Deleterious effects of soluble amyloid-β oligomers on multiple steps of synaptic vesicle trafficking

Highlights

• Aβ oligomers alter the ratio of recycling to resting vesicle pools.

• They cause multiple defects in presynaptic endocytic recycling.

• The release probability of the RRP is enhanced.

• The PtdIns(4,5)P₂ and calpain-CDK5 pathways are involved in the presynaptic dysfunction.

Abstract

Growing evidence supports a role for soluble amyloid-β oligomer intermediates in the synaptic dysfunction associated with Alzheimer's disease (AD), but the molecular mechanisms underlying this effect remain unclear. We found that acute treatment of cultured rat hippocampal neurons with nanomolar concentrations of Aβ oligomers reduced the recycling pool and increased the resting pool of synaptic vesicles. Endocytosis of synaptic vesicles and the regeneration of fusion-competent vesicles were also severely impaired. Furthermore, the release probability of the readily-releasable pool (RRP) was increased, and recovery of the RRP was delayed. All these effects were prevented by antibody against Aβ. Moreover reduction of the pool size was prevented by inhibiting calpain or CDK5, while the defects in endocytosis were averted by overexpressing phosphatidylinositol-4-phosphate-5-kinase type I-γ, indicating that these two downstream pathways are involved in Aβ oligomers-induced presynaptic dysfunction.

Introduction

Alzheimer's disease (AD) is the most common form of neurodegenerative dementia. Its hallmarks are neurofibrillary tangles and amyloid plaques, caused by hyperphosphorylated tau and amyloid beta (Aβ) protein, respectively (Lee et al., 2001). Growing evidence indicates that the 42-residue amyloid beta protein plays an essential role in the pathogenesis of AD. The amyloid cascade hypothesis proposes that the abnormal accumulation of Aβ inhibits synaptic function, gradually induces neuronal and glial changes, and initiates the process of neurodegeneration (Hardy and Selkoe, 2002).

Reduced synaptic function is one of the earliest measurable defects in AD (Walsh et al., 2002b). Although different assembly forms of Aβ may affect AD pathogenesis at different stages (Haass and Selkoe, 2007, Lambert et al., 1998, Walsh et al., 2002a), plaque numbers and insoluble Aβ levels do not appear to correlate with disease progression in the early stages of AD (McLean et al., 1999). Instead, many aspects of early AD pathogenesis are closely correlated with cerebral levels of soluble Aβ oligomers. For example, Aβ oligomers trigger various pathophysiological events associated with AD, such as alteration of synaptic plasticity by inhibiting long-term potentiation (LTP), facilitating long-term depression (LTD), causing the loss of dendritic spines, modulating the expression of 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptors, and disturbing calcium homeostasis (Demuro et al., 2005, Green and LaFerla, 2008, Haass and Selkoe, 2007, Hsieh et al., 2006, Shankar et al., 2007, Snyder et al., 2005). They also have acute effects on synaptic function. For example, injection of soluble forms of human Aβ oligomer disrupts cognitive function in a rapid and transient manner (Cleary et al., 2005), and endogenous or exogenous Aβ antibodies can restore synaptic function and reverse the acute memory deficits in AD animal models (Billings et al., 2005, Dodart et al., 2002, Kotilinek et al., 2002).

Most previous studies have focused on the effects of Aβ oligomers on postsynaptic function, and it is still unclear which step(s) in presynaptic function is affected (Abramov et al., 2009, Berman et al., 2008, Calabrese et al., 2007, Moreno et al., 2009, Nimmrich et al., 2008, Puzzo et al., 2008). Synaptic vesicle components are recycled through a local exo-endocytic pathway in presynaptic nerve terminals, and recent studies have revealed some of the molecular details of this process (Dittman and Ryan, 2009, Murthy and De Camilli, 2003). The synaptic vesicle pool consists of a recycling pool, which includes a readily-releasable pool (RRP) and a reserve pool, and a resting pool that does not normally recycle. The size of the pool of vesicles that recycles and the kinetics of each of the recycling steps all play important roles in determining the efficacy of synaptic function. Therefore, uncovering the step(s) in synaptic vesicle trafficking that are affected by Aβ oligomers and the intracellular signaling pathways involved could identify suitable drug targets and lead to the development of new therapeutic interventions.

To examine the effect of soluble Aβ oligomers on presynaptic function, we treated cultured rat hippocampal neurons with nanomolar concentrations of Aβ oligomers. Using a quantitative kinetic analysis of the trafficking of synaptic vesicles, we were able to detect subtle alterations in multiple steps of trafficking and the intracellular signaling pathways involved, thus revealing how Aβ oligomers cause presynaptic dysfunction.