Elsevier

Neurobiology of Disease

Volume 20, Issue 2, November 2005, Pages 187-198
Neurobiology of Disease

Beta-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses

https://doi.org/10.1016/j.nbd.2005.02.008Get rights and content

Abstract

Synaptic dysfunction is increasingly viewed as an early manifestation of Alzheimer's disease (AD), but the cellular mechanism by which β-amyloid (Aβ) may affect synapses remains unclear. Since cultured neurons derived from APP mutant transgenic mice secrete elevated levels of Aβ and parallel the subcellular Aβ accumulation seen in vivo, we asked whether alterations in synapses occur in this setting. We report that cultured Tg2576 APP mutant neurons have selective alterations in pre- and post-synaptic compartments compared to wild-type neurons. Post-synaptic compartments appear fewer in number and smaller, while active pre-synaptic compartments appear fewer in number and enlarged. Among the earliest changes in synaptic composition in APP mutant neurons were reductions in PSD-95, a protein involved in recruiting and anchoring glutamate receptor subunits to the post-synaptic density. In agreement, we observed early reductions in surface expression of glutamate receptor subunit GluR1 in APP mutant neurons. We provide evidence that Aβ is specifically involved in these alterations in synaptic biology, since alterations in PSD-95 and GluR1 are blocked by γ-secretase inhibition, and since exogenous addition of synthetic Aβ to wild-type neurons parallels changes in synaptic PSD-95 and GluR1 observed in APP mutant neurons.

Introduction

The progressive accumulation of β-amyloid (Aβ) in vulnerable brain areas is a neuropathological hallmark of Alzheimer's disease (AD), but the mechanism by which Aβ is involved in AD pathogenesis remains unclear. Loss of synapses, as seen by loss of synaptophysin immunoreactive pre-synaptic terminals (Mucke et al., 2000, Sze et al., 1997), occurs early in AD and is considered the best pathological correlate of cognitive decline (Masliah et al., 2001, Terry et al., 1991). Several lines of evidence suggest a relationship between Aβ accumulation and synaptic dysfunction. Loss of synaptophysin has been reported to correlate well with Aβ accumulation (Mucke et al., 2000, Oddo et al., 2003). More directly, a recent report demonstrated that neuronal activity increased secretion of Aβ, which in turn appeared to depress subsequent synaptic transmission (Kamenetz et al., 2003). Exogenous Aβ42 inhibits long-term potentiation (LTP) (Rowan et al., 2003, Selkoe, 2002). Oligomers of Aβ42 are especially able to impair LTP (Walsh et al., 2002). Depressed synaptic transmission, including impaired LTP, has been reported in AD mouse models of β-amyloidosis and occurs concomitantly with the appearance of intraneuronal Aβ accumulation even prior to appearance of extracellular plaques (Oddo et al., 2003).

Tg2576 mice harbor the human APP transgene with the familial AD Swedish mutation and develop AD-like cerebral amyloidosis (Hsiao et al., 1995). Aβ accumulates and oligomerizes within neurons of Tg2576 mice with time in culture and with aging in vivo, especially within neuronal processes and pre- and post-synaptic compartments (Takahashi et al., 2004). APP is known to be transported down neuronal processes to synapses by fast axonal transport (Koo et al., 1990), and changes in the Aβ domain can alter the sorting of APP in neurons (Tienari et al., 1996). Lesioning of the perforant path, the major axonal projection system into the hippocampus, has been demonstrated to reduce plaques in the hippocampus of APP mutant transgenic mice (Lazarov et al., 2002, Sheng et al., 2003). This implies that synaptic input is essential for the development of plaques. Presumably, this is due to the transport of APP to and generation of Aβ at synapses. Since we previously demonstrated that cultured primary neurons of APP transgenic mice recapitulate with time in culture the accumulation of Aβ and associated ultrastructural degeneration in distal processes and synapses seen in vivo (Takahashi et al., 2004), we set out to characterize the consequences of this aberrant Aβ42 accumulation on synapses. We now report selective alterations in pre- and post-synaptic proteins, in Tg2576 cultured primary neurons compared to wild-type neurons, alterations that progress between 12 and 19 days in culture. Tg2576 neurons have early reductions in levels of PSD-95, a membrane-associated guanylate kinase involved in recruiting and anchoring glutamate receptors to the post-synaptic density (Ehrlich and Malinow, 2004). PSD-95 is known to regulate the surface expression of glutamate receptors. In agreement with these known actions of PSD-95, we also observed reduced surface expression of the AMPA receptor subunit GluR1. Glutamate receptors are critical determinants for LTP and long-term depression (LTD), which are impaired in transgenic mouse models of AD. Our results support the scenario that Aβ accumulation leads to synaptic dysfunction by reducing PSD-95 and GluR1 at synapses.

Section snippets

Antibodies

We employed the following antibodies to: drebrin (monoclonal, IF 1:200, WB 1:2000; Stressgen), α-tubulin (monoclonal, WB 1:10000; SIGMA), actin (monoclonal, 1:5000; SIGMA), synaptophysin (monoclonal, WB 1:1000; Chemicon), synapsin I (polyclonal, IF 1:500, WB 1:3000; SIGMA), GluR1 (polyclonal, WB 1:1000; Upstate), PSD-95 (monoclonal, IF 1:200; Chemicon), PSD-95 (monoclonal, WB 1:3000; Upstate), spinophilin (polyclonal, IF 1:500, WB 1:1000, (Feng et al., 2000), Zif268 (polyclonal, WB 1:1000;

Alterations in pre- and post-synaptic proteins in Tg2576 neurons at 19 days in vitro

We evaluated by Western blot the levels of synapsin I and synaptophysin, two important pre-synaptic proteins involved in recycling of synaptic vesicles (Fig. 1A). Post-synaptically, we analyzed the levels of GluR1, PSD-95, drebrin, and spinophilin (Fig. 1A). GluR1 is an AMPA receptor subunit, and PSD-95 is a post-synaptic density protein. Both are important for synaptic plasticity (Malinow and Malenka, 2002). Drebrin is an actin-binding protein involved in spine morphogenesis (Hayashi and

Discussion

Alterations in synapses appear to be among the earliest events in the initiation of the cognitive decline that characterizes AD and have long been considered the best pathological correlate of cognitive decline in AD (Coleman and Yao, 2003). Interestingly, while Aβ plaques are a poor correlate of cognitive decline, levels of soluble Aβ are considered the best Aβ-correlate of cognitive decline in AD (McLean et al., 1999, Naslund et al., 2000). Based on our previous electron microscopy studies in

Acknowledgments

We thank Drs. Sushmita Mukherjee, Timothy Ryan, and Frederick Maxfield (Department of Biochemistry, Weill Medical College of Cornell University) for helpful discussions. We thank Fangmin Yu and Patrick Kearney for expert technical assistance.

This study was supported by grants from the National Institutes of Health (AG09464 to P. Greengard and G.K. Gouras; NS002037 and NS045677 to G.K. Gouras), Beeson Awards (to M.T. Lin and G.K. Gouras), and the Alzheimer's Association (to G.K. Gouras). C.G.

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