Amyloid beta 42 oligomers induce neuronal and synaptic receptor dysfunctions.

Abstract

Amyloid beta 42 (Aβ42) oligomers are considered a major hallmark of Alzheimer's disease (AD) pathology (Mattson 2004). They are produced through the processing of the amyloid precursor protein (APP) via the amyloidogenic pathway. The amyloidogenic cascade is initiated through cleavage of APP by β-secretase, followed by sequential cleavage via γ-secretase, thus producing Aβ42 peptides (Mattson 2004). Aβ42 peptides are highly prone to aggregation in the extracellular space of neurons, and in the case of AD, are overproduced. Accumulation and aggregation of Aβ42 peptides results in the formation of oligomers, which are associated with neuronal dysfunction and death (Mattson 2004). Interestingly, Aβ42 has also been shown to promote dysregulation of calcium (Ca2+) homeostasis, eventually leading to synaptic dysfunction. The disruption of Ca2+ homeostasis has a profound effect on the processes of learning and memory formation that are implicated in AD pathology (Mattson 2004). A recent study published in The Journal of Physiology by Marcantoni et al. (2020) sought to investigate the impact of Aβ42 oligomers on NMDA synapses in the context of Ca2+ dysregulation. NMDA receptors (NMDARs) are particularly important in the promotion of neuronal plasticity and memory formation. Within AD pathology, NMDARs are found to be upregulated, resulting in an observed increase in intracellular Ca2+ ([Ca2+]i) levels and dysregulation of Ca2+ homeostasis. NMDAR upregulation is implicated in compromised synaptic function leading to neuronal death and therefore alterations in the normal physiological functions of NMDARs, which can significantly contribute to AD pathology. Also, ryanodine receptors (RyRs) are of particular interest due to their role in regulating synaptic activity as they partly control [Ca2+]i levels. Coupled with NMDARs, RyRs are involved in a mechanism which thereby increases [Ca2+]i levels, known as the calcium-induced calcium release (CICR) mechanism. To examine the impact of Aβ42 oligomers, Marcantoni et al. (2020) used hippocampal neurons isolated from 18-day-old C57BL/6 mouse embryos. NMDARs are known to be involved in spontaneous network excitability through activating glutamatergic synapses. In the presence of Aβ42 oligomers, glutamatergic synapses are impaired, which compromises their functional capacity. Given that Aβ42 oligomers have pre- and post-synaptic effects on NMDA synapses, it was deemed important to initially investigate the post-synaptic effects. The results showed that Aβ42 oligomers significantly reduce the inward currents associated with NMDARs (Marcantoni et al. 2020). However, the stoichiometry and binding affinity of those receptors were not impacted by the oligomers, which would indicate that glutamate was able to bind normally to NMDARs despite the Aβ42 treatment. Although the inward current of NMDARs experienced a decrease, the results also show that the unitary current of post-synaptic NMDARs was unaffected. This was explained by the idea that Aβ42 oligomers impair neuronal functioning by acting on NMDA synapses. Additionally, it was found that upon treatment with Aβ42, the total number of post-synaptic NMDARs was reduced, which may explain the reduction in inward current and could result in damage to synaptic plasticity and neuronal survival. These findings are relevant to AD pathology as the Aβ42-induced reduction in NMDARs reduces the influx of Ca2+, which as per previous research, reduces spine density and compromises learning and memory formation systems. Interestingly, it was found that the [Ca2+]i release facilitated by NMDARs and RyRs remained unchanged in Aβ42-treated neurons. This observed effect was explained by the fact that NMDA can also mediate [Ca2+]i release through a CICR coupling mechanism with RyR. The study confirmed the proposed explanation by showing that the proportion of [Ca2+]i released through the CICR mechanism remained unchanged despite the treatment with Aβ42, and when treated with the RyR inhibitor dantrolene, it was found that half of the CICR effects on [Ca2+]i can be attributed to RyRs (Marcantoni et al. 2020). In the study by Marcantoni et al. (2020), Aβ42 treatment of neurons was found to increase the frequency as well as size of electrically evoked EPSCs (eEPSCs) despite Gavello et al.’s (2018) finding of decreased eEPSC amplitude when treated with caffeine or Aβ42 when the eEPSCs are dependent on AMPAR activation. Consequently, Aβ42 oligomers were also involved in significantly increasing the pulse paired depression (PPD) of eEPSCs, as well as the synaptic depression during episodes of high-frequency (10 Hz) stimulation, and it is through the estimation of PPD that it is possible to measure the presynaptic neurotransmitter release probability. Therefore, it can be concluded that Aβ42 oligomers are the driving force behind the Ca2+-dependent potentiating effect facilitated by glutamatergic synapses. Additionally, following the Aβ42-induced reduction in Ca2+ influx, neurons reacted by activating compensatory mechanisms aiming to restore the total [Ca2+]i balance. In particular, it was deduced that these compensatory mechanisms have an indirect impact on the firing rate, resulting in increased regularity of spontaneous EPSCs. However, previous work by Gavello et al. (2018) showed conflicting findings in which Aβ42-treated neurons possessed a decreased synchronism of spontaneous firing of the hippocampal network. This discrepancy was postulated to be a result of differential SK channel contributions among the various neuronal properties, such as excitability and synaptic activity. Marcantoni et al. (2020) also found that treatment with Aβ42 resulted in a significant compromise of NMDA's capacity to maintain prolonged stimulation despite the observed increase probability of glutamate release, ready releasable pool size and number of release sites. The study by Marcantoni et al. (2020) is a strong continuation of the work of Gavello et al. (2018) but with more focus towards the functional link between NMDARs and RyRs. The study by Gavello et al. (2018) was broader, having included extensive testing of Aβ42 on voltage-gated calcium channels (VGCCs) and large-conductance Ca2+ activated potassium (BK) channels, as well as the effect of their respective inhibitors nifedipine and paxilline. VGCCs were previously found to be responsible for a similar proportion of the Ca2+ current as NMDARs, which the present authors also included in their study, as it was found that Aβ42 treatment resulted in a pre-synaptic potentiation of Ca2+ influx via L-type calcium channels (Gavello et al. 2018). Both Gavello et al. (2018) and Marcantoni et al. (2020) used the same in vitro protocol consisting of 18-day-old embryonic C57BL/6 hippocampal neurons treated with Aβ42, with the intention to simulate early AD. Utilization of this primary neuronal culture was beneficial for looking at the mechanistic effects of Aβ42 and could be replicated in the future with cortical neurons and a human cell line to further highlight the applicability of these findings to a human model of AD. The RyR inhibitor used by Marcatoni et al. (2020) was dantrolene, a drug that is primarily used in the periphery as a muscle relaxant in spastic disorders and malignant hyperthermia. Currently, the primary mode of administration for dantrolene is oral and there is significant evidence against its ability to readily cross the blood–brain barrier. If true, it is possible that the large and chronic doses required to elicit the potent effects displayed in this research may damage hepatocytes and alter liver function among a variety of other acute side-effects (Wang et al. 2020). Very recent testing has begun on dantrolene via the intranasal route with promising results for sustaining greater concentrations of the drug in the brain without observed side effects. Assuming chronically high levels of dantrolene are able to be tolerated by the brain, the ability of the drug to limit the increase of [Ca2+]i pre-synaptically will prove very therapeutically relevant. Current AD therapeutics, such as memantine, act post-synaptically by blocking NMDARs, and cannot prevent excess glutamate release. Cognitive effects of dantrolene could not be tested in the present study, and therefore the true potential of this pharmaceutical to combat AD in the early stages of the disease remains unknown, highlighting a future direction for this research (Wang et al. 2020). In the current report, Marcantoni et al. (2020) found that the number of post-synaptic NMDARs, as well as their functionality, was reduced in the presence of Aβ42 oligomers in primary hippocampal cultures. Also, with the utilization of dantrolene administration, the authors provided mechanistic evidence that the involvement of RyRs is approximately half of the NMDA/CICR effects on [Ca2+]i. Extensive quantification of these effects was performed at the synapse with eEPSC testing. It was found that Aβ42 had an inhibitory effect on post-synaptic NMDA synapses that opposes the pre-synaptic upregulation of glutamate release probability, among other findings secondary to elevated eEPSCs, in the same environment. This novel cellular evidence for the role of RyR inhibitors, such as dantrolene, highlights a new potential for these pharmaceuticals in diseases that require RyR regulation of Ca2+, such as AD. The findings from this study have also laid the groundwork for future investigations into Aβ42's effects on Ca2+ function in aged neurons and animal models of AD. This will help provide a complete picture of the role of Aβ42 in neuronal and synaptic function throughout the different stages of AD. None. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None. The authors would like to thank Dr Rebecca MacPherson for providing critical comments on the manuscript.