Immunohistochemical identification of kynurenine aminotransferases in corpora amylacea in the human retina and optic nerve

Introduction

Corpora amylacea (CAm) are homogeneous or laminated oval structures frequently found in the brain and peripheral nerves. In the eye, CAm are observed in the optic nerve head, nerve fibre layer, ganglion cell layer, but also in the inner plexiform layer and inner nuclear layer [13,31]. Ultrastructurally, CAm consist of a mass of filamentous tangles within an axonal swelling [1,31]. Formation of CAm was suggested to result from impaired axonal flow [16].
In the central nervous system, CAm are regarded as a hallmark of ageing, and are thought to be associated with neurodegeneration [6,8,17], but little is known about their role in normal and pathological circumstances. Studies on the structure of CAm have shown that their rich acid polysaccharide content makes them best demonstrable by the PAS (periodic acid-Schiff) stain. CAm contain, in addition to glucose polymers, ageing, stress and proinflammatory proteins [4]. But, previous studies emphasised the surprising lack of their immunoreactivity by using many other antigens [14].
Importantly, data from various brain studies suggest that CAm possess a relatively high affinity to accumulate to some extent ‘protective’ substances which could rescue nerve cells from the devastating effects of ischaemia or ageing [4,6]. To test this hypothesis it was reasonable to extend studies on potential involvement of CAm in mechanisms of endogenous protection in the nervous system.
Kynurenine aminotransferases (KAT I and II) are pivotal to the synthesis of kynurenic acid (KYNA), the only known endogenous antagonist of glutamate [21], acetylcholine a7 nicotinic receptors [11] and neuro-protectant [29]. Presence of KATs and KYNA in the CNS and retina has been well documented [12,22-27, 30,33]. Investigations on KYNA are important because alterations of its synthesis are involved in the pathophysiology of several brain [3,18,32] and retinal disorders [22,23].
Therefore, to gain new insight into the role of CAm, this study is the first to examine the presence and patterns of KAT I and II immunoreactivity in CAm in the human retina and optic nerve.


Material and methods

Twenty-three human eyes from twenty-three patients [13 female, 10 male, age: 56-90] enucleated because of choroidal malignant melanoma were used for this study. The patients did not suffer from other ocular diseases. The study protocol was approved by the Human Ethics Committee of the University of Erlangen-Nuernberg.
All globes were fixed immediately after enucleation in a solution of 4% formaldehyde and 1% glutaraldehyde in 0.1% phosphate buffer (pH 7.2). Consecutive 5-µm sections including the centre of the disc and the pupil (P-O sections) were stained with PAS (periodic acid-Schiff), HE (hematoxilin-eosin) or subjected to immunohistochemistry.
Each of the eyes was stained with an anti-KAT I or anti-KAT II polyclonal antibody (1:50) [19,20] at least twice, using the streptavidin-biotin-method, as described previously [7]. Briefly, after deparaffini-zation and rehydration, sections were digested with proteinase K (Dako) before incubation with peroxidase for 10 minutes. Sections were then incubated with primary antibody (30 minutes) and horseradish peroxidase (HRP)-conjugated secondary antibody before development with 3-amino-9-ethyl-carbazole (AEC)+ substrate (red reaction product). Finally, the sections were counterstained with Mayer haemalaun (Chroma, Münster, Germany) and mounted in an aqueous-based medium (Faramount; Dako). Preimmune serum was included as the negative control and showed no staining of corpora amylacea. Sections were photographed with a micro-scope (Axiophot; Carl Zeiss, Oberkochen, Germany) using colour film (Ektachrome 64 T; Eastman Kodak, Rochester, NY).


Results

Light microscopically, all studied eyes revealed collateral retinal detachment and degeneration of the retina overlying the choroidal malignant melanoma. In PAS-stained sections CAm occurred as round, oval, smooth or laminated bodies with dense centres. CAm were observed in all cases in the optic nerve head and prelaminary, laminary and retrolaminary regions of the optic nerve (Fig. 1: R1, PL1, L1, RL1). In the retina, CAm were found in the inner plexiform layer, inner nuclear layer, ganglion cell layer and nerve fibre layer (data not shown). These findings are in agreement with previous results of Kubota and colleagues [13].
Immunohistochemistry showed the presence of both KAT I and KAT II immunoreactivity in CAm (Figs. 1, 2, 3, 4). KAT I immunoreactivity was observed in CAm in the retina (Fig. 1 R2) and prelaminary (Fig. 2 PL2), laminary (Fig. 3 L2) and retrolaminary (Fig. 4 RL2) regions of the optic nerve, and the pattern of its staining in most cases was intense. In general, there was more pronounced staining of KAT I in the retrolaminar part of the optic nerve (Fig. 4 RL2).
The presence of KAT II expression was observed in CAm localised in both the retina and optic nerve (Fig. 1 R3; Fig. 2 PL3; Fig. 3 L3; Fig. 4 RL3). Some CAm showed only faint KAT II immunoreactivity and occasionally there was no staining (data not shown). Immunoreactivity of KAT II was less pronounced than KAT I. There was no association of staining variety of KAT II and localisation of CAm. Also, there was no correlation between size of CAm and immunoreactivity of both KAT I and KAT II.
Moreover, our studies revealed cellular expression of both isoforms of KAT in the human retina (Fig. 5). KAT I was preferentially localised on Müller cell endfeet while KAT II was expressed in retinal ganglion cells. These results parallel our previous observations in the rodent retina [22,23,26].


Discussion

CAm are the only light microscopically visible structures in the retina and optic nerve associated with degeneration and are still of mysterious nature. Up to now, only limited data are found in the literature concerning the mechanisms of their formation.
The present study is the first to demonstrate the immunoreactivity of KAT I and KAT II in CAm in the human retina and optic nerve. CAm expressing both enzymes were observed in all cases in the retina and in the prelaminary, laminary and retrolaminary regions of the optic nerve.
A variety of staining patterns of KAT I depending on the location of CAm were found. In general, there was more pronounced staining in the retrolaminar part of the optic nerve. Immunoreactivity of KAT II was less pronounced than KAT I, with no association of staining variety and localisation of CAm. By showing that all CAm expressing KATs are PAS-
-positive we were able to prove that KAT-stained structures are CAm. The findings in PAS-stained sections are in agreement with previous results of Kubota and colleagues [13].
It has been well documented that CAm have no pathognostic significance, but that they accumulate in certain conditions and pathological processes [15,17]. Numerous factors have been suggested to contribute to the formation of CAm, such as the components of the degraded cells, metabolites originated from the cerebrospinal fluid, blood and the mesenchyma of pia mater and adventitia of the vessel wall [14]. Importantly, hypoxic/ischaemic injury has been shown to potentiate the enigmatic biological pathway leading to the formation of CAm during ageing. The authors speculated that damaged mitochondria and de novo induced or overproduced proteins during cellular insults may be sequestrated by CAm [4]. Assuming that the formation of CAm represents an arrangement for the management of products escaping normal cell catabolism [5], greatly increased numbers of CAm in the optic nerve and retina may reflect increased metabolic work caused by repetitive cellular stress [4], and possibly the presence of KAT I and II in CAm might suggest a potential role of those enzymes in mechanisms of endogenous cellular protection against insult.
Interestingly, there are some data from brain studies suggesting that the CAm possess a relatively high affinity to accumulate to some extent ‘protective’ substances (such as Bcl2, AP1, heat shock proteins, etc.) which could rescue nerve cells from the devastating effects of ischaemia or ageing [4,6,9]. So far, immunohistochemical investigations have demonstrated anti-tau-2 immunoreactivity in CAm in the retina, optic nerve and brain tissue [16].
Only recently, the age-dependant decrease of cellular expression of both KAT I and II was observed in the retina of DBA/2J mice, a model for ocular hypertension [22]. Moreover, we have already shown that KYNA deficiency is causally related to the pathology of excitotoxic retinal diseases and that NMDA-induced retinal ganglion cell loss may cause alterations of KYNA content in the rat retina [25]. Importantly, there are data suggesting that alterations of KYNA synthesis are involved in the pathomechanisms of several brain disorders, e.g. Parkinson’s disease [18], Huntington’s disease [3], Alzheimer’s disease [2] and epilepsy [32].
In vitro studies have revealed that KAT II is responsible for most of the KYNA formation in the brain, although changes in the relative importance of the two enzymes may occur in various patho-physiological situations [10]. Moreover, dysfunction of KYNA synthesis in the brain was suggested to be one of the factors contributing to neuronal dege-neration [3,18,32]. It was reported that in several regions of Alzheimer’s disease brain activity of KAT I was significantly increased while only a minor increase of KAT II was observed [2]. Since increased CAm formation was described in Alzheimer’s disease brain [28] we speculate that it may explain stronger KAT I immunoreactivity in CAm as compared to KAT II, which was observed in the present study.
Only recently, KYNA content and enzymatic activities of its synthesising enzymes in the human retina and vitreous body have been characterized using biochemical methods [33]. Cellular expression of KATs in neurons and glial cells of CNS is well described [12,23,26,27]. The present study, similarly to results of our previous studies in rodents [22,23,26], showed that both KATs were present in the human retina. KAT I was preferentially localised on Müller cell endfeet while KAT II was expressed in cells within the ganglion cell layer. Interestingly, unequivocal representation of KAT I and II immuno-reactivity in CAm demonstrated in this study may suggest extracellular expression of both enzymes or extracellular accumulation via a specific transport outside the cell body. The question arises whether presence of these proteins is a primary event in CAm formation or a secondary mechanism induced by some products of a degenerative process (ageing, neurodegeneration) or by recurrent functional disturbances of the cellular barriers. It might be hypothesised that the enzymes are released from cells dying due to degeneration and are accumulated in CAm. However, mechanisms leading to extra-cellular occurrence of both enzymes reported here need further investigations.
The presence of KATs in CAm in the human retina and optic nerve suggest that KYNA synthesis might be involved in the mechanisms of retinal ageing and neurodegeneration leading to CAm formation. Future extended experiments are necessary to provide a better understanding of the involvement of tryptophan metabolism in the development of degenerative retinal products, which also might help to understand the biological role and significance of CAm.


Acknowledgements

Supported by ELAN Funds of the University of Erlangen-Nuernberg and Kerstan Foundation.


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