Possible Role of Chitin-Like Proteins in the Etiology of Alzheimer’s Disease

INTRODUCTION

Chitin is a β-linked straight chain carbohydrate mono-polymer prominent in invertebrates. Surprisingly, chitin is now documented in vertebrates [1]. In mice and humans, genes that share a high degree of homology to those encoding chitin synthase in plants and fungi have been reported [2]. Specifically, these genes appear to be expressed early during development. It is an unexpected component of vertebrate physiology that has been neglected until now. Chitin, as well as chitinases and chitin lectins, occur at elevated levels in Alzheimer’s disease (AD) patients. The discussion that follows attempts to demonstrate that chitins and their associated proteins and enzymes involved in synthesis, deposition, and degradation may be responsible for AD. A systematic review of the available data may help to support this hypothesis.

CHITIN

Chitin is a simple straight chain sugar polymer consisting of repeating units of N-acetyl glucosamine in β-1,4 linkages. It is the second most abundant insoluble polymer in the biosphere, exceeded only by cellulose. It is a key structural element in fungi, mollusks, arthropods, and other invertebrates. The β-linkage of chitin is similar to that in cellulose. However, one hydroxyl group of each chitin monomer is replaced with an acetyl amine group. This gives increased hydrogen bonding between adjacent polymers and provides vastly increased strength [3]. Combined with calcium carbonate, it is an extremely strong material, as exemplified by the shells of oysters and clams.

It was once assumed that chitin does not occur in vertebrates; however, recent evidence confirms that it does occur, even in mammals [1, 2]. Chitin found in mammals consists of two synthetic sources. One in particular, the short chain oligomers that precede hyaluronan (HA) synthesis, is of particular interest, as described below. These should be distinguished from the long chitin chains found in invertebrates and in many vertebrates, including mammals.

Chitin, chitinases, and chitinase-like proteins, the chitinase lectins, participate in the pathophysiology of a growing number of human diseases. There are immune disorders such as asthma, neurological diseases such as AD, genetic problems such as Gaucher’s disease, and several disorders of unknown origin that include sarcoidosis [4–7].

CHITIN SYNTHASE

Chitin synthases, the enzymes that synthesize chitin, have recently been documented in zebra fish and involve several gene isoforms [1]. Chitin synthesis was also shown in developing frog embryos several decades ago [8, 9] with gene homologs identified in the mouse. However, these observations were considered a curiosity at the time and were not further pursued. It is only now appreciated how important and prescient those observations were. The high diversity of animal chitin synthases and their broad distribution indicate that there is much to be learned about these enzymes and why they occur in vertebrates [10].

It is also intriguing to note that the synthases of all three major β-chain carbohydrate polymers of the biosphere, cellulose, chitin, and HA, evolved from an ancient common precursor, based on sequence homologies [11, 12].

CHITINASE AND CHITIN LECTINS

Chitinases are the enzymes that degrade chitins. Levels of circulating chitinase activity occur in a wide range in humans. It follows that the corresponding ability or inability to eradicate chitin chains varies widely in the human population.

Chitinases are involved in human immune processes, a subject that is little understood. Chitinases are among the most abundant proteins of human monocytes and macrophages [13, 14]. The functions of these chitinases are entirely unknown. It was once assumed that they served as protection against chitinous parasites; however, it is now documented that enzyme activity levels increase following immune stimulation. The chitinase enzymes must have assumed new functions through evolution. We know so little about this entire class of important enzymes.

HYALURONAN AND THE DG42 CONUNDRUM

Hyaluronan (HA, hyaluronic acid) is also a straight chain β-linked sugar polymer containing two sugars, N-acetyl glucosamine alternating with glucuronic acid. HA resembles chitin more closely than it does other glycosaminoglycans (GAGs). Chitin may be an evolutionary precursor of HA [15].

Both polymers are synthesized by enzymes embedded on the inner surface of the cell plasma membrane. Both polymers become extruded out of the cell as they are being synthesized. Intracellular retention of these polymers would be disastrous for the integrity of the cell. All other GAGs, in particular, the sulfated and epimerized GAGs, are synthesized on the Golgi apparatus and are often attached to core proteins, structures that are termed proteoglycans.

A controversy arose decades ago regarding whether the DG42 gene product expressed early in frog embryogenesis synthesized HA or chitin [8, 16]. Both sets of investigators were correct [17], depending on how the experiments were conducted. It is only possible now to understand that dichotomy. Synthesis of small chitin fragments occurs before HA chain synthesis commences. HA turns over rapidly, but chitin is difficult to degrade and does so with great variability. It accumulates and can cause pathology. Despite previous assumptions, vertebrate tissues do contain chitin, albeit in varying amounts. Such chitin is particularly toxic to neurons. As discussed below, this may account for the cognitive decline in AD.

HA synthesis occurs on a chitin platform. Recent data establishes that HA synthesis is initiated with a short run of a chitin primer, seven to nine saccharides in length [18, 19]. These small chitin chains resist degradation, in marked contrast to HA’s ability to be rapidly degraded [20, 21]. These chitin platforms for HA synthesis can accumulate with age.

Chitinase levels vary widely in the human population, and chitin accumulation is inversely proportional to chitinase activity levels. Some AD patients are postulated to have relatively low levels of circulating chitinase, while it is extraordinarily high in some individuals.

CHITIN IN ALZHEIMER’S DISEASE

AD is the most common of the human dementias. There are 5.4 million cases in the US today, and 0.5 million new cases are added yearly. Of the 5.4 million cases, 5.2 million occur after age 65. It is thus a major problem among the elderly, the most rapidly growing component of the population.

Despite the fact that AD was described in detail over 100 years ago, that billions of dollars have been spent on research, and that thousands of scientists have devoted time and effort exploring this disorder, we still have no information regarding the etiology of AD. The hypothesis proposed here is that chitin accumulation in neurons is the basis of AD and underlies the cognitive decline that characterizes the disorder.

AD in its sporadic form constitutes 95% of cases. The far less frequent familial form of AD has a much earlier onset. Both forms of AD, however, have a steady downhill course, ending in death within 10 years following diagnosis. Studies indicate that subtle manifestations can occur years before actual diagnosis.

The histopathologic features of AD include: 1) extracellular senile neuritic plaques containing dystrophic neurites and an amyloid core, 2) intracellular neurofibrillary tangles, referred to by pathologists as “spaghetti and meatballs,” and 3) amyloid-rich blood vessels, termed amyloid angiopathy.

The amyloid plaques of AD contain chitin. Such deposits are present in sporadic cases of AD but not in the familial cases [22–25]. Such toxicity has been documented in vitro in neuron cell cultures [25]. The presence of chitin fragments in AD brains has now been documented [22, 23]. Such chitinous deposits are present in the brains of the sporadic form of AD but not in the rarer familial form. The putative toxicity of these chitin fragments in the brain may contribute to the dementia that characterizes AD patients.

In cell culture studies, incubation of neuronal cell lines with the sugar that comprises chitin, GlcNAc, and its precursor, UDP-GlcNAc, induces significant cell death. Similar results are obtained with primary cultures of hippocampal neurons and in hippocampal slices. Phagocytosis of the chitin chains by microglia is also observed [22].

As mentioned, chitin is tolerated in much of the body but may be particularly toxic to neurons. Accumulation continues over years and eventually may trigger the cognitive decline that characterizes AD patients. The onset however must certainly occur years before there are actual changes in mental status.

Additional evidence that supports the role of chitins in AD are the observations that chitinase, as well as the chitin-binding protein, the chitin lectin, chitinase 3-like 1 (CH3L1), also known as YKL-40, are elevated in the plasma and in the cerebrospinal fluid of AD patients [26–28].

GAUCHER’S DISEASE

Gaucher’s disease is a genetic disorder in which glucocerebroside, a sphingolipid, accumulates in cells and certain organs. It is the most common of the lysosomal storage diseases and is caused by a hereditary deficiency of the enzyme glucocerebrosidase, also known as glucosylceramidase. The disease is caused by a recessive mutation in a gene located on chromosome 1. About one in 100 people are carriers of Gaucher’s disease. The carrier rate among Ashkenazi Jews is approximately 9% with a birth incidence of one in 400.

The wide range of chitinase activity in the human circulation is best exemplified by Gaucher’s disease patients. Such individuals have a thousand-fold increase in circulating chitinase enzyme activity [29–31]. This is often used to follow disease progression and effectiveness of treatment. Curiously, both cerebroside and chitinase occur on chromosome 1. The heterozygote relatives of Gaucher’s disease patients with only one copy of the defective gene also have elevated levels [32]. Why there is coordinate or linked expression of chitinase and the Gaucher’s disease gene is unknown, except that both loci are located on chromosome 1.

The relative inability to degrade chitins because of low levels of chitinase activity may be the source of vertebrate chitins that accumulate in variable amounts with age throughout the body, wherever HA synthesis occurs. Indeed, chitin deposition has been documented in sporadic cases of AD, but not in the familial forms of the disorder.

Further research could be considered involving patients with Gaucher’s disease. Such patients as well as their heterozygote relatives would be predicted to have a lower incidence of disease. Indeed, it can be postulated that Gaucher’s disease may be a form of balanced polymorphism that protects against AD.

NEW ANIMAL MODELS AND POTENTIAL TREATMENT MODALITIES

There are several murine models for AD, but none accurately reflect the human disorder [33]. A new murine model might be the deletion of the gene for the chitinase enzyme. This would be a direct test of the hypothesis that lack of chitinase activity leads to an AD-like disorder.

Another and more immediate approach is to administer allisomidin to animals [34]. This inhibitor of chitinase activity would increase the buildup of chitin in tissues, particularly in the central nervous system, and could lead to an AD-like disorder.

Another testable hypothesis would be to assay levels of circulating chitinase activity in patients with AD, to demonstrate that individuals with lower activities have a higher incidence or have greater risk for developing the disorder.

Recombinant chitinase enzyme administration has the potential of becoming a future therapy for the treatment of AD and would perhaps even be able to reverse some of the cognitive decline. Consideration of a clinical trial could certainly be justified. Since the enzyme is a naturally occurring human protein in the circulation and in tissues, the likelihood of an immune reaction is very low. This has the potential of becoming the first effective therapeutic measure for this devastating human disorder.

BRAIN CHITIN MAY REFLECT REMOTE FUNGAL INFECTIONS

A caveat in our understanding of the role of chitin in AD is the documentation of fungal infections. Such infections have been implicated in AD brains, while absent from normal controls [35–38]. This may be an additional or alternative explanation for chitin deposition in AD. The presence of fungal infections in AD brains is not a new concept, but was proposed over 100 years ago [39].

Chitin in the brain may be a manifestation of remote and not totally resolved fungal infections, as chitin constitutes the cell walls of yeast and other fungi. This can reflect the inability of tissue chitinases, and particularly brain chitinases to remove traces of such infections.

PCR analysis reveals a variety of fungal species are involved [35]. In one such study, fungal proteins and DNA were found in the brains and neurovascular tissue of eleven AD patients examined and none in control patients [36]. These studies are further confirmed by direct visualization using a variety of anti-fungal antibodies [37].

Remnants of fungal infections can remain in human and animal tissues long after active infections have apparently been cleared. Chitinases are not efficient enzymes, and may not be able to eradicate all traces of remote infections. Staining for chitins is not routine in neuropathology examination of the brain.

Such chitins may previously have been overlooked. Indeed, fungal infections may be more ubiquitous than previously assumed, as could be documented by staining brains for remnants of chitin deposition, cast fragments of fungal cell walls, spores and hyphae, all straightforward procedures. A rapid and convenient staining procedure that could easily be included into routine examinations of the brain has been described [40].

CONCLUSIONS

In summary, chitin, chitin synthases, chitinases, and the chitin lectins are a neglected area of human pathophysiology. They do not give up their secrets easily. A number of possible scenarios are available for the presence of chitins in the human brain, and as a possible etiology for AD.

HA turns over very rapidly in vertebrate tissues as a result of the hyaluronidase family of enzymes. These are extremely efficient enzymes with catalytic rates one log greater than conventional globular enzymes [21]. The HA components of the initiating HA chains are easily degraded by hyaluronidases, but the small chitin oligomers at the origins of the lengthy HA chains may remain relatively intact, particularly in individuals with intrinsically low levels of chitinase activity. With time, chitin oligomers can accumulate, among the processes that may occur with aging. Most tissues can withstand such buildup of chitin fragments, but they may be particularly toxic to neurons.

This is a new concept that could easily be tested as a possible contributor to the etiology of this devastating disorder.