The results presented herein indicated that Aβ impaired synaptic vesicle endocytosis, and hence the normal recycling of these organelles, during sustained synaptic activity in cultured hippocampal neurons. In addition, our data provided further evidence suggesting that these Aβ effects could be mediated, at least in part, by the significant depletion of dynamin 1 induced by this peptide. Together, these findings identify potential mechanisms underlying synaptic dysfunction in early stages of AD.
Synapse loss correlates with the severity of cognitive decline in AD (Davies et al. 1987
; Terry et al. 1991
). However, it has become increasingly clear that synaptic dysfunction might also play an important role in the earliest stages of this disease (Davies et al. 1987
; Terry et al. 1991
). This hypothesis has been further supported by studies using animal models of AD. These transgenic mice developed cognitive impairments that preceded any significant neuronal loss in affected brain areas (Hsiao et al. 1996
; Giacchino et al. 2000
; Takeuchi et al. 2000
; Jacobsen et al. 2006
). In addition, data obtained recently indicated that the intracellular injection of Aβ disrupted cognitive function in rodents (Clearly et al., 2005). Conversely, immunization with antibodies raised against this peptide reverse memory defects in AD animal models (Billings et al., 2005
; Dodart et al., 2002
; Kotilinek et al., 2002
). Research on the potential molecular mechanisms that could lead to synaptic dysfunction in the context of this disease has focused in several synaptic proteins. As a result of this work, it has been shown that dynamin 1 was significantly reduced not only in the hippocampus of an AD mouse model and in cultured hippocampal neurons treated with Aβ but also in the brain of AD patients (Yao et al. 2003
; Kelly et al. 2005
; Kelly and Ferreira 2006
). The mechanisms leading to this dynamin 1 depletion seem to involve calpain, a protease system that is active in AD (Tsuji et al. 1998
; Lee et al. 2000
; Battaglia et al. 2003
; Tomizawa et al. 2003
; Chen and Fernandez 2005
; Demuro et al. 2005
; Gong et al. 2005
; Kelly et al. 2005
; Fifre et al. 2006
; Kelly and Ferreira 2006
). Furthermore, we have reported that this Aβ-mediated dynamin 1 degradation was the result of calpain activation induced by the sustained calcium influx mediated by N-methyl-D-aspartate (NMDA) receptors in hippocampal neurons (Kelly and Ferreira 2006
). However, the mechanisms by which Aβ increases NMDA receptor-mediated calcium influx are not known. Aβ could induce the expression of these receptors or their membrane localization. Results obtained recently showing an Aβ-induced decrease in the levels of NMDA receptor availability in hippocampal neurons seem to argue against this possibility (Snyder et al., 2005
). This mechanism does not seem to mediate the effects of Aβ in our model system either since we did not detect changes in the levels or localization of these receptors in Aβ-treated cultured hippocampal neurons (Kelly and Ferreira, unpublished observations). Alternatively, the aggregated peptide could interact directly or indirectly with these receptors modifying different aspects of their function. Further analysis would provide additional insights into the role of NMDA receptors in the increase in calcium influx triggered by Aβ.
Little is known also about the functional consequences of Aβ-induced dynamin 1 depletion in central neurons. The role of dynamin 1 in synaptic vesicle endocytosis has been well documented in Drosophila. In the absence of dynamin 1, synapses lose their ability to successively transmit signals under stimulation due to incomplete endocytosis of the synaptic vesicles (Delgado et al. 2000
). Our results provide evidence suggesting that Aβ-induced dynamin 1 depletion caused similar deficiency in synaptic vesicle recycling in mammalian hippocampal neurons. When placed in culture, hippocampal neurons form functional synapses that are structurally and molecularly identical to synapses formed in vivo
(Bartlett and Banker 1984b
; Ferreira et al. 1997
). Synaptic vesicle recycling appeared to be functional and efficient in these neurons, as there were no signs of synaptic vesicle depletion or incomplete endocytosis in the presynaptic terminals of stimulated cells. The treatment of hippocampal neurons with Aβ prior to stimulation did not seem to cause synaptic retraction or synapse loss. The integrity of the synaptic contacts between the presynaptic terminal and the postsynaptic element were well preserved and no apparent severance between these two structures was observed. However, the ultrastructural analysis of synapses from these treated neurons showed signs of impaired synaptic vesicle endocytosis following driven synaptic activity including depleted pools of synaptic vesicles and invaginated pits at the plasma membrane. It is worth mentioning that similar morphological abnormalities have been described in the Drosophila dynamin 1 loss-of-function model (Koenig and Ikeda 1989
). Interestingly, several studies have shown that presynaptic terminals are enlarged both in human AD brains and in cultured neurons from AD animal models (Bertoni-Freddari et al. 1988
; DeKosky and Scheff 1990
; Games et al. 1995
; Snyder et al. 2005
). The enlargement of the presynapse in these cases might be the consequence of incomplete synaptic vesicle endocytosis and integration of synaptic vesicle membrane into the plasma membrane.
The results obtained from the biochemical analysis performed in this study provided further evidence consistent with functional abnormalities due to dynamin 1 depletion in hippocampal neurons cultured in the presence of Aβ. Synaptic vesicle endocytosis involves a number of proteins that translocate from the cytoplasm to the plasma membrane to form the proper endocytic complex (reviewed by Murthy and De Camilli 2003
). One of these proteins, amphiphysin, specifically serves as a dynamin 1 binding partner to recruit it to the membrane. Our results showed that a normal redistribution of amphiphysin from the cytosol to the membrane occured during stimulation in cultured hippocampal neurons. This redistribution is likely the result of amphiphysin translocating to the membrane to support the need for increased synaptic vesicle endocytosis (David et al. 1996
). However, we showed here that pretreatment with Aβ caused an abnormal accumulation of amphiphysin in membrane fractions and a depletion from cytosol fractions upon stimulation of these neurons. There are two possible explanations for the excessive membrane association of amphiphysin. First, the synaptic vesicles could be endocytosed, but the amphiphysin may not be released from the synaptic vesicles. Alternatively, the synaptic vesicles might not be endocytosed normally causing amphiphysin to become trapped and accumulate at the plasma membrane. The abnormal morphology detected in the synapses of these Aβ-treated hippocampal neurons seems to support the notion that endocytosis is impaired under these experimental conditions. Our results showing that Aβ caused decreased FM1-43 dye uptake during stimulated synaptic activity in cultured hippocampal neurons further support this view. This dye is normally taken up as synaptic vesicles are endocytosed after exposure to the extracellular environment. Low levels of dye uptake were detected even under control conditions. These results suggested the presence of basal spontaneous synaptic activity in control unstimulated cultured hippocampal neurons. These data were in agreement with a previous report showing spontaneous release of neurotransmitter and synaptic vesicle endocytosis under these cultured conditions (Sara et al. 2005
). This basal level of endocytosis was also reflected by the appearance of amphiphysin in the membrane fraction of control neurons as detected by quantitative Western blot analysis. Addition of Aβ prior to potassium stimulation did not reduce the amount of dye uptake attributed to spontaneous synaptic activity under control conditions, suggesting that Aβ did not cause any deficit in synaptic vesicle endocytosis when these hippocampal neurons were in a resting, basal state. On the other hand, after these neurons were pretreated with Aβ and stimulated to drive sustained synaptic activity, a significant inability to take up the dye emerged. Taken collectively, these data suggested that this deficiency emerges when the level or speed of synaptic vesicle recycling is increased under sustained synaptic activity. In contrast, no decrease in dye uptake was observed when hippocampal neurons overexpressing APP were cultured as microislands (Ting et al., 2007
). Possible explanations of this discrepancy are the different age (embryonic vs. postnatal), species (rats vs. mice), and culture conditions (dissociated vs. microisland cultures) used in these two studies. More importantly, while we used tightly control amounts of Aβ and aggregation times, Ting and coworkers used cells that overexpressed APP and were kept in culture for 10 to 17 days (Ting et al., 2007
). Under these experimental conditions, the undertermined amounts of Aβ could accumulate in the culture medium for long periods of time and could be aggregated beyond the oligomeric forms into the less toxic fibrils, and hence, fail to elicit changes in synaptic vesicle recycling similar to the ones reported in this study.
Finally, it is worth noting that similar ultrastructural and functional results were obtained when dynamin 1 depletion was achieved using a specific dynamin 1 inhibitor. The parallel effects of these two treatments further support the hypothesis that Aβ-induced depletion of dynamin 1 could play a key role in synaptic dysfunction in AD. However, because Aβ can affect a number of proteins involved in synaptic vesicle recycling, we cannot completely rule out that changes in other proteins may contribute to altered synaptic vesicle endocytosis in this disease. Aβ could also induced changes in postsynaptic elements that might contribute to synaptic dysfunction. Thus, it has been recently shown that APP over expression led to a selective reduction of α-amino-3-hydroxy-5methyl-4-isoxazole-propionic acid (AMPA) receptors, and hence, disruption in excitatory transmission in hippocampal neurons (Chang et al., 2006
; Ting et al., 2007
). However, it is unlikely that these defects are mediated by dynamin 1 depletion since these proteins are localized in different subcellular compartments.
Collectively, our results showed that Aβ caused a disruption of synaptic vesicle endocytosis in stimulated hippocampal neurons. They also suggested that this disruption of synaptic vesicle endocytosis was dependent, at least in part, on dynamin 1 loss-of-function in these neurons. Even discrete disruptions of synaptic vesicle endocytosis could be significant in the context of early-stage AD, resulting in a wide-ranging decline of cognition at the behavioral level. Thus, the process of Aβ-induced dynamin 1 depletion could be a viable therapeutic target to treat synaptic dysfunction in the earliest stages of AD.