé les degrés d’intensité de l’immunofluorescence des rétines glaucomateuses avec des rétines témoins. On a enfin la protéine tau anormale AT8 et la parvalbumine, marqueur spécifique des cellules horizontales, en utilisant les techniques de double immunofluorescence pour en établir la colocalisation.
From *the Department of Ophthalmology & Vision Sciences, †the Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Ont.; ‡Keenan Research Centre at the Li Ka Shing Knowledge Institute, Toronto, Ont.; §Glaucoma & Nerve Protection Unit, St. Michael’s Hospital, University of Toronto, Toronto, Ont.; ||the Department of Pathology, Neuropathology, University of Western Ontario, London, Ont.; and ¶Ophthalmic Pathology Laboratory, Department of Ophthalmology & Vision Sciences, University of Toronto, Toronto, Ont.
Correspondence to: Neeru Gupta, MD, Glaucoma & Nerve Protection Unit, St. Michael’s Hospital, University of Toronto, 30 Bond Street, Cardinal Carter Wing, Suite 8-072, Toronto, ON M5B 1W8; [email protected] This article has been peer-reviewed. Cet article a été évalué par les pairs. Can J Ophthalmol 2008;43:53–60 doi:10.3129/i07-185
Presented at the annual meeting of the American Academy of Ophthalmology in New Orleans, La., November 2007 Originally received Mar. 23, 2007. Revised July 24, 2007 Accepted for publication Aug. 15, 2007 Published online Jan. 21, 2008 CAN J OPHTHALMOL—VOL. 43, NO. 1, 2008
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Retinal tau pathology in human glaucomas—Gupta et al. Résultats : Chez les spécimens chirurgicaux de glaucome, la protéine tau normale avait baissé dans le nerf optique et la rétine comparativement aux témoins du même âge. La protéine tau anormale AT8 s’est avérée évidente dans la partie postérieure de la rétine et prédominante dans la couche nucléaire interne des spécimens glaucomateux chirurgicaux, alors qu’on n’en a pas observé chez les groupes témoins ni les spécimens oculaires post-mortem. Les techniques quantitatives d’immunofluorescence ont démontré que la protéine tau anormale AT8 avait augmenté façon substantielle par rapport aux groupes témoins. La protéine tau anormale AT8 était colocalisée avec la parvalbumine dans les cellules horizontales de la rétine. Interprétation : La protéine tau anormale AT8, marqueur de lésions dans diverses maladies neurologiques, est présente dans le glaucome humain à cause d’une pression intraoculaire non maîtrisée. La présence de la protéine tau anormale dans les cellules horizontales de la rétine peut être reliée a une forte pression intraoculaire et (ou) à une dégénérescence neurale avancée dans le glaucome. L’anormalité de la protéine tau dans le glaucome met en évidence le partage de voies avec d’autres maladies dégénératives.
G
laucoma is a leading cause of irreversible blindness, with elevated intraocular pressure (IOP) as a major risk factor. Elevated IOP may lead to retinal ganglion cell (RGC) death, characteristic optic nerve head changes, and central visual pathway degeneration.1–8 Blocked axon transport following IOP elevation may contribute to neural injury by deprivation of essential nutrient and growth factor supplies.9,10 Tau protein is a microtubule-associated protein associated with axon transport in healthy nerve cells. The abnormal phosphorylation of tau may disrupt microtubules, interfere with axon transport mechanisms,11 and be toxic to neurons.12 Abnormal tau protein is a hallmark of a number of neurodegenerative diseases collectively known as tauopathies and these include Alzheimer’s disease, supranuclear palsy, corticobasal degeneration, Pick’s disease, argyrophilic grain disease, and frontotemporal dementia and parkinsonism linked to chromosome 17 with tau mutations, among others.13,14 Abnormal tau AT8 with the phospho-epitope at serine 202 is well characterized, and AT8 antibody is used routinely to stage human neurodegenerative diseases in neuropathology.15–18 It is not known whether abnormal tau protein AT8 plays a role in glaucomatous neural degeneration. Using immunocytochemical and quantitative methodologies, this study investigates normal tau protein and abnormal tau AT8 in the human retina in glaucomas with uncontrolled IOP, and incidental primary open-angle glaucoma, compared with age-matched controls. METHODS Specimens and processing
Following institutional research ethics board approval, normal and glaucoma human eye specimens were obtained from the Eye Bank of Canada, Ontario Division, and the Ophthalmic Pathology Laboratory, University of Toronto. Inclusion criteria for surgical glaucoma specimens were a history of glaucoma necessitating enucleation for uncontrolled IOP and histopathological demonstration of optic nerve head excavation, a patho-
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logical hallmark of glaucomatous damage. Incidental glaucoma cases were based on a history of glaucoma at the time of death. Exclusion criteria included chronic central nervous system disorder, diabetes, and intraocular tumor. Eleven glaucoma surgical eye specimens enucleated because of uncontrolled IOP from different subjects and 10 control eyes from different age-matched control subjects were studied (Table 1). The mean ages of surgical glaucoma patients and control patients were 42.7 (SD 20.0) years and 49.3 (SD 20.6) years, respectively. To assess whether these changes were related to uncontrolled IOP, 6 incidental open-angle glaucoma specimens and 3 age-matched control eyes were also studied, with mean ages of 77 (SD 5.55) years and 74 (SD 14.45) years, respectively (Table 2). The specimens were fixed in 10% neutral-buffered formalin, processed routinely, embedded in paraffin, and sectioned with a rotatory microtome at 7 μm. Sections were deparaffinized in Histoclear II (National Diagnostics, Atlanta, Ga.) and rehydrated in a Table 1—Details of surgical glaucoma and control eyes Sex
Eye
Pathological diagnosis/ cause of death
Time to fixation* (h)
19 19 24 29 37 38 49 52 57 66 80
M M M M M M M M M M M
OD OS OD OS OS OD OS OD OS OD OD
Angle closure Juvenile glaucoma Juvenile glaucoma Angle closure Angle closure Angle closure Angle closure Angle closure Angle recession Primary open-angle glaucoma Angle closure
<1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
18 26 27 35 58 59 59 68 69 74
M M M M F M F M M M
OD OD OS OS OD OS OD OD OD OD
Head trauma Heart transplant rejection Liver failure Myocardial fibrosis Intracerebral hemorrhage Ischemic bowel, sepsis Cardiac arrest, trauma Arteriosclerotic heart disease Pancreatic cancer Myocardial infarction
>48 9 >24 21 15 >48 21 8 >24 10
Patient no. Age (y) Glaucoma 1 2 3 4 5 6 7 8 9 10 11 Control 1 2 3 4 5 6 7 8 9 10
*Time from surgery or death to fixation. Note: y, years; h, hours; F, female; M, male.
Retinal tau pathology in human glaucomas—Gupta et al. graded alcohol series prior to immunocytochemical stainings. Experiments of all glaucoma and age-matched control retinas were performed simultaneously under similar conditions, and experiments were performed at least twice. Negative controls were obtained by omitting the primary antibodies. Brain sections from patients with Alzheimer’s disease with AT8-positive neurofibrillary tangles were used as positive controls. Normal tau and abnormal tau Immunoperoxidase labeling
Sections were washed in 0.05 mol/L tris-buffered saline and incubated in 3% hydrogen peroxide for 10 minutes to quench endogenous peroxide. Sections were incubated in citrate–ethylenediaminetetraacetic acid (EDTA) buffer (pH 6.2) and heated for 30 seconds in a microwave oven (Danby Products Ltd, Guelph, Ont.). After incubating in Power Block (BioGenex, San Ramon, Calif.) for 10 minutes at room temperature, they were washed in 0.01 mol/L phosphate-buffered saline (PBS). Primary antibody, BT2 diluted 1:400 (monoclonal antibody, Pierce Biotech Inc, Rockford, Ill.) or AT8 diluted 1:800 (monoclonal antibody, Pierce Biotech Inc) in blocking reagent, was applied for 1 hour at room temperature. BT2 antibody recognizes normal tau between residue 194 and 198, whereas AT8 antibody recognizes abnormally phosphorylated tau at the serine 202 residue (numbering according to human Tau40). The antibody–antigen complexes were detected with a supersensitive detection kit (BioGenex) that included horseradish peroxidase-conjugated streptavidin and 3,3-diaminobenzidine. Sections were then counterstained with Mayer’s hematoxylin and rinsed in water. Finally, they were dehydrated in a graded alcohol series and mounted before applying a coverslip using Histomount (National Diagnostics). Negative controls were obtained by omitting the primary antibodies.
heated in a pressure cooker (Biocare Medical DC2002, Concord, Calif.) for 15 seconds for antigen retrieval. After incubating the sections in PBS with 10% normal goat serum, 1% bovine serum albumin, and 0.1% Triton X-100 for 1 hour at room temperature, the primary antibody, either BT2 (1:100 dilution) or AT8 (1:200 dilution) in PBS with 3% normal goat serum, was applied for 1 hour at room temperature. The secondary antibody, Alexa 488conjugated goat anti-mouse immunoglobulin (Ig) G (Invitrogen Canada Inc, Burlington, Ont.) diluted 1:400 in PBS with 1% normal goat serum, was applied for 1 hour at room temperature. Sections were coverslipped with polyvinyl alcohol (PVA) (Sigma, Oakville, Ont.) and 1,4diazabicyclo[2,2,2]octane (DABCO) (Sigma) antifade mounting medium. Double labeling for abnormally phosphorylated tau and parvalbumin
Parvalbumin is a well-established marker for horizontal cells,19,20 and double labeling with anti-parvalbumin antibody was used to demonstrate that hyperphosphorylated tau is located in horizontal cells. Sections were prepared as above and incubated overnight at 4 oC with the first primary antibody, AT8 diluted 1:200 in PBS with 3% normal goat serum. The secondary antibody, Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc, West Grove, Penn.) diluted 1:200 in PBS with 1% normal goat serum, was applied for 1 hour at room temperature. Sections were then washed in PBS and incubated for 1 hour at room temperature with the second primary antibody against parvalbumin polyclonal antibody (Swant Inc, Bellinzona, Switzerland) diluted 1:1000 in PBS with 3% normal goat serum. This was followed by application of the second secondary antibody, Alexa 488conjugated goat anti-rabbit IgG (Invitrogen Canada Inc) with 1:400 dilution in PBS with 1% normal goat serum, for 1 hour at room temperature. Sections were coverslipped using PVA-DABCO antifade mounting medium.
Immunofluorescence labeling
After several washes in 0.05 mol/L tris-buffered saline, sections were incubated in citrate-EDTA buffer (pH 6.0) Table 2—Details of postmortem eyes with incidental glaucoma and control eyes Sex
Eye
Pathological diagnosis/ cause of death
Time to fixation* (h)
81 81 68 77 73 82
F F F F M F
OS OD OS OD OD OD
Primary open-angle glaucoma Primary open-angle glaucoma Primary open-angle glaucoma Primary open-angle glaucoma Primary open-angle glaucoma Primary open-angle glaucoma
7 17 21 21 6 9
57 83 81
F M M
OS OD OD
Cerebrovascular accident Natural causes Cerebrovascular accident
6 1 17
Patient no. Age (y) Glaucoma 1 2 3 4 5 6 Control 1 2 3
*Time from surgery or death to fixation. Note: y, years; h, hours; F, female; M, male.
Confocal laser scanning microscopy
Immunofluorescence-labeled sections were viewed using a confocal laser scanning microscope (Bio-Rad Radiance 2000, Hercules, Calif.). An acousto-optical tunable filter was used to maintain spectral separation and prevent bleedthrough. Pixel images (512–512) of the retina were obtained using LaserSharp 2000 software (Bio-Rad Cell Science Division, Hernel Hempstead, U.K.) and exported in *.TIF format. Immunofluorescence intensity measurements
Confocal images of the retina sections immunolabeled for BT2 or AT8 were obtained by a reader without knowledge of glaucoma or control status. Anterior and posterior retinal images were captured at one-third of the distance from the pars plana to the equator, and at one-third of the distance from the optic disc to the equator, respectively, for CAN J OPHTHALMOL—VOL. 43, NO. 1, 2008
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Retinal tau pathology in human glaucomas—Gupta et al. consistency. During this procedure, gain and exposure settings were adjusted and kept constant for all sections. Digital images were imported into ImageJ (v. 1.37, National Institutes of Health, Bethesda, Md.) for immunofluorescence intensity analysis. A constant threshold value was applied to all images. Using the “measure” tool on ImageJ, the number of pixels that matched or exceeded the set threshold value was calculated for each image. This value was divided by the total number of pixels in the image to determine the fluorescence intensity index, a measure of the level of immunofluorescence. Immunofluorescence intensity index values in glaucoma retinas were compared with controls using a two-tailed t test, using GraphPad Prism (v. 3.02, GraphPad Software
Inc, San Diego, Calif.). A p value of less than 0.05 was considered statistically significant. Colocalization study of dual-color confocal images
Confocal images of the retina were shown using ImageJ in their corresponding channel windows, channel 1 (red) and channel 2 (green). The background was subtracted from the images. Manders’ overlap coefficient and Pearson’s correlation coefficient were calculated. Manders’ coefficient ranges in value from 0 to 1, and is independent of pixel intensity. It represents the number of colocalized pixels expressed as a fraction of the number of pixels in the dualcolor image. Pearson’s coefficient ranges in value from –1 to 1, and is an index to determine pattern similarity.21,22 To determine whether colocalization occurred as a matter of chance, random images were generated as previously described.23 Blocks of pixels from channel 2 were rearranged at random to create a set of scrambled images. These were compared with channel 1, and Pearson’s correlation coefficient was calculated for each new random image. For each comparison, 100 such random images were generated, and the values of the random Pearson’s coefficient were compared with the actual value. RESULTS Normal tau protein in control and glaucoma
All control eyes showed normal tau immunoreactivity (BT2) in the inner nuclear and inner plexiform layers, with variable staining in RGC and nerve fiber layers (Fig. 1A).
Fig. 1—In the control retina (A), normal tau protein (brown) was observed in the inner nuclear and inner plexiform layers and the RGC layer (BT2 immunoreactivity with hematoxylin counterstain). In glaucoma (B), normal tau protein was absent. Immunofluorescence BT2 stain (bright green) confirms normal tau protein in control (C), and reduced tau protein in the glaucoma (D) inner retinas. Calibration bar indicates 25 μm.
Fig. 2—Normal tau protein immunofluorescence by mean intensity index in control (grey) and glaucoma (black). The difference in normal tau protein between control and glaucoma groups was statistically significant (*, p < 0.05).
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Fig. 3—In the normal optic nerve head, normal tau protein (BT2) (brown) was observed (A) and in glaucoma, it was mainly absent (B). Calibration bar indicates 250 μm.
Retinal tau pathology in human glaucomas—Gupta et al. In all surgical glaucoma retinas, normal tau profiles were either significantly reduced or absent (Fig. 1B). BT2 immunofluorescence labeling confirmed the presence of normal tau in the inner nuclear and inner plexiform layers in control retinas (Fig. 1C) and decreased normal tau in surgical glaucoma retinas (Fig. 1D). The mean immuno-
fluorescence intensity index for BT2 tau protein immunoreactivity in the surgical glaucoma group was significantly decreased compared with the control group (0.008 [SD 0.014] vs. 0.107 [SD 0.056]; p = 0.0002) (Fig. 2). Incidental postmortem glaucoma cases were not different from age-matched controls (0.156 [SD 0.081] vs. 0.154 [SD 0.078]; p > 0.05). Control optic nerves showed abundant normal tau (BT2) in the prelaminar and postlaminar portions of the optic nerve (Fig. 3A), and this was similar to most incidental postmortem glaucoma cases. In contrast, immunoreactivity for normal tau was absent in all surgical glaucomatous optic nerves studied (Fig. 3B). Abnormal tau (AT8) in control and glaucoma
Fig. 4—AT8 immunoreactivity was not observed in control posterior retinas. Calibration bar indicates 25 μm.
Control eyes showed very weak to absent immunoreactivity for abnormally phosphorylated tau AT8 in the posterior retina (Fig. 4), similar to incidental open-angle glaucoma eyes. All surgical glaucoma eyes were immunoreactive for tau AT8 at the outer border of the inner nuclear retinal layer (Fig. 5). AT8 immunoreactivity was also noted in the inner plexiform layer in the rare case. Optic nerve heads examined in control and glaucoma cases did not show evidence of AT8 immunoreactivity.
Fig. 5—Glaucomatous optic nerve head excavation was evident in glaucoma cases (left column). Abnormal tau AT8 (brown) in the retina was consistently observed in glaucoma (middle column). Boxed areas in middle column are seen at higher power in the right column. Red arrows indicate AT8 immunoreactivity observed at the outer border of the inner nuclear layer. Calibration bars indicate 250 μm (left column), and 25 μm (middle and right columns). (Row A, patient no. 10; B, no. 9; C, no. 8; D, no. 7.) CAN J OPHTHALMOL—VOL. 43, NO. 1, 2008
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Retinal tau pathology in human glaucomas—Gupta et al. Double labeling with AT8 and parvalbumin in the retina
AT8 immunofluorescence labeling in red confirmed the presence of abnormal tau at the outer border of the inner nuclear layer in surgical glaucoma cases (Fig. 6D). Minimal to no AT8 signal was observed in posterior control retinas (Fig. 6C). The same sections were labeled also for parvalbumin-immunofluorescence, a horizontal cell marker. Figures 6A and B show the presence of parvalbumin labeling in green in control and glaucoma retinas, respectively. AT8 immunoreactivity colocalized to parvalbumin-immunoreactive horizontal cells in glaucoma (Fig. 6F), and this was not seen in the control (Fig. 6E). Colocalization analysis of glaucoma retina images generated values for Manders’ overlap and Pearson’s correlation coefficients that were close to 1. Manders’ overlap coefficient ranged in value from 0.709 to 0.881, and Pearson’s correlation coefficient ranged from 0.514 to 0.716, indicating a high degree of colocalization for abnormal tau (AT8) and parvalbumin. Costes’
Fig. 6—Parvalbumin immunofluorescence in green labels horizontal cells in control (A) and glaucoma (B) retinas. Abnormal tau AT8 immunofluorescence in red is absent in controls (C), and present in horizontal cells in glaucoma (D). Double labeling with parvalbumin (green) and abnormal tau AT8 (red) shows only parvalbumin in control (E) and colocalization of parvalbumin and abnormal tau protein (yellow) in glaucoma (F).The calibration bar indicates 25 μm.
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analysis generated random values for Pearson’s correlation coefficient that were lower than the actual value in 100% of iterations, indicating that this colocalization is not due to chance alone. The immunofluorescence intensity index for abnormal tau AT8 in the posterior retina in glaucoma was significantly increased compared with controls (0.030 [SD 0.020] vs. 0.003 [SD 0.003]; p = 0.0012) (Fig. 7). In control and surgical glaucoma groups, mild AT8 staining adjacent to pars plana was observed in some cases, with no significant difference in immunofluorescence intensity index observed between the surgical glaucoma and control groups (0.016 [SD 0.014] vs. 0.018 [SD 0.015]; p = 0.79). INTERPRETATION
Normal tau protein has been described in normal human retina, and the present study confirms this in the inner nuclear retinal layers, including the RGCs and their nerve fibers.24 The decrease in normal tau in the inner retinal layers in glaucoma compared with controls is consistent with the advanced glaucoma seen in the surgical glaucoma enucleation specimens. The finding of abnormal tau protein was observed across a broad age range, from 19 years to 80 years, in cases of glaucoma from primary and secondary causes with uncontrolled elevated IOP. Abnormal tau protein is implicated in altered microtubule stability,11 axonal transport,25,26 and neurodegenerative diseases.14 Abnormal tau AT8 detected in these cases of glaucomas from causes of elevated IOP and (or) transneuronal degeneration is likely to be a posttranslational modification secondary to insult, as seen in various types of neuron injury.27,28 The absence of hyperphosphorylated AT8 in postmortem eye specimens with incidental open-angle glaucoma at the time of death further supports the theory that abnormal tau is related to uncontrolled elevated IOP and/or advanced glaucomatous damage. The specific localization of abnormal tau protein in
Fig. 7—Mean immunofluorescence intensity index in the retina for abnormal tau AT8 in control (grey) and glaucoma (black). The difference between glaucoma and control groups is statistically significant (*, p < 0.05).
Retinal tau pathology in human glaucomas—Gupta et al. human glaucoma to retinal horizontal cells was an unexpected finding. Despite known variation among human tissue specimens, the positive finding of abnormal tau in horizontal cells was consistent for each surgical glaucoma eye, across all ages. Horizontal cell pathology was noted in 2 cases of secondary glaucoma.29 Horizontal cells influence the spatial-chromatic organization of the receptive fields of RGCs.30 Recent experimental evidence in nonhuman primates suggests that the surrounds of parasol ganglion cells are generated mainly by horizontal cell negative feedback to the photoreceptors.31 One hypothesis for the horizontal cell abnormality in glaucoma takes into account the unique lateral arrangement of horizontal cell processes, possibly predisposing them to IOP-induced retinal stretch injury, inducing structural changes and tau protein alterations. It is also possible that the remodelling of horizontal cell processes observed following retinal degeneration and retinitis pigmentosa might also play a role following RGC loss in glaucoma.32–34 In experimental glaucoma, a number of molecules have been implicated in microtubule stability and intraneuronal transport, including kinesin and dynein,35 amyloid precursor protein,36 kinases,37–40 and phosphatases.41–44 Further studies of abnormal tau protein in experimental ocular hypertension models are needed to determine a possible relationship between tau changes and elevated IOP and (or) glaucomatous RGC injury. In addition, access to human cases with mild and moderate disease states and better characterized clinical history as it relates to visual field loss and IOP elevation would be helpful. The possible relationship of horizontal cell injury to glaucoma is intriguing, and whether horizontal cells contribute to visual dysfunction in glaucoma remains to be determined. Tau protein pathology in glaucoma underscores the presence of shared pathways with other neurodegenerative diseases, and may add to our understanding of the neuropathological processes leading to vision loss from glaucoma.45 Furthermore, treatment strategies directed at preliminary steps in the molecular cascades causing tauopathies may also be relevant to glaucomatous disease. Eye specimens were obtained from the Eye Bank of Canada, Ontario Division, and Ophthalmic Pathology Laboratory, University of Toronto. We thank Judy Trogadis of the St. Michael’s Hospital Bioimaging Centre. We are grateful to Eileen Girard, MLT, for her excellent technical assistance. This work was supported by the Glaucoma Research Society of Canada (Neeru Gupta, Yeni Yücel), the Thor Eaton Fund (Neeru Gupta) and Roy Foss fund (Neeru Gupta), and the Institute of Medical Science Summer Research Program, University of Toronto (Jessica Fong).
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Key words: microtubule-associated protein, neurodegenerative diseases, Alzheimer’s, tauopathy, horizontal cells, optic nerve