Amyloid, Tau, and Cell Death in Alzheimer's Disease

Introduction

An extensive search is on for the factors responsible for the neuronal damage, cell death, and brain atrophy underlying the development of minimal cognitive impairment and full-blown Alzheimer's disease (AD).

Among such factors, mitochondrial abnormalities, disturbances in amyloid precursor protein (APP) production, function and cleavage, the accumulation of proamyloidogenic peptides, and alterations in tau activity are at the forefront of many researchers' efforts.[1,2] (The association of apolipoprotein E isoforms with AD and the progress made in understanding its significance are discussed in a separate article).

Prominent researchers convened at the 9th International Conference on Alzheimer's Disease and Related Disorders, recently held in Philadelphia, Pennsylvania, to present the latest results of their research in these fields. Neurosciences and cell biology, with the help of suitable disease models, hold the key to unravel the origin of AD and open new therapeutic avenues.

Biological Functions of APP

Accumulation of amyloid beta (Abeta) peptides in amyloid plaques is the hallmark of AD. Cleaved by secretases from the cell-bound APP, soluble Abeta peptides aggregate in oligomers to form fibrillar structures.[1,2] Initially described in 1987, the gene encoding for APP has been known for about 18 years.

Although indicted in the ethiopathogenesis of AD, how does APP contribute to normal neuronal homeostasis and function? A clear knowledge of its characteristics is important to understand its role in AD and the outcomes of newly proposed strategies to interfere with APP cleavage and deposition. How would such blocking steps affect the expression and function of APP? Would all consequences of manipulation be to the advantage of targeted neurons?

As illustrated by Dr. Edward Koo,[3] of the University of California, La Jolla, more and more physiologic functions are being ascribed to APP. It can interact with multiple components of the nervous system and thus mediate trophic functions, cell adhesion, neurite outgrowth, neuronal migration, synaptic functions, and the induction of apoptosis.

Overexpression of APP was found in subjects with Down's syndrome, and engineered overexpression of the C-terminal fragment of APP in mice was associated with neurodegeneration and abnormal axonal transport. APP homologues also have been recently identified in other species, such as mouse and Drosophila melanogaster. Abnormal expression of APP was associated with lethality in Caenorhabditis elegans and reduced axonal transport in D melanogaster.[4,5]

Molecular mapping of the AD-type dementia seen in subjects with Down's syndrome revealed a duplication of the distal arm of chromosome 21 on the proximal arm, which, however, did not include the APP gene. In segmental trisomy, on the other hand, endosomal abnormalities were related to the APP gene dosage.

APP actively traffics within neurons, contributing to transcription in the nucleus and apoptosis in the cytoplasm. Axonal transport of APP has been demonstrated in specific structures, such as the optic nerve. Of note, a rapid accumulation of APP has been observed in the axons of patients affected by the shaken baby syndrome.[3]

Such anterograde transport of APP is powered by the kinesin-based motor, either by direct interaction with the kinesin light chain or through an adaptor molecule. At fluorescence analysis, APP molecules appear in the axolemma as stable, segmental patches colocalizing with adhesion molecules. Double knockout mice for APP and APLP showed neuromuscular junctions that were abnormal in structure and function, with a reduction in miniature potentials. APP, internalized at the terminal with other synaptic markers, can also traffic back to the neuron's body.

APP binds to quite a number of proteins, such as Fe65, mDAP, JIP18, and X11. After cleavage at the epsilon site, the AICD fragment is quite unstable, but it can be stabilized by interaction with Fe65. APP, Fe65, and Tip60 form a transcriptionally active complex that participates in gene transcription, thus making of APP a gene regulator. The interaction of APP with AICD is required to render Fe65 signaling competent, and AICD is thought to function as a facilitator for Fe65. Although confirmed with different reporter genes, these results have been reported only from in vitro experiments. Evidence of physiologic activity in vivo is still to come.[3]

Proteolytic enzymes of the caspase family (cysteine aspartyl proteases) can mediate cleavage of APP with the release of a toxic C-terminal fragment called C31.[6-8] Mutation of the identified caspase site prevented cleavage of APP and restored transfected cells to control levels of viability.

In dynamic terms, APP is thought to self-dimerize and recruit suitable adapters, with the activation of caspase 3, release of C31, and induction of apoptosis. Results consistent with this hypothesis were obtained in transgenic mice expressing an AD-associated Swedish variant of APP (mtAPP) with a wild type or a mutated caspase site. Synaptic transmission was reduced in mice expressing "caspase-sensitive" mtAPP, but it was restored in those animals expressing the caspase-non-cleavable form of mtAPP.[3]

Thus, Dr. Koo concluded that we can talk of the ying and yang of APP. Too little APP is bad, but also too much APP is bad. Necessary functions played by APP, in fact, include neuronal trophism, cell adhesion, neuronal migration, neurite outgrowth, synapse formation and plasticity, and cell-cell signaling. Too much APP, on the other hand, may lead to APP oligomerization, caspase activation, and neuronal apoptosis.

Tau and Tauopathies

In addition to APP and the amyloidogenic Abeta peptides, neurofibrillary tangles, formed by filamentous aggregates of the microtubule-associated protein tau, are at the epicenter of AD lesions in the brains of affected patients.

How do the neurofibrillary tangles occur? Are they related to the amyloid deposits and the cellular damage induced by APP and Abeta peptides? A better insight into how tau functions in normal cells and why it undergoes pathologic aggregation in AD lesions may help in our search for specific inhibitors, as noted by Dr. Eva-Maria Mandelkow,[9] of the Max Planck Institute, Hamburg, Germany.

The microtubule-associated protein tau has a number of physiologic functions in normal neurons, stemming from its ability to stabilize microtubules during axonal transport and to help in neurite growth.[10] Tau is regulated by phosphorylation, and once phosphorylated it tends to aggregate in filaments, thus inducing breaks in the microtubular tracks and neuronal death.[11] Although critically involved in the formation of paired helical filaments (PHF), tau can be toxic to cells also before these catastrophic events.

Expressed in 6 different isoforms and containing 441 amino acid residues, tau is a basic, highly hydrophilic, highly soluble protein that is natively unfolded. In other words, it has no structure, and harsh, denaturing treatments do not affect its function. Compared with the kinesin motors, tau molecules occupy a lot of space on the microtubules.[10]

Owing to its unfolded nature, tau can be phosphorylated very easily by many enzymes, including MAPK and GSK3beta. Following phosphorylation, tau detaches from the microtubules, and as a consequence of tau detachment, the microtubules fall apart.

Tau presents abnormal features in AD cells. It is, in fact, hyperphosphorylated, dissociated from the microtubules, and aggregated in PHFs. Specific sites appear to be preferentially phosphorylated early on in patients with AD, as, for example, the KXGS motifs targeted by the enzyme MARK. The MARK/PAR-1 kinase is a large serine-threonine protein kinase important for maintaining a polar network of microtubules and, thus, cell polarity in neurons.[9]

Although tau mutants in the KXGS site inhibited neurite outgrowth in the neuroblastoma Neuro2a cells, transfection of MARK induced the spontaneous growth of neurites. Consistently, reduction in MARK expression by siRNA inhibition prevented phophorylation of tau and neurite outgrowth.

Endogenous MARK colocalized with phosphorylated tau in neurons, which in turn colocalized with active growth cones in hippocampal neurons. In this model, tau is associated with microtubules, becomes phosphorylated, and then switches from the microtubule to the actin network waiting for further growth -- with the overall effect of establishing cell polarity.[9]

Overexpression of tau was found to inhibit the kinesin-dependent transport of mitochondria in Neuro2a cells. The mitochondria were trapped in the cell bodies and unable to reach the synapses. Up to 45% of the anterograde mitochondrial transport mediated by kinesin motors was inhibited by overexpression of tau in retinal ganglion axons. Tau, in a way, achieved this effect by "clogging" the microtubular tracks and preventing attachment of the smaller kinesin molecules. Cotransfection of the MARK enzyme induced tau phosphorylation and relief of the inhibition mediated by tau on axonal traffic.[12]

Of note, neurites of Neuro2a cells overexpressing tau were highly vulnerable to oxidative stress. Further analysis of tau function in a transgenic model, generated with an inducible form of tau, showed degeneration of cortical neurons upon the induction of tau expression. It still remains to be determined whether tau mediates its toxic effects through aggregation, and whether compounds able to inhibit tau aggregation would prevent such tau-associated neuronal toxicity.[9]

Thus, as seen with APP, too little tau is bad and too much tau is bad. A lack of tau leads to impairments of the microtubule network with deficits in axonal growth and transport. An excess of tau promotes self-aggregation and the formation of PHFs.

Mitochondria and Oxidative Damage

Mitochondrial alterations and oxidative damage may be involved in the neurodegeneration associated with AD. As illustrated by Dr. Flint Beal,[13] of Cornell University, Ithaca, New York, mitochondria can induce disease through mutations of mitochondrial DNA and alterations in the respiratory chain mediated by defective mitochondrial enzymes.[14]

Acquired mutations of mitochondrial DNA, which consists in a circular molecule encoding for 13 peptides and 22 tRNAs, have been associated with aging and neurodegenerative diseases.[15,16] The extraction of mutant DNA and polymerase chain reaction analysis of cloned sequences showed an up to 3-fold increase in point mutations in mitochondrial brain DNA, with increasing age. Do these point mutations have functional consequences?

Approximately 60% of the mitochondrial DNA mutations observed led to an amino acid change, and 52% had substantial effects on the function of the proteins involved, such as cytochrome b or ATP synthase. Decreased enzyme activity, measured independently, correlated with increasing age and the presence of point mutations.[13]

Defective activity of mitochondrial DNA polymerase has been recently shown to lead to premature aging in knockout mice. At 12 months, the mice showed cardiac dilation with the degeneration of cardiomiocytes, chifosis, osteoporosis, thinning of the skin, progressive weight loss, and a 50% decrease in survival.

How are mitochondrial alterations involved in the aging process? The mechanism responsible for these damages is believed to be related to the oxidative, molecular damages that are being inflicted over time, and a lack of neutralization of oxygen-reactive species (ROS) that increase with age and can be associated with functional deficits.[14]

The ROS theory of aging, in fact, ascribes to these mediators a critical role in compromising cell homeostasis and survival of the individual as a whole. Experiments conducted in D melanogaster have, for instance, shown that an overexpression (up to 70% of baseline) of methionine sulfoxide reductase A increased survival in the fruit flies (both male and females), with preservation of fertility, that is often lost in other attempts at increasing life span, such as caloric and protein restrictions. Along a similar line, another group reported very recently that 8-hydroxy-2'-deoxyguanosine was substantially increased in individuals older than 70 years of age.[13]

In an analysis of DNA damage and gene regulation in aging human brains, a number of genes were overexpressed or underexpressed when compared with younger controls. Among the genes that were less functional with aging were those encoding for molecules involved in synaptic regulation, vesicular transport, synaptic plasticity, and mitochondrial genes. Genes involved with DNA repair, protection against ROS-mediated damage, showed, on the other hand, an increased expression in older individuals, perhaps as in the attempt to provide compensation.[13]

What might be the relevance of these findings for AD?

Mutations occurring in mitochondrial DNA may affect mitochondrial function as well as transcription. Sequencing of 4-point mutations showed that 3 of them were found only in 8 AD brains and appeared therefore specific for this condition. Some of the mitochondrial DNA mutations found in AD patients were associated with defects in oxidative phosphorylation. In addition, there was a 50% increase in mutations in control vs coding regions in the mitochondrial DNA of AD patients. This was associated with a 50% decrease in total mitochondrial vs total cellular DNA in AD brain cells.[13,15,16]

The hypothesis of a mitochondrial toxicity also has been put forward by Lustbader and colleagues[17] who reported that inhibition of the interaction between Abeta and the Abeta-binding alcohol dehrydrogenase (ABAD) affected the removal of Abeta. Conversely, overexpression of ABAD led to increased neuronal stress and memory impairment.[17] Other investigators have claimed a role for the antioxidant vitamin E in reducing the risk of AD. Morris and Engelhardt reported, for example, a beneficial effect in 70% of subjects taking vitamin E and other vitamin supplements.[18-22]

More recent studies with the analysis of isoprostanes in the cerebrospinal fluid and magnetic resonance imaging (MRI) reported a significant increase, with AD progression, of F2, which is considered a marker of lipid peroxidation. Other redox enzymes and compounds implicated in AD by experimental data are heme oxygenase and 8-hydroxy-2-deoxy-guanosine. Of note, ROS have been detected in amyloid plaques.[23]

The first direct evidence of oxidative damage in AD has, however, been given by heterozygous mice expressing APP and mutant manganese superoxide dismutase (MnSOD), which showed a substantial increase (8 to 9-fold) in the Abeta plaque burden in the motor cortex and other brain regions, when compared with mice expressing wild-type MnSOD.[24] At extraction, there was a 25% increase in amyloid recovery (which is probably an underestimate) associated with reduced MnSOD activity and an increase in protein-associated carbonyl groups, which are a sign of increased oxidative stress.

Thus, Dr. Beal[13] concluded:

• Mutations can occur in mitochondrial DNA that are associated with normal aging and an increased production of ROS;

• Oxidative damages may be linked to downregulation of specific genes;

• Incidence of mutations in mitochondrial DNA (control regions) was found increased in AD patients; and

• Oxidative damage (eg, mediated by a deficit in MnSOD) was associated with more extensive deposition of amyloid.

Conclusion

Efforts are being devoted to acquire a clear understanding of how all these "indicted" molecules and structures work in normal neurons, and what goes haywire in the subjects that go on to develop AD.

Although apparently diverging in pinpointing the cause of this complex disease, analysis of these molecules and the physiologic/pathologic functions they mediate all come together when we look at the complex "daily life" of neuronal networks (one of our most sophisticated and precious tissues kept hidden behind our thick skulls).

So it is not surprising, in a way, that in such a complex disease, the damages occurring in degenerating neurons are multiple and of a different nature. More data are needed to assess how each of these damages occurs, what is the temporal relationship among them, and whether any of these deficits or excesses plays a critically unique role in inducing neurodegeneration and dementia.

AD research is acquiring momentum, and contributions from different perspectives and insights can only make it more rewarding for all.