Trisomy 21 and the Brain

Abstract

In fetuses with Down syndrome, neurons fail to show normal dendritic development, yielding a “tree in winter” appearance. This developmental failure is thought to result in mental retardation. In adults with Down syndrome, neuronal loss is dramatic and neurofibrillary and neuritic Aβ plaque pathologies are consistent with Alzheimer disease. These pathological changes are thought to underlie the neuropsychological and physiological changes in older individuals with Down syndrome. Two chromosome 21-based gene products, β-amyloid precursor protein (βAPP) and S100B, have been implicated in these neuronal and interstitial changes. Although not necessary for mental retardation and other features, βAPP gene triplication is necessary, although perhaps not sufficient, for development of Alzheimer pathology. S100B is overexpressed throughout life in Down patients, and mice with extra copies of the S100B gene have dendritic abnormalities. S100B overexpression correlates with Alzheimer pathology in post-adolescent Down syndrome patients and has been implicated in Aβ plaque pathogenesis. Interleukin-1 (IL-1) is a non-chromosome-21-based cytokine that is also overexpressed throughout life in Down syndrome. IL-1 upregulates βAPP and S100B expression and drives numerous neurodegenerative and self-amplifying cascades that support a neuroinflammatory component in the pathogenesis of sporadic and Down syndrome-related Alzheimer disease.

INTRODUCTION

Trisomy 21 is unique among human diseases in producing a viable, functional human being with triplication of an autosomal chromosome. Individuals affected with Down syndrome manifest a number of abnormalities, ranging from the characteristic facies to congenital heart malformations to susceptibility to leukemias and infections. The most consistent and striking alterations, however, involve the brain. These changes, still incompletely understood, fall into 2 categories. The first is a subtle developmental abnormality that results in lifelong mental retardation. The second is a less subtle, almost absolute predisposition to severe Alzheimer disease that commences at relatively young ages (forties, or even thirties) and results in demonstrable cognitive decline in these already impaired individuals.

Brains from Down syndrome patients are smaller than normal, often around 1,000 g in weight, with corresponding reductions in neuronal content. They show a characteristic rounding of contour and shortening in the anterior-posterior axis with a steeply rising, almost vertical occipital outline. A small superior temporal gyrus is also characteristic and the cerebellum and brainstem are also often disproportionately small. Subtle abnormalities such as focal heterotopias, polymicrogyria, and other abnormal convolutional patterns may also be found (1, 2).

The most striking finding in young Down syndrome brains is apparent only with special Golgi preparations. Neuronal dendritic trees are normal in Down syndrome fetuses, but these fail to show the progressive increase in dendritic number and complexity that is seen in normal individuals from birth to young adulthood (3, 4). This results in a striking “tree in winter” appearance of neuronal dendrites that persists into adulthood, and that immediately suggests a morphological substrate for mental retardation.

Premature Alzheimer-type neuropathological changes in Down syndrome were noted as early as 1948 (5), long before Alzheimer disease was recognized as a common cause of senile dementia. The association has been documented in detail by Wisniewski et al (6).

THE GENETICS OF DOWN SYNDROME

John Langdon Down described “Mongolian” features among patients under his care at the Earlswood Asylum for Idiots in 1866 (7), but it was not until 1959 that trisomy for chromosome 21 was described in these individuals (8). Complete triplication of the entire chromosome is not necessary to produce the clinical phenotype; triplication of just a portion—the so-called Down syndrome region—of the distal long arm, especially 21 q22.2, has been identified as sufficient (9). The entire sequence of human chromosome 21 is now available and there are 225 predicted chromosome 21 genes, almost all of which are located on the long arm. More than half of all human genes are thought to be involved in brain development or function, suggesting that there are 110 to 150 candidate genes responsible for Down syndrome (10). There are at least 10 known chromosome 21 genes that are involved in brain structure or function, but only 3 have been implicated in the neuropathology of Down syndrome. These are the genes for the Aβ precursor protein (βAPP), for superoxide dismutase (SOD1), and for the astrocyte-den ved neurotrophic factor S100B.

βAPP is, of course, the precursor protein for the Aβ peptide that is deposited in amyloid plaques in the brain of Alzheimer patients and in middle-aged individuals with Down syndrome, suggesting its importance in Alzheimer pathogenesis. Identification of specific mutations in the βAPP gene that cause Alzheimer disease also implicated this gene in disease pathogenesis. As further evidence for this idea, triplication of the gene for βAPP is not necessary for the development of phenotypic characteristics of Down syndrome, but does seem to be necessary for the appearance of Alzheimer disease in Down syndrome patients (11, 12), as discussed in detail below.

SOD1 catalyzes the dismutation of superoxide radicals, a product of normal oxidative metabolism, to produce hydrogen peroxide and molecular oxygen. As such, it is generally thought of as an anti-oxidative agent that is neuroprotective. Indeed, decreased SOD1 activity, associated with certain pathogenic mutations, can cause a familial form of motor neuron disease (13, 14). However, in addition to the anti-oxidation effects of degrading superoxide radicals, SOD1 generates hydrogen peroxide, which is itself a potential source of oxidative damage. SOD1 is overexpressed in the brains of adult Down syndrome patients (15) but not in the brains of Down syndrome fetuses (16), suggesting that SOD1 dysregulation is not involved in the abnormal neurological development in Down syndrome. In adult Down syndrome patients, brain levels of this protein actually decline with the appearance of dementia (17).

S100B, a small, soluble, astrocyte-derived homodimeric cytokine, which is encoded by a gene mapped to the Down syndrome region, has trophic actions at picomolar concentrations on neurons—including promotion of neurite outgrowth, and on glia—including proliferation and differentiation (18). The neuritogenic and survival promoting actions of S100B are associated with intracellular calcium modulation, resulting in mobilization of calcium stores by S100B stimulation of phosphoinositide hydrolysis by phospholipase C (19). Calcium elevation is associated with a number of development-related events (19). S100B is overexpressed (i.e. at greater levels than 1.5 times that expected from gene loading) in the brains of Down syndrome fetuses (20), and this overexpression continues throughout life (21). Transgenic mice overexpressing human S100B show increased density of hippocampal dendrites at young ages, followed by decreased density of these dendrites as the animals age (22). This, of course, recalls the arrested dendritic development in young Down syndrome patients that follows initially normal dendritic morphology in human fetuses with Down syndrome (3, 4). Young S100B transgenic mice also show impaired learning (22) and altered social behaviors (23) that suggest further analogies to Down syndrome. In addition to a possible role in the neurodevelopmental abnormalities of Down syndrome, overexpression of S100B has been implicated in the midlife appearance of Alzheimer disease in Down syndrome, as described below (24).

DOWN SYNDROME AND ALZHEIMER DISEASE: THE ROLE OF βAPP

Alzheimer disease is virtually inevitable in Down syndrome (6), making Down syndrome a “natural model” for studying the origins and pathogenesis of Alzheimer disease. Mapping of the gene that encodes the precursor protein of Aβ to chromosome 21 (25, 26) and finding mutations there that cause Alzheimer disease (27, 28) immediately suggested that the triplication of this particular chromosome 21 gene is important in the genesis of Alzheimer pathology in Down syndrome. This idea has been confirmed by the exceptional case of a 78-year-old woman with partial trisomy 21 and Down syndrome, but without clinical or pathological evidence of Alzheimer disease (11). This woman had only 2 copies of the gene for the Alzheimer amyloid precursor protein βAPP, providing confirmation that triplication of this gene is not necessary for the development of Down syndrome, but is necessary for the development of Alzheimer disease in Down syndrome patients. Necessary, but not sufficient, as suggested by studies tracing the earliest βAPP-associated abnormalities in Down syndrome.

Actual deposition of fibrillar Aβ in brains of Down syndrome patients does not occur until adolescence (29), but more subtle Aβ changes have been described in younger patients. These consist of activation of endocytic uptake and recycling of Aβ, with consequent endosomal enlargement. These changes are found in early cases of sporadic Alzheimer disease and in young patients with Alzheimer disease-causing mutations in the βAPP gene, but not in young patients with Alzheimer disease-causing mutations in presenilin (30). Similar changes are found in Down syndrome fetuses by 28 weeks of gestation (31), suggesting that the initial stages of Alzheimer disease can be traced back to early development, a theme that we will return to in examining the role of S100B (see below). These early endosomal abnormalities are also seen in an animal model of Down syndrome, the Ts65 mice, which is partially trisomic for mouse chromosome 16 (the mouse homolog of human chromosome 21). These early abnormalities vanish when 1 copy of the βAPP gene is deleted from these same trisomic mice, confirming the association between βAPP triplication and Alzheimer pathology. However, triplication of the βAPP gene alone does not reproduce the phenotype, suggesting that additional factors are necessary for the generation of Alzheimer pathology in partial trisomy 16 mice, and in trisomy 21 humans (32). In mice transgenic for mutated human βAPP, S100B overexpression precedes the appearance of Aβ plaques (33).

DOWN SYNDROME AND ALZHEIMER DISEASE: THE ROLE OF S100B

S100B possesses a number of trophic actions on neurons, neurites, and glia that are important in the pathogenesis of Alzheimer disease (24). In Down syndrome, S100B expression, but not βAPP, is elevated in brain even at fetal stages (20, 21), making S100B overexpression one of the earliest Alzheimer-associated brain alterations in this disorder. This S100B overexpression increases progressively throughout life in Down syndrome (21) and, in post-adolescent Down syndrome patients, the degree of this S100B overexpression correlates with the degree of Aβ deposition in brain (34), suggesting a continuing role for S100B overexpression in either maintenance of or promotion of progression of Alzheimer neuropathological changes. The early increases in S100B expression are not accompanied by astrocytic expression of glial fibrillary acidic protein (21), a structural protein that is commonly used as a marker of gliosis. This finding is similar to observations made in brain tissue from patients with chronic intractable epilepsy (35), suggesting an uncoupling of S100B expression from gliosis.

S100B is overexpressed by activated astrocytes associated with the Aβ plaques of Alzheimer disease (20, 36, 37), and tissue levels of homodimeric, biologically active S100B are markedly increased in Alzheimer brain (38). Moreover, this increase in S100B tissue levels correlates with degree of neuropathological involvement in specific brain regions (39) as well as with the density of neuritic plaques (36). This, together with the importance of S100B as a neurite growth-promoting cytokine (40, 41), suggests a role for S100B in the generation of dystrophic neurites in Aβ plaques. Such a role is supported by our demonstrated correlation between the abundance of these dystrophic neurites in individual plaques and the number of adjacent activated astrocytes, overexpressing S100B, in Alzheimer brain (37). Moreover, S100B induction of βAPP expression in neurons is associated with growth of neurites, suggesting that growth of these processes may be dependent on increased expression of βAPP (42). In addition to these trophic effects, S100B has potentially neurotoxic actions. S100B induces astrocytic expression of nitric oxide synthase with release of nitric oxide (43), and chronic overexpression of S100B could result in neuron cell death due to S100B mobilization of calcium stores in neurons (19). S100B-mediated increases in free calcium levels are toxic in vitro (44), suggesting that S100B overexpression in Down syndrome, and in Alzheimer disease, may contribute to neuronal injury and loss. Such progressive neuronal injury in Alzheimer brain accompanies plaque progression and locally elevated levels of S100B (45). Progressive neuronal injury also accompanies neurofibrillary tangle formation in neurons (46) and responsivity to glutamate (47), and this progression is also accompanied by increasing numbers of adjacent activated astrocytes, overexpressing S100B (48).

DOWN SYNDROME AND ALZHEIMER DISEASE: THE ROLE OF INTERLEUKIN-1

Interleukin-1 is not encoded by chromosome 21-based genes, but overexpression of IL-l occurs in brains of Down syndrome patients and—as is the case for S100B—this overexpression is demonstrable even during fetal development (20). This IL-1 overexpression likely represents secondary upregulation induced by some chromosome 21-based gene(s). Two such genes have been implicated in IL-1 overexpression: βAPP has been shown to activate microglia and induce excessive expression of IL-1 (49), and S100B has been shown to induce expression of IL-1 in astrocytes (50). S100B is also a potent regulator of IL-1 expression in neurons as well as microglia and astrocytes; this induction of IL-1 is differentially regulated in neurons and glia via Sp1 in neurons and principally via NFκB in microglia and astrocytes (51). This cell-specific differential regulation of IL-1 by S100B may contribute to switching of S100B effects between neurotrophic and neurotoxic. Conversely, IL-1 induces excessive expression of both S100B and βAPP. This may explain the amplified expression (beyond 1.5 times that attributable to gene loading alone) that is seen for both βAPP (25) and S100B (20) in Down syndrome fetuses and, indeed, throughout life in Down syndrome brain (21).

As it is increasingly clear that IL-1 is a key orchestrating cytokine in the pathogenesis and progression of the neuropathological changes in Alzheimer disease, it follows that the lifelong overexpression of IL-1 in Down syndrome is a significant contributor to the generation and progression of Alzheimer pathological changes in Down syndrome (52). Like S100B, IL-1 has neurotrophic effects as well as neurotoxic effects. For instance, IL-1 induces excessive expression of βAPP (53, 54), an acute-phase response that may be beneficial in short-term injury response situations. However, chronic IL-1 overexpression, together with consequent chronic neuronal βAPP overexpression (54), translation (55), and processing (56) of neuronal βAPP, is likely to have detrimental effects. Chronic release of secreted βAPP fragments, for instance, would precipitate further microglial activation and over-expression of IL-1 (49), and thus could favor Aβ deposition.

Chronic overexpression of IL-1 may also explain other detrimental outcomes, both in Alzheimer disease and in Down syndrome. For instance, IL-1 promotes both neuronal acetylcholinesterase expression and activity (57), suggesting that the overexpression of IL-1 in Down syndrome contributes to the cholinergic deficits associated with Alzheimer disease. IL-1 at high concentrations is directly toxic to cortical neurons (58), an effect that may also contribute to plaque-associated neurotoxicity in Alzheimer disease (45). The possibility that IL-1 plays a role in neurofibrillary tangle pathology in Alzheimer disease is supported by a number of findings in Alzheimer brain as well as in cell culture and in experimental animal studies: i) in Alzheimer disease there are activated microglia overexpressing IL-1 closely associated with neurofibrillary tangle-bearing neurons, and the numbers of these associated microglia increase steadily with progressive stages of neurofibrillary tangle formation (48); ii) intrathecal IL-1 levels in Alzheimer disease have been shown to correlate with those of the tangle-associated protein tau (58); iii) IL-1 has been shown to promote expression of neurofilament proteins and of tau in vitro and in vivo (59, 60), further implicating IL-1 in tangle formation and progression; and iv) more concrete evidence is provided by our demonstration that IL-1 induces increases in neuronal levels of phosphorylated tau, which is dependent on simultaneous increases in neuronal levels of phosphorylated (activated) MAPK-p38 (61, 62). These potentially deleterious increases in phosphorylation of tau occur concurrently with IL-1-induced decreases in synaptophysin expression in these neurons, suggesting that early and sustained overexpression of IL-1 in Down syndrome precipitates multiple events that cascade into numerous degenerative consequences in susceptible neurons.

Taken together, these data suggest that the lifelong overexpression of IL-1 in Down syndrome brain promotes a complex cascade of IL-1-mediated glial and neuronal effects that collectively promote the Alzheimer-type neuropathological changes and cognitive decline of middle-aged Down syndrome patients: neuropathologically through IL-1 regulation of βAPP overproduction and processing, contributing to Aβ plaque generation, and through IL-1 regulation of tau phosphorylation, contributing to neurofibrillary pathology; and cognitively through IL-1 dysregulation of cholinergic processes and through downregulation of synaptic proteins production.

In addition to its effects on neurons, IL-1 also manifests important autocrine gliotrophic actions. These include promotion of microglial activation and further promotion of IL-1 expression (63, 64). IL-1 has numerous trophic effects on astrocytes, including increased survival and differentiation (18) as well as potential increases in astrocyte activation and promotion of S100B expression (65). This latter effect provides a potential feedback loop between microglial IL-1 overexpression and astrocytic S100B overexpression, and, as noted above, may explain in part the greater than 1.5-fold increase in S100B expression that is found in brain of Down syndrome patients. Certain polymorphisms in the genes for the 2 IL-1 isoforms have been associated both with IL-1 overexpression and with increased risk for sporadic Alzheimer disease (66–70). Whether these same polymorphisms modulate disease onset or severity for Alzheimer disease in Down syndrome patients is not known.

The established functions and gene polymorphisms of IL-1 suggest that this cytokine may play a key role in the pathogenesis of Alzheimer disease, and there is evidence for this from studies of Alzheimer disease itself (71). IL-1 is markedly overexpressed in Alzheimer brain, as evidenced by increased tissue levels of IL-1 and by increased numbers of IL-1-immunoreactive microglia (20). There are also important morphological patterns to this overexpression in Alzheimer brain. Microglia overexpressing IL-1 show close associations with Aβ plaques, and the distribution of these activated microglia across brain regions reflects the distribution of Aβ plaque pathology (72). Moreover, the pattern of Aβ distribution across cerebral cortical layers in Alzheimer brain mirrors the distribution of microglia within cerebral cortical layers in brains of normal controls (73), suggesting that the normal distribution of microglia within the brain determines in part the distribution of Aβ plaques in Alzheimer disease.

Microglia overexpressing IL-1 are present at all stages of Aβ plaque formation and progression in Alzheimer disease (74). Such microglia are also found associated with Aβ plaques in transgenic animal models of Alzheimer disease (75, 76). Even early “pre-amyloid” or “diffuse” deposits of A peptides, which are not associated with discernible evidence of neuritic or neuronal damage, contain microglia overexpressing IL-1 in Alzheimer brain (72). Significantly, similar diffuse “pre-amyloid” deposits of Aβ peptide are sometimes found in brains of elderly individuals that are not demented and that do not show other pathological features of Alzheimer disease. These apparently benign deposits, in contrast to those found in Alzheimer disease, do not have associated activated microglia (77), suggesting that the presence of these microglia and the concomitant overexpression of IL-1 are important in the evolution of diffuse amyloid deposits to neuritic plaque forms, and consequently in pathogenesis of Alzheimer neuropathology and of cognitive decline.

We have conceptualized the myriad biological effects of IL-1 and of the downstream molecular consequences of IL-1 overexpression in the brain in the form of the cytokine cycle (78). This complex of self-propagating IL-1-driven cascades forms the basis for a glia-derived neuroinftammatory process that may drive both plaque progression and tangle progression in Alzheimer disease, and which underlies the progressive neurotoxicity and neuronal loss associated with disease progression. The early and continuing overexpression of IL-1 in brains of individuals with Down syndrome thus likely reflects and drives the lifelong activation of these innate neuroinftammatory processes in these patients.

In Down syndrome, the cytokine cycle may be driven by βAPP and S100B gene triplication and consequent increased production of their gene products, which, in turn, induce excessive IL-1 expression and microglial activation. The IL-1 generated cascades outlined here could explain the prominent Alzheimer-type neuropathological changes associated with Down syndrome: the genesis and development of neuritic Aβ plaques (driven by the self-enhancing cycle of βAPP, S100B, and IL-1 overexpression); the accumulation of dramatic neurofibrillary tangles (driven by IL-1-promotion of excessive production of phosphorylated tau and phosphorylation-related paired helical filament formation); and neuritic pruning with concomitant synaptic loss (driven by IL-1 downregulation of synaptophysin expression). The latter two effects appear to be linked through IL-1-induced activation of MAPK-p38.

In contrast to our increasing understanding of the neurodegenerative events of Alzheimer-type changes in middle-aged Down syndrome patients, the pathogenesis of the myriad developmental anomalies of Down syndrome—mental retardation, somatic structural differences, and personality traits—is yet to be defined. With a clearer understanding of Alzheimer-type changes, however, we would predict that these developmental effects involve complex interactions between the overexpression of chromosome 21 gene products and non-chromosome-21 gene products.