Amyloid β oligomers enhance presynaptic exocytosis via CaV2.1 to drive disease progression in Alzheimer’s models

doi:10.21203/rs.3.rs-1638917/v1

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Amyloid β oligomers enhance presynaptic exocytosis via Ca_V2.1 to drive disease progression in Alzheimer’s models

https://doi.org/10.21203/rs.3.rs-1638917/v1

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Mixed outcomes in clinical trials of amyloid β-lowering agents for Alzheimer’s disease (AD) make the identification of alternative candidate molecular targets for therapy a priority. However, limited understanding of molecular pathways mediating the effects of amyloid β on synaptic and cognitive function hampers these efforts. Here, we uncover an ENaC-Ca_V2.3-PKC-GSK-3β signal transduction pathway that is engaged by oligomeric amyloid β (Aβ_o)to enhance presynaptic Ca_V2.1 voltage-gated Ca²⁺ channel activity, resulting in pathological potentiation of action potential-evoked synaptic vesicle exocytosis. Normalization of presynaptic function by pharmacological Ca_V2.1 inhibition or genetic Ca_V2.1 haploinsufficiency rescues Aβ_o-induced loss of dendritic spines and synaptic long-term potentiation ex vivo, and prevents spine loss, memory deficits and premature mortality in vivo, demonstrating a critical role for enhanced Ca_V2.1-driven presynaptic exocytosis in synaptic and cognitive decline. These findings reveal a previously unrecognized mechanism driving disease progression in AD, and identify multiple tractable potential therapeutic targets.

Biological sciences/Neuroscience/Cell death in the nervous system

Biological sciences/Neuroscience/Synaptic transmission/Synaptic vesicle exocytosis

Health sciences/Diseases/Neurological disorders/Neurodegeneration

Biological sciences/Neuroscience/Molecular neuroscience

Alzheimer’s disease (AD) is increasing in prevalence in the aging population, and the identification of effective disease-modifying treatments remains a priority. Numerous lines of evidence suggest that amyloid β (Aβ) peptides, in particular Aβ oligomers (Aβ_o), are a key trigger of the synaptic toxicity and cognitive decline that characterize AD¹. Exposure to Aβ_o produces multiple effects on different cell types², with alterations in neuronal synaptic transmission being among the earliest observed³. These alterations include enhanced excitatory glutamatergic synaptic activity in cortico-hippocampal neuronal networks³ and impaired synaptic plasticity at glutamatergic synapses¹, both of which precede the appearance of overt histopathology and are considered to be the key cellular substrates of early cognitive impairment in AD. Cognitive decline later in the disease process, on the other hand, is most strongly associated with synapse loss⁴.

Because of the critical role of Aβpeptides in AD pathogenesis, Aβ depletion has long been seen as a promising treatment strategy for AD, and substantial effort and resource has been deployed in the development of anti-amyloid therapeutics. A number of these have now reached clinical trials but the results have been mixed, with many agents failing while a small number have shown modest benefits, albeit with significant side-effect profiles. While these results therefore support a central role for Aβ in pathogenesis, they also indicate that it is a highly challenging therapeutic target⁵. An alternative strategy is to elucidate the pathogenic signaling events downstream of Aβ, which are currently incompletely understood, in the hope of identifying novel candidate drug targets for therapeutic intervention. To this end, we set out to investigate the cellular mechanisms underlying the effects of Aβ_o on synaptic transmission in well-characterized AD model systems, and to explore the significance of these mechanisms in the development of the canonical AD-associated synaptic and cognitive phenotypes. We found that Aβ_o engage a novel, ENaC-directed presynaptic signal transduction pathway that upregulates the activity of Ca_V2.1 voltage-gated channels, thereby increasing action potential-evoked Ca²⁺ influx and exocytotic neurotransmitter release. Normalization of presynaptic function by pharmacological or genetic suppression of Ca_V2.1 rescues key synaptic and cognitive phenotypes in ex vivo and in vivo AD models, indicating that Ca_V2.1-mediated dysregulation of presynaptic exocytosis is a critical event in AD pathogenesis. Our results provide mechanistic insight into how Aβ_o elicit canonical disease-related phenotypes, and identify a number of potential targets for therapeutic intervention in AD.

Aβ _o induce a robust enhancement of evoked synaptic vesicle exocytosis in hippocampal neurons due to increased Ca²⁺ entry through presynaptic Ca_V2.1 channels

We first studied the effects of acute applications of a pathologically relevant Aβ_o preparation (Supplementary Fig. 1a) on synaptic transmission using patch-clamp recordings in cultured rat hippocampal pyramidal neurons. A two hour incubation with 200 nM Aβ_o induced an increase in overall excitatory glutamatergic synaptic activity, while inhibitory activity was unchanged (Supplementary Fig. 1b-d). This could potentially be explained by either changes in the amplitude or duration of action potentials in excitatory glutamatergic neurons, or a change in neuronal excitability. However, we found that none of these parameters were altered by Aβ_o treatment (Supplementary Fig. 1e-i), although we note that these recordings were made at the soma and it remains formally possible that the voltage waveform at the presynaptic terminal, which is not feasible to record directly, could be different. We then focused on the regulation of neurotransmitter release at the presynaptic terminal, which we imaged in neurons expressing a high-resolution fluorescent reporter of synaptic vesicle exocytosis, synaptophysin-pHluorin (SypHy)⁶ (Fig. 1a). Incubation with Aβ_o produced a > 40% potentiation of exocytosis in response to a 10 Hz stimulus train (Fig. 1b-d), an effect that was similar across a wide range of Aβ_o concentrations (Supplementary Fig. 2a,b). To confirm that this effect does not reflect changes at inhibitory terminals, we expressed a pHluorin fused to the vesicular GABA transporter (vGAT-pHluorin) under control of the GAD67 promoter, which restricts expression to GABAergic synapses⁷. This showed no change in exocytosis following Aβ_o incubation (Supplementary Fig. 2c-e).

During the repetitive action potential firing elicited by stimulus trains, the total amount of exocytosis at each synapse is regulated by multiple parameters including the rate of replenishment of the pool of available synaptic vesicles and the probability that any single action potential will drive the exocytosis of one or more release-competent vesicles, known as the probability of release. Therefore, to refine possible targets of Aβ_o action, we examined exocytosis in response to single action potential stimuli directly using SypHluorin 2x (SypH 2x), a derivative of SypHy with enhanced sensitivity⁸. The average single stimulus SypH 2x response measured over 10 trials was around 40% greater in neurons exposed to Aβ_o, indicating that this treatment robustly enhances probability of release (Fig. 1e,f).

Probability of neurotransmitter release at hippocampal boutons is set by action potential-evoked Ca²⁺ influx through presynaptic voltage-gated Ca²⁺ channels (VGCC)⁹. We studied boutonal Ca²⁺ transients using the presynaptically-localized Ca²⁺ reporter SyGCaMP5 (Fig. 1g). Focusing on single stimulus-evoked events, which lie within the linear range of this probe¹⁰, we observed an approximately 10% increase in Ca²⁺ influx in the presence of Aβ_o (Fig. 1h,i). We used optical fluctuation analysis, which analyses trial-to-trial variation in boutonal Ca²⁺ transients to determine whether changes in Ca²⁺ influx are due to changes in the number or properties of VGCC¹¹. This demonstrated that the enhanced presynaptic Ca²⁺ influx was due to an increase in unitary Ca²⁺ channel currents rather than the number of available channels or their opening probability (Fig. 1j,k). In addition to altered Ca²⁺ influx, changes in probability of release can also be driven by changes in the proximity of VGCC to the neurotransmitter release machinery⁹. We tested this using the slow Ca²⁺ chelator EGTA. If the chelator is present in excess, and therefore non-saturable, % inhibition of exocytosis will depend on the distance between the Ca²⁺ channel pore and the release sensor (synaptotagmin), as well as the chelator’s Ca²⁺ binding kinetics¹². Importantly, it will not be sensitive to the total Ca²⁺ influx through the channel¹², which changes following Aβ_o exposure. We found that a brief incubation in a set concentration of cell-permeable EGTA-AM (200 µm for 90 seconds) depressed exocytosis in response to a single stimulus to a lesser extent following Aβ_o incubation (Supplementary Fig. 2f,g), indicating tighter physical coupling of VGCC to release machinery. Together, these changes can account fully for the enhancement of synaptic vesicle exocytosis by Aβ_o, particularly given the highly non-linear, co-operative relationship between Ca²⁺ influx and vesicular neurotransmitter release at mammalian hippocampal synapses¹³.

Ca²⁺ influx at glutamatergic hippocampal terminals is predominantly dependent upon a combination of Ca_V2.2 and Ca_V2.1 VGCCs¹⁴, and we used the specific Ca²⁺ channel blocking peptides ω-conotoxin GVIA (Ca_V2.2) and ω-agatoxin IVA (Ca_V2.1) to dissect the relative contribution of these channels to the Aβ_o-mediated enhancement of Ca²⁺ entry. We found that the enhancement is abolished only in the presence of ω-agatoxin IVA (Fig. 1h,i), indicating that it is mediated by Ca_V2.1, but not Ca_V2.2 VGCCs. There was no difference in the Ca²⁺ signal following Aβ_o incubation when both Ca_V2.1 and Ca_V2.2 were blocked, indicating that non-Ca_V2.1/Ca_V2.2 Ca²⁺ sources do not contribute (Fig. 1h,i). To confirm that enhanced Ca²⁺ entry specifically mediated via Ca_V2.1 drives the increase in synaptic vesicle exocytosis, we used VGCC-blocking peptides with SypHy-expressing neurons to show that Ca_V2.1, but not Ca_V2.2, channels are necessary for Aβ_o-mediated potentiation of the response to a 10 Hz stimulus train (Fig. 1l,m).

Ca_V2.1 function is potentiated via a presynaptic ENaC-Ca_V2.3-PKC signaling axis

We set out to identify the molecular signaling pathway engaged by Aβ_o to enhance Ca_V2.1 function. We previously reported that a similar specific upregulation of Ca_V2.1 function without change to Ca_V2.2 underlies the potentiation of evoked synaptic vesicle exocytosis in the context of homeostatic synaptic plasticity (HSP)¹⁵, a physiological process that regulates the strength of synapses to maintain activity in neuronal networks within set bounds¹⁶. Potentiation of synaptic vesicle exocytosis by HSP at both Drosophila and mammalian synapses requires the insertion of a pre-existing intracellular pool of the epithelial sodium channel ENaC into the presynaptic membrane^17,18, and we hypothesized that this might be a conserved mechanism for Ca_V2.1-dependent regulation of presynaptic strength that could also underlie the presynaptic effects of Aβ_o. Using immunofluorescence on non-permeabilized and permeabilized neurons in culture to disclose surface and total presynaptic ENaC respectively, we found that while the total presynaptic ENaC level did not change over the time course of the experiments, there was robust insertion of channels into the presynaptic membrane in response to Aβ_o (Fig. 2a-c). To then confirm that ENaC activity is necessary for the ability of Aβ_o to enhance exocytosis, we showed that the specific ENaC blocker amiloride¹⁹ was able to rescue the effects of Aβ_o treatment in SypHy-expressing neurons (Fig. 2d). In these and subsequent experiments, we chose to assay Ca_V2.1-driven changes in synaptic vesicle exocytosis rather than examining Ca²⁺ influx directly because the power law relationship between these two parameters¹³ lends experimental measurements of exocytosis much greater sensitivity and robustness.

ENaC channels carry a Na⁺ leak current that causes a modest, chronic depolarization of the bouton¹⁸. This could directly augment action-potential evoked depolarization and thereby Ca²⁺ influx through VGCC or, alternatively, ENaC could enhance Ca_V2.1 function indirectly via recruitment of a signaling pathway. We sought to distinguish between these two possible modes of action by performing a second set of ENaC blockade experiments in which amiloride was not present during the Aβ_o incubation, allowing potential signaling events downstream of ENaC depolarization to take place, but was applied immediately before stimulation and imaging and stimulation to abolish any direct contribution of ENaC-mediated depolarization to exocytosis. This failed to rescue the effects of Aβ_o (Fig. 2d), suggesting that signaling downstream of channel insertion is necessary. This result is in keeping with the specific enhancement of Ca_V2.1, but not Ca_V2.2, function by ENaC insertion; if direct effects of depolarization on channel function were responsible, a similar enhancement of both VGCC types would be expected since they have similar gating properties²⁰.

Chronic, subthreshold depolarization similar to that mediated by ENaC can initiate intracellular signaling by mobilizing intra- or extracellular Ca²⁺ sources to raise cytoplasmic [Ca²⁺]. Resting [Ca²⁺] was determined at SyGCaMP5-expressing boutons from the GCaMP5 signal²¹, and we found that Aβ_o elicited a clear [Ca²⁺] increase (Fig. 2e). We considered possible Ca²⁺ source(s) that could support this rise including intracellular stores and presynaptic VGCCs, particularly subtypes with a low activation voltage that are more efficiently opened by subthreshold depolarization such as Ca_V3 (T-type) or Ca_V2.3 (R-type)²⁰; we focused on the latter as Ca_V3 is not expressed at glutamatergic hippocampal terminals²². We found that incubation with either BAPTA, to chelate extracellular Ca²⁺, or the Ca_V2.3 blocking peptide SNX-482 abolished the Aβ_o-mediated rise in resting cytoplasmic [Ca²⁺], indicating that intracellular stores are not sufficient for this but Ca_V2.3 are required (Fig. 2e). We then directly linked Ca_V2.3 to the effects of Aβ_o on synaptic vesicle exocytosis by using SypHy-expressing neurons to show that SNX-482 abolishes potentiation of the response to a 10 Hz stimulus train (Supplementary Fig. 2h). Previous work has suggested that Aβ_o can activate presynaptic mGluR5 receptors²³, which could potentially also contribute to a rise in cytoplasmic [Ca²⁺]. We therefore tested the potential involvement of this mechanism, finding that mGluR5 blockade with the specific antagonist SIB-1757 did not prevent the increase in exocytosis seen following Aβ_o exposure (Supplementary Fig. 2h).

Intracellular Ca²⁺ rises can activate Ca²⁺-sensing proteins, which in turn can trigger functional changes in a variety of substrates. Aside from the exocytotic Ca²⁺ sensors that mediate excitation-secretion coupling, the predominant Ca²⁺-sensing proteins expressed presynaptically are protein kinase C (PKC)²⁴ and calmodulin (CaM), which directly regulates the activity of a wide variety of proteins including CaM kinase II, calcineurin and Ca_V2.1²⁵. We have shown that Aβ_o exposure produces a rise in presynaptic cytoplasmic [Ca²⁺] of approximately 60nM (Fig. 2e), far short of the [Ca²⁺] elevation required to activate CaM, which is well into the micromolar range²⁶. PKC, on the other hand, is activated by nanomolar presynaptic [Ca²⁺] rises very comparable to that we have demonstrated²⁷. To ask whether Aβ_o activate presynaptic PKC, we prepared synaptosomes, which comprise the complete presynaptic terminal along with the postsynaptic density, from cultured hippocampal neurons and subjected these to various treatments before assessing PKC activity in synaptosomal lysates with a specific ELISA-based assay. We found that Aβ_o treatment increased PKC activity in the synaptosomes (Fig. 2f), and this effect was blocked by the inclusion of SNX-482 (Supplementary Fig. 2i), corroborating an essential upstream role of Ca_V2.3 in Aβ_o-linked PKC activation. Finally, to confirm that PKC activity is necessary for the ability of Aβ_o to enhance exocytosis, we applied the PKC inhibitor Go 6976 to SypHy-expressing neurons, showing that it rescued the enhancement of synaptic vesicle exocytosis by Aβ_o consistent with an essential role for PKC in this process (Fig. 2d). Since resting presynaptic [Ca²⁺] varies among even boutons on the same axon²⁸, this mechanism would be consistent with independent regulation of release probability at individual synapses.

Upregulation of Ca2.1 activity by Aβ requires GSK-3β and α7-nAChR

Next, we sought a mechanistic link between PKC activation and enhanced Ca_V2.1 function. Our data show that following exposure to Aβ_o, the increased in Ca_V2.1-driven neurotransmitter release is due to changes in both unitary Ca²⁺ currents and physical coupling of the channels to release machinery. In VGCC of the Ca_V2 family, both can be achieved via the phosphorylation of a partially conserved intracellular amino acid sequence known as the synprint (synaptic protein interaction) site, which interacts directly with the SNARE proteins that mediate synaptic vesicle exocytosis⁹. While a number of kinases are known to phosphorylate the Ca_V2.2 synprint site⁹, the Ca_V2.1 synprint sequence is different and just one Ca_V2.1 synprint kinase has been reported to date, glycogen synthase kinase-3β (GSK-3β), which phosphorylates Ca_V2.1 to suppress both Ca²⁺ currents and SNARE protein binding²⁹. It is itself inactivated via phosphorylation at residue serine 9 by a variety of kinases including PKC³⁰. To test its involvement, we first used immunofluorescence to assess the proportion of GSK-3β within hippocampal presynaptic terminals that is S9-phosphorylated and therefore inactivated, and found that this increases following Aβ_o treatment (Fig. 3a,b). We then co-expressed SypHy together with a constitutively active mutant form of GSK-3β that is not subject to phosphoregulation at serine 9³¹ and showed that Aβ_o was no longer able to enhance exocytosis (Fig. 3c,d), indicating that negative phosphoregulation of GSK-3β is necessary for this effect.

To complete the pathway linking Aβ_o to Ca_V2.1, we sought to connect Aβ_o to the mobilisation and membrane insertion of ENaC, a process which is generally poorly understood, although it is known to be Ca²⁺-dependent³². While the Ca²⁺ source driving ENaC insertion in neurons is not known, nicotinic acetylcholine receptors containing the Ca²⁺-permeable α7 subunit (α7-nAChR) are a strong candidate since they have been reported to bind Aβ peptides with picomolar affinity and are expressed on hippocampal presynaptic boutons, where they can be directly activated by Aβ_o to produce a Ca²⁺ rise³³. To study the binding of Aβ_o to presynaptic α7-nAChR, we used CRISPR/Cas9 gene editing to knock out α7-nAChR expression in cultured hippocampal neurons, confirming the efficacy of the CRISPR/Cas9 construct in abolishing expression of the protein at the cell surface with a fluorescently tagged high-affinity ligand, α-bungarotoxin CF594 (Fig. 3e). We showed that axons and boutons of neurons lacking surface α7-nAChR bound significantly less Aβ_o conjugated with the fluorophore tetramethyl rhodamine (TAMRA) (Fig. 3e,f), a tag that has previously been shown to have no effect on the proportions of different Aβ assembly states present in Aβ_o preparations made according to our protocol³⁴. We also used untagged α-bungarotoxin to test whether α7-nAChR function is necessary for the ability of Aβ_o to enhance exocytosis, and found that while α-bungarotoxin treatment alone did not alter exocytosis in response to a 10 Hz stimulus train, it completely prevented the effects of Aβo (Fig. 3g). Thus, an initial nAChR-mediated Ca²⁺ rise is converted, via the recruitment of ENaC, to a chronic, depolarization-driven cytoplasmic [Ca²⁺] elevation that ensures efficient activation of the Ca²⁺ sensor, in this case PKC, in order to sustain a chronic effect on Ca_V2.1 activity.

Together, the data support a signaling pathway in which Aβ_o elicits presynaptic Ca²⁺ entry via α7-nAChR that drives the insertion of an intracellular pool of ENaC channels into the presynaptic membrane. These channels mediate a chronic depolarization that, by activating Ca_V2.3, elevates basal cytoplasmic [Ca²⁺] to activate PKC; this then phosphorylates and inactivates the negative Ca_V2.1 regulator GSK-3β, thereby potentiating Ca_V2.1 function and neurotransmitter release (Fig. 3h).

Ca _V 2.1 suppression prevents Aβ _o -induced synaptic depression, inhibition of long-term potentiation and dendritic spine loss in the hippocampus

We then addressed the critical question of whether enhanced Ca_V2.1 activity and the resultant increase in synaptic vesicle exocytosis could play a causal role in AD pathogenesis. Specifically, we hypothesized that an increase in presynaptic neurotransmitter release mediated by enhanced Ca_V2.1 activity might, via alterations in synaptic activity, drive pathological changes in the postsynaptic neuron that could account for key synaptic and cognitive phenotypes of AD. This mechanistic scheme would place upregulation of Ca_V2.1 activity as one of the most upstream events in the pathological cascade triggered by Aβ_o. First, we used our dissociated neuronal culture system to examine the effect of two interventions that mitigate the impact of enhanced Ca_V2.1 activity and neurotransmitter release, either Ca_V2.1 blockade with ω-agatoxin IVA or pan-glutamate receptor blockade, on the depression of postsynaptic AMPAR number and synaptic strength that follow exposure to Aβ_o and represent the early stages of the postsynaptic pathway leading to synapse loss³⁵. As expected, incubating cultured hippocampal neurons with Aβ_o for 24–36 hours produced a reduction in surface synaptic AMPAR, and this was prevented by addition of either ω-agatoxin IVA or a cocktail of glutamate receptor blockers (Fig. 4a,b). To verify that the loss of surface AMPAR corresponds to a depression in the postsynaptic AMPA response, we recorded miniature excitatory postsynaptic currents (mEPSCs), choosing these rather than evoked currents so that Aβ_o-induced changes in presynaptic strength would not bias the results. To study changes in NMDAR function, we recorded mixed mEPSCs and took advantage of the different kinetics of AMPAR and NMDAR responses to separate the currents (Supplementary Fig. 3a)³⁶. Aβ_o induced decreases in both AMPAR and NMDAR-mediated mEPSC amplitudes, which were prevented by the addition of ω-agatoxin IVA (Supplementary Fig. 3b-f). These data indicate that Ca_V2.1 is required for the Aβ_o-mediated depression of postsynaptic function in cultured neurons.

Field EPSP recordings at CA3-CA1 (Schaffer collateral) synapses in Aβ_o-incubated acute hippocampal slices allowed us to explore a potential causal role of Ca_V2.1 activity in important disease-associated phenotypes in a more physiologically intact setting (Fig. 4c). To first validate our findings in cultured neurons in this system, we examined the effects of Aβ_o treatment on basal synaptic transmission and paired-pulse ratio (PPR), an electrophysiological index of the probability of release. Because Aβ_o cause desensitization of synaptic AMPA receptors³⁷, which can profoundly affect measurement of both synaptic transmission and PPR at CA3-CA1 synapses^37–39, we included cyclothiazide, an inhibitor of AMPAR desensitization, in these initial experiments. Application of Aβ_o produced a rapid increase in fEPSP responses (Fig. 4d,e) and a reduction in PPR (Fig. 4f) that together indicate elevated synaptic transmission due to enhanced probability of release. These effects were both prevented by inhibition of Ca_V2.1 with ω-agatoxin IVA (Fig. 4d,e and g). We then asked whether the well-established ability of Aβ_o to inhibit synaptic long-term potentiation (LTP)⁴⁰, a form of synaptic plasticity that is required for learning⁴¹, could be dependent on Ca_V2.1-enhanced release. Because we were concerned that full Ca_V2.1 inhibition could weaken the inducing stimulus sufficiently to alter the magnitude of the resulting LTP, we titrated a lower dose of ω-agatoxin IVA that reduced probability of release (Supplementary Fig. 4a,b) to mitigate the effect of Aβ_o, yet did not affect LTP (Fig. 4h,i). We found that, as expected, Aβ_o treatment alone severely diminished LTP. However, the inclusion of ω-agatoxin IVA completely prevented this (Fig. 4h,i). To examine the role of the presynaptic Ca_V2.1 regulatory pathway we uncovered in cultured neurons in these effects, we tested the involvement of ENaC using the blocking drug amiloride. We found that treatment with this drug alone had no effect on either PPR, and consequently release probability, or LTP in acute slices, yet it was able to rescue the effects of Aβ_o on both, indicating a critical role for ENaC-mediated release enhancement in the Aβ_o-mediated inhibition of LTP (Supplementary Fig. 4c-e).

Finally, we studied Aβ_o-associated synapse loss, for which we turned to organotypic hippocampal slices that enable more chronic interventions. Prolonged (> 5 days) Aβ_o incubation leads to a loss of spines, which represent the sites of excitatory glutamatergic synapses, on tertiary dendrites of CA1 pyramidal neurons in organotypic hippocampal slices⁴². Consistent with this, we found a reduced density of spines on tertiary dendrites of GFP-expressing CA1 neurons in slice cultures following a 7-day Aβ_o incubation. This was fully rescued in the presence of ω-agatoxin IVA, which itself had no effect on spine density (Fig. 4j,k). Together, these results indicate that the enhancement of neurotransmitter release, inhibition of LTP and loss of dendritic spines mediated by Aβ_o in the hippocampus are all dependent on Ca_V2.1 activity.

Synaptic vesicle exocytosis is enhanced at CA3-CA1 synapses in hAPP transgenic mice

To explore the role of Ca_V2.1-mediated alterations in synaptic function in AD pathogenesis in vivo, we used an established and well-characterized AD model mouse line, J20, which carries a human APP transgene bearing two pathogenic mutations⁴³. We first sought to confirm whether hippocampal neurons in 4–8 month old hAPP transgenic mice show evidence of elevated neurotransmitter release by measuring PPR at CA3-CA1 synapses in acute hippocampal slices, using a variety of interpulse intervals to increase the sensitivity of the study. As previously, we included cyclothiazide in these experiments to prevent confounding effects due to AMPA receptor desensitization³⁷. We found that PPR at 25 and 50 ms intervals was significantly reduced in hAPP mice compared to wild-type littermate controls, indicating enhanced probability of release (Fig. 5a). We also recorded the synaptic input-output relationship as a measure of the strength of basal synaptic transmission and found that this was impaired (Fig. 5b). This suggests that a depression of postsynaptic function and/or a loss of synapses, both of which are well-documented in in vitro and in vivo models of AD¹, is superimposed on the increased probability of release and is of sufficient magnitude to outweigh its effects on overall synaptic strength.

To circumvent any potential confounding effects of postsynaptic changes that could influence our measurements, we then used the styryl dye FM 1–43 to image synaptic vesicle exocytosis directly in native hippocampal tissue⁴⁴. Acute hippocampal slices were prepared from 4–8 month old mice and CA3 axons were stimulated while the dye was applied to CA1 to label the total recycling pool of synaptic vesicles (Fig. 5c). A subsequent 5 Hz stimulus train revealed significantly faster dye unloading in hAPP vs. littermate control slices (Fig. 5d,e). These results corroborate the electrophysiological data indicating enhanced neurotransmitter release in hAPP transgenic mice.

Lowering Ca _V 2.1 expression normalizes neurotransmitter release in hAPP mice and preserves synaptic plasticity following Aβ _o exposure

To examine the role played by Ca_V2.1 in enhancing neurotransmitter release in AD model mice in vivo, we crossed the hAPP J20 line with transgenic mice bearing a heterozygous deletion of the gene encoding the pore-forming α1 subunit of Ca_V2.1 VGCC (Cacna1a^+/−)⁴⁵. While homozygous Cacna1a ablation results in severe ataxia, seizures and premature mortality⁴⁵, mice carrying a heterozygous deletion lack any readily apparent phenotype⁴⁶ and, importantly, display normal synaptic properties including input-output relationships, probability of release, short-term synaptic facilitation and excitation-spike coupling (Supplementary Fig. 5a-e). Therefore, partial suppression of Ca_V2.1 activity seems to be very well-tolerated by mice over their lifespan. However, synaptic transmission in Cacna1a^+/− slices shows a reduced dependence on Ca_V2.1 (Supplementary Fig. 5f), and we therefore hypothesized that if enhanced Ca_V2.1 activity mediates the effects of Aβ_o on neurotransmitter release, these effects would be ameliorated by the Cacna1a^+/− genotype. We confirmed that this is the case using FM 1–43 in acute hippocampal slices, finding that dye unloading, and therefore evoked synaptic vesicle exocytosis, at CA3-CA1 synapses in both Cacna1a^+/− and hAPP/Cacna1a^+/− mice was nearly identical to that seen at wild-type synapses (Fig. 5d,e). We also tested the effects of Aβ_o on LTP in acute slices from Cacna1a^+/− mice. While incubation with Aβ_o strongly inhibited hippocampal LTP in wild-type slices, it had no effect on LTP in Cacna1a^+/− slices (Supplementary Fig. 5g,h). This result underscores the critical importance of increased Ca_V2.1 activity and evoked synaptic vesicle exocytosis in Aβ_o-mediated LTP impairment, extending our earlier findings using a pharmacological manipulation (Fig. 4h,i).

Evidence of enhanced Ca2.1 function in hAPP mouse and human AD brains

We asked whether Ca_V2.1 function is enhanced in human AD brains. Because it is not possible to assess this directly in post mortem material, we instead took advantage of the tighter physical coupling of Ca_V2.1 to the neurotransmitter release machinery that is part of the upregulation of Ca_V2.1 function by Aβ_o (Supplementary Fig. 2f,g). The strength of Ca_V2.1 binding to syntaxin 1, a SNARE protein that mediates vesicular neurotransmitter release, provides a measure of this coupling that can be assessed by co-immunoprecipitation. Accordingly, Ca_V2.1 was immunoprecipitated from cortical synaptosomal membrane samples prepared from a cohort of AD patients and non-demented control subjects (Supplementary Table 1). Immunoprecipitates were subjected to electrophoresis and Western blotting with antibodies against Ca_V2.1 and syntaxin 1. This showed that Ca_V2.1 binds more syntaxin 1 in AD brains than those of control subjects (Fig. 5f,g), indicating a stronger Ca_V2.1-SNARE association. We corroborated this observation in aged hAPP mice, supporting their relevance to human disease (Supplementary Fig. 6a,b). We noted incidentally that our Western blots showed an apparent global reduction in the expression of presynaptic proteins in AD patients (Fig. 5f), which is expected since extensive loss of presynaptic terminals is a feature of advanced AD⁴⁷.

Ca2.1 reduction prevents cognitive impairments, synapse loss and premature mortality in hAPP mice

We then tested whether Ca_V2.1-mediated enhancement of neurotransmitter release is necessary for the development of disease-relevant cognitive phenotypes in AD model mice. The hAPP/Cacna1a^+/− mouse line is well suited for this purpose as the Cacna1a^+/− genotype normalizes evoked synaptic vesicle exocytosis via suppression of Ca_V2.1 function (Fig. 5d,e), but does not otherwise impact synaptic function (Supplementary Fig. 5a-e). It also confers resistance to Aβ_o-mediated inhibition of LTP (Supplementary Fig. 5g,h), which is widely considered to be a key cellular correlate of memory⁴¹. Consequently, we assessed hippocampus-dependent learning and memory phenotypes in naïve mice at 6–8 months of age using three different protocols. Habituation of locomotor activity to a novel environment, measured by a reduction in exploratory activity, provides a simple measure of nonassociative learning⁴⁸ that has been shown to be deficient in AD model mice⁴⁹. The spatial novelty preference Y-maze and aversively-motivated Y-maze swim escape task assess hippocampus-dependent short-term spatial memory⁵⁰ and associative long-term spatial memory⁵¹, respectively. In all tests, the performance of hAPP mice was significantly impaired, while Cacna1a^+/− and, crucially, hAPP/Cacna1a^+/− mice performed at a similar level to wild-type (Fig. 6a-f). Memory deficits in hAPP mice are therefore dependent upon the Ca_V2.1-mediated enhancement of presynaptic function.

We used Golgi-Cox staining to assess dendritic spine density in CA1 pyramidal cells of the hippocampus in the cohort of animals used for behavioral testing. The hAPP mice showed a significant reduction in dendritic spine density on tertiary branches compared to wild-type that was not seen in either Cacna1a^+/− or hAPP/Cacna1a^+/− mice (Fig. 6g,h), confirming both the dependency of synapse loss on Ca_V2.1 function and the synapto-protective effect of Ca_V2.1 reduction. Finally, we assessed the effects of Cacna1a haploinsufficiency on survival. Many hAPP mouse lines, including J20, die early, possibly from seizure activity⁵². Heterozygous knockout of Cacna1a completely prevented premature mortality (Fig. 6i).

Alzheimer’s disease represents a major unmet need in human disease therapeutics. We set out to investigate molecular mechanisms underlying the dysregulation of synaptic transmission by Aβ_o, one of the earliest events in AD, in the hope of identifying novel candidate targets for therapeutic engagement. We uncover a presynaptic Aβ_o signaling pathway that converges on the Ca²⁺ channel Ca_V2.1, enhancing action potential-evoked Ca²⁺ currents and thereby potentiating evoked synaptic vesicle exocytosis. Partial suppression of Ca_V2.1 normalizes synaptic function and confers robust protection against a full sequence of pathophysiological events including synaptic plasticity deficits, synapse loss, cognitive changes and premature mortality. Our data demonstrate a critical role for enhanced Ca_V2.1 activity in AD-relevant phenotypes in vivo and provide a robust basis for the development of therapies targeting Ca_V2.1 to prevent or delay synaptic decline and cognitive impairment in AD.

Current evidence supports a view of AD as the final outcome of a complex series of events representing interactions between a variety of cell types in the brain, including neurons, astrocytes and inflammatory microglia². However, the nature and sequence of these events is incompletely understood, and it is unclear which of the many changes that can be observed at relatively early stages in AD models play a seminal role in pathogenesis, and are therefore potentially therapeutically relevant. In particular, it has not been known whether the reported derangements in synaptic transmission triggered by Aβ, which include a number of functional changes in neurotransmitter release^23,53–56, play an active role in driving synaptic and cognitive decline¹. In this study, we show that Aβ_o-mediated upregulation of the probability of release via the inappropriate functional enhancement of a single Ca²⁺ channel subtype drives postsynaptic depression, leading eventually to synapse loss and learning and memory deficits. This represents a novel paradigm of AD pathogenesis, although it is supported by a report of VGCC-dependent enhancement of synaptic vesicle exocytosis in a Drosophila model of Huntington’s disease that is linked to neurodegeneration⁵⁷. Although Ca_V2.1 VGCC drive release at both excitatory and a subset of inhibitory synapses, we show that the effects of Aβ_o on presynaptic function are restricted to the former. The most likely reason for this relates to known differences in the regulation of Ca_V2.1 function at excitatory vs. inhibitory synapses; this is evident in the observation that certain human disease-associated Ca_V2.1 mutations produce, similar to the effects of Aβ_o described here, an enhancement of excitatory neurotransmission with no change in GABA release, although the underlying mechanisms are not well understood⁵⁸.

How might such a change in presynaptic function lead to AD-associated synaptic and cognitive phenotypes? Increased neurotransmitter release across populations of synapses can lead to more efficient activation of postsynaptic NMDA receptors in extrasynaptic locations via glutamate escape from the synaptic cleft⁵⁹. One function of extrasynaptic NMDA receptors is to mediate the induction of long-term depression (LTD)³⁷, a physiological process of activity-induced synaptic weakening, and we suggest that increased glutamate release following Aβ_o exposure activates these receptors to generate a chronic drive towards LTD that initially causes weakening of synapses, potentially progressing to synapse loss (Supplementary Fig. 7). Consistent with a role for LTD in AD pathogenesis, we recently demonstrated that the inappropriate or excessive induction of LTD in vitro can drive the pathological hyperphosphorylation of tau, a critical downstream step in Aβ_o-mediated synaptic toxicity⁶⁰.

We identify a signaling pathway linking Aβ_o to enhanced Ca_V2.1 activity that implicates a number of ion channels and signaling kinases in novel or non-canonical functional roles. While in vivo regulation of Ca_V2.1 by a variety of different presynaptic interactors including calmodulin, G protein-coupled receptors and SNAREs is well-recognised⁹, regulation via GSK-3β-mediated phosphorylation of the synprint site has only been described as a consequence of non-physiological manipulations in vitro²⁹ and has not previously been implicated in a specific physiological or pathological pathway. Here, we begin to fill this knowledge gap by demonstrating a role for GSK-3β regulation of Ca_V2.1 in AD pathogenesis. Further upstream in the signaling pathway, we implicate Ca²⁺ entry via Ca_V2.3, itself opened by ENaC-mediated depolarization, in the activation of PKC, which then phosphorylates and negatively regulates GSK-3β. The distinct molecular properties of Ca_V2.3, which opens at significantly more negative potentials than the principal presynaptic VGCC subtypes Ca_V2.1 and Ca_V2.2, suggest that it may be particularly amenable to activation by modest, subthreshold depolarization²⁰ such as that mediated via ENaC at the presynaptic terminal¹⁸. Presynaptic Ca_V2.3 has previously been implicated mainly as a trigger of spontaneous neurotransmitter release, a role that also exploits its greater probability of opening at close to resting membrane potential⁶¹. Here, however, its special properties allow it to function in the novel context of an intracellular signal transduction pathway, where it serves to link subthreshold membrane depolarization to the upregulation of kinase activity.

The identification of a role for ENaC in AD pathogenesis has particular significance. While ENaC is just beginning to be explored in the brain, it is relatively well-studied in the periphery, where it is expressed in a number of tissues including renal tubular epithelium. Here, it is the target of potassium-sparing diuretic drugs, such as amiloride, that are widely used in clinical practice for the management of hypertension¹⁹. Epidemiological studies have revealed that this particular class of anti-hypertensive agents confers robust protection against the development of AD^62,63, although no mechanistic basis for this effect has ever been identified. Our data now suggest a potential explanation for this important observation. While further studies will be required to gain a fuller understanding of ENaC function in the context of the CNS, our results provide a conceptual basis for this work and identify ENaC as a potential therapeutic target in AD. This raises the possibility that drugs with a protective effect against disease development, a critical goal of AD research, could already be licensed and available.

Competing Interests

The authors declare no competing interests.

Author Contributions

AFJ designed experiments, performed experiments, analyzed data and wrote the paper. ZP, HT, CMW, HC, SA and WJF performed experiments and analyzed data. DB designed and supervised behavioral experiments. WLK characterized and contributed Aβ_o preparations. AMJMvdM contributed the Cacna1a knockout mouse line. NJE designed experiments and wrote the paper. All authors revised the manuscript.

Acknowledgements

We thank Leon Lagnado and Yongling Zhu for gifts of plasmids, and the members of the Emptage laboratory for comments on the manuscript. We also thank Chris Barkus for help with the behavioral experiments. This work was supported by an MRC (UK) Clinician Scientist Fellowship (G0802812) and Centenary Award to AFJ.

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Preparation, characterization and use of Aβ oligomers

Oligomers were prepared from Aβ_1-42peptide (American Peptide) as previously described⁶⁴. Briefly, solid Ab_1-42 was dissolved in cold hexafluoro-2-propanol (HFIP; Sigma). The peptide was incubated at room temperature for at least 1 hour to establish monomerization and randomization of structure. The HFIP was aliquoted and allowed to evaporate overnight, followed by 10 minutes in a Savant Speed Vac. The resulting peptide was stored as a film at -80 °C. The film was dissolved in anhydrous dimethylsulfoxide (Sigma) to 5 mM, diluted to 100 mM with Ham’s F12 (without phenol red, with glutamine; Caisson Laboratories) and briefly vortexed. The solution was incubated at 4 °C for 22-24 hours and soluble oligomers obtained by centrifugation at 14,000 g for 10 minutes at 4 °C. Protein concentration was estimated using Coomassie Plus Protein Assay (Thermo Scientific) and a bovine serum albumin (BSA) standard. For shipping purposes, small aliquots of the soluble oligomers were dried using a Savant Speed Vac and reconstituted with cold sterile water and gentle pipetting immediately prior to use. Tetramethyl rhodamine (TAMRA)-conjugated Aβ_1-42(Cambridge Bioscience/AnaSpec) was subjected to the same protocol to prepare fluorescently-tagged oligomers.

Hippocampal cell culture, adenoviral infection and plasmid transfections

Dissociated hippocampal cultures were prepared from E18 Wistar rat embryos (Charles River Laboratory) as already described¹⁵. SypHy (kind gift of Prof. L. Lagnado) was cloned into an adenoviral expression vector using the Ad-HQ system (Vector Biolabs, Philadelphia, PA, USA), and packaged to produce an active human Adenovirus Type 5 (dE1/E3). This was used to infect cultured hippocampal neurons 8 days after plating at a multiplicity of infection (MOI) of 0.2. Other plasmids were introduced into neurons 8 days after plating using Lipofectamine 2000. For each well, DNA and Lipofectamine were added to 200 mL Neurobasal at a ratio of 3 mg:3 mL and incubated for 20 minutes before transfection, which was carried out in 2 mL of medium per well. Transfection mix was incubated with the cultures for 1 hour before being removed and replaced with a 2:1 ratio of conditioned to fresh medium. The SypH 2x plasmid was a gift of Dr. Y. Zhu, the GAD67^pro-vGAT-pH plasmid was a gift of Prof. S. H. Kim and SyGCamP5 was a gift of Prof. L. Lagnado. The CRISPR/Cas9 plasmids were generated by the Weatherall Institute of Molecular Medicine Genome Engineering Facility (University of Oxford) and based on the vector px458 into which sequences coding for sgRNA directed against either the firefly luciferase gene (control) or mouse Chrna7 (g13)⁶⁵ were cloned.

Patch-clamp electrophysiology in cultured hippocampal neurons

Whole-cell patch recordings were made using either an Axoclamp 900A or an Axoscope 2B amplifier (both Axon Instruments). Data was low-pass filtered at 3k Hz, sampled at >10kHz. Data was acquired using WinWCP and analyzed using Clampfit software. Patch electrodes (4-8 MΩ) contained (in mM): 120 CsMeSO₄, 10 KCl, 10 NaPhosphocreatine, 10 HEPES, 4 MgATP and 0.4 Na₃GTP. Spontaneous activity was recorded in Tyrode’s solution containing (in mM): 120 NaCl, 30 Glucose, 25 HEPES, 5.4 KCl, 1.5 CaCl₂and 0.5 MgCl₂. Miniature excitatory postsynaptic currents (mEPSC) comprising of mixed AMPAR and NMDAR currents were recorded at -70 mV in Mg²⁺-free conditions using Tyrodes solution containing (in mM): 120 NaCl, 30 Glucose, 25 HEPES, 5.4 KCl, 4 CaCl_2,0 MgCl₂, to which 1 µM of TTX and 100 µM of picrotoxin was added. For each cell recorded, 30 isolated mEPSCs were chosen for analysis. As previously described³⁶, AMPAR currents were measured at the peak of the mEPSC trace and NMDAR currents were measured by averaging the peak current across a 5 ms window, 15 ms following the mEPSC peak.

Live cell imaging and analysis

Experiments were performed 14-21 days after plating (6-13 days after transfection) when synapses are mature. Coverslips were mounted in a Chamlide EC-B18 stimulation chamber (Live Cell Instrument) on the stage of an Olympus IX-71 inverted microscope fitted with a 100X, NA 1.40 UPlanSApo objective and an Andor iXon EM CCD camera, and imaging of live neurons was carried out as described¹⁵. Phluorin imaging was carried out at 1 Hz for 100 AP stimulation and 5 Hz for 1 AP stimulation; Ca²⁺ imaging was carried out at 10 Hz. All time series images were acquired with 2 X 2 pixel binning. Unless otherwise specified, Aβ_o were applied at 200 nM (monomer equivalent) in culture medium in the incubator for 2 hours. Where cells had been incubated with Aβ_o or vehicle, these were present throughout the experiment. Where the following were used they were added 10 minutes before Aβ_o or vehicle treatment and were present throughout the incubation and experiment: w-Agatoxin IVA (100 nM, Alomone); w-conotoxin GVIA (250 nM, Alomone); amiloride (100 μM, Sigma); BAPTA (5 mM tetrapotassium salt, Molecular Probes); SNX-482 (500 nM, Alomone); SIB-1757 (3 μM, Tocris); Go 6976 (100 nM, Abcam); α-bungarotoxin (100 nM, Life Technologies). In experiments measuring basal Ca²⁺ concentration, ionomycin (Calbiochem) was used at 10 μM following initial image acquisition to elicit maximal Ca²⁺ entry; the maximal signal value obtained is used to normalize the basal Ca²⁺signal measurements to account for differences in SyGCamP5 expression. In experiments imaging binding of either α-bungarotoxin CF594 (100 nM, Cambridge Bioscience/Biotium) or TAMRA-conjugated Aβ_o (200 nM), which were applied in the incubator for 10 minutes or 2 hours respectively, the image acquisition system described above was used but with a 900 ms exposure time and no pixel binning.

Time series images were analyzed in ImageJ (http://rsb.info.nih.gov/ij) using the Time Series Analyzer plugin (http://rsb.info.nih.gov/ij/plugins/time-series.html). All visible varicosities were selected for analysis with a 2 mm diameter ROI. Terminals were excluded from analysis in pHluorin experiments if their maximum response to 40 AP or 100 AP was less than 2 SD of baseline noise; for Ca²⁺ experiments, terminals were excluded if their peak response to 1 AP averaged over 5 trials was less than 2 SD of baseline noise, or of they did not show a response to ionomycin application. Data exported from ImageJ were background adjusted and pHluorin data were normalized to the peak signal obtained following NH₄Cl application (mean value of plateau over 5 seconds). Ca²⁺ indicator data were normalized to either the basal, unstimulated signal or to the signal elicited by ionomycin. Peak fluorescence in all experiments was taken at the end of the stimulation period. For analysis of single images generated during Aβ_o binding experiments, segmentation was first carried out using inbuilt ImageJ functionality to generate a mask from a 50 μm section of axon in the GFP channel, and this was then used to measure mean fluorescence in the red fluorophore channel with additional background adjustment. All analysis was performed using custom-written macros or scripts in Microsoft Excel or MatLab.

Optical fluctuation analysis

Optical fluctuation analysis analyses trial-to-trial variation in Ca²⁺transients through VGCC to determine whether changes in Ca²⁺influx are due to changes in the number (N), open probability (p) or unitary channel currents (q) of functional channels¹¹. We used the experimentally derived inverse squared coefficient of variation (CV^-2) of boutonal Ca²⁺ transients along with a published value for p at cultured hippocampal terminals⁶¹ to calculate the mean N at each terminal under control conditions (N = 19.5). We then extrapolated both N and p to values expected if the experimentally observed increase in mean Ca²⁺transient size following Aβ_o treatment were solely a result of an increase in either of these parameters alone, and used these to calculate the CV^-2that would be expected after Aβ_o treatment in each instance. Note that in optical fluctuation analysis, changes in N or p (or some combination of these) are associated with a change in CV^-2, while changes in q are not.

Immunofluorescence and quantification

Cultured neurons were subjected to relevant treatments before being washed twice with PBS; where used, Aβ_o were applied at 200 nM for 2 hours. For all experiments except surface ENaC staining, coverslips were then treated with 0.1% saponin on ice for 15 minutes, before incubation in 10% FCS in PBS at room temperature for 30 minutes. Cells were then incubated with guinea pig anti-synaptophysin (1:1000; Synaptic Systems) together with either anti-GSK-3β (CST 9832, 1:1000; Cell Signaling Technology) and anti-phospho-GSK-3β (S9) (ab131097 1:500; Abcam), or anti-ENaC α-subunit (SPC-403, 1:1000, StressMarq). For surface ENaC staining, the procedure was identical except that the primary antibody incubations were carried out in succession, anti-ENaC antibody first, and the permeabilization step was carried between the two. Following primary antibody incubation, coverslips were washed, incubated for 1 hour at room temperature with various AlexaFluor-conjugated secondary antibodies (Abcam), all used at 1 in 400 dilution, washed again and mounted using ProLong Gold antifade (Thermo Fisher). For double immunostaining of surface GluR1 receptors and synaptophysin, live neurons were incubated with rabbit anti-GluR1 (pc246, Calbiochem) diluted with well medium for 1 hour in a 5% CO₂ incubator at 37 °C, followed by 1 hour with 2 ml of medium to wash out unbound and nonspecifically bound antibodies. The cells were then fixed in 4% paraformaldehyde for 15 minutes on ice before incubating with an anti-rabbit Cy-3 conjugated IgG (Jackson Immunoresearch) for 1 hour at room temperature. This was followed by block/permeabilisation in 10% foetal calf serum/0.1% Triton X-100 for 60 minutes at room temperature and incubation with guinea pig anti-synaptophysin (Synaptic Systems) at 4 C overnight. Alexa 488-conjugated anti-guinea pig IgG (Jackson Immunoresearch) was then applied at room temperature for 1 hour, and coverslips were mounted using ProLong Gold antifade.

Images were collected using an Olympus Fluoview FV1000 confocal system with an Olympus IX-81 inverted microscope, and either a 100X, NA 1.40 UPlanSApo or a 60X, NA 1.35 UPlanSApo oil immersion objective. Images were acquired in Olympus Fluoview software and analyzed using ImageJ as described in the main text or caption. Image segmentation was carried out using inbuilt ImageJ functionality to generate a mask from the synaptophysin (green) channel, and this was then used to measure mean fluorescence in the red fluorophore channel. For GluR1 labelling experiments, 50 μm sections of dendrite were examined. For all experiments, image acquisition parameters, as well as any image thresholding applied, were fixed within an experiment to allow for comparison between conditions.

Protein kinase C activity assay

Synaptosomes were prepared 14 days after plating from individual 35 mm culture wells of hippocampal neurons cultured as described above. Syn-PER reagent (Thermo Fisher) was used according to the manufacturer’s instructions, and each pellet representing synaptosomes from a single well was resuspended in 100 μl Syn-PER, before this was then divided into three parts that were exposed to either a vehicle, Aβ_o (200 nM) or Aβ_o + SNX-482 (500 nM) treatment for 2 hours before immediate freezing at -80 °C. Protein concentration in each sample was assessed with a BCA assay, and PKC activity in the samples was then assessed with an ELISA-based kit (ADI-EKS-420A, Enzo Life Sciences) used according to manufacturer’s instructions. Based on preliminary experiments with a purified PKC standard included with the kit, a 0.1 μg total protein equivalent of each sample was loaded per assay well, as this amount gave results within the dynamic range of the assay, and all samples were assayed in duplicate.

Preparation of acute hippocampal slices and slice electrophysiology

Field excitatory postsynaptic potentials (fEPSPs) were recorded in 300 μm thick acute hippocampal slices prepared as described⁶⁶ from 7 - 8 week old C57BL/6J mice, or from aged, genotyped mice as specified. Slices were placed in an interface recording chamber perfused with oxygenated ACSF at 1 - 2 mL/min, and a bipolar stimulating electrode (FHC Inc., Bowdoin, ME, USA) was placed in Schaffer collaterals to deliver test and conditioning stimuli. For most experiments, a borosilicate glass recording electrode filled with artificial cerebrospinal fluid was positioned in stratum radiatum of CA1; for the fEPSP–population spike coupling experiment, the recording electrode was placed in stratum pyramidale to better capture population spike firing. Test responses, to stimuli delivered at 0.067 Hz, were recorded for at least 10 minutes prior to beginning experiments to ensure stable responses. Field potentials were amplified using a Digitimer NeuroLog amplifier, filtered below 3 Hz and above 3 KHz and digitized with a BNC-2090A converter (National Instruments). Recording was carried out on WinWCP software and and analyzed using the Clampfit program. LTP was induced using a 20X theta-burst protocol comprising a block of 4 stimuli at 100 Hz repeated 20 times over 20 seconds. For drug treatments, slices were incubated for > 2 hours prior to the experiment and drugs were maintained in the perfusing ACSF for the duration of the recording. Concentrations used were as follows: w-agatoxin IVA (Alomone Labs) 400 nM, or 50 nM for LTP experiments only; Ab oligomers 10 nM; cyclothiazide (Abcam) 100 μm; amiloride (Sigma) 100 μM. Other than these, drugs were not included in the experiments in order to preserve intact neuronal circuits. The magnitude of fEPSPs was determined as the gradient of the rising slope to avoid population spike contamination. Paired-pulse ratios were obtained by delivering two stimuli at intervals as specified and expressed as fEPSP2/fEPSP1.

Organotypic hippocampal slice preparation, adeno-associated virus infection and dendritic spine quantification

Organotypic hippocampal slices were prepared from day 7 postnatal male Wistar rats as previously described⁶⁷ and used at 21-28 DIV. We took advantage of the Cre-lox expression system, which gives sparse but strong expression of eGFP, for spine counting. Accordingly, a mixture of two adeno-associated viruses was used: AAV1.CAG.Flex.eGFP.WPRE.bGH (2.35 x 10¹³ GC/mL) and AAV1.hSyn.Cre.WPRE.hGH (1.47 x 10⁸ GC/mL). Both were obtained from the University of Pennsylvania Vector Core. Slices were infected at 14 DIV using a patch pipette to deliver ~1 μl aliquots into CA1 directly, and left for 7-10 days to allow strong expression. Aβ_o were applied at 200 nM for chronic incubation. Slices were imaged on an Olympus BX50WI microscope fitted with a BioRad Radiance 2000 confocal scanhead. For each dendrite, a z stack of 21 images at 512 x 512 pixels was acquired at 1 μm intervals using Zeiss LaserSharp software, and 50 μm sections were analyzed with ImageJ.

Mouse lines

All mouse work was carried out in accordance with the Animals (Scientific Procedures) Act, 1986 (UK) and under project and personal licenses approved by the Home Office (UK). Both the Cacna1a knockout (from Prof. A. van den Maagdenberg, Leiden University Medical Centre) and J20 hAPP (kind gift of Prof. D. Anthony, University of Oxford) transgenic mouse lines were on a C57BL/6J background, and the lines were maintained by crossing heterozygotes or hemizygotes with wild-type C57BL/6J mice. Crossing hAPP hemizygote with Cacna1a^+/- mice yielded all four of the genotypes used in in vivo assays, and animals used in these experiments were therefore littermates. For co-immunoprecipitation experiments only, another mouse line carrying, like J20, an APP transgene with Swedish and Indiana mutations, was used. Line B6.Cg-Tg(tetO-APPSwInd)102Dbo/Mmjax was crossed with a CamKIIα-tTA line to activate hAPP expression⁶⁸, and 20 month old double transgenic (tTA/hAPP) individuals along with accompanying non-transgenic littermate controls, a kind gift of Dr. Mariana Vargas-Caballero, were sacrificed for removal of the cortex and hippocampus. All genotyping was carried out by Transnetyx Inc.

FM dye loading and unloading in acute hippocampal slices

Slices of 300 μm thickness were transferred to a custom-made recording chamber mounted on an Olympus BX50WI microscope fitted with a BioRad Radiance 2000 confocal scanhead (BioRad/Zeiss) and were superfused at 35°C with oxygenated aCSF supplemented with 10 μM NBQX and 50 μM APV (both Tocris) to block recurrent activity. A patch pipette was filled with a 20 μM solution of the styryl dye FM1-43 (Molecular Probes) in aCSF and placed in stratum radiatum of CA1 at a depth of approximately 100 μm. The dye was pressure applied for 3 minutes using a custom-made picospritzer before a 10 Hz train of 1200 stimuli (100 μA) was delivered to Schaffer collaterals using a glass stimulating electrode (4-8 MΩ) filled with 150 mM NaCl placed within 70 μm of the dye-filled pipette; the stimulating electrode was under the control of WIN WCP software (Strathclyde Electrophysiology Software) and a DS3 stimulation box (Digitimer). Pressure application of dye was maintained throughout the loading stimulus and for 2 minutes afterwards to ensure completion of endocytosis. Slices were then perfused continuously in fresh aCSF containing 0.2 mM ADVASEP-7 (Cambridge Bioscience) for 15-20 minutes to wash residual FM dye from extracellular membranes. Imaging of labeled terminals was performed using a 60X, NA 1.1 LUMFL N objective (Olympus), a 488 nm Argon laser for excitation and a 500 nm long-pass emission filter. Image stacks were acquired every 15 seconds throughout the unloading stimulus (3000 stimuli at 10 Hz). Each image stack comprised 6 images of 512 x 512 pixels acquired at 1 μm intervals in the z-axis, and a digital zoom of 3X and 2X Kalman averaging were applied. Images were acquired using Zeiss LaserSharp software and analyzed using ImageJ and custom-written scripts in MatLab.

Human tissue

Samples of human frontal cortex were obtained from the MRC London Neurodegenerative Diseases Brain Bank, King’s College, London (part of the Brains for Dementia Research initiative) with full ethical approval; 5 AD cases, all BrainNet Europe stage or modified Braak stage VI, and 5 age-matched controls, all BrainNet Europe or Braak stage I or II, were used (see Table S1).

Co-immunoprecipitation

For co-immunoprecipitations, approximately 500 mg of either human or mouse brain tissue was homogenized using a Dounce tissue grinder into 2 mL buffer A containing (in mM): 320 sucrose, 5 Tris base, pH 7.4, 2 EDTA, on ice; note that all buffers used throughout the immunoprecipitations included HALT protease and phosphatase inhibitor cocktail (Thermo Scientific) at 1:100. After a 10 minute centrifugation at 750 rpm, the supernatant was removed and centrifuged at 17,000 rpm for 1 hour. This yielded a membrane pellet which was resuspended in 1 mL buffer A + 1% CHAPS, and 300 μL of this was rotated for 3 hours at 4°C with Novex magnetic Protein G Dynabeads (Life Technologies) to which anti-Ca_V2.1 or antibody had previously been bound (see below). After incubation, the beads were retrieved by magnetic separation and washed twice in buffer B containing (in mM): 150 NaCl, 25 Tris base, pH 7.4, 2 EDTA, to which 0.5% BSA and 0.4% CHAPS had also been added. Beads were then washed once in buffer B alone before being resuspended in 100 μL 2X Laemmli sample buffer with 5% β–mercaptoethanol for Western blotting. For binding of antibodies to Protein G Dynabeads, the supplied buffer was removed from 50 μL of bead suspension, and beads were washed once in buffer A + 0.5% BSA + 0.4% CHAPS. 5μL of anti-Ca_V2.1 antibody (as used for Western blotting) was then added, and the bead/antibody mixture was rotated for 10 minutes at room temperature before beads were washed twice in buffer A + 0.5% BSA.

Western blotting

Samples were dissolved in 2X Laemmli sample buffer with 5% β–mercaptoethanol, heated to 60°C for 3 minutes and run on a precast 4-20% gradient SDS-PAGE gel (Thermo Scientific). The separated samples were transferred to a nitrocellulose membrane (Bio-Rad) before blocking with 5% milk and 1% horse serum in TBS with 0.05% Tween-20 (TBST) and subsequent probing with a mixture of anti-Ca_V2.1 (ACC-001, 1:200; Alomone Labs) and anti-stx1 (110 011, 1:1000; Synaptic Systems) antibodies. After three TBST washes, bound antibodies were detected with IRDye 680LT donkey anti rabbit IgG (Li-Cor, 1:20,000) and IRDye 800CW goat anti-mouse IgG (Li-Cor, 1:15,000) fluorescent secondary antibodies, washed three times in TBST and imaged on a Li-Cor Odyssey system. Image analysis was performed in Li-Cor Image Studio Lite software.

Mouse behavioral testing

Locomotor activity in a novel environment was assessed in clear plastic cages (26 x 16 x 17 cm), containing clean sawdust. Mice were allocated a different cage each day. Activity levels were assessed by an array of infrared photobeam sensors that covered the x- and y-axes of the cages (San Diego Instruments). Beam breaks were recorded in 12 bins of 5 minutes over a one hour session to assess total ambulatory activity and this was repeated over three days. Results for each animal over the three days were averaged.

To assess hippocampus-dependent spatial novelty preference, a perspex Y-maze with arms of 30 x 8 x 20 cm was placed into a room containing a variety of extramaze cues. Mice were assigned two arms (the ‘start’ and the ‘other’ arm) to which they were exposed during the first phase (the exposure phase), for 5 minutes. This selection of arms was counterbalanced with respect to genotype. Timing of the 5 minute period began only once the mouse had left the start arm. The mouse was then removed from the maze and returned to its home cage for a one minute interval between the exposure and test phases. During the test phase, mice were allowed free access to all three arms. Mice were placed at the end of the start arm and allowed to explore all three arms for 2 minutes beginning once they had left the start arm. An entry into an arm was defined as a mouse placing all four paws inside the arm. Similarly, a mouse was considered to have left an arm if all four paws were placed outside the arm. The times that mice spent in each arm were recorded manually and a novelty preference ratio was calculated for the time spent in arms [novel arm/(novel + other arm)].

To assess hippocampus-dependent spatial reference memory, mice were trained on an aversively motivated, Y-maze swim-escape task. Like the well-known Morris water maze (MWM), performance in this task relies on the ability to associate a particular spatial location with the hidden escape platform. However, we have shown previously that it is more sensitive to disruption of hippocampal CA1 synaptic plasticity than the classical open-field MWM⁶⁹, and it is also less susceptible to a variety of confounds. Performance is unlikely to be influenced by innate differences in preferred swimming distance from the side walls (thigmotaxis)⁵¹, which have been reported in mouse models of AD⁷⁰, or by apathy or floating behavior, which we observed in some of our mouse lines in preliminary experiments in the MWM. As it uses a fractional choice-accuracy measure, it is also insensitive to confounds associated with locomotor activity differences. The apparatus was a transparent acrylic Y-maze, with each arm measuring 30 cm in length and 8 cm in width. The maze was surrounded by a 20 cm high wall. The maze was filled with water, rendered opaque with white paint, to a depth of 12 cm. The water temperature was 20 ± 2°C. A variety of distal extramaze cues were positioned around the laboratory, which remained constant across all sessions. Mice could escape from the water by climbing onto a hidden platform positioned 1.5 cm beneath the water surface. The platform was consistently positioned at the end of one of the three arms, with the allocation of target arm counterbalanced across genotype groups. In all trials, mice were released at the end of one of the two non-target arms, facing towards the wall. The release arm was varied pseudo-randomly across trials and test sessions. Training trials were 90 seconds in duration and mice that did not locate the platform within this time were guided towards the platform by the experimenter. All mice completed a single training session each day for six consecutive days. Each session consisted of five consecutive trials, with an inter-trial interval of 45 seconds (30 seconds spent on the platform and 15 seconds drying time). During each training trial, first choice accuracy (i.e. whether the first arm entered was the target arm) was recorded. A probe trial was performed on day 7, during which mice were able to swim freely for 60 seconds with the platform removed, and the percentage of total time spent in each of the three maze arms was recorded.

Golgi-Cox staining

We used the FD Rapid Golgi Stain Kit (FD Neurotechnologies, Inc.) to label neurons in the CA1 region of hippocampus. Animals were sacrificed by cervical dislocation and brains were immediately removed and rinsed in distilled water. Brains were then sagittally bisected and one half of each was treated according to kit instructions before embedding in agarose and cutting. Brains were sectioned in the coronal plane at 100 μm thickness on a Leica VT 1000M vibratome. Sliced tissues were transferred onto gelatin-coated slides and were air dried at room temperature in the dark. After drying, sections were rinsed with distilled water, stained in the developing solution and dehydrated with 50%, 75%, 95%, and 100% ethanol. Finally, the sections were defatted in xylene substitute and coverslipped with Permount (Fisher Scientific). All images were collected using an Olympus Fluoview FV1000 confocal system with an Olympus IX-81 inverted microscope and a 100X, NA 1.40 UPlanSApo oil immersion objective was used. For each dendrite, a 7 μm pseudostack comprising 11 differential interference contrast (DIC) images was acquired using Olympus Fluoview software and 50 μm sections were analyzed using NeuronStudio (http://research.mssm.edu/cnic/tools-ns.html).

Statistical analysis

Statistical analysis was performed using GraphPad Prism software. Data were assessed for normality using the Shapiro-Wilk test, and analyzed using parametric or non-parametric tests accordingly. Unless otherwise stated, the two-tailed unpaired Student’s t-test was used to determine the statistical significance of observed differences between various conditions. Where other tests were used, this is clearly stated in the caption of the appropriate figure. P values greater than 0.05 were regarded as non-significant. For optical fluctuation analysis, we determined whether changes in N, p or q best described our data using the Bayesian Information Criterion (BIC), with a BIC difference of 10 or more providing strong evidence in favor of the model with the lowest BIC score⁷¹.

References

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Padamsey, Z. et al. Activity-Dependent Exocytosis of Lysosomes Regulates the Structural Plasticity of Dendritic Spines. Neuron 93, 132-146 (2017).
De Simoni, A. & Yu, L.M. Preparation of organotypic hippocampal slice cultures: interface method. Nat Protoc 1, 1439-45 (2006).
Jankowsky, J.L. et al. Persistent amyloidosis following suppression of Abeta production in a transgenic model of Alzheimer disease. PLoS Med 2, e355 (2005).
Bannerman, D.M. et al. Dissecting spatial knowledge from spatial choice by hippocampal NMDA receptor deletion. Nat Neurosci 15, 1153-9 (2012).
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Amyloid β oligomers enhance presynaptic exocytosis via Ca_V2.1 to drive disease progression in Alzheimer’s models

Status:

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Abstract

Figures

Main

Results

Ca_V2.1 function is potentiated via a presynaptic ENaC-Ca_V2.3-PKC signaling axis

Upregulation of Ca2.1 activity by Aβ requires GSK-3β and α7-nAChR

Synaptic vesicle exocytosis is enhanced at CA3-CA1 synapses in hAPP transgenic mice

Evidence of enhanced Ca2.1 function in hAPP mouse and human AD brains

Ca2.1 reduction prevents cognitive impairments, synapse loss and premature mortality in hAPP mice

Discussion

Declarations

Competing Interests

Author Contributions

Acknowledgements

References

Methods

Additional Declarations

Supplementary Files

Status:

Version 1

Amyloid β oligomers enhance presynaptic exocytosis via CaV2.1 to drive disease progression in Alzheimer’s models

Status:

Version 1

Abstract

Figures

Main

Results

CaV2.1 function is potentiated via a presynaptic ENaC-CaV2.3-PKC signaling axis

Upregulation of Ca2.1 activity by Aβ requires GSK-3β and α7-nAChR

Synaptic vesicle exocytosis is enhanced at CA3-CA1 synapses in hAPP transgenic mice

Evidence of enhanced Ca2.1 function in hAPP mouse and human AD brains

Ca2.1 reduction prevents cognitive impairments, synapse loss and premature mortality in hAPP mice

Discussion

Declarations

Competing Interests

Author Contributions

Acknowledgements

References

Methods

Additional Declarations

Supplementary Files

Status:

Version 1

Amyloid β oligomers enhance presynaptic exocytosis via Ca_V2.1 to drive disease progression in Alzheimer’s models

Ca_V2.1 function is potentiated via a presynaptic ENaC-Ca_V2.3-PKC signaling axis