Alzheimer disease is characterized by three dramatic abnormalities of the brain, all of which Alzheimer described. First, the brain is atrophied, with narrowed gyri, widened sulci, reduced brain weight, and enlarged ventricles (Figure 59–9). These changes, seen in mild form in cognitively intact elderly people who die from other causes, are severe in advanced AD. Moreover, neuron death is widespread in AD, whereas in normal aged brains it is minimal. Thus AD is a neurodegenerative disease.
Overt pathological changes in the brain of individuals with Alzheimer disease.
When compared to age-matched normal brains, the brain of an Alzheimer patient displays marked shrinkage and ventricular enlargement. (Whole brain photos from P. Anderson, University of Alabama at Birmingham, Department of Pathology; brain slice photos from A. C. McKee; all reproduced with permission.)
Second, sections of brains of AD patients obtained at autopsy reveal extracellular plaques of dense material called amyloid, large aggregates of fibrillar peptides arranged as sheets (Figure 59–10). Amyloid can be detected when stained with dyes such as Congo red, and is refractive when viewed in polarized light or when stained with thioflavin and viewed with fluorescence optics. The extracellular deposits of amyloid are surrounded by swollen axons and dendrites. These neuronal processes in turn are associated with processes of astrocytes and microglia (inflammatory cells). Amyloid plaques also occur in the walls of cerebral blood vessels in the Alzheimer brain.
Plaques and tangles in the Alzheimer brain.
A section of cerebral cortex from the brain of an individual with severe Alzheimer disease shows typical plaques and neuro-fibrillary tangles. (Images from James Goldman, reproduced with permission.)
Left: The diagram shows a neuron containing neurofibrillary tangles, composed of bundles of paired-helical filaments in the cell body and more paired helical filaments in the axon. Amyloid plaques are shown in the neuropil, one of them surrounding a dendrite, which displays an altered, swollen shape. Tangles are composed of abnormal polymers of hyperphosphorylated tau protein, and amyloid plaques are extracellular deposits of polymers of the amyloid beta (A4) peptide.
Middle: A section of neocortex from a patient with Alzheimer disease has been treated with a silver stain. The tissue shows neuronal cell bodies containing neurofibrillary tangles and neuropil containing amyloid plaques.
Right: A higher magnification of the cortex shows neurofibrillary tangles in neuronal cell bodies and, in contrast, a healthy neuron without a tangle. Note the many thin silver-positive cell processes in the neuropil; these are neuronal processes that contain paired helical filaments.
Third, neurons that are affected but still alive have cytoskeletal abnormalities, the most dramatic of which is the accumulation of neurofibrillary tangles (Figure 59–10). These tangles are filamentous inclusions in the cell bodies and proximal dendrites that contain paired helical filaments and 15 nm straight filaments. Other cytoskeletal abnormalities occur in axons and terminals (dystrophic neurites) and dendrites (neuropil threads). Both types of lesions include intracellular paired helical filaments, suggesting that these fibrillar inclusions result from common mechanisms.
In AD these alterations do not occur uniformly throughout the brain, but rather affect specific regions. The entorhinal area, the hippocampus, the neocortex, and the nucleus basalis are especially vulnerable (Figure 59–11). Alterations in the entorhinal cortex and hippocampus are likely the structural underpinnings of problems with declarative memory that are the first symptoms of AD. These alterations contrast with those in frontostriatal circuits that correlate with age-related cognitive decline in normal subjects. Abnormalities in the basal forebrain cholinergic systems may contribute to cognitive difficulties and attention deficits that appear later in the progression of the disease. These anatomical differences, along with widespread neuronal death, argue against the idea, once prevalent, that AD is an extreme form of the normal aging processes.
Neurofibrillary tangles and senile plaques are concentrated in different regions of the Alzheimer brain.
Lateral and medial views of the cerebral hemispheres. (Adapted, with permission, from Arnold et al. 1991.)
Amyloid Plaques Contain Toxic Peptides That Contribute to Alzheimer Pathology
To characterize amyloid plaques, George Glenner, Konrad Beyreuther, and their colleagues isolated the plaques by centrifugation, based on their low solubility, and determined some of their components. The principal constituent turned out to be a group of small peptides that together were named Aβ.
Two main forms of the peptide were found. The predominant peptide is 40 amino acids in length and the minor one 42 amino acids (the original 40 residues plus two additional amino acids at the carboxy terminal end). Biochemical studies showed that the Aβ42 peptide nucleates more rapidly than Aβ40 into amyloid fibrils. In individuals with AD, amyloid deposition begins with Aβ42, while Aβ40 accumulates later. Moreover, when applied to neurons in culture, the Aβ42 peptide is more toxic than Aβ40. These results implicate Aβ42 as a key component of amyloid plaques.
In general, short peptides are formed by cleavage from a precursor protein, so researchers set out to isolate the protein from which Aβ was derived. The precursor was soon found and molecularly cloned, and named amyloid precursor protein or APP (Figure 59–12). APP is a large transmembrane glycoprotein that is present in the dendrites, cell bodies, and axons of many types of neurons as well as a variety of nonneuronal cells. Despite intensive study, the normal functions of APP in the brain remain poorly understood.
Processing of the amyloid precursor protein.
The amyloid precursor protein (APP) is a transmembrane protein of uncertain function. Through proteolytic cleavage it generates many distinct proteolytic fragments. Proteolytic cleavage by γ- and β-secretases generate an intracellular fragment, an Aβ peptide, which can be 40 or 42 residues in length, and an amino-terminal fragment (N-APP) that may have distinct effects.
Once APP was isolated it became possible to ask how it is processed to form Aβ peptides. The answer has turned out to be complex. Three proteolytic activities—α-, β-, and γ-secretases—cut APP into pieces. The β- and γ-secretases cleave APP to generate a soluble extracellular fragment that is released into the interstitial fluid, the Aβ peptides that include part of the transmembrane segment, and a cytosolic or intracellular fragment (Figure 59–12). The cleavage by γ-secretase is unusual in that it occurs in a membrane-spanning portion of APP, a region long thought to be immune from hydrolysis because it is surrounded by lipids rather than water. In nonneuronal cells α-secretase cleaves APP in the middle of the Aβ sequence. This cleavage prevents the formation of Aβ peptides and helps explain why Aβ peptides are largely confined to the nervous system even though APP is present in neuronal and nonneuronal cells alike.
The enzymes that account for α-, β-, and γ-secretases have been isolated and characterized. α-Secretase is a member of a large family of extracellular proteases called ADAM (A Disintegrin and Metalloproteinase) that are responsible for degrading many components of the extracellular matrix. β-secretase, called β-site APP cleaving enzyme 1 or BACE1, is a transmembrane protein in central neurons and concentrated in synaptic areas. Brain cells derived from mutant mice lacking BACE1 do not produce Aβ peptides, proving that BACE1 is indeed the neuronal β-secretase. γ-Secretase, the most complicated of the three, and the most recently isolated, is actually a multiprotein complex that cleaves several different transmembrane proteins. As expected, given its peculiar ability to act within the membrane, γ-secretase itself includes membrane proteins. Two are called presenilin-1 and presenilin-2, reflecting their association with AD. Other components of the complex are nicastrin, Aph-1, and Pen-2, also transmembrane proteins.
Although the biochemical properties of Aβ and APP appeared interesting, the critical question remained: Are they causally related to the debilitating symptoms of AD? One might imagine that the disease is caused by Aβ accumulation, but Aβ might also form as a result of another pathological process or even be an innocuous correlate. Genetic evidence in humans and experimental animals has been critical in demonstrating that APP plays a central role in AD.
The first clue came from the observation that the APP gene lies on chromosome 21. Chromosome 21 is present in three copies (rather than the normal two) in people with Down syndrome. For this reason Down syndrome is also referred to as trisomy 21. Interestingly, it had long been known that most people with Down syndrome who live to the age of 40 develop AD. This association is consistent with the idea that excessive APP predisposes to AD.
More direct genetic evidence came from the analysis of patients with familial AD. Only a small number of cases of AD are familial, usually those in which onset occurs early, younger than age 60 years, and most of these are inherited as simple dominant mutations. As new methods of molecular cloning became available in the late 1980s, several groups began using them to identify the genes mutated in familial AD. Remarkably, the first three genes identified were those encoding APP, presenilin-1, and presenilin-2 (Figure 59–13). By now many different mutations have been found in all three genes, and the majority of them influence cleavage of APP, increasing the production of Aβ peptides or the proportion of the more toxic Aβ42 species.
Environmental and genetic factors in Alzheimer disease.
A. Environmental and genetic factors. (ApoE, apolipoprotein E; APP, amyloid precursor protein; PS1, presenilin-1; PS2, presenilin-2.)
B. Specific genes involved in early-onset Alzheimer disease (AD).
C. Presenilin-1 is associated with APP proteins within the plasma membrane.
Some APP mutations are amino acid substitutions flanking or within the Aβ region. Cells that express these mutant sequences secrete several-fold more Aβ peptide than cells expressing wild-type APP. Another APP mutation influences γ-secretase to selectively generate Aβ42 rather than Aβ40. Likewise, in most presenilin mutants the mutant γ-secretase has higher than normal activity or generates peptides with an increased ratio of Aβ42 to Aβ40. Thus mutants in the APP or presenilin genes do not lead to loss of the proteins but rather to increased production of Aβ42.
These human studies offer compelling evidence that cleavage of APP plays a key causative role in at least some cases of familial, early-onset AD, and point to a role for Aβ42. Because Aβ42-rich amyloid plaques are a cardinal feature of the far larger group of patients with sporadic, late-onset AD, it is likely that APP cleavage is also involved in generating symptoms in this larger group.
Finally, genetic studies of mice have strengthened the case that APP cleavage contributes to AD. Transgenic mice that express relatively high levels of wild-type or mutant APP exhibit structural, physiological, and behavioral abnormalities associated with AD. Transgenic expression of mutant APP forms identical to those found in familial AD leads to appearance of amyloid plaques in the hippocampus and cortex, swollen neurites in proximity to Aβ deposits, decreased density of synaptic terminals in the forebrain, decrements in synaptic transmission, and degeneration of neurons. Moreover, these transgenic mice are deficient in tasks assessing spatial and episodic-like memory. Alterations are more severe in transgenic mice that express altered forms of both APP and presenilin-1. These lines of mice are invaluable tools for addressing mechanistic issues about the pathogenesis of AD and for testing potential therapies. Moreover, AD-like alterations in transgenic mice appear in a year or less rather than over decades, as in humans.
Given the strong evidence that APP cleavage is involved in the pathogenesis of AD, the next question is how the accumulation of cleavage products contributes to the symptoms. There are three sets of cleavage products: the secreted extracellular region (ectodomain), the Aβ peptide, and the cytoplasmic fragment. Greatest attention has been paid to the Aβ peptides, which were the first to be discovered. One view holds that Aβ in plaques, especially Aβ42, poisons neurons in the vicinity, leading to synaptic dysfunction, degeneration of axon terminals, and eventually death of neurons.
An alternative explanation is that aggregation of soluble forms of Aβ into plaques is the body's incompletely successful attempt to sequester toxic protein fragments. The Aβ peptides can bind to synaptic proteins and affect trafficking of postsynaptic neurotransmitter receptors, including glutamate receptors. Regulated trafficking of these receptors may be essential for forms of synaptic plasticity such as long-term depression and potentiation. As a result, memory defects in AD could involve interference by Aβ in plasticity. In turn, interference in synaptic function could lead to withdrawal and loss of synapses.
Involvement of Aβ peptides in AD does not mean that the other two cleavage products of APP, the cytoplasmic and extracellular fragments (Figure 59–12), have no role in AD. The cytoplasmic fragment can form a complex with other proteins, including Fe65, translocate to the nucleus, and influence transcription in ways that could be deleterious. Although there is little evidence that this mechanism contributes to AD, it is well established that Notch, a critical regulator of neurogenesis (see Chapter 53), is activated by γ-secretase cleavage and that its cleaved cytoplasmic fragment is transported to the nucleus, where it acts as a transcription factor. Likewise, the secreted extracellular fragment of APP appears to have toxic effects on nearby neurons. Thus it is possible that APP is a potent instigator of AD because the fragments generated from it by proteolytic cleavage damage neurons in different ways.
Neurofibrillary Tangles Contain Microtubule-Associated Proteins
Most research on the molecular and cellular basis of AD has focused on amyloid plaques but the neurofibrillary tangles have also been implicated in disease progression (Figure 59–10). Molecular analysis revealed that these abnormal inclusions in cell bodies and proximal dendrites contain aggregates of hyperphosphorylated isoforms of tau, a microtubule-binding protein that is normally soluble (Figure 59–14). Tau plays a key role in intracellular transport, particularly in axons, by binding to and stabilizing microtubules. Impairments in axonal transport compromise synaptic stability, trophic support, and other interactions. Eventually, affected nerve cells die and the neurofibrillary tangles remain in the extracellular space as tombstones of the cells destroyed by disease.
Neurofibrillary tangles contain mutant tau proteins.
A. In healthy neurons tau protein associates with normal microtubules but not paired helical filaments, and contributes to the structural integrity of the neuron.
B. In a diseased neuron the tau protein becomes hyperphosphorylated. As a consequence, phosphorylated tau loses its association with normal microtubules, which begin to disassemble, and instead associates with paired helical filaments, which become sequestered in neurofibrillary tangles (NFT).
Although tangles are a defining feature of AD, it remains unclear what role they, and the hyperphosphorylated tau of which they are made, play in the pathogenesis of the disease. Whereas mutations of APP and presenilin genes can lead to AD, no mutations of the tau gene have been found in familial AD. This difference leads some to view tangles as a consequence or correlate, but not a cause, of AD symptoms.
Other observations suggest a more causal relationship. First, filamentous deposits of hyperphosphorylated tau are seen in a variety of neurodegenerative disorders. Second, mutations in the tau gene have been found to underlie another form of inherited dementia, frontotemporal dementia with Parkinson disease type 17 (FTPD17), which shares some features with AD but lacks plaques. Third, symptoms of AD correlate better with the number and distribution of tangles than that of plaques seen in autopsy material. For example, tangles are usually first evident in neurons of the entorhinal cortex, the likely site of early memory disturbance, before plaques appear in this area.
For many years controversy has raged between those who believe that Aβ is the main causal agent of AD and those who believe that tau-rich tangles play a major role. These advocates have been called "Baptists" and "Taoists," respectively. It now seems most likely that both Aβ and tau are involved, and that pathology results from their combined effects. For example, transgenic mice that express both mutant APP and mutant tau develop more severe AD-like behavioral deficits than mice overexpressing either alone. Moreover, there appears to be interplay between plaques and tangles. Thus injection of Aβ42 into specific brain regions of transgenic mice that express a mutant tau protein increases the number of tangles in nearby neurons, and a manipulation that reduces the number and size of plaques leads to a decrease in levels of hyperphosphorylated tau. Thus, even if tangles are not sufficient to "cause" AD, they are likely to play a role in symptoms and disease progression.
Risk Factors for Alzheimer Disease Have Been Identified
A few individuals develop AD because they bear mutant alleles of the APP or presenilin genes, but in most cases no genetic or environmental causes are obvious. Can we, then, predict who will get AD?
The major risk factor is age. The disease is present in a vanishingly small fraction of people younger than age 60 (these being mostly familial cases), 1–3% of those between ages 60 and 70, 3–12% of those between ages 70 and 80, and 25–35% of those older than age 85. Thus one safe prediction is that elderly people are prime candidates for AD. However, this statistical association is of little therapeutic use because modern medicine can do nothing to slow the passage of time. There has therefore been intense interest in other factors that affect the incidence of AD.
The most significant genetic risk factor discovered to date in sporadic late-onset AD is an allele of the gene ApoE. The ApoE protein is the major carrier of cholesterol and other lipids in the blood. The gene is expressed as three alleles, ApoE2, ApoE3, and ApoE4, which differ from each other at only a few amino acids (Figure 59–15). People with the ApoE4 allele are at risk for AD. This allele is present in only a few percent of the general population but in 40–50% of those with AD. Put another way, carrying one ApoE4 allele increases by about fourfold one's chance of developing AD, compared to people who carry only ApoE2 or ApoE3 alleles. The mechanism by which ApoE4 predisposes one to AD is not known. Moreover, ApoE4 is a risk factor for several neurological diseases, including Parkinson disease and multiple sclerosis, so it may act in ways that do not involve APP or Aβ directly.
Polymorphisms in the ApoE gene are a prominent risk factor for Alzheimer disease.
A. Polymorphisms in the ApoE gene involve changes in amino acid sequence at two critical residues (112 and 158).
B. Changes in amino acid sequence in the three ApoE alleles.
C. Prevalence of ApoE genotypes in the general population.
D. ApoE alleles are risk factors for Alzheimer disease. Inheritance of one ApoE4 allele increases by about fourfold the probability of progressing to Alzheimer disease. Inheritance of the ApoE2 allele reduces the risk.