Journal of Undergraduate Research
Volume 4, Issue 1 - September 2002

The Role of Microglial Cells in Alzheimer's Disease

Allison Stanton

ABSTRACT

Alzheimer's disease (AD) is a major health problem that affects approximately four million Americans. It is estimated that 22 million Americans will have Alzheimer's disease by the year 2025. Alzheimer's disease is a progressive, degenerative disease of the brain and the most common form of dementia. People that suffer from this disease experience cognitive dysfunction, intellectual impairment, and disorientation.

One of the prevalent current theories is that chronic inflammation contributes to AD pathogenesis. Specifically, it is thought that chronically activated microglial cells in the brain produce neurotoxic substances that cause neurons to degenerate. The β-amyloid protein (Aβ) which accumulates in AD is viewed as the agent that stimulates microglial activation. On the other hand, it is known from experimental studies that microglial cells are neuroprotective and can aid in regenerative processes in the central nervous system. In this study, I will study the relationship between microglial cells and neurons using tissue samples from AD and age-matched non-demented subjects. The purpose will be to determine histologically the spatial relationship between activated microglia, Aβ deposits, and neurodegenerative changes. It is my hypothesis that neurodegeneration occurs in AD because microglia are becoming dysfunctional and lose their neuroprotective properties. Thus, I expect to see no correlation between presence of activated microglia and presence of Aβ or neurodegenerative changes.

INTRODUCTION

Alzheimer's disease (AD) is a progressive, irreversible, degenerative disease of the brain and the most common form of dementia. AD affects an estimated 4 million people in the United States and approximately 100,000 victims die, while 360,000 new cases are diagnosed each year. People that suffer from this disease experience cognitive and behavioral dysfunction, intellectual impairment, and disorientation. Cognitive dysfunction associated with AD includes severe and debilitating memory loss, disorientation, confusion, and problems with reasoning and thinking. Behavioral dysfunction, which is also associated with AD, includes agitation, anxiety, delusions, depression, hallucinations, insomnia, and wandering.

Although the cause or causes of AD are still unknown, there are two pathological features that appear in the brains of AD victims. These microscopic features are amyloid plaques or senile plaques (SP) and neurofibrillary tangles (NFTs). Senile Plaques are thought to contribute to neurodegeneration in the brain and NFTs actually represent the neurodegenerative change that has occurred. Senile plaques consist of a central core of amyloid-like (A_42) material surrounded by swollen abnormal nerve cell processes. Amyloid is a protein fragment that accumulates between neurons in the brain forming dense deposits outside and around the nerve cells. In a healthy brain, these protein fragments (A_42) would be broken down and eliminated. In AD they accumulate and form hard, insoluble plaques. Another hallmark pathological feature of AD are NFTs. NFTs consist of insoluble twisted bands of fibers that are found inside the nerve cells. NFTs primarily contain an abnormal protein called tau. The dense bundles of long, unbranched filaments in the NFTs displace cellular organelles and the nucleus, leading to neurodegeneration (Graham et al, 1997). It is thought, in the amyloid cascade hypothesis, that A_42 protein is toxic to neurons and eventually causes formation of NFTs (Hardy et al., 1998; Selkoe, 1996). The question associated with this hypothesis is how does the A_42 cause neurodegenerative changes?

In order to answer this question, the supporting cells for neurons, glial cells, must be examined. There are three types: microglia, astrocytes, and oligodendrocytes. Microglia are of particular interest because the A_ deposits within the senile plaques are thought to cause chronic microglial inflammation, which is accompanied by the production of microglial neurotoxins. Microglial neurotoxins in turn are thought to cause neurodegeneration, which becomes evident as NFTs (see Figure 1) (Akiyama et al., 2000). Between this chronic microglial inflammation hypothesis and the amyloid cascade hypothesis, some observations still cannot be explained. For instance, microglial activation increases with normal aging even in the absence of A_ (Streit and Sparks, 1997; Sheng et al., 1998). In addition, the amyloid cascade hypothesis cannot explain how A_ was deposited in the first place. It has long been suspected (Frautschy et al., 1992) and was recently confirmed through A_ immunization experiments (Schenk et al., 1999) that microglia are involved in clearing A_. Thus, if for some reason microglia became unable to clear A_, this could explain why A_ protein accumulates in AD. With the continued accumulation of extracellular A_ protein, microglial function becomes compromised even further due to this amyloid overload and therefore the cells may be less able to perform their normal functions, which include providing trophic support to distressed or injured neurons. The lack of trophic support could cause neurons to undergo degenerative changes, neurofibrillar degeneration, and the formation of tangles.

Figure 1. The Amyloid Cascade Hypothesis

By examining spatial relationships of microlgial cells, dense extracellular deposits, and neurofibrillary tangles, we will be able to determine whether or not our hypothesis that amyloid deposits result from inadequate clearance of this protein by dysfunctional microglia and neurofibrillary tangles from inadequate trophic support, is correct. In order to view microglia cells, an immunohistochemical staining technique was performed using LN-3. LN-3 is a monoclonal antibody that recognizes a protein that is found on the cell surface microglial cells. To view NFTs an antibody against the tau protein was used. Another antibody against _-internexin was used to view neurons. Internexin is a neurofilament, structural, and cytoskeletal protein that is present in most CNS neurons. To view the extracellular deposits of A_, an antibody, 10D5, was used with the same immunohistochemical staining procedure.

MATERIALS AND METHODS

Human Subjects

In this study, human AD brain tissue and age-matched non-AD brain tissue were obtained (Table 1). The tissue blocks were sent from the Sun Health Research Institute, Sun City, AZ. The tissue was fixed in 4% buffered PFA for about 48 hours, then cryoprotected for 48 hours in each of the following: buffered 2% DMSO/10% glycerol and buffered 2%DMSO/20% glycerol. The blocks were then stored in a 30% glycol (antifreeze) solution.

Histochemical Procedures

Vibratome sections were cut at 50mm and were quenched with 3% H2O2/PBS solution for 10 minutes. The samples were then blocked with 10% normal goat serum (NGS) in PBS for 60 minutes. The primary antibody was then added in 5% serum/PBS solution overnight at 4ºC (Table 2). The next day the samples were rinsed three times in PBS for 20 minutes each. The secondary antibody (vector antimouse, biotinylated 1:500) in 5% NGS:PBS was placed on the sections for 60 minutes and the sections were rinsed again three times in PBS for 20 minutes. The Avidin-D:HRP conjugate, (1:500) in PBS was applied to the sections for 30 minutes. The sections were rinsed again and were then ready to be developed. The sections were developed with 0.5 mg/ml diaminobenzidine (DAB) solution in PBS and the solution was activated with a 3% H₂O₂, 1µl of 3% H₂O₂/ml DAB solution prior to use. The sections were incubated no longer than 10 minutes and were rinsed quickly three times each (see Figure 2). The tissues were then mounted on subbed slides and selected sections were counterstained with cresyl violet (Nissl) by first soaking them for 30 seconds in acetate buffer, followed by three minutes in cresyl violet, and for one minute in two of each of the following mixtures: 70% ethanol, 90% ethanol, 100% ethanol, and finally, xylene. The slides were then cover slipped.

Figure 2. Principle of the avidin-biotin immunoperoxidase method

Double Labeling

The procedure was the same as the above, except 1% Cobalt Chloride in H₂O₂ (250 ml/10ml) was added in with the DAB solution and the sections were not quenched for the second antibody. The procedure was then repeated for the second antibody beginning with the blocking using 10% NGS.

Figure 3a-d. Staining of microglia with LN-3 anitbody in the cerebral cortex gray matter. a,b. Non-demented individual with microgilia even dispersed. c,d. Microglia are in clusters in the Alzheimer's diseased individuals. a,c x10; b,d x40

RESULTS

All sections studied were from the cerebral cortex gray matter, which includes all cell bodies. After staining for microglia with LN-3, a noticeable difference between AD and ND brains was apparent. As seen in Figures 3a and b, microglia are evenly dispersed throughout the gray matter. All microglia are ramified with long processes and all contain a single nucleus (Figure 3b). In contrast, the AD brain shows clusters of microglia (Figures 3c,d). Using 10D5, amyloid deposits were only viewed in the AD brain (Figure 4c), but not in neurodegenerating brains (Figure 4a). When combining 10D5 and LN-3, it is obvious that microglia cluster around the amyloid deposits, while the surrounding area is devoid of microglia (see Figures 4b, d). Figure 5a shows the degenerating neurons of an AD patient using anti-tau 2 immunohistochemical staining (arrow). In contrast, Figure 5b displays the presence of surviving neurons in the AD brain using a-internexin. The absence of microglial clusters or plaques around degenerating, tau-positive neurons is demonstrated in the AD brain in Figure 6a, through the use double-staining with anti-tau 2 and anti-LN-3. Figure 6b shows a cluster of microglia, but no surrounding tau-positive neurons. The presence of normal, healthy neurons next to a cluster of microglia is seen in Figure 6c.

Figure 4. Staining of amyloid deposits using Nissl and 10D5 anitbody. b,d Staining of microglia and amyloid deposits using 10D5 and LN-3 antibodies. a. No amyloid deposits are seen in the non-demented brain. c. Amyloid deposits are seen in the AD brain. b,d. Microglia are clustered around the amyloid deposits, while surrounding areas are devoid of microglia. a,b, c x10; d x40

 

 

Figure 5 a,b. Adjacent sections of an AD brain. a. Staining of degenerating neurons using anti-tau 2 (arrow). b. Surviving neurons are displayed using α-internexin. a,b x40

Figure 6 a,b. Staining of microglia and degenerating tau-positive neurons using anti-LN-3 and anti-tau 2. c. Staining of microglia and neurons using anti-LN-3 and anti-α-internexin. a. Microglia clusters are absent around degenerating tau-positive neurons. b. Here is a microglia cluster, but degenerating neurons are not present. c. Normal, healthy neurons are present next to a cluster of microglia. a-c x40

DISCUSSION

The objective of this study was to examine the spatial relationship between activated microglial cells and the histopathological features of AD, in order to reexamine the amyloid/neuroinflammation hypothesis of AD pathogenesis. Single immunohistochemical staining was performed to visualize microglia, and double immunohistochemistry was used to visualize the relationship between microglia and normal/abnormal neurons, as well as the relationship between microglia and β-amyloid.

This study has shown that microglial cells cluster around β-amyloid deposits in AD brains, while ND brains show no signs of β-amyloid deposits or clustering of microglia. The clustering of microglia at sites of amyloid deposition suggests that microglia are drawn to these sites and attempt to remove the amyloid protein. However, due to the insoluble nature of amyloid and the fact that it is present in great quantities, it is likely that microglia are unable to clear it. Thus, amyloid deposits remain and continue to attract microglial cells over prolonged periods of time ultimately causing the cells to "burn out" and die. In fact microglial cell death has been reported in AD (Lassmann et al.,1995). The results also show that there is no clear-cut spatial relationship between activated microglia and NFTs. This suggests that the amyloid hypothesis is insufficient to explain AD pathogenesis. The amyloid hypothesis states that activated microglia, chronically activated by amyloid, produce toxic substances that cause neurons to degenerate (Hardy et al., 1998; Selkoe, 1996). With this hypothesis, one would expect the neurons to degenerate around plaques, but when we looked for tau-positive neurons around microglia clusters, none were to be found. This suggests that neurodegeneration is not, as would be predicted by the hypothesis, specific to plaques.

Moreover, we were able to observe normal (α-internexin-positive) neurons in the immediate vicinity of clustered (activated) microglia, further suggesting that activated microglia do not cause bystander damage in their immediate vicinity. The evidence obtained calls for an alternate hypothesis to explain AD pathogenesis and we hypothesize that neurodegeneration occurs because microglial cells become dysfunctional with aging. This hypothesis could explain the close association between occurrence of AD and aging. It could also explain why amyloid protein accumulates in the aging and AD brain, namely, because of an impaired ability of microglia to clear this protein. The appearance of neurodegenerative changes could be attributed to an impaired ability of microglia to perform their normal, neuron-sustaining functions. Thus, the present study suggests that neurodegeneration may occur as a result of neglect rather than as a result of neurotoxic aggression

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