Many studies have postulated that the aggregation of Aβ in both soluble and insoluble forms in the brain is likely a key initiating factor in AD pathogenesis. Thus, influences on the aggregation process are potentially major treatment targets. Since the process of Aβ aggregation is concentration-dependent, understanding factors that regulate Aβ levels in the location that it aggregates (e.g. in the brain extracellular space) is likely to provide critical insights into the biology of AD. While it has been demonstrated that APP endocytosis is a key step in Aβ generation and release in vitro, we provide direct evidence that inhibition of clathrin-mediated endocytosis reduces ISF Aβ levels in awake mice, both transgenic and wild-type, under normal conditions. Importantly, in addition to endocytosis, our work shows that synaptic activity, and specifically synaptic vesicle exocytosis, is linked with neuronal Aβ release. Utilizing two distinct pharmacological interventions, we found that increasing synaptic activity increases ISF Aβ levels in a manner dependent on endocytosis. Clathrin-mediated endocytosis of APP is necessary for activity-dependent regulation of ISF Aβ levels in APP transgenic mice as well as in wild-type mice. This emphasizes that these results elucidate a normal pathway for regulating ISF Aβ levels. Blockade of endocytosis primarily affected Aβ generation with no detectable change in ISF Aβ elimination. We estimate that the majority of ISF Aβ in vivo is generated through endocytosis of APP and that in the brain, synaptic activity plays a primary role in that process. Overall, these findings define a pathway by which synaptic activity influences Aβ release into brain ISF that is likely relevant to AD pathogenesis.
One major pathway for Aβ generation is endocytosis of full length APP from the plasma membrane into the endocytic compartment, where BACE and γ-secretase act to produce Aβ. Aβ is then subsequently secreted from the neuron into the extracellular space. Our hypothesis is that synaptic activity drives more APP into the endocytic compartment, thus increasing Aβ generation and release. Action potential invasion of the synaptic terminal causes calcium influx (). Calcium causes synaptic vesicles to fuse with the plasma membrane and to release neurotransmitter into the synaptic cleft (). This is the basis of synaptic transmission. As more synaptic vesicles fuse with the plasma membrane, there is an increase in the amount and overall rate of endocytosis that recycles the vesicular membrane from the cell surface. When the synaptic vesicle membrane is recycled from the plasma membrane via clathrin-mediated endocytosis, more APP is internalized as well (). It is possible that calcium entry may also cause endocytosis through other mechanisms, such as synthesis of PI(4,5)P2
, which recruits the clathrin assembly and mediates clathrin-mediated endocytosis (Wenk et al., 2001
). Increased endocytosis increases APP internalization into the endocytic compartment where Aβ is generated (). Once generated, Aβ can be secreted from the neuron into the brain extracellular space ().
Model of synaptic-dependent release of Aβ
Our previous studies provide direct evidence that Aβ is generated due to presynaptic mechanisms; causing synaptic vesicle exocytosis with α-latrotoxin in the absence of postsynaptic activation is still capable of generating Aβ (Cirrito et al., 2005
). We cannot rule out a postsynaptic mechanism as well however. α-Latrotoxin in the presence of postsynaptic activation has a greater effect on extracellular Aβ than α-latrotoxin in the absence of the postsynaptic activation. The difference between these two conditions (~20% of Aβ generation) is likely due to postsynaptic mechanisms that also influence Aβ generation (Cirrito et al., 2005
Two mechanistically distinct agents increased synaptic activity and increased ISF Aβ levels. Picrotoxin inhibits ionotropic GABAA receptors, thus increasing synaptic transmission by reducing inhibition of excitatory neurons. In contrast, LY341495 blocks perisynaptic G-protein coupled mGluR2/3s, thus increasing excitatory neurotransmission by directly enhancing glutamate release at the synapse. Both of these pathways increased Aβ levels by modestly increasing synaptic activity. In both cases, inhibition of endocytosis completely blocked the elevation the ISF Aβ levels. This phenomenon was also observed with wild-type murine Aβ40 and Aβ42 levels. Interestingly, inhibition of endocytosis had a larger affect on Aβ42 levels than Aβ40, perhaps suggesting that a greater percent of extracellular Aβ42 comes from endosomes than does Aβ40.
Dynamin-DN reduced ISF Aβ levels by over 60%. However, washout of dynamin-DN caused a transient surge in ISF Aβ levels. This suggests that APP may have been retained on the cell surface during inhibition of endocytosis and comprised a large pool of substrate capable of generating Aβ when endocytosis resumed. In the in vivo paradigm, for Aβ production to be elevated when endocytosis resumed, much of the APP retained on the plasma membrane must have remained as the full length, uncleaved protein.
Several studies have demonstrated that Aβ, particularly oligomeric species, can modulate various aspects of synaptic transmission (Hsieh et al., 2006
; Shankar et al., 2007
; Ting et al., 2007
; Townsend et al., 2006
). Our studies demonstrate that inhibition of endocytosis has no effect on spontaneous EEG activity or evoked potentials in vivo. Interestingly, blocking endocytosis reduced ISF Aβ levels by 70% but did not appear to modulate transmission. Given that Aβ levels were not reduced to zero, it is possible that the remaining 30% of ISF Aβ was sufficient to retain Aβ’s effect, if any, on synaptic transmission. It is also likely that the majority of ISF Aβ present in 3 month old Tg2576 mice is monomeric and not oligomeric Aβ. Further studies are required to address what role Aβ has on synaptic activity in vivo.
The data suggest there are at least three cellular mechanisms that contribute to ISF Aβ production. First, a pathway that is synaptic activity-dependent (TTX sensitive) and endocytosis-dependent which contributes to approximately 60% of ISF Aβ levels in Tg2576 mice. Second, a pool of ISF Aβ that requires endocytosis, but is independent of synaptic activity. This pathway is responsible for another 10% of ISF Aβ levels. Interestingly, inhibition of endocytosis does not reduce ISF Aβ levels to zero. The remaining pool of Aβ, comprising 30% of total ISF Aβ levels, may be the product of several mechanisms, including Aβ produced within the secretory pathway (Busciglio et al., 1993
) or Aβ diffusing from brain areas that are not affected by dynamin-DN or TTX. Alternatively, this last pool may be a factor of incomplete inhibition of endocytosis or some small contribution of altered Aβ elimination. These values provide rough estimates for each of these pools and provide an interesting framework to consider the various pathways that contribute to the overall pool of ISF Aβ. A source of 70% of ISF Aβ in vivo is a tantalizing target for therapeutic development. While inhibiting all clathrin-mediated endocytosis is unlikely to be a feasible strategy for lowering extracellular Aβ levels, it may be possible to modulate individual components of the endocytic machinery or synaptic transmission to selectively affect Aβ generation.
Several molecules influence the rate and amount of APP endocytosis. LRP1 expression increases the rate of APP endocytosis from the plasma membrane whereas LRP1b expression reduces the rate of internalization (Cam et al., 2004
; Cam et al., 2005
). Recent evidence also implicates another LDL-R family member, LR11 or SorLa, in modulating APP trafficking through the endosomal compartment (Andersen et al., 2005
; Dodson et al., 2006
; Offe et al., 2006
; Rogaeva et al., 2007
). LR11 overexpression increases APP co-localization within the Golgi and reduces Aβ generation in culture whereas deletion of the LR11
gene in mice increases brain Aβ levels (Andersen et al., 2005
). Given that several LDL-R family members modulate Aβ endocytosis, it will be important to elucidate if and how these molecules affect synaptic activity-dependent Aβ generation.
While our studies do not directly assess where Aβ is produced or released, it is likely that Aβ generation that is caused by events within the synaptic terminal would occur at or near the synapse. Kamenetz and colleagues demonstrated that neurons virally expressing APP can depress synaptic transmission in nearby neurons (Kamenetz et al., 2003
). This also suggests that Aβ released from a neuron might even be able to feed back to inhibit activity within the same neuron. We have identified a mechanism of Aβ production and release near the synapse that is intimately associated with synaptic activity. This phenomenon produces a situation whereby synaptically-released Aβ is positioned to modulate synaptic activity. The conformation of Aβ that is released following synaptic activity will likely be a critical factor for modulation of neurotransmission. For instance, Aβ oligomers appear to be much more potent at depressing synaptic transmission than Aβ monomers (Townsend et al., 2006
). The Aβ conformations that are released at synapses and whether those Aβ species change with age or disease stage is unknown.
Synaptic activity appears to affect Aβ levels in humans as well. First, a fraction of patients with temporal lobe epilepsy develop diffuse Aβ deposits in seizure-prone brain regions as early as 30 years of age (Gouras et al., 1997
; Mackenzie and Miller, 1994
). Second, there are several links between stress, neuronal activity, and AD. Individuals that are prone to psychological distress are more likely to develop AD (Wilson et al., 2005
; Wilson et al., 2003
). Also, plasma levels of the stress hormone, cortisol, are correlated with the rate of dementia progression in patients with AD (Csernansky et al., 2006
). In APP transgenic mice subjected to restraint stress, ISF Aβ levels are significantly higher than control animals, however the acute increase in Aβ levels is blocked in the absence of neuronal activity (Kang et al., 2007
). Final support for the idea of a casual link between synaptic activity and Aβ levels in humans comes from recent brain imaging studies. Brain regions that contain the most metabolic activity throughout life, and presumably have the highest levels of neuronal activity, are also the regions most vulnerable to Aβ accumulation and aggregation in AD patients (Buckner et al., 2005
). In each of these cases, synaptic activity appears to play a role in regulating Aβ levels under physiologic conditions. We now identify a cellular pathway, endocytosis, that likely links synaptic activity and Aβ production.