At the age of 65 years, we carry a 2–3% risk of developing Alzheimer‘s disease (AD), the single major cause of dementia in middle and old age. This risk increases mercilessly during the retirement years, doubling every 5 years after the age of 65 years to almost 50% at 85 years of age. With improvements in modern medicine, the lifespan continues to increase, and, thus, so does the prevalence of AD. Currently, 18–25 million individuals worldwide suffer from AD and this number will multiply several-fold by the end of the 21st Century unless drugs to prevent, inhibit or slow the progression of this disease, which are unavailable at present, are developed.
AD is multifactorial, and is both clinically and histopathologically heterogeneous. Owing to clinical heterogeneity, the diagnosis of AD remains most likely until postmortem histopathological examination of the brain. Development of therapeutic drugs requires the ability to accurately diagnose the disease and its specific subtypes, in addition to the availability of specific outcome measures.
In fewer than 1% of cases, the disease co-segregates with certain mutations in amyloid-β (Aβ) precursor protein, presenilin-1 or -2 genes Citation[1]. The cause of the disease in the remaining cases is not currently understood Citation[2]. Independent of cause, AD is characterized clinically by progressive dementia and histopathologically by the presence of numerous neurofibrillary tangles and neuritic (senile) plaques with neurofibrillary changes in the dystrophic neurites Citation[3]. While plaque amyloid is largely made up of Aβ peptide Citation[4], the neurofibrillary tangles, both in the neuronal cell body and its dystrophic neurites, including those surrounding the amyloid core in the plaque, are made up of abnormally hyperphosphorylated tau protein Citation[5,6]. Tau is ubiquitinated in a large number of the mature tangles Citation[7–9].
Owing to clinical heterogeneity, the diagnosis of AD remains likely until postmortem histopathological examination and is based, primarily, on the exclusion of other causes of dementia Citation[10]. AD histopathology shows considerable qualitative and quantitative heterogeneity. AD can be either neocortical, limbic or plaque-dominant type, and it may present with numerous neurofibrillary tangles exclusively confined to the hippocampus and entorhinal cortex Citation[11]. The two most common confounding diagnoses are cerebral vascular disease (multi-infarct dementia) and dementia with Lewy bodies.
Increased ventricular volume and rates of whole brain atrophy have been demonstrated in AD Citation[12]. The whole brain atrophy in AD results in a loss of brain mass of as much as approximately 2–3% per year, compared with approximately 0.4–0.5% in age-matched control subjects Citation[13]. A number of animal and human studies have suggested that Aβ1–42 levels in cerebrospinal fluid (CSF) reflect the Aβ pathology in the brain. Reduction of Aβ1–40 and Aβ1–42 in the brain of adult rats treated orally with γ-secretase inhibitors has been found to result in decreased levels of Aβ in both brain and CSF Citation[14,15]. An inverse relationship between in vivo amyloid load and CSF levels of Aβ1–42 has been found in humans Citation[16]. Antemortem CSF levels of Aβ1–42, total tau and phosphotau-Thr231 have been reported to reflect the histopathological changes observed postmortem in the brains of AD cases Citation[17,18]. The CSF levels of tau have been demonstrated to be markedly increased in patients with diffuse axonal injury in head trauma, and these revert with clinical improvement Citation[19].
We postulate that more than one disease mechanism and signaling pathway are involved in producing the AD pathology, especially the neurofibrillary degeneration of abnormally hyperphosphorylated tau, and that various subgroups of AD can be identified based on the CSF levels of proteins associated with senile (neuritic) plaques and neurofibrillary tangles. To test this hypothesis, we immunoassayed the levels of tau, ubiquitin and Aβ1–42 in retrospectively collected lumbar CSFs of 468 patients diagnosed clinically as AD (353 CSFs) and as non-AD neurological and non-neurological cases (115 CSFs). Based on the level of these molecular markers, all subjects were subjected to the latent profile analysis to determine the assignment of each subject to a particular cluster. We found that AD subdivides into at least five subgroups based on the CSF levels of Aβ1–42, tau and ubiquitin, and that each subgroup presented a different clinical profile Citation[20]; these five subgroups are:
AELO: AD with low Aβ1–42, high incidence of apolipoprotein (Apo)E4 and late onset;
ATEO: AD with low Aβ1–42, high tau and early onset;
LEBALO: AD with high incidence of Lewy bodies, low Aβ1–42 and late onset;
HARO: AD with high Aβ1–42 and recent onset;
ATURO: AD with low Aβ1–42 high tau, high ubiquitin and recent onset.
Subgroups AELO, ATEO, HARO and ATURO accounted for approximately 50, 22, 5 and 1 of the AD cases studied, respectively. The subgroup LEBALO, which contained the majority of AD cases with Lewy bodies, accounted for approximately 19% of the AD cases.
To classify diagnosed AD cases into the proposed subgroups, we sought a simple set of rules using only one indicator protein at any stage in the classification process. Ideally, it would classify cases with a sensitivity and a specificity of no less than 90% for each category and a comparable overall level of correct classification. The algorithm must categorize all cases unambiguously. A decision tree based on the algorithm was derived from the examination of cluster characteristics and experimental runs that came closest to fulfilling those criteria. The respective sensitivities and specificities were: AELO: 90%, 92%; ATEO: 90%, 95%; LEBALO: 88%, 99%; HARO: 100%, 99%; and ATURO: 100%, 100%. This study demonstrated that CSF levels of Aβ1–42, tau and ubiquitin could diagnose AD in five different subgroups at sensitivities and specificities of greater than 88% and, overall, 86% of cases were classified correctly. This rate of diagnostic accuracy is not only superior to theb use of one of these markers individually or in a combination of two, but also exceeds the biomarker criteria of the Consensus Report Citation[21].
Our recent studies have revealed that more than one signaling pathway is involved in neurofibrillary degeneration. We have demonstrated that tau can be abnormally hyperphosphorylated to self-assemble into bundles of paired helical filaments with more than one combination of protein kinases and that this phosphorylation of tau can be regulated by protein phosphatases (PP), especially PP-2A Citation[22]. Thus, it is likely that additional subgroups of AD will be identified from phosphorylation patterns of CSF tau in AD patients in the future.
CSF analysis not only helps to identify a specific subgroup of AD patients, but can also serve as the outcome measure for a drug treatment. We discovered that memantine inhibited abnormal hyperphosphorylation of tau in rat hippocampal slices in culture Citation[23], and that this inhibitory effect of the drug occurred through the disinhibition of PP-2A activity Citation[24], which previously demonstrated to be downregulated in AD brain Citation[25]. Based on this finding, Gunnarsson and colleagues investigated and observed a significant decrease in the CSF phosphotau levels of patients 1 year after treatment with memantine Citation[26].
Owing to the involvement of different etiopathogenic mechanisms in AD, the identification of different subgroups of this single major cause of age-associated dementia is critical for the development of potent and specific drugs to prevent and cure this disease. Currently, several hundred drugs for AD are under development by the pharmaceutical industry. Stratification of the test subjects in clinical trials by disease subgroups may increase the chance of success by several-fold. The future of therapeutic drugs for AD may depend upon the recognition of different subgroups of the disease.
Acknowledgements
We are grateful to Janet Murphy for secretarial assistance, including preparation of this manuscript. Our laboratory is supported in part by the New York State Office of Mental Retardation and Developmental Disabilities, National Institutes of Health grant AG019158 and Alzheimer‘s Association grant IIRG-06–25836. Authors have a patent pending on the subgrouping of Alzheimer‘s disease.
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