Synaptic activity amyloidp and Alzheimers disease

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John R. Cirrito1'2'4 and David M. Holtzman1'3'4'5

An unsolved mystery for most neurodegenerative disorders, including Alzheimer's disease (AD), is why the underlying pathology that occurs is region-specific. In other words, why are some brain regions vulnerable and others not? Several interesting findings and events have come together over the last six years that have led us to hypothesize that one of the reasons that the amyloid-p (Ap) peptide deposits in aregion-specific fashion in AD is related to the overall synaptic activity that occurs over many years in areas vulnerable to Ap deposition. If this assumption is correct, it has important implications for both AD pathogenesis and potentially for future therapies.

John Cirrito began his PhD thesis in the Holtzman lab in 2000. Because of the key role that Ap appears to play in the pathogenesis of AD, we thought it was critical to better understand its metabolism in the brain. Since Ap aggregation and deposition occurs in the extracellular space of the brain, we thought it would be very useful to be able to dynamically and specifically measure the concentration of Ap in the brain extracellular space over time. John accomplished this by developing a microdialysis system to measure Ap in the interstitial fluid (ISF) of the brain. This was a difficult task, as microdialysis enables one to assess small molecules such as neurotransmitters much more readily than larger molecules such as peptides. Using a large pore size microdial-ysis probe (e.g., 38 kDa molecular weight cut-off) enabled us to recover Ap (4.4 kDa) from the ISF. Addition of bovine serum albumin (BSA) to the perfusion buffer was also required to prevent Ap from sticking to the tubing and collection vials. While albumin-containing buffers are generally very simple to prepare, handling and preparing the BSA just right for this application was a pain- staking process, however necessary, so that the BSA would not clog the small diameter microdialysis probe or membrane pores. Once the technique was optimized, we were able to measure the concentration of Ap in the ISF of the hippocampus and striatum in mouse models of AD up to every 30 minutes for 24-36 hours in the same mouse (Cirrito et al. 2003,2005a). Additionally, specially designed cages allowed the mice to be awake and behaving throughout the experiment, allowing us to show that the half-life of total Ap species in the hippocampal ISF of human amyloid precursor (APP) transgenic mice was very short, 1-2 hours. This finding was interesting given that, once Ap forms plaques, some of the Ap within these structures may have a half-life of months or even years. We also showed that, in mice with plaques, there was a prolongation of ISF Ap clearance, most likely secondary to the fact that there is a dissociable pool of Ap in plaques that can re-enter the soluble

Dept. of Neurology1, Psychiatry2, Molecular Biology & Pharmacology3, the Hope Center for Neurological Disorders4, and Alzheimer's Disease Research Center5, Washington University School of Medicine, St. Louis, MO 63110

phase when ISF Ap levels decline. The microdialysis technique has also proven very useful in determining whether endogenous and exogenous molecules influence ISF Ap metabolism in vivo. For example, we found that endogenous apolipoprotein E (an Ap chaperone) affects the overall level of ISF Ap and Ap half-life (DeMattos et al. 2004). In addition, it was shown that an inhibitor of the molecule P- glycoprotein was able to increase ISF Ap over hours, suggesting that this molecule is involved in Ap transport out of the brain via the blood-brain barrier (Cirrito et al. 2005b).

In addition to our own findings, several pieces of information convinced us that it would be important to determine whether neuronal or synaptic activity in some way was linked with the concentration of ISF Ap in vivo. First, in the 1990s, Gouras, Gandy, and colleagues (1997), studying patients who had a temporal lobe removed as part of surgery for temporal lobe epilepsy, found that a large percentage of these individuals who were less than 50 years of age had amyloid plaques in their hippocampus (Gouras et al. 1997). This finding is otherwise very uncommon before the age of 50, unless one has Down's syndrome or an autosomal dominant form of familial AD. While there are many possible reasons for this finding, one is that deposition of Ap was somehow linked to excessive electrical activity over time. Second, in 2003, Roberto Malinow's lab made several observations with organotypic brain slices cultures, including the fact that drugs that decrease neuronal activity decrease Ap in cell culture media over 24-48 hours and that drugs that increased neuronal activity had the opposite effect (Kamenetz et al. 2003). Third, findings from Buckner and colleagues demonstrated that the areas of the human brain that develop the most Ap plaques also have the highest basal rates of metabolic and synaptic activity, as measured by positron emission tomography (PET) and functional magnetic resonance imaging (fMRI), when individuals are not performing a specific mental task, the so-called "default state" (Raichle et al. 2001; Buckner et al. 2005). If activity in brain regions that make up the "default state" network over a lifetime are the most active in the brain, the high level of activity in these areas may make them particularly susceptible to Ap deposition if there is a positive relationship between synaptic activity and Ap levels. Fourth, physical and cognitive activity can alter plaque burden later in life. In APP transgenic mice, it was found that physical and cognitive enrichment results in altered Ap plaque burden (Jankowsky et al. 2003, 2005a; Adlard and Cotman 2004; Lazarov et al. 2005b). One of several possibilities for this effect is that physical and cognitive enrichment alters synaptic activity (and hence Ap levels) in specific brain regions.

With these facts as a backdrop, we asked whether neuronal or synaptic activity was in some way dynamically linked with extracellular levels of Ap in the brain in vivo. We utilized one of the most studied APP transgenic mouse models for these experiments, Tg2576 (APPsw) mice (Hsiao et al. 1996). Young mice (three- to four-months old), several months prior to the appearance of plaques, were studied to assess Ap metabolism without the complexity of plaques being present. We thought a key experiment was to determine whether direct electrical stimulation of a defined anatomical pathway would alter ISF Ap. Through a collaboration with Bob Sloviter at the University of Arizona and both Kel Yamada and Steve Mennerick at Washington University, John Cirrito worked out a method whereby he could electrically stimulate the perforant pathway to cause a focal seizure in the synaptic terminal zone of this pathway (the hippocampus) while simultaneously monitoring hippocampal EEG activity and ISF Ap (Fig. 1a). Immediately after the onset of electrical stimulation that elicited a focal

Eeg Pathway

Fig. 1. Electrical stimulation of the perforant pathway increases ISF Ap levels. (a) Diagram of the hippocampus showing an electrode stimulating the perforant pathway and both recording electrodes and a microdialysis probe in the hippocampus. (b) Representative traces of basal EEG activity (top) and epileptiform discharges during electrical stimulation of the perforant pathway (bottom) in three- to five-month-old Tg2576 mice. (c) When EEG activity was elevated, ISF Ap levels increased by 133.3 ± 19.7% (p = 0.05; n = 5). Modified with permission from Cirrito et al., 2005. p.p., perforant pathway

Fig. 1. Electrical stimulation of the perforant pathway increases ISF Ap levels. (a) Diagram of the hippocampus showing an electrode stimulating the perforant pathway and both recording electrodes and a microdialysis probe in the hippocampus. (b) Representative traces of basal EEG activity (top) and epileptiform discharges during electrical stimulation of the perforant pathway (bottom) in three- to five-month-old Tg2576 mice. (c) When EEG activity was elevated, ISF Ap levels increased by 133.3 ± 19.7% (p = 0.05; n = 5). Modified with permission from Cirrito et al., 2005. p.p., perforant pathway seizure (Fig. 1b), there was a 30% increase in ISF Aß (Fig. 1c). We then determined the effects of decreasing neuronal and synaptic activity. Direct administration into the hippocampus by reverse microdialysis of tetrodotoxin, which blocks action potentials but not all synaptic activity, resulted in a very rapid 30% decrease in ISF Aß (Fig. 2a). Tetanus toxin decreases synaptic activity by blocking synaptic vesicle release. Direct hippocampal infusion of tetanus toxin resulted in an 80% decrease in ISF Aß by 18 hours after infusion (Fig. 2b). Finally, using brain slices derived from APPsw transgenic

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Fig. 2. Synaptic activity and synaptic vesicle release are linked with ISF Ap levels and neuronal release of Ap. (a) Following local treatment with tetrodotoxin (TTX), ISF Ap1—C levels declined, reaching 70.4 ± 4.5% of baseline at 16 hours (p < 0.0001; n = 5). (b) 0.2 ^g tetanus toxin was injected directly into the hippocampus surrounding the microdialysis probe to inhibit synaptic vesicle release. By 18 hours following treatment, ISF Ap levels declined significantly compared to baseline (p = 0.0003, n = 4). The lag time between treatment and an effect on Ap levels is likely due to the time necessary for the toxin to enter the cell and effectively cleave the synaptic vesicle associated protein VAMP2. (c) Acute brain slices were made from four- to five-week-old Tg2576 mice. To determine the affect of synaptic vesicle exocytosis on extracellular Ap levels in the absence of synaptic activity, Tg2576 brain slices were cultured for two hours in the presence of 0.5 nM a-latrotoxin and/or a cocktail of activity inhibitors including 100 nM TTX, 10 ^M NBQX, and 50 ^M APV. a-Latrotoxin alone caused a 35 ± 6.9% increase in Ap levels whereas the inhibitor cocktail lowered Ap levels by 18.0 ± 4.1% compared to untreated slices. a-Latrotoxin plus the inhibitor cocktail resulted in 13.3% more extracellular Ap as compared to untreated slices and 38.3 ± 6.2% more Ap compared to the inhibitor cocktail alone (n = 12-15 per group). Modified with permission from Cirrito et al. 2005

mice, we administered a-latrotoxin, which results in massive synaptic vesicle release without depolarizing the cell. a-Latrotoxin was administered in the absence or presence of blockers of neuronal activity and neurotransmitter receptors to differentiate between the effects of vesicle exocytosis and neuronal activation. Even in the presence of blockers of neuronal and synaptic activity, there was a rapid 30% increase in Ap in the media surrounding the brain slices (Fig. 2c). Together, these results strongly argue that synaptic vesicle release is linked, either directly or indirectly, with the release at the synapse of Ap in vivo. Current evidence suggests that APP/Ap is not in synaptic vesicles (Ikin et al. 1996; Marquez-Sterling et al. 1997). APP is co-internalized from the plasma membrane with synaptic vesicle integral membrane proteins such as synap-tophysin and synaptotagmin, then sorted away from those proteins and incorporated into distinct vesicles (Marquez- Sterling et al. 1997). This suggests that synaptic vesicle membrane recycling and APP endocytosis are linked. APP endocytosis is directly linked to Ap generation and release (Koo and Squazzo 1994). While synaptic vesicle exocytosis can rapidly modulate extracellular Ap levels, it may actually be an associated event, such as membrane recycling or another process, that is directly responsible for the rapid modulation of extracellular Ap levels.

Taken together, these findings suggest that synaptic activity, and specifically synaptic vesicle release, directly results in Ap release into the brain extracellular space and is likely an important mechanism regulating the ISF level of Ap in vivo. The implications of these findings are several. First, neurotransmitter and neurotransmitter receptor modulators are likely to directly regulate ISF Ap levels through modulation of synaptic activity, suggesting that such modulators may provide new drugs to alter ISF Ap and the downstream process of Ap aggregation. Second, synaptic activity that occurs within specific vulnerable networks of the brain such as the "default state" network may be very relevant to the onset and amount of Ap deposition that occurs there and hence the pathogenesis of AD. Overall, further exploration into the relationship between synaptic activity, Ap levels, and development of AD pathology may lead to new insights into the underpinnings of AD and future therapies.

Acknowledgements. This work was supportedby National Institutes of Health grants AG13956, AG11355 (DMH), and DA07261 (JRC), an Alzheimer's Association Zenith Award (DMH), the Blanchette Hooker Rockefeller Fund, MetLife Foundation (DMH), and Eli Lilly and Co.

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