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From the Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts.
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Abstract |
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METHODS. Reverse transcription–polymerase chain reaction (RT-PCR) and Western blot analysis were performed on rat retinal tissue, and the immunophilin FKBP12 was found to be present in retina. Immunohistochemical studies showed the presence of FKBP12 in retinal ganglion cells (RGCs). In rats, optic nerve crush was performed on one side and a sham operation on the other side. By gavage, animals were given 5 mg/kg per day of the FKBP12 ligand FK506 in sterile phosphate-buffered saline (PBS) or in PBS alone. Eight days after nerve crush, the total number of back-labeled RGCs was estimated from retinal wholemounts.
RESULTS. In control eyes, the number of labeled ganglion cells was 74,104 ± 4,166 (mean ± SEM) in rats receiving vehicle and 74,993 ± 3,098 in animals receiving FK506 daily. Eight days after optic nerve crush, 27,775 ± 3,332 labeled ganglion cells were counted in retinas of animals receiving vehicle (n = 11), whereas 33% more ganglion cells (37,118 ± 2,475) were counted in animals receiving FK506 daily (n = 11). This difference was statistically significant (P < 0.05).
CONCLUSIONS. The data presented demonstrate that the immunophilin FKBP12 is present in retina and specifically in RGCs. In addition, the FKBP12 ligand FK506 confers neuroprotection on RGCs after optic nerve crush. This neuroprotection may occur as a result of FK506’s ability to interfere with apoptotic mechanisms after optic nerve crush.
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Introduction |
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Immunophilins, including the prototypical molecule FK506-binding protein (FKBP12), are a large group of small cytoplasmic proteins, characterized by their ability to bind and mediate the actions of FK506 and related immunosuppressant agents. They were first described in immune-competent cells such as T cells. FK506 is in widespread clinical use after organ transplantation.4 The FK506–FKBP12 complex inhibits the phosphatase activity of calcineurin, a Ca2+-calmodulin–dependent phosphatase. This leads to the decreased dephosphorylation of nuclear factor of activated T-cell (NF-AT) preventing its entry into the nucleus and the production of interleukin-2.5
Steiner et al.6 reported in 1992 that immunophilins are also very abundant in the nervous system, although their role in the brain and peripheral nervous system remains uncertain. Recent data suggest that FK506 has properties that modulate neural damage in a variety of paradigms. FK506 reduces ischemic brain damage after middle cerebral artery occlusion when administered up to 60 minutes after occlusion.7 Also, FK506 has been shown to increase the levels of axotomy-induced growth-associated protein (GAP43) mRNA in dorsal root ganglion,8 decrease the activation of microglia–macrophages in the white matter of the optic nerve after chronic cerebral ischemia, and promote protection against white matter lesions.9 Recent data have also shown that FK506 protects hippocampal neurons in culture against glutamate-induced excitotoxicity and apoptosis.10
Although immunophilins have been demonstrated in the brain and peripheral nervous system, the presence of FKBP12 in retina has not yet been examined. We present 4 lines of evidence that FKBP12 is present in retina and is physiologically active: detection of mRNA by reverse transcription–polymerase chain reaction; detection of a 12-kDa band on Western blot analysis; immunostaining of RGCs with FKBP12 monospecific antisera; and pharmacologic protection by FK506 against RGC death after experimental optic nerve crush.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA Isolation and Reverse Transcription–Polymerase Chain Reaction
Rats were killed with an overdose of chloral hydrate and
transcardially perfused with phosphate-buffered saline (PBS). The
retinas and brain were removed and immediately stored at -80°C.
Total cell RNA was isolated from retina and brain and reverse
transcribed into cDNA (Reverse Transcription System; Promega, Madison,
WI), according to the manufacturer’s instructions, using 1 µg
isolated mRNA. Polymerase chain reaction (PCR) was then performed using
oligonucleotide primers. Forward primer 5'-GAG AAA AGC CAG CAT AAA
GC-3' and reverse primer 5'-TCT AGA ACT TCG GTG GAA AG-3' were used to
amplify 321 bp of the FKBP12 cDNA gene. The PCR product was then
subjected to electrophoresis on a 3% agarose gel, and fragments were
visualized by ethidium bromide staining.
Protein Isolation and Western Blot Analysis
Animals received overdoses of chloral hydrate and were
transcardially perfused with PBS. Eyes were enucleated, retinas
removed, and retinas and brain immediately stored at -80°C. Rat
cortex and rat retina were homogenized in 50 mM Tris (pH 7.6), 150 mM
NaCl, 12 mM CHAPS, 2 mM EDTA, 1% Nonidet P-40, 200 µM
phenylmethylsulfonyl fluoride. Human cortex protein (the generous gift
of Bradley Hyman, Boston, MA) was prepared as previously
described.11
Total protein was measured by the
bicinchoninic acid assay (Sigma, St. Louis, MO). Protein
isolated from approximately one half of one retina was analyzed in
immunoblots. Human brain extract (50 µg), rat brain extract (100
µg), and rat retinal extract (150 µg) were separated by 10% to
20% polyacrylamide gel electrophoreses (Tris-Tricene gel; Novex, San
Diego, CA) and transferred to a membrane (Immobilyn-P; Millipore,
Bedford, MA) The membrane was probed with a rabbit polyclonal
anti-FKBP12 antibody (1:2000, Affinity Bioreagents, Golden, CO) and
developed by chemiluminescence (Amersham Pharmacia Biotech, Piscataway,
NJ).
Immunohistochemistry
Animals were killed as described and perfused with PBS followed by
4% paraformaldehyde (PFA). Eyes were enucleated and placed in 4%
PFA/3% sucrose solution overnight at 4°C. Eyes were then incubated
in 5%, 10%, 15%, and 20% sucrose for 60 to 90 minutes each,
embedded in optimal cutting temperature compound, and maintained at
-80°C. Cryostat sections (16-µm thick) were cut and sections
mounted onto slides (SuperFrost; VWR, West Chester, PA).
Mounted sections were soaked in 0.5% Triton X-100 in Tris-buffered
saline (TBS) for 20 minutes and rinsed three times in TBS. They were
then incubated for 1 hour, with shaking, in 3% nonfat dry milk in TBS.
After one rinse in TBS, slides were incubated in 1.5% normal goat
serum in TBS with a polyclonal, rabbit anti-FKBP12 antibody (1:500;
Affinity Bioreagents) at 4°C overnight in a humidified chamber. The
slides were rinsed three times in TBS for 5 minutes each and incubated
in goat anti-rabbit Cy3 antibody (1:250; Jackson ImmunoResearch, West
Grove, PA) in a humidified chamber for 1 hour at room temperature.
Slides were then rinsed again three times in TBS for 5 minutes each,
allowed to dry, and coverslipped with antifade medium (Slowfade;
Molecular Probes, Eugene, OR), according to the manufacturer’s
directions. In negative control experiments, primary antibody was
omitted. Sections were viewed with a confocal microscope (model 1024;
Bio-Rad, Hercules, CA).
Optic Nerve Crush
Adult male Wistar rats (250–300 g) were anesthetized with a
cocktail of ketamine-xylazine-acepromazine and their intraorbital optic
nerves exposed after a lateral canthotomy and limbal peritomy. Three
millimeters from the globe, on one side only, the nerve was crushed in
three 10-second intervals with no. 5 jeweler’s forceps.12
The tissues were reposited and antibiotic ointment applied to the eye.
The retinal vasculature was then checked by direct ophthalmoscopy, and
those animals with interrupted blood supply were excluded. A sham
operation was performed in the fellow eye exposing the optic nerve but
not crushing it. Two days before death, the RGC were back-labeled using
a modification of the technique reported by Koeberle and Ball for
axotomy.13
Each optic nerve was re-exposed and the dural
sheath incised longitudinally 1 mm from the globe in both control eyes
and eyes that had received an optic nerve crush. Crystals of
fluorescein dextran (3000 MW, Molecular Probes) were applied to the
exposed optic nerve, and a slurry of crystals dissolved in 10%
dimethyl sulfoxide in water was injected into the optic nerve. A piece
of gelfoam soaked in this mixture was then applied to each optic nerve,
the tissues reposited, antibiotic ointment applied, and the animal
allowed to recover.
Eight days after optic nerve crush, the animals received an overdose of chloral hydrate intraperitoneally and were transcardially perfused with PBS and then 4% PFA in 0.1 M PBS. The eyes were enucleated and soaked in 4% PFA for 30 minutes. The retinas were then removed, flatmounted on slides, and coverslipped with antifade medium. Retinal wholemounts were counted in a masked fashion.
Sterologic Assessment of RGC Number
Estimates of total RGC number were performed using statistically
unbiased stereologic protocols chosen to be advantageous for use in
circumstances in which there is heterogeneity among different
subregions of the structure under study.14
15
In the
retina, for example, there are substantial differences in the density
of RGCs between the central retina and the periphery, without clear
cutoffs identifying individual subregions. Three stereologic tools were
used. Sampling was performed using a systematic random sampling
protocol that ensured that each part of the retina had an equal, but
random, chance of being sampled. A grid was placed over each retina,
and 1 of 66 grids was assessed for estimating the total number of
back-labeled RGC. The location of the first grid to be counted was
determined by choosing the first location between 1 and 66 from a
random number table; thereafter, every 66th grid was assessed
throughout the entire retina. A stereologic counting chamber with
extended exclusion lines was used to perform the counts. This ensured
that cells were not double counted, by excluding cells that intersected
with two (e.g., top and right) lines of the counting chamber but
counting cells that intersected with the other two lines (e.g., bottom
and left). The exclusion lines are extended to avoid double counting of
curved features. The fractionater approach was taken to
calculating total RGC number. By this method, the average number of
RGCs counted per counting chamber is multiplied by the total number of
possible counting chambers in the structure. The fractionater provides
estimates that are unaffected by shrinkage before, during, and after
processing of the tissue. These counting rules do not presuppose any
assumptions about the size, shape, or distribution of RGCs in the
retina and therefore avoid potential biases in counting. The sampling
intensity was targeted to yield a precision of the individual estimates
of approximately 5%, in accord with the stereologic
literature.16
Using our protocol, the counting precision,
or the coefficient of error, is 4%. This low coefficient of error
suggests that the sampling strategy was sufficient to estimate the
total number accurately.
Drug
FK506 (Fujisawa, Deerfield, IL) was administered daily by gavage
at a dose of 5 mg/kg, beginning the day before the crush procedure.
This dose of FK506 was chosen because it was found to be the optimal
dose in peripheral nerve regeneration experiments.17
The
contents of FK506 capsules were dissolved in sterile PBS and given to
the rats. The control rats were given an equal volume of PBS. There
were 11 rats in each group.
Statistics
Groups were compared using unpaired Student’s t-test.
Significance was set at 0.05. Power analysis using the variability in
RGC number observed in preliminary studies showed that 10 to 12 rats
per group would be necessary to demonstrate statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although FKBP12 was originally described in its role in immunosuppression, it has subsequently been shown to be widely distributed in the brain.6 In our initial studies, we extended these observations by showing, by multiple techniques, the robust presence of FKBP12 in retina. Moreover, by back labeling RGCs, we were able to localize the FKBP12 to RGCs unambiguously. This report represents the first description of FKBP12 in the retina and RGCs and prompts consideration that FKBP12 is the immunophilin involved in the recently described in vitro evidence of FK506-mediated protection against glutamate toxicity in retinal cell cultures18 and hippocampal neurons.10
Evidence supports both an excitotoxic and an apoptotic component to the molecular mechanisms underlying RGC death. Glutamate, an excitatory neurotransmitter, has been shown to be elevated after optic nerve crush in rats,19 suggesting that glutamate may contribute to the death of RGCs. FK506 has been shown to have a protective effect on N-methyl-D-aspartate–treated cortical neurons in culture by inhibiting the production of nitric oxide through a calcineurin-mediated process.20 However, numerous markers of apoptosis, including TdT-dUTP terminal nick-end labeling (TUNEL) staining, appear in RGCs after optic nerve injury.21 The pharmacologic effects of FK506 and of the FK506-immunophilin complex appear to be directly related to programmed cell death pathways. A recent report suggests that one target of the FK506-immunophilin complex, calcineurin, mediates a critical step in the apoptotic pathway by dephosphorylating BAD, a proapoptotic member of the Bcl-2 family which translocates to the mitochondria after dephosphorylation and forms heterodimers with Bcl-xL.10 Thus, by inhibiting the calcineurin-mediated dephosphorylation of BAD, the FK506-immunophilin complex promotes cell survival. In hippocampal neurons in culture, for example, FK506 inhibited calcineurin and suppressed mitochondrial targeting of BAD after L-glutamate treatment, suppressing apoptosis.10
Our current data are consistent with the hypothesis that RGCs die through an apoptotic pathway after optic nerve crush, and that this is modulated by a novel therapeutic approach using immunophilin ligands. These data raise the possibility that immunophilin binding agents may have a role in disease conditions characterized by RGC degeneration, including glaucoma.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 28, 1999; revised October 12, 1999; accepted November 16, 1999.
Commercial relationships policy: N.
Corresponding author: Cynthia L. Grosskreutz, Glaucoma Consultation Service, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114. cynthia_grosskreutz{at}meei.harvard.edu
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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