Ординатура / Офтальмология / Английские материалы / Aging and Age Related Ocular Diseases_Lutjen-Drecoll_2000

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The phenomenon of aging is characterized by various degenerative changes which differentially affect the highly specialized structures within the eye, such as the purely cellular lens, the brain-derived retina and the connective tissue of the uvea and sclera. The eye can therefore serve as an excellent model system to study age-related degenerative diseases.

Glaucoma is a common age-related ocular disease. Studies in glaucomatous eyes have shown that there is a correlation between optic nerve fiber loss and changes in the outflow tissues, with the latter being presumably responsible for increased intraocular pressure (IOP). Another finding in glaucoma is that increased expression of the stress protein ·B-crystallin occurs in TM of glaucomatous eyes. ·B-crystallin is one of the proteins normally protecting lens fiber proteins from unfolding due to various stress factors. In this volume, an overview article deals with the molecular biology of this molecule, and original articles give further insights into the distribution and possible functional significance of nonlenticular ·B- crystallin. A better understanding of the role of protective molecules in the eye might open new perspectives in the treatment of degenerative diseases.

The commonest causes of blindness are the tapetoretinal degenerations and age-related macular degeneration.

Retinal transplantation studies have been undertaken that may ultimately lead to therapy for these diseases. New kinds of laminated transplants with promising survival times have been implanted and studied using different immunohistochemical methods. Further, an attempt has been made to investigate factors that influence the development of the retinal tissues, since less differentiated embryonic retina has the potential to be well integrated into degenerated retina. In the essentially cellular lens, it is difficult to define when development ends and ageing begins. An overview article examines cells of the deep lens fibers. These cells lose organelles and finally undergo denucleation, even though they remain viable for the life of the lens. The membranous channel system of the lens fibers plays a role in retaining transparency of the lens. In vitro, oxidative damage is one factor responsible for inducing changes in these lenticular ion channels. Protection against oxidative damage might therefore protect against cataract formation.

The studies were kindly supported by the European Commission as part of the Biomed project BMH4-CT96- 1593 (Brussels).

We thank especially Mr. Kallasvaara for his help and his patience.

E. Lütjen-Drecoll, Erlangen

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Key Words

·B-Crystallin W Neurodegenerative diseases W Heat shock protein

Abstract

·B-Crystallin, which has homology with the small heat shock proteins, is the basic subunit of ·-crystallin, a major component of the vertebrate eye lens. These crystallins have for a long time been thought to be absolutely lens specific. However, about a decade ago ·B-crystallin has been detected extralenticularly in many tissues among which the central nervous system. Under pathological conditions the expression level of ·B-crystallin frequently increases. For this reason it is considered to be a useful marker in a variety of neurodegenerative diseases. In this mini-review, a number of typical neurodegenerative disorders is dealt with in which ·B-crystallin may play a role.

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Introduction

·B-Crystallin was discovered as the basic subunit of ·-crystallin, a major structural protein found in the eye lens of vertebrates [1, 2]. ·-Crystallin is an aggregate with a molecular mass of about 800 kD that is composed of two closely related subunits, ·A- and ·B-crystallin, both hav-

ing a molecular mass of approximately 20 kD. These subunits which share the ability to form protein aggregates with each other or on their own show a 57% sequence homology.

For a long time it has been thought that crystallins were the prototype of organ-specific proteins. However, this idea became doubtful when these proteins were found occasionally outside the lens [3±5]. The real extralenticular detection of ·B-crystallin, the basic subunit of ·-crys- tallin, came when this protein was discovered in muscle, heart, brain and kidney [6]. Also ·A-crystallin, the acidic subunit of ·-crystallin, was detected extralenticularly in brain, spleen, thymus and retina, albeit to a far lesser extent [7]. In order to explain the occurrence of crystallins outside the lens, it is now assumed that during evolution normal metabolic proteins were recruited to function as structural proteins of the eye lens in order to maintain its transparency.

Ingolia and Craig [8] showed for the first time that ·- crystallin belongs to the large family of small heat shock proteins (sHSPs). Heat shock proteins (HSPs) are preferentially synthesized in organs exposed to heat or other types of physiological stress. These proteins are classified as several families: HSP100, HSP90, HSP70, HSP60, respectively, and the sHSP family that enjoy a wide phylogenetic distribution.

The sHSPs, including ·B-crystallin, prevent protein unfolding or aggregation, a property called chaperone-like activity, while others are involved in refolding or proteo-

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lytic destruction. HSPs play an important role because they mediate many cellular processes. The chaperone property of ·-crystallin has been discovered by Horwitz [9].

In most if not all cells HSPs exist in low, sometimes extremely low, concentrations. As a rule the level of expression is enhanced following different kinds of stress. This holds true too for ·B-crystallin, which in contrast to ·A-crystallin is stress inducible. However, both proteins contain the so-called ·-crystallin domain that is shared by all members of the sHSP family [10] and that presumably is responsible for the chaperone-like activity. sHSPs as well as ·B-crystallin appeared to be involved in intracellular changes during disease progression [11, 12]. Besides, in disease progression, ·B-crystallin is also involved in reactive processes of astrocytes and oligodendrocytes in the central nervous system [13], development of astrocytic tumors [14] and in the development of benign tumors associated with tuberous sclerosis. ·B-Crystallin and HSP27 are expressed at elevated levels in the brains of patients suffering from Alexander's [15] and Alzheimer's disease [16]. Therefore, ·B-crystallin is indeed a useful biochemical marker for studying the pathogenesis of various types of human neurodegenerative disorders and brain tumors [17]. Moreover, this protein is also a major component of ubiquitinated inclusion bodies in human degenerative diseases [18].

In this mini-review we shall restrict ourselves to the discussion of the expression of ·B-crystallin in some selected neurodegenerative diseases.

·B-Crystallin in Patients with Alexander's Disease

Alexander's disease [19] is a rare sporadic nonfamilial leukodystrophy occurring in early childhood. There is no evidence of inherited enzyme deficiency or other genetic factors [20±23]. Megalencephaly, psychomotor retardation, spastic paresis and epileptic seizures are the clinical symptoms. Also extensive proliferation of abnormal astrocytes, the formation of inclusions in astrocytes and Rosenthal fibers (RFs) are typical of this disease. They are predominantly found in perivascular, subependymal and subpial regions [24±26]. The disease already manifests itself during the first months of life and progresses during the next 2 years, culminating in death. A definite diagnosis can only be assessed after death by brain biopsy. The disease is characterized by a diffuse leukodystrophy, which histopathologically can be seen as subcortical de-

myelination involving the brainstem, cerebellum and spinal cord together with the presence of RFs, osmiophilic dense abnormal inclusions in astrocytes. Recently it has been shown that RFs which are characteristic of Alexander's disease, are composed of glial acidic fibrillary protein (GFAP), ·B-crystallin (some of which is ubiquitinated) and the sHSP HSP27, which is closely related to ·B-crystallin [15, 27]. Since ·B-crystallin and HSP27 are stress proteins, they might be induced in response to stress caused by the disease per se. Indeed it is assumed that the formation of RFs is due to a chronic stress to a yet unknown stimulus [28]. The overproduction of ·B-crys- tallin in astrocytes as a response to stress may also be the cause of RF accumulation in some neurological disorders other than Alexander's disease. The interaction of ·B- crystallin with other sHSPs, GFAP or ubiquitin may play an important role in RF accumulation. Ubiquitin is a highly conserved protein 76 amino acids long that is involved in the nonlysosomal degradation of proteins in eukaryotic cells by forming conjugates with abnormal proteins [29]. Since it has been shown by immunocytochemical analysis that the RF matrix reacts with antibodies to ·B-crystallin and GFAP and in addition with antibodies to ubiquitin it might well be that all those proteins together form the RFs [15].

Goldman and Corbin [30] provided evidence that RFs contain monoand polyubiquitinated conjugates of ·B- crystallin. The binding of the two proteins together could be shown by using both the antibodies to ·B-crystallin and ubiquitin on identical Western brain samples from diseased people. ·B-Crystallin contains a number of lysines that are potential substrates for isopeptide bond formation with ubiquitin. The finding of ubiquitin-·B-crys- tallin conjugates in pathological inclusions raises a number of questions. One is whether or not ·B-crystallin normally becomes ubiquitinated, possibly as an intermediate in the turnover of the protein, or whether the conjugates in RFs are only seen as part of a pathological process. But if ubiquitin conjugation plays a role in the metabolism of ·B-crystallin, then it seems paradoxical that the conjugates accumulate. Several explanations are possible for the accumulation. Either ·B-crystallin might be altered by posttranslational modifications in a way that it becomes a poor substrate for proteolytic enzymes, or ·B-crystallin in RFs might be more stable than soluble ·B-crystallin. Another option is that the proteolytic system in astrocytes is changed. Although ubiquitin-·B-crystallin complexes are formed, most of the ·B-crystallin in RFs is not ubiquitinated. This might be due to overloading of the ubiquitin conjugating system or to ·B-crystallin not being accessible

for conjugation. By definition, RFs are abnormal inclusions accumulating in cells because they are not degraded efficiently. Ubiquitin has been implicated in the breakdown of proteins [31], but despite the ubiquitination of ·B-crystallin in RFs, the affected astrocytes still accumulate ·B-crystallin and its ubiquitinated conjugates [30].

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a human progressive neurodegenerative lethal disease with the onset in midlife. It is characterized by the degeneration of large motor neurons of the spinal cord, brainstem and motor cortex. Death of these neurons leads to weakness, atrophy and spasticity [32±34]. ALS is one of the most devastating neurodegenerative disorders since it leads to death in less than 5 years. The etiology of ALS consistently shows an early axonal degeneration, increased remyelination and a preponderance of fibers with smaller diameters [35]. Motor neurons in the motor cortex, spinal cord and brainstem are targets, with death ensuing once respiratory functions are paralyzed.

ALS occurs both sporadically and in familial forms, which are clinically and pathologically similar [36]. Mutations in the Cu/Zn-dependent superoxide dismutase (SOD-1) gene are associated with the latter type of the disease and are found in approximately 20% of patients with familial ALS [37, 38]. This means that 80% of familial ALS cases may be associated with a genetic locus yet to be identified [39]. SOD has been considered to be an excellent buffer against free concentrations of copper ions in the cytosol. Unbound copper ions have the potential to cause oxidative injury to tissues or to initiate an apoptotic process in motor neurons [40]. A defective copper binding site in SOD-1 could cancel or limit buffering capacity. For example, the H4R6 mutation in SOD-1 from ALS patients has been shown to impair the binding of copper to SOD [41].

ALS belongs to the group of neurodegenerative disorders which are characterized by the occurrence of socalled ballooned (swollen) neurons. A comparative study has recently been reported on the expression of ·B-crys- tallin, other stressresponse proteins and phosphorylated neurofilament protein. ·B-Crystallin was expressed in the ballooned neurons of Pick's disease (a rare dementing disorder) and Creutzfeldt-Jakob disease, but not in those of ALS and other cases of neuropathology. In contrast, phosphorylated neurofilament protein was detected in most abnormal neurons [42]. Skein or skein-like inclusions in

motor neurons detected by ubiquitin immunohistochemistry are characteristic of ALS. A first report on these inclusions has been published recently. The results strongly support the notion that ALS is a multisystem disease and not simply a pathology of the motor neurons [43]. Eosinophilic fibrillary neuronal inclusions have been described in patients with sporadic ALS. Aberrant phosphorylation of neurofilament protein paralleled by induction of ·B-crystallin was shown to exist in ballooned neurons bearing eosinophilic fibrillary neuronal inclusions [44].

Alzheimer's Disease

Alzheimer's disease (AD) is the most common form of dementia and results in the loss of intellectual abilities in an age-dependent manner. Pathologically, AD is defined by extracellular cortical amyloid deposits called senile plaques and intraneuronal bundles of paired helical filaments referred to as neurofibrillary tangles [45]. The main proteins of the amyloid plaques are the so-called ß-amy- loid peptides (Ab1±40 amino acids), proteolytic fragments of the ·-amyloid precursor protein (APP). Those peptides are normal products of the APP metabolism. However, in patients with familial forms of AD either the overall production of Ab peptides or the generation of elongated peptides (Ab1±42 and Ab1±43 amino acids) is increased [46±49].

Expression of ·B-crystallin in normal human brain is found in oligodendroglia and subpial astrocytes [13, 16]. On the other hand in AD patients, ·B-crystallin expression was found to be increased. The protein was detected in reactive astrocytes, microglia and oligodendrocytes, indicating that all types of glia overexpress ·B-crystallin in response to stress associated with AD. However, in comparison to the large increase in the number of GFAPpositive astrocytes the increase in ·B-crystallin-express- ing cells seems minor [50]. Nevertheless it may be concluded that under pathological conditions the expression of ·B-crystallin increases, suggesting that this protein together with GFAP may serve as a marker for gliosis in neurodegenerative diseases. ·B-Crystallin might also be a marker for neurons at the edge of areas of cerebral infarction, since it is found in cells regenerating upon damage. The presence of ·B-crystallin in those ballooned neurons and its close association with filaments suggests that ·B- crystallin may be involved in aggregation and remodeling of neurofilaments in this particular disease. In families in which AD is inherited as an autosomal dominant trait,

four genes that are identified with the development of the disease have been characterized [51]. Mutations in three genes account for virtually all early-onset familial AD: APP on chromosome 21, presenilin 1 on chromosome 14 and presenilin 2 on chromosome 1. One allele of apolipoprotein E on chromosome 9 is associated with late-onset AD, in patients with and without a strong family history [52].

Another example for the connection of ·B-crystallin with AD stems from patients with Down's syndrome (DS). Those people over 40 years develop features of dementia of AD [53]. The idea behind it is not fully understood, but it is probably a genetic effect. The APP gene seems very important since people with DS have trisomy for the chromosome containing the APP gene. Disturbance of the organization of microtubules might underlie both AD and DS [54]. The neuropathological similarities and biochemical changes in people with DS and people suffering from AD suggest a common mechanism underlying the degenerative process in the two conditions [55, 56]. The expression of ·B-crystallin in people with DS appears to be related to the presence of dementia and AD neuropathological structures. Virtually no ·B-crystallin could be found in DS people without AD. As in people without DS, the ·B-crystallin in AD brains was found in reactive glia [57]. The significance of the expression of ·B-crystallin in AD brains is not yet known. The elevation of the protein as a response to stress might influence the progression of AD. It might protect other cells from stressful attacks or influence the toxicity of the Ab peptides [58]. Recently a scheme to screen out cases of AD, dementia with Lewy bodies, has been developed in order to discriminate between nonAD degenerative dementias and AD. Besides ubiquitin and Ù protein, ·B-crystallin plays a key role in the diagnosis assessed by immunochemical detection of these marker proteins [59].

·B-Crystallin in Other Neurodegenerative

Diseases

The results of an ultrastructural and immunohistochemical study on ballooned cortical neurons in Creutz- feldt-Jakob disease suggested that the expression of phosphorylated neurofilament proteins and synaptophysin may reflect axonal impairment and that the presence of ·B-cystallin in addition to HSP27 and ubiquitin might be related to the degenerative processes that neurons undergo in Creutzfeldt-Jakob disease [60].

By immunochemical analysis ubiquitinated ·B-crys- tallin has been found in glial cytoplasmic inclusions obtained from the brains of patients with multiple system atrophy [61].

Multiple sclerosis (MS) is a major but poorly understood neurological disease of young adults in the Western world; in fact, it is a demyelinating disorder. In a review van Noort [62] discussed functional and therapeutic implications of his finding that ·B-crystallin is a single immunodominant myelin antigen to human T cells expressed at enhanced levels in MS-affected myelin [63]. Using immunohistochemical techniques, oligodendrocytes as well as astrocytes were shown to express ·B-crys- tallin in MS lesions. Different regulatory pathways for ·B- crystallin have been suggested in either type of glia cells [64]. It seems that alterations of the normal myelin structure or normal oligodendrocytes might be the primary events that affect the susceptibility to MS [65]. Once triggered the immune system attacks and damages myelinforming cells. Oligodendrocytes respond to the attack of the immune cells and their secreted products, through modulation of their metabolism and gene expression. As part of this process, induction of HSPs, including ·B-crys- tallin, may take place in the oligodendrocytes as a kind of survival response.

Corticobasal degeneration is another example of a neuropathological aberration. In this disease there is a marked neuronal loss and gliosis in the substantia nigra. Again the ballooned neurons are positively stained for phosphorylated neurofilament protein, synaptophysin and ·B-crystallin [66]. Ballooned neurons are also present in the cerebral cortex of these patients. It is concluded that the cytopathology of the subcortical gray matter and brainstem in corticobasal degeneration patients resembles that of progressive supranuclear palsy [67]. These swollen neurons which were positive for phosphorylated neurofilament protein and ·B-crystallin appeared to be abundant in 2 out of 6 patients. Cortical degeneration was observed in the precentral cortex.

Ballooned neurons are a constant feature of amygdaloid nuclei in patients with argyrophilic grain disease [68]. Those ballooned neurons appeared to be strongly labeled with antibodies against ·B-crystallin and phosphorylated Ù and neurofilament protein. In contrast nonballooned neurons that were immunoreactive with anti-Ù remained consistently unstained with the crystallin antibody. The conclusion is drawn that two different pathological mechanisms may be operative in limbic neurons.

As final example of a neurodegenerative disorder we want to mention Parkinson's disease that is the second

largest neurodegenerative disorder progressively destroying part of the brain which regulates coordinated motion. In Europe the number of patients suffering from this disease amounts to more than 1 million persons. With increasing numbers of aged people a substantial increase in Parkinson's disease has to be anticipated. Major symptons are resting tremor, rigidity, gait disturbance, postural instability, vegetative and psychiatric aberrations. A prominent feature of Parkinson's disease is a gradual loss of dopaminergic neurons in the midbrain. The role of ·B- crystallin in the development and progression of this

pathology is far from being clear, and the amount of sound data in the literature is not overwhelming. It is reported that a familial parkinsonism is accompanied by the presence of ballooned neurons and ·B-crystallin [69].

A similar expression pattern of sHSPs as observed in AD has also been described for Parkinson's disease, a disorder without dementia [70]. At any rate the putative role of enhanced ·B-crystallin production in the cases discussed is still not much more than a matter of speculation.

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