Introduction Alzheimer s disease AD is a complex neurologica
Alzheimer's disease (AD) is a complex neurological disorder characterized by a progressive dementia resulting from synapse failure (Selkoe, 2002, Tanzi, 2005). The amyloid hypothesis maintains that the pivotal event in AD is the production of amyloid-β (Aβ) peptides following the proteolytic cleavage of the amyloid precursor protein (APP) (De Strooper et al., 2010, Hardy and Selkoe, 2002). The accumulation of C-terminal fragments (Aβ) within the PPADS tetrasodium salt is thought to cause the synapse dysfunction and the memory loss that is characteristic of AD. Aβ has the capacity to self-aggregate, and consequently is found in different forms ranging from monomers and small soluble oligomers to much larger fibrils and plaques. The soluble Aβ oligomers are currently considered to be the principal mediators of synapse damage (Yang et al., 2017).
The mechanisms of Alzheimer's-related synapse damage can be examined by incubation of cultured neurons with Aβ. Since the loss of synapses and synaptic proteins is a feature of AD that strongly correlates with cognitive decline in AD (Counts et al., 2006, Masliah et al., 1991, Reddy et al., 2005) synapse density was measured by determining the amounts of synaptophysin and cysteine string protein (CSP) in cultured neurons (Lipton et al., 2001). Soluble forms of Aβ derived from the brains Alzheimer's patients caused synapse degeneration in neurons (Yang et al., 2017). The biological effects of these Aβ oligomers occurred at picomolar concentrations, similar concentrations to those found in the cerebrospinal fluid of Alzheimer's patients (Bibl et al., 2007, McLean et al., 1999, Mehta et al., 2000). The role of Aβ monomers that are found in high concentrations in the brain are poorly understood; while some studies report that Aβ monomers are not toxic (Shankar et al., 2007, Walsh et al., 2002) another study suggested that they were neuroprotective (Giuffrida et al., 2009). Confusion may arise due to the heterogeneity of Aβ in monomer preparations, in addition to Aβ40 and Aβ42 peptides there are other APP fragments in cerebrospinal fluid (Brinkmalm et al., 2012) and that some of these carboxy-terminal fragments of APP have biological activity (Willem et al., 2015). In the present study Aβ monomer preparations and Aβ oligomers were isolated from soluble brain extracts and their biological activity studied.
Increasing evidence suggests that aberrant activation of cytoplasmic phospholipase A2 (cPLA2) plays an important role in Aβ oligomer-induced synapse damage. For example, Aβ activates cPLA2 (Anfuso et al., 2004, Shelat et al., 2008) and pharmacological inhibition of cPLA2 protects cultured neurons against Aβ-induced synapse damage (Bate et al., 2010). In addition, cPLA2 inhibitors ameliorated cognitive decline in a mouse model of AD (Sanchez-Mejia et al., 2008). Here we demonstrate that whereas Aβ oligomers increased synaptic cholesterol concentrations, activated cPLA2 and caused synapse damage in cultured neurons, Aβ monomers had none of those effects. The cellular prion protein (PrPC) mediates Aβ-induced memory defects (Lauren et al., 2009) and aggregation of PrPC by Aβ oligomers caused the activation of cPLA2 and synapse damage (Bate and Williams, 2011). Here we report that Aβ monomers did not cause aggregation of PrPC; rather that they reduced the aggregation of PrPC by Aβ oligomers. The presence of Aβ monomers significantly reduced the Aβ oligomer-induced activation of cPLA2 and synapse damage.
Materials and methods
Discussion The principal finding of this study is that Aβ monomer preparations reduced the Aβ oligomer-induced synapse damage. This protection was associated with Aβ monomers reducing the Aβ oligomer-induced aggregation of PrPC and the subsequent increase in synaptic cholesterol concentrations and the activation of cPLA2. Soluble brain extracts were used in this study since they contain a mixture of monomers and low-n oligomers, that are similar in size, solubility, stability and potency to the Aβ previously reported in the CSF from AD patients (Bibl et al., 2007, McLean et al., 1999, Mehta et al., 2000). Thus, the presence of brain extracts containing picomolar concentrations of Aβ42 caused synapse damage to cultured neurons. The initial observations, that the removal of Aβ monomers from brain extracts increased synapse damage, suggested that Aβ monomers have a protective role. This hypothesis was supported by experiments using Aβ monomers collected by filtration. At the maximum concentration of monomers used (containing 20nM Aβ42) they did not cause synapse damage. In contrast, the oligomer preparation that reduced the synaptophysin content of neurons by 50% contained approximately 0.2nM Aβ42 (Fig. 3C). We note that in addition to Aβ40 and Aβ42 the preparations used in this study are likely to contain other APP fragments which may contribute to the activity reported and consequently the word “monomers” was used to include all Aβ fragments passing through a 10kDa filter. The activity of monomer preparations was removed by immunodepletion with mAb 6E10, indicating that the bioactive peptides contained a specific epitope. However the methods used in this study do not differentiate whether those peptides were Aβ40, Aβ42 or other APP fragments sharing this epitope.