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. 2021 Oct 8;13(1):165.
doi: 10.1186/s13195-021-00901-9.

Activation of PLCβ1 enhances endocannabinoid mobilization to restore hippocampal spike-timing-dependent potentiation and contextual fear memory impaired by Alzheimer's amyloidosis

Jaedong Lee  1 , Jeehyun Kwag  2
Affiliations

Activation of PLCβ1 enhances endocannabinoid mobilization to restore hippocampal spike-timing-dependent potentiation and contextual fear memory impaired by Alzheimer's amyloidosis

Jaedong Lee et al. Alzheimers Res Ther. .

Abstract

Background: Accumulation of amyloid beta oligomers (AβO) in Alzheimer's disease (AD) impairs hippocampal long-term potentiation (LTP), leading to memory deficits. Thus, identifying the molecular targets of AβO involved in LTP inhibition is critical for developing therapeutics for AD. Endocannabinoid (eCB) synthesis and release, a process collectively called eCB mobilization by hippocampal CA1 pyramidal cells, is known to facilitate LTP induction. eCB can be mobilized either by postsynaptic depolarization in an intracellular Ca2+ concentration ([Ca2+]i)-dependent pathway or by group 1 metabotropic glutamate receptor (mGluR) activation in a phospholipase Cβ (PLCβ)-dependent pathway. Moreover, group 1 mGluR activation during postsynaptic depolarization, which is likely to occur in vivo during memory processing, can cause synergistic enhancement of eCB (S-eCB) mobilization in a PLCβ-dependent pathway. Although AβO has been shown to disrupt [Ca2+]i-dependent eCB mobilization, the effect of AβO on PLCβ-dependent S-eCB mobilization and its association with LTP and hippocampus-dependent memory impairments in AD is unknown.

Methods: We used in vitro whole-cell patch-clamp recordings and western blot analyses to investigate the effect of AβO on PLCβ protein levels, PLCβ-dependent S-eCB mobilization, and spike-timing-dependent potentiation (tLTP) in AβO-treated rat hippocampal slices in vitro. In addition, we assessed the relationship between PLCβ protein levels and hippocampus-dependent memory impairment by performing a contextual fear memory task in vivo in the 5XFAD mouse model of AD.

Results: We found that AβO treatment in rat hippocampal slices in vitro decreased hippocampal PLCβ1 protein levels and disrupted S-eCB mobilization, as measured by western blot analysis and in vitro whole-cell patch-clamp recordings. This consequently led to the impairment of NMDA receptor (NMDAR)-mediated tLTP at CA3-CA1 excitatory synapses in AβO-treated rat hippocampal slices in vitro. Application of the PLCβ activator, m-3M3FBS, in rat hippocampal slices reinstated PLCβ1 protein levels to fully restore S-eCB mobilization and NMDAR-mediated tLTP. In addition, direct hippocampal injection of m-3M3FBS in 5XFAD mice reinstated PLCβ1 protein levels to those observed in wild type control mice and fully restored hippocampus-dependent contextual fear memory in vivo in 5XFAD mice.

Conclusion: We suggest that these results might be the consequence of memory impairment in AD by disrupting S-eCB mobilization. Therefore, we propose that PLCβ-dependent S-eCB mobilization could provide a new therapeutic strategy for treating memory deficits in AD.

Keywords: Alzheimer’s disease; Contextual fear memory; Endocannabinoid mobilization; Hippocampus; Long-term potentiation; PLCβ1.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
AβO disrupts synergistic enhancement of endocannabinoid (S-eCB) mobilization. a Schematic of the S-eCB mobilization protocol: whole-cell voltage-clamp recording in CA1 pyramidal cells (PCs) to measure Schaffer collateral (SC) stimulation-evoked inhibitory postsynaptic potentials (eIPSCs) in rat hippocampal slices in vitro. S-eCB mobilization was induced by postsynaptic CA1 PC spikes (spike, at 1 Hz) with DHPG (50 μM) for 60 s. b, c Time course of the changes in mean eIPSC amplitudes following DHPG (empty circle, n = 8, b), DHPG + AM251 (3 μM) (filled circle, n = 7, b), spike + DHPG (empty circle, n = 7, c), and spike + DHPG + AM251 (filled circle, n = 8, c) in DMSO-treated rat hippocampal slices. d Mean of normalized eIPSC amplitudes of the first 10 s after S-eCB mobilization protocol in b and c. DHPG or spike + DHPG (empty bar), DHPG + AM251, or spike + DHPG + AM251 (dotted bar). e Same as c, but in the presence of LY367385 (100 μM) in DMSO-treated (black circle, n = 7), AβO-treated (red circle, n = 7), and scrambled AβO-treated rat hippocampal slices (empty circle, n = 7). f The mean of the normalized eIPSC amplitudes in e. DMSO (black bar), AβO (red bar), and scrambled AβO (empty bar). g–h Same as e and f, but in the presence of MPEP (10 μM) in DMSO-treated (black, n = 7), AβO-treated (red, n = 7), and scrambled AβO-treated rat hippocampal slices (empty, n = 7). Inset: b, c, e, and g representative eIPSC traces at the indicated time points (1, 2) in each condition. Statistical tests: d Unpaired Student’s t test, *p < 0.05, ***p < 0.001; f, h one-way ANOVA with post hoc Tukey’s test, #p < 0.05, ns: p > 0.05. Data are represented as mean ± SEM
Fig. 2
Fig. 2
m-3M3FBS, a PLC activator, restores PLCβ-dependent S-eCB mobilization and PLCβ1 protein levels disrupted by AβO. a Time courses of the changes in mean eIPSC amplitudes following a S-eCB mobilization paradigm consisting of 60-s-long postsynaptic CA1 PC spikes (spike, at 1 Hz) with DHPG (50 μM) and LY367385 (100 μM) application in the presence of a PLCβ blocker, U73122 (5 μM) (+ U73122) in DMSO-treated (black circle, n = 9), AβO-treated (red circle, n = 10), and scrambled AβO-treated rat hippocampal slices (empty circle, n = 8). b The mean of the normalized eIPSC amplitude of the first 10 s after S-eCB mobilization protocol in rat hippocampal slice treated with DMSO (black bar), AβO (red bar), and scrambled AβO (empty bar). c–d Same as a and b but for eIPSCs in the presence of a PLC activator, m-3M3FBS (30 μM) (+ m-3M3FBS) in DMSO-treated (black, n = 7), AβO-treated (magenta, n = 8), and scrambled AβO-treated rat hippocampal slices (empty, n = 8). Inset: a, c representative eIPSC traces at indicated time points (1, 2). e Representative photomicrograph of western blots of PLCβ1 (top, 150 kDa) and GAPDH (down, 35 kDa) proteins from DMSO-treated (left lane), AβO-treated (middle lane) rat hippocampal slices, and in the presence of m-3M3FBS (30 μM) in AβO-treated rat hippocampal slices (right lane). f PLCβ1 protein levels normalized to the GAPDH protein levels in DMSO-treated (black, n = 8), AβO-treated rat hippocampal slices (red, n = 8), and in the presence of m-3M3FBS AβO-treated rat hippocampal slices (magenta, n = 7). Statistical tests: b, d, f One-way ANOVA with post hoc Tukey’s test, # p < 0.05, ns: p > 0.05. Data are represented as mean ± SEM
Fig. 3
Fig. 3
m-3M3FBS restores spike-timing-dependent potentiation (tLTP) impaired by AβO. a Experimental schematic. Whole-cell current-clamp recordings in CA1 pyramidal cells (PCs) and Schaffer collateral (SC) stimulation for tLTP induction at CA3-CA1 excitatory synapses in rat hippocampal slices in vitro. b tLTP induction paradigm consisting of S-eCB mobilization protocol with SC stimulation-evoked presynaptic CA3 PC spikes (SC stim, top) 10 ms before postsynaptic CA1 PC spikes (spikes, middle), repeated 200 times at 1 Hz with DHPG (50 μM) application (bottom). Boxed inset: enlarged presynaptic SC stim-evoked EPSPs paired with postsynaptic CA1 PC spikes during tLTP induction. c–g EPSP slopes normalized to the mean of the 10-min baseline after tLTP in DMSO-treated slices (n = 11, c), in the presence of D-AP5 (50 μM) in DMSO-treated slices (n = 8, d), in the presence of AM251 (3 μM) in DMSO-treated slices (n = 5, e), in AβO-treated slices (n = 11, f), and in the presence of m-3M3FBS (30 μM) in AβO-treated slices (n = 9, g). Filled circles: test pathways, empty circles: control pathways. Inset: representative EPSP traces at indicated time points (1, 2 or 1’, 2’). h The mean of normalized EPSPs slopes from the last 5 min of the test (filled bars) and control pathways (empty bars) in DMSO (black), + D-AP5 in DMSO-treated slices (gray), + AM251 in DMSO-treated slices (dotted), in AβO-treated slices (red), and + m-3M3FBS in AβO-treated slices (magenta). Statistical tests: h Paired Student’s t test for comparison between the test and control pathways within the same group, *p < 0.05, ns: p > 0.05; one-way ANOVA with post hoc Tukey’s test for comparison among the test pathways in five different groups (DMSO vs. + D-AP5 in DMSO, # p < 0.05; DMSO vs. + AM251 in DMSO, † p < 0.05; DMSO vs. AβO, & p < 0.05; DMSO vs. AβO + m-3M3FBS, ns: p > 0.05). Data are represented as mean ± SEM
Fig. 4
Fig. 4
m-3M3FBS restores hippocampus-dependent contextual fear memory impaired in 5XFAD mice. a Experimental schematic. DMSO or m-3M3FBS (50 μM) was injected into the dorsal and ventral hippocampal CA1 regions of wild type (WT) control mice and 5XFAD mice. b Representative photomicrograph of western blots of PLCβ1 protein (top, 150 kDa) and GAPDH protein (bottom, 35 kDa) in WT mice (left lane), 5XFAD mice (middle lane), and m-3M3FBS-injected 5XFAD mice (+ m-3M3FBS in 5XFAD, right lane). c PLCβ1 protein levels normalized to GAPDH protein levels in WT mice (black, n = 7), 5XFAD mice (red, n = 7), and m-3M3FBS-injected 5XFAD mice (magenta, n = 7). d Experimental schematic for contextual fear conditioning (CFC). CFC was performed 3 days after DMSO or m-3M3FBS injection. On day 1, mice were habituated for 20 min in the conditioning chamber (habituation). On day 2, electrical foot shocks (0.5 mA, 1s) were delivered three times every 120 s (conditioning). On day 3, the mice were returned to the conditioning chamber for memory recall test for 2 min (Recall). e The mean of the percentage of freezing, a fear memory index, in WT mice (black, n = 7), 5XFAD mice (red, n = 9), and m-3M3FBS-injected-5XFAD mice (magenta, n = 7). Statistical tests: c, e One-way ANOVA with post hoc Tukey’s test, # p < 0.05, ## p < 0.01, ### p < 0.001, ns: > 0.05. Data are represented as mean ± SEM
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References

    1. Price DL. New perspectives on Alzheimer's disease. Annu Rev Neurosci. 1986;9(1):489–512. doi: 10.1146/annurev.ne.09.030186.002421. - DOI - PubMed
    1. Selkoe DJ. Toward a comprehensive theory for Alzheimer's disease. Hypothesis: Alzheimer's disease is caused by the cerebral accumulation and cytotoxicity of amyloid beta-protein. Ann N Y Acad Sci. 2000;924:17–25. doi: 10.1111/j.1749-6632.2000.tb05554.x. - DOI - PubMed
    1. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science. 2002;297(5580):353–356. doi: 10.1126/science.1072994. - DOI - PubMed
    1. Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A. 1998;95(11):6448–6453. doi: 10.1073/pnas.95.11.6448. - DOI - PMC - PubMed
    1. McLean CA, Cherny RA, Fraser FW, Fuller SJ, Smith MJ, Beyreuther K, Bush AI, Masters CL. Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann Neurol. 1999;46(6):860–866. doi: 10.1002/1531-8249(199912)46:6<860::AID-ANA8>3.0.CO;2-M. - DOI - PubMed

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