Originally Posted on Cell Reports | 20 March 2014
Alzheimer’s disease (AD) diagnosis is hampered by the lack of early, sensitive, and objective laboratory tests. We describe a sensitive method for biochemical early diagnosis of Alzheimer’s disease based on specific detection of misfolded Ab oligomers, which play a central role in AD pathogenesis. The protein misfolding cyclic amplification assay (Ab-PMCA), exploits the functional property of Ab oligomers to seed the polymerization of monomeric Ab. Ab-PMCA allowed detection of as little as 3 fmol of Ab oligomers.
Most importantly, using cerebrospinal fluid, we were able to distinguish AD patients from control individuals affected by a variety of other neurodegenerative diseases or nondegenerative neurological diseases with overall sensitivity of 90% and specificity of 92%. These findings provide the proof-of-principle basis for developing a highly sensitive and specific biochemical test for AD diagnosis.
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly population and one of the leading causes of death in the developed world (Hebert et al., 2003). The disease is typically characterized by a progressive amnestic disorder followed by impairment of other cognitive functions and behavioral abnormalities associated with speciﬁc neuropathological changes, in particular accumulation of protein aggregates in the form of amyloid plaques and neuroﬁbrillary tangles (Terry, 1994). Although the etiology of the disease is not yet clear, compelling evidence suggests that misfolding, oligomerization, and accu-mulation of amyloid aggregates in the brain is the triggering fac-tor of the pathology (Selkoe, 2000; Haass and Selkoe, 2007; Soto, 2003). Amyloid aggregates are composed predominantly of a 42-residue peptide called amyloid-b (Ab), which is the product of the enzymatic processing of a larger amyloid precur-sor protein (Selkoe, 2000). Ab misfolding and ﬁbrillar aggregation follow a seeding-nucleation mechanism that involves the forma-tion of several intermediates, including soluble oligomers and protoﬁbrils (Caughey and Lansbury, 2003; Soto et al., 2006; Jar-rett and Lansbury, 1993). Recent ﬁndings have shown that Ab oligomers, rather than large amyloid ﬁbrils, might be the culprit of neurodegeneration in AD (Walsh and Selkoe, 2007; Haass and Selkoe, 2007; Glabe and Kayed, 2006; Klein et al., 2004).
AD belongs to a large group of diseases associated with misfolding, aggregation and tissue accumulation of proteins (Soto, 2003). These diseases, termed protein misfolding disorders (PMDs), include Parkinson’s disease, type 2 diabetes, Hunting-ton’s disease, amyotrophic lateral sclerosis, systemic amyloidosis, prion diseases, and many others (Soto, 2003; Luheshi and Dobson, 2009). In all these diseases, misfolded aggregates composed of different proteins are formed by a similar mechanism resulting in the accumulation of toxic structures that induce cellular dysfunction and tissue damage (Caughey and Lansbury, 2003; Soto et al., 2006; Jarrett and Lansbury, 1993).
One of the major problems in AD is the lack of a widely accepted early, sensitive, and objective laboratory diagnosis to support neuropsychological evaluation, monitor disease progression, and identify affected individuals before they display the clinical symptoms (Parnetti and Chiasserini, 2011; Urbanelli et al., 2009). For diseases affecting the brain, a tissue with low regeneration capacity, it is crucial to intervene before irreversible neuropathological changes occur. Therefore, early diagnosis of AD is of utmost importance. Several lines of evidence point that the process of Ab misfolding and oligomerization begins years or decades before the onset of clinical symptoms and substantial brain damage (Braak et al., 1999; Buchhave et al., 2012). Recent studies have shown that Ab oligomers are naturally secreted by cells and circulate in AD biological ﬂuids (Gao et al., 2010; Head et al., 2010; Walsh et al., 2002; Klyubin et al., 2008; Georganopoulou et al., 2005; Fukumoto et al., 2010). Thus, detection of soluble Ab oligomers might represent the best strategy for early and speciﬁc biochemical diagnosis of AD. The challenge of this approach is that the quantity of Ab oligomers is likely very small in tissues other than the brain. An additional difﬁculty for speciﬁc detection of Ab oligomers is that their sequence and chemical structure is the same as the native Ab protein.
Our strategy to detect misfolded oligomers is to use their functional property of being capable of catalyzing the polymeri-zation of the monomeric protein. For this purpose, we invented the protein misfolding cyclic ampliﬁcation (PMCA) technology in order to achieve the ultrasensitive detection of misfolded aggregates through ampliﬁcation of the misfolding and aggregation process in vitro (Saborio et al., 2001). So far, PMCA has been applied to detect minute quantities of oligomeric misfolded prion protein (PrPSc) implicated in prion diseases (Morales et al., 2012). Using PMCA, we were able to detect the equivalent of a single particle of misfolded PrP oligomer (Saa´ et al., 2006b) and strikingly to identify PrPSc in the blood and urine of infected animals at symptomatic and presymptomatic stages of the disease (Castilla et al., 2005; Saa´ et al., 2006a; Gonzalez-Romero et al., 2008). The basis for the PMCA technology is the fact that the process of misfolding and aggregation of Ab, PrP, and the other proteins implicated in PMDs follow a seeding-nucleation mechanism (Soto et al., 2002, 2006).
In a seeded-nucleated polymerization, the limiting step is the formation of stable oligomeric seeds that, depending on the conditions, may take a very long time to form or not occur at all. Once formed, oligomers grow exponentially by recruiting and incorporating protein monomers into the growing polymer. Addition of preformed seeds into a solution containing the monomeric protein accelerates protein misfolding and aggregation (Soto et al., 2006; Jarrett and Lansbury, 1993). Thus, measuring seeding activity could be used to estimate the presence and quantity of oligomers in a given sample. To increase the sensitivity of detection, PMCA combines steps of growing polymers with multiplication of oligomeric seeds to reach an exponential increase of misfolding and aggregation (Soto et al., 2002). In this study, we describe the implementation and optimization of PMCA for highly sensitive detection of misfolded Ab oligomers and show its application to detect these structures in the cerebrospinal ﬂuid (CSF) of AD patients.