A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains

Journal of Neuroscience Research 31:75-83 (1992)

A Histochemical Study of Iron, Transferrin, and Ferritin in Alzheimer’s Diseased Brains J.R. Connor, S.L. Menzies, S.M. St. Martin, and E.J. Mufson Department of Neuroscience and Anatomy, M.S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey (J.R.C., S.L.M. , S.M.S.), and Institute for Biogerontology Research, Sun City, Arizona (E.J.M.)

Immunohistochemical and histochemical staining were performed on Alzheimer’s diseased brain tissue obtained at autopsy. The iron-regulatory proteins transferrin and ferritin as well as iron are, in general, found predominantly in oligodendrocytes similar to that previously reported for normal brain tissue. However, in the vicinity of senile plaques, the staining pattern is altered for both proteins and iron. Trans- ferrin is homogenously distributed around the senile plaques and is apparently extracellular. In addition, transferrin is found in astrocytes in the cerebral cortical white matter of the Alzheimer’s tissue rather than its normal distribution in oligodendrocytes. A robust ferritin immunoreaction accompanies senile plaques and many blood vessels in the Alzheimer’s brain tissue. Although many ferritin-positive oligodendrocytes are present in the Alzheimer’s tissue, most of the ferritin-containing cells associated with senile plaques and blood vessels are microglia. Iron can also be demonstrated in the senile plaques. The iron reaction product is observed both diffusely in proximity of the plaques and in cells associated with the plaques. These data strongly suggest a disruption in brain iron homeostasis in Alzheimer’s disease as demonstrated by alterations in the normal cellular distribution of iron and the proteins responsible for iron regulation. These data will contribute to under- standing both the potential for oxidative damage and the potential for metal neurotoxicity in Alzheimer’s disease.

Key words: aging, oligodendrocytes, CNS disorders

INTRODUCTION A number of neurodegenerative disorders such as

Parkinson’s disease (Earle, 1968) and Alzheimer’s disease (Goodman, 1953) and others (reviewed in Yehuda and Youdim, 1988) have been suggested to result from or involve an imbalance in brain iron metabolism. How- ever, little is understood regarding iron regulation and metabolism in the brain. Much of the iron in the brain is

acquired during early postnatal development (Dallman et al., 1975; Taylor and Morgan, 1990), although iron uptake across the blood-brain barrier is continuous throughout adulthood (Fishman et al., 1987; Pardridge et al., 1987). Considerable levels of iron do accumulate in the brain, particularly in the basal ganglia, in which the iron concentration is similar to that found in the liver (Hall- gren and Sourander, 1958). How iron homeostasis in the brain is maintained is an area of increasing interest in neurobiology .

Our laboratory has focused on the possibility that the altered iron distribution observed in neurodegenerative disorders results from a malfunction of the iron regulatory proteins in the brain, namely, transferrin (for iron mobilization) and ferritin (for iron storage). Transferrin (Tf) accounts for approximately 0.4% of the total protein in the brain (Connor et al., 1992) and is found predominantly in white matter or areas high in white matter (Connor et al., 1987, 1992). At the cellular level, Tf is localized mainly in oligodendrocytes (Connor and Fine, 1986; Connor et al., 1990) although neuronal immunostaining has also been reported (Dwork et al., 1988; Oh et al., 1986). Transfenin mRNA is expressed in the brain (Levin et al., 1984); indeed at a level that is second in the body only to that found in the liver (Adrian et al., 1990). The Tf mRNA in the brain is expressed by oligodendrocytes (Bloch et al., 1985) and the choroid plexus (Aldred et al., 1987).

Ferritin levels in the human brain are 10 times higher than transferrin (Connor et al., 1992) and approximately one-third the levels found in the liver (Fleming and Joshi, 1987). Brain ferritin levels are altered in Par- kinson’s disease, but the direction of change is inconsis- tent (Riederer et al., 1989; Dexter et al., 1990).

Received January 14, 1991; revised April 3, 1991; accepted April 22, 1991.

Address reprint requests to James R. Connor, Ph.D., Department of Neuroscience and Anatomy, M. S. Hershey Medical Center, Hershey, PA 17033.

0 1992 Wiley-Liss, Inc.

Figs. 1-7,

Iron Regulatory Proteins in AD Brains 77

ascending concentrations of sucrose, sectioned at 40 pm on a freezing microtome, and stored in 30% glycol until use. Some tissue blocks were sectioned immediately af- ter fixation on a vibratome. The brain areas represented in this study are the superior temporal gyrus, precentral gyrus, occipital pole, and hippocampus. A total of ten AD brains were examined.

Immunhistochemistry and iron histochemistry were performed as previously reported (Connor et al., 1990). For immunohistochemistry , tissue sections were pre- treated with 3.0% hydrogen peroxide, 5% dimethyl sulf- oxide, and blocked in either sheep serum (1:30) or 5% powdered milk. Incubation with the primary antibody occurred overnight. The antisera used were to human transferrin (Boehringer-Mannheim, 1: 100, or Cappel 1: 250) or to human ferritin (ICN Immunobiologicals, 1: 500). Control sections were either incubated in sheep serum (1: loo), in preadsorbed antiserum, or in 5% powdered milk. The immunohistochemical reaction was completed using the PAP method with 3,3’ diaminoben- zidine as the chromogen. All rinses and dilutions were performed in 0.1 M PBS. For the iron reaction, the Perls stain with DAB intensification was used as described previously (Nguyen-Legros et al., 1980; Connor et al., 1990). The iron stain was performed on free-floating sections.

Immunofluorescence was performed using a mono- clonal antibody specific for astrocytes (GFAP; Boeh- ringer-Mannheim, 1: 100) or a fluorescein conjugated lectin, Ricinus Communis Agglutinin 1 (RCA 1; Vector Laboratories, 1:50) for a microglia (Mannoji et al., 1986; Suzuki et al., 1988) in combination with either Tf or ferritin antiserum.

Some tissue sections were processed for Congo Red staining following immunohistochemistry or iron staining to detect amyloid associated with senile plaques (Puchtler et al., 1962). The senile plaques were also identified using birefringence polarized light microscopy or thioflavin-S (Schwartz, 1972).

It is clear from these observations that the brain has a high storage capacity for iron and also has the ability to mobilize iron. The purpose of this study is to examine Alzheimer’s diseased (AD) brains immunohistochemi- cally and histochemically for transferrin, ferritin, and iron. The general hypothesis under investigation is iron mobilization and storage are disrupted in the AD brain. A manifestation of this disruption in iron mobilization and storage would be an altered histological distribution of iron-regulatory proteins and iron in the AD brain.

MATERIALS AND METHODS The work reported here is part of a larger study on

the distribution of iron-binding proteins and iron in the human brain and tissue collection and procedural details have been reported earlier (Connor et al., 1990). It was decided to present the observations on AD brain tissue separately when it became apparent that brains with a diagnosis of AD (by Dr. Harold Civin, according to NIH/AARP guidelines [Katchaturian, 19851) could be identified without prior knowledge of their condition based on the immunohistochemical patterns of ferritin and transferrin and the iron staining in the senile plaques.

Brain tissue was collected at autopsy (within 2-12 hr of death), blocked into appropriate areas, and im- mersed into either 4% paraformaldehyde, formalin-eth- anol-acetic acid (Luna, 1968), or 10% formalin overnight. The blocks of tissue were cryoprotected in

Fig. 1. Diffuse Tf immunostaining (arrows) around senile plaques in cerebral cortical gray matter from AD brain. b, blood vessel. X 750.

Fig. 2. The same tissue section as in Figure 1 was reacted with thioflavin-S following the Tf immunoreaction to demonstrate the presence of amyloid (arrows) in the center of the senile plaques. b, blood vessel. x 750.

Fig. 3,4,5. In cerebral cortical white matter, Tf-immunoreactive cells, with the morphological appearance of astrocytes (straight arrows) and an occasional oligodendrocyte (feathered arrow), are visible. Using double label immunofluorescence, the Tf + cells (arrows in Fig. 4) are identified as astrocytes (arrows in Fig. 5 ) because they also express GFAP. The bands of fluorescence seen in Figure 4 are Tf+ blood vessels. All figures x750.

Figs. 6,7. In the alveus of the hippocampus a strong Tf immunoreaction is always present and the Tf+ positive cells have a morphological appearance of oligodendrocytes (arrow). Tf + astrocytes are not seen in this white matter tract despite the abundant presence of GFAP + astrocytes and processes (Fig. 7). x 900.

RESULTS No immunostaining was observed in any of the

control sections for any of the antisera in this investigation.

Transferrin In AD brain tissue, the Tf immunoreactive pattern

of cell immunostaining is similar to normal (Connor et al., 1990). That is, the Tf reaction product is in small, round cells and is confined to the perikaryal cytoplasm of these cells. Tf positive cells are found in perineuronal and perivascular positions. In some white matter tracts the immunostained cells are more rectangular in shape and aligned in rows. However, there are two major dif-

Figs. 8-14.


Ferri tin In the cerebral cortical and hippocampal AD tissue,

numerous small, round ferritin immunostained cells are distributed throughout the gray matter in perineuronal and perivascular locations in a manner similar to normal (Connor et al., 1990). However, the distribution pattern for ferritin is clearly disturbed in the gray matter by the presence of senile plaques (Fig. 8). Ferritin containing cells accumulate around the periphery of the core of the plaque and the majority of these cells have relatively thick processes which contain reaction product (Figs. 8, 9, 10). Most of the ferritin-positive cells associated with the plaques are microglial cells as indicated by the colocalization of ferritin (Fig. 11) and RCA-1 (Fig. 12). Very few ferritin containing cells associated with plaques are GFAP positive (Figs. 13, 14); none of the ferritin-positive cells contain Tf.

The morphological appearance of ferritin immunoreactivity in the white matter of AD tissue was not dramatically different from normal, i.e., fairly numerous, aligned in rows, and the reaction product confined to the perikaryal cytoplasm. The normal patchwork appearance of the white matter following ferritin immunostaining was less noticeable in the AD tissue compared to normal (Connor et al., 1990) in all the cerebral cortical regions. However, there did not appear to be an increase in ferritin-containing microglial cells in the AD white matter nor was ferritin immunoreactivity detected in astrocytes .

A striking feature of the AD cerebral cortical and hippocampal gray matter is the immunostaining of ferritin containing cells associated with many of the blood vessels. Ferritin positive cells and numerous processes are seen in proximity with blood vessels (Figs. 15, 16). Some of the ferritin-containing processes of these cells are traceable to blood vessels, but few of these cells are GFAP positive. Similar to those ferritin-positive cells associated with senile plaques, the abnormal ferritin-positive cells associated with blood vessels are RCA-1 positive and are presumably microglia (Figs. 17, 18). This robust ferritin immunostaining in the proximity of the blood vessels is not at all similar to the type of ferritin immunostaining normally seen in association with blood vessels (Connor et al., 1990).

ferences in the Tf immunostaining pattern that is specific to AD: (1) the area surrounding senile plaques, and (2) the cerebral cortical white matter. A homogeneous, diffuse reaction product surrounds the core of the senile plaque following immunoreaction with Tf antiserum (Figs. 1, 2). Only rarely are Tf-immunoreactive cells present within the diffuse immunostained area. No reaction product associated with senile plaques (identified with Congo red staining) is visible in the control sections.

In the cerebral cortical white matter of AD tissue in addition to the normally appearing Tf + oligodendrocytes, many Tf + astrocytes are present (Fig. 3). There is an inverse relationship between astrocytic and oligodendrocytic Tf-immunoreactivity; the more Tf-positive astrocytes, the fewer Tf-positive oligodendrocytes are seen. Most of the Tf-positive astrocytes have processes that can be traced to blood vessels. The presence of Tf in astrocytes was verified by immunofluorescent colocalization with GFAP (Figs. 4, 5). The white matter immunostaining pattern for Tf+ astrocytes can be demonstrated to the same degree in AD brains in all of the cerebral cortical areas examined.

In the hippocampus, the Tf immunoreaction product in addition to the normal distribution reported earlier (Connor et al., 1990) is also associated with the senile plaques. However, unlike the cerebral cortical white matter, the alveus is remarkable for the absence of Tf + astrocytes. The alveus consistently contained numerous Tf + oligodendrocytes (Fig. 6) even though many astrocytes (non-Tf containing) were present (Fig. 7).

Fig. 8 . Ferritin-positive cells with thick processes are associated with senile plaques (e.g., broad arrows) in the presence of normal oligodendrocytic ferritin immunostaining (e.g., thin arrow) in this micrograph, which is counterstained with hema- toxylin. x 600.

Figs. 9,lO. Ferritin immunostained cells are closely applied to the senile plaque (feathered arrow) and are morphologically different from the normal appearing ferritin positive oligodendrocytes (split arrow). The amyloid core of the senile plaque is demonstrated by reacting the section shown in Figure 9 with thioflavin-S (arrow in Fig. lo). X 750.

Figs. 11,12. These figures demonstrate that many of the ferritin-positive cells (e.g., at arrow in Fig. 11) associated with a senile plaque are RCA-1 positive (e.g., at arrow in Fig. 12). x 750.

Figs. 13,14. The arrowhead in Figure 13 indicates a ferritin- positive cell associated with a senile plaque. This cell also contains GFAP (arrowhead in Fig. 14). A ferritin-containing cell which is not associated with a senile plaque and does not contain GFAP is shown in Figure 13 (arrow). X 750.

Iron In AD cerebral cortical gray matter, the iron-posi-

tive cells are small, round, and found in perineuronal and perivascular locations similar to normal (Connor et al., 1990) with the exception of the area around senile plaques (Figs. 19, 20). Two types of iron staining are associated with plaques: cellular staining (Fig. 19) and a diffuse, homogeneous surround (Fig. 20). The iron-pos-

80 Connor et al.

Figs. 15-18. Femtin-positive cells with ferritin containing processes (arrows) associated with blood vessels (b) are seen in Figures 15 and 16. A normal fenitin containing oligodendrocyte is present in Figure 15 (feathered arrow). Colocalization

of ferritin (Fig. 17) and RCA-1 (Fig. 18) demonstrate that many of the ferritin-containing cells around blood vessels (e.g., at arrow) are microglial cells (e.g., at arrow). X750.

itive cells associated with plaques have thick, iron-containing processes. Identification of these cells is difficult because the protocol for iron precludes colocalization studies. However, based on their morphology and the RCA-1 and GFAP immunostaining seen in other sections, the iron-containing cells around the plaques are presumably astrocytes and/or microglia.

DISCUSSION

staining associated with senile plaques. The iron-positive cells have iron-containing processes that extend into the core of the plaque and are thought to be microglia. U1- trastructural analysis of these cells provided equivocal information because of inadequate fixation combined with the harshness of the Perls’ reaction. Microglial cells containing iron were not identified in the normal tissue or identified in areas not associated with senile plaques in the AD tissue.

Quantitative studies on iron in the human brain The observations on iron staining in the AD cere-

bra1 cortex are consistent with those reported previously (Goodman, 1953), including both the diffuse and cellular

have found that in AD tissue, iron is 67% higher in gray matter and 27% higher in white matter compared to con- trols (Ehmann et al., 1986). The quantitative results


Fig. 19. Iron staining cells associated with senile plaques (feathered arrow) have thick iron-containing processes. x 300.

showing an increase in iron with AD in conjunction with the histological observations of increased iron staining around blood vessels and plaques would suggest the increased iron in AD brains has extravasated from the blood.

The ferritin containing cells around senile plaques and many blood vessels were robustly immunoreactive for ferritin and were only seen in the AD brain tissue. These ferritin-positive cells are microglial cells and in- frequently, astrocytes. The ferritin-containing cells in the vicinity of senile plaques are similar in appearance to the microglial cells reported by Kaneko et al. (1989). The increased ferritin immunostaining of microglia around senile plaques and blood vessels in the AD brain tissue likely represents increased ferritin synthesis by these cells to sequester iron that is either entering the CNS from the blood or is available from cellular degeneration. Iron may not be the only metal that elicits the increased ferritin and microglial response. Aluminum content of ferritin is increased in AD brain tissue relative to normal (Fleming and Joshi, 1987). The response of microglial cells clearly represents an important attempt at detoxi- fication and helps decrease the potential for oxidative damage by free iron. However, it is also clear that iron which is being sequestered by the microglia is not being utilized in a normal manner by the brain and subsequent decreased neuronal and glial cell function would be expected to follow.

As with ferritin, Tf oligodendrocytic immunostaining is present in the AD tissue. However, there are two differences in the pattern of Tf immunoreactivity compared to normal. First, in the area of senile plaques, the Tf immunostaining is diffuse and homogeneous. Only

Fig. 20. Iron is also found in the vicinity of senile plaque with a more diffuse, homogeneous staining pattern (feathered arrows). X 300.

rarely is a Tf-positive cell seen in the vicinity of a plaque. The diffuse immunostaining suggests that plasma Tf has extravasated from blood vessels contained within the plaque. This Tf is likely the source of iron associated with plaques and may also be the source of aluminum associated with plaques (Roskams and Connor, 1990). The second difference between AD and normal tissue is the presence of Tf-positive astrocytes in the white matter of the brain areas examined except in the alveus of the hippocampus. It is not known if the Tf in astrocytes is being synthesized by the astrocytes or is being taken up from the plasma. Recently, Tf mRNA was reportedly detected in astrocytes grown in culture (Espinosa et al., 1990). Tf expression (accumulation) by astrocytes is not a concomitant response with astrogliosis (Connor et al., 1987), nor has Tf been observed in astrocytes which are involved in the phagocytosis of iron (Connor and Men- zies, 1990).

The observation that Tf in astrocytes is specific to white matter in the AD cerebral cortex suggests that iron uptake in the white matter is also affected in AD. The involvement of white matter in AD has been underesti- mated (Miller et al., 1980; Englund et al., 1988; Wallin et al., 1989) and the loss of white matter in AD may actually exceed that of gray matter (Perry, 1986). There is no evidence to our knowledge that the population of oligodendrocytes is decreased or dedifferentiated in AD and the histological observations in the present study with ferritin and iron do not suggest a loss of or change in oligodendrocytes. Thus, the absence of immunode- tectable Tf in these cells indicates a specific dysfunction in the iron mobilization protein in this cell type. The role of Tfhron in myelin production and maintenance is under

82 Connor ef al.

investigation, but the presence of Tf in oligodendrocytes seems to be a sine qua non for myelination (Connor et al., 1987). A loss of white matter would be expected to follow a loss of Tf in oligodendrocytes.

The results of this study are consistent with the hypothesis that iron homeostasis is disrupted in the AD brain. Iron accumulates around and within senile plaques. Ferritin and transferrin immunostaining patterns are altered in the vicinity of plaques and, in the case of Tf, in the cerebral cortical white matter. It cannot be determined from the present data whether the iron and iron-regulatory proteins associated with the plaques result from breakdown within the CNS or from leaking across the blood-brain barrier or both. The extracellular nature of the diffuse transferrin reaction around the plaques as well as the diffuse iron staining suggests ex- travasation. The cellular ferritin and iron staining associated with plaques could represent active phagocytosis of extravasated iron (or other toxic metals) and active synthesis of ferritin. The abnormal robust ferritin immunostaining along blood vessels in the AD tissue supports the hypothesis that the uptake of iron into the brain in AD is compromised.

Iron is important for lipid synthesis, synthesis of a number of neurotransmitters, and neurotransmitter- receptor interaction (Yehuda and Youdim, 1988). Fur- thermore, iron is considered the most probable agent responsible for lipid peroxidative damage in the brain (Arai et al., 1987) and oligodendrocytes are particularly sensitive to apparently iron-initiated peroxidative damage in vitro (Griot et al., 1990). Thus, a breakdown in brain iron homeostasis in AD as indicated by the results of this investigation could play a role in the wide range of neuronal and glial activities that are known to be dys- functional in AD.

ACKNOWLEDGMENTS The authors are grateful to Dr. Javad Towfighi for

his assistance with tissue collection and the staining and identification of senile plaques. Ms. Debra Hinton provided expert technical assistance. This work was sup- ported by funds from Alzheimer’s Disease Research, a program of the American Health Assistance Foundation, Rockville, MD (JRC, EJM), and U.S. Public Health Service grants HL-07477 (SSM) and AGO9063 (JRC).

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A histochemical study of iron, transferrin, and ferritin in Alzheimer's diseased brains

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