Amyloid-β Protein Precursor Deficiency Changes Neuronal Electrical Activity and Levels of Mitochondrial Proteins in the Medial Prefrontal Cortex

Abstract

Background:

Neuropathological features of Alzheimer’s disease are characterized by the deposition of amyloid-β (Aβ) plaques and impairments in synaptic activity and memory. However, we know little about the physiological role of amyloid-β protein precursor (AβPP) from which Aβ derives.

Objective:

Evaluate APP deficiency induced alterations in neuronal electrical activity and mitochondrial protein expression.

Methods:

Utilizing electrophysiological, biochemical, pharmacological, and behavioral tests, we revealed aberrant local field potential (LFP), extracellular neuronal firing and levels of mitochondrial proteins.

Result:

We show that APP knockout (APP^-/-) leads to increased gamma oscillations in the medial prefrontal cortex (mPFC) at 1-2 months old, which can be restored by baclofen (Bac), a γ-aminobutyric acid type B receptor (GABA_BR) agonist. A higher dose and longer exposure time is required for Bac to suppress neuronal firing in APP^-/- mice than in wild type animals, indicating enhanced GABA_BR mediated activity in the mPFC of APP^-/- mice. In line with increased GABA_BR function, the glutamine synthetase inhibitor, L-methionine sulfonate, significantly increases GABA_BR levels in the mPFC of APP^-/- mice and this is associated with a significantly lower incidence of death. The results suggest that APP^-/- mice developed stronger GABA_BR mediated inhibition. Using HEK 293 as an expression system, we uncover that AβPP functions to suppress GABA_BR expression. Furthermore, APP^-/- mice show abnormal expression of several mitochondrial proteins.

Conclusion:

APP deficiency leads to both abnormal network activity involving defected GABA_BR and mitochondrial dysfunction, suggesting critical role of AβPP in synaptic and network function.

Keywords

Amyloid-β precursor protein gamma oscillations L-methionine sulfonate medial prefrontal cortex mitochondria spikes

INTRODUCTION

Amyloid-β protein precursor (AβPP) belongs to a highly expressed type I membrane protein family in the central nervous system (CNS). AβPP cleavage by α-, β-, and γ-secretase gives rise to secreted AβPP (sAβPPα, sAβPPβ, and sAβPPγ), respectively [1, 2]. AβPP cleaved by β- and γ -secretase also leads to the production of amyloid-β (Aβ) [3], which is involved in the pathogenesis of Alzheimer’s disease (AD) [4, 5]. Physiologically, AβPP is distributed at both presynaptic terminals [6, 7] and postsynaptic dendrites [8, 9], where it plays a key role in synaptic transmission [10 –12] and contributes to the maintenance of mitochondrial function [13, 14]. Previous studies have focused on the pathological significa-nce of AβPP as a precursor of Aβ, but little is known about the physiological role of AβPP itself in the synapse. In particular, how AβPP mediates γ-aminobutyric acid (GABAergic) inhibitory synaptic function remains mostly unclear, despite the fact that AβPP is required for normal GABAergic activities [15 –17] and for the survival of adult-born neurons via maintenance of proper GABAergic transmission [18].

GABA is the major inhibitory neurotransmitter in the vertebrate CNS: it mediates fast synaptic in-hibition through GABA_A ionotropic receptors (GABA_ARs) as well as slow and prolonged synaptic inhibition through γ-aminobutyric acid type B receptors (GABA_BRs) [19]. GABA_BRs are metabotropic transmembrane receptors that are linked to potassium channels via G-proteins to regulate neuronal activity [20]. GABA_BRs sense chronic perturbations in GABA levels resulting in homeostatic changes in synaptic strength [21]. Abnormal GABAergic function has been seen in several neuronal disorders, including AD [22, 23], where this involves abnormal AβPP overexpression and subsequent Aβ production [24, 25]. A recent study shows that the sAβPP extension domain directly interacts with the presynaptic sushi domain of GABA_BR subunit 1a (GABA_BR1a), resulting in suppression of hippocampal synaptic transmission [26]. These studies suggest a critical role for AβPP in the regulation of synaptic activity via GABA_BR, but this needs further investigation. Furthermore, GABA inhibits the selective autophagy pathways, mitophagy, and pexophagy, which leads to oxidative stress [27], indicating a possible link between GABA signaling and mitochondrial function that has not been examined in AD models. Utilizing electrophysiological, biochemical, pharmacological, and behavioral tests, this study aimed to further determine the molecular, cellular, and systematic features of the upregulation of GABA_BR expression, and possible alterations in mitochondrial protein levels, in the medial prefrontal cortex (mPFC) of APP-deficient (APP^-/-) mice. Our data reveal pivotal roles for AβPP in the regulation and maintenance of normal mPFC function.

MATERIALS AND METHODS

Animals and surgery

All experimental procedures were approved by the Animal Protection and Use Committee of South China Normal University. APP^-/- mice [28] were obtained from the Model Animal Research Center of Nanjing University. One-to-two month-old APP^-/- and littermate wild type (WT) mice were given free access to water and food and maintai-ned under a 12 h dark/light cycle. Mice were anesthetized by intraperitoneal (IP) injection of chloral hydrate (400 mg/kg). The body temperature of experimental animals was kept at 37±0.5°C using a homeothermic heating device. Mice were placed in stereotactic devices (RWD Life Sciences) for surgery, extracellular recording, and drug injection. Bilateral craniotomy (diameter 1 mm) was performed 1.78 mm anterior to the bregma and located just above the mPFC. Cisternal cerebrospinal fluid (CSF) drainage was used to relieve brain pulsation. The dura of the cerebral hemispheres was dissected and retracted bilaterally. The skull was filled with warm sterile saline (0.9%NaCl) and petroleum jelly [16].

In vivo extracellular recordings in the mPFC of anesthetized mice

Mice were anesthetized by IP injection with chloral hydrate (400 mg/kg). During the experiment, whenever necessary, a one third dose of chloral hydrate was used to maintain anesthesia at a level that did not cause withdrawal reflex. During experiments with a heating pad (Harvard Apparatus, USA), rectal temperature was kept at 37±0.5°C and animals were self-breathing. The heads of the anesthetized mice were positioned in a stereotaxic apparatus (RWD Life Science, China) for extracellular recordings. Extracellular recordings were obtained from the mPFC region located AP: 1.98 mm; ML: 0.25 mm; DV: 1.3 mm.

In vivo local field potential (LFP) was conduceted as indicated [29] in the mPFC (AP: 1.98 mm; ML: 0.25 mm; DV: 1.3 mm) using glass electrodes (0.8–1.0 MΩ) containing 0.5 M NaCl. Data were collected using Spike2 software (Cambridge Electronic Design, CED), sampled at 10 kHz, and filtered at 1 kHz and amplified×1000 (A-M Systems, Model 3000) before acquisition, then visualized and stored in a PC through a D-A converter (CED micro 1401; Cambridge Electronic Design). Extracellular data were analyzed using Spike2, MATLAB 2012a (MathWorks) and GraphPad Prism 8.0.

Micro- and IP-injection

GABA_BR agonist, baclofen (Bac) (Sigma), and tertiapin-Q (Teq) (Tocris) were dissolved in saline to a final concentration of 100μM. After recording extracellular baseline activity for 10 min, drugs (both at 100μM) were focally microinjected using a syringe infusion/withdrawal pump (53210, Stoelting) into the mPFC through a glass pipette (tip diameter: 5–10μm) at an injection rate of 0.2μL/min. For chronic treatments, methionine sulfoximine (MSO) was purchased from Sigma (M5379). Mice were IP-injected daily for one week with either 0.9%NaCl or MSO (25 mg/kg body weight) dissolved in 0.9%NaCl.

Plasmids, cell culture, and western blots

Full-length human APP cDNA (hAPP695) was ob-tained from the Hui Zheng laboratory at Baylor College of Medicine. Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, containing L-glutamine (Invitrogen, Carlsbad, CA) supplemented with 10%(v/w) fetal calf serum (Invitrogen) in a humidified incubator with 5%CO₂/ 95%N₂. Cells were passaged every three days at a ratio of 1 : 10. APP-His, hAPP695, GABA_B1bR, and GABA_B2R plasmids were transiently transfected into HEK293 cells according to the requirements of individual experiments as follows. DNA-Lipofectamine 2000 reagent (Invitrogen) was used for transfection, acc-ording to the manufacturer’s instructions. Briefly, cells were seeded to be 70%–90%confluent at transfection. DNA (0.8μg) and lipofectamine 2000 (2μl) were diluted into 50μl Opti-MEM medium. After incubation for 5 min, the DNA-lipid complex was added to cells. The amounts of DNA and lipofectamine 2000 were adjusted according to the area of culture wells. For molecular and biochemistry experiments, the cells were collected 24–72 h after transfection.

Mouse mPFC samples were homogenized with micro tissue grinders (Kimble) in 2 ml tubes, using lysis buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA pH 8.0, 1%SDS, and prote-ase inhibitor (Complete Mini; Roche). After 1 min homogenization, cellular debris was removed by centrifugation at 13600×g for 10 min at 4°C, and the supernatant was collected for denaturation for 20 min at 75°C. Tissue lysate was treated with SDS-PAGE (Bio-Rad) and transferred to nitrocellulose membrane. Then, 5%skim dry milk (TBS) containing 0.5%Tween-20 (TBST) was used to block membranes for 0.5 h. Specific primary antibodies against GABA_B1R (Neuromab), glutamine synthetase (GS) (Abcam, ab49873), dynamin-like protein-1 (Drp-1) (Abcam, ab184247), mitofusin-1 (Mfn-1) (Abcam, ab57602), mitofusin-2 (Mfn-2) (Abcam, ab56889), nitrotyrosine (Santa Cruz, sc-32757), ATP-5A (Abcam, ab14748) and PTEN-induced kinase-1 (Pink-1) (Santa Cruz, sc-517353) were used to detect target proteins, with an anti γ-tubulin antibody (Sigma, T6557) used as a loading control. After three washes with TBST, HRP-labeled secondary antibody (CWS) was added at room temperature (RT), 5%milk was washed in TBST for 1 h, and then TBST was used for three extra washes. The Immobilon Western ECL system (CWS) was used to visualize bands. Analysis was performed using Gel Pro Analysis Software [17].

Immunofluorescent staining

Brains of WT and APP^-/- mice were removed, perfused transcardially with saline and 4%parafor-maldehyde, postfixed overnight, sucrose-protected in 30%sucrose overnight and then sectioned at 40μm intervals using a freezing microtome (Leica). Free-floating sections were washed three times with PBS and incubated with rabbit anti-parvalbumin (PV) antibody (Abcam, ab11427) diluted in PBS supplemented with 0.3%Triton X-100 and 3%BSA. After primary antibody incubation for 24 h at 4°C, sections were washed in PBS three times for 10 min each and incubated with secondary antibody (Invitrogen, A21207) diluted in PBS supplemented with 10%Triton X-100 and 3%BSA for 1 h at RT. Sections were washed, incubated with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Vectashield) for 15 min at RT, washed again and mounted on gelatin-coated slides, and then imaged on a fluorescence microscope (EVOS).

Eight-arm radial maze test

Animals were trained on a radial eight-arm maze using a delayed-nonmatching-to-place (DNMP) paradigm [30]. After visiting an arm (i.e., study arm) for a reward, each animal was confined in a bucket on the center platform for a 10 s delay period during which doors for both an adjacent arm (i.e., choice arm) and the visited study arm were opened. The animal had to choose the unvisited choice arm to receive a reward. If the mice selected the study arm again either by completely traversing the study arm to the end or when both of the rear paws touched the study arm, it was recorded as an error. On each trial, a study arm was randomly selected, and a choice arm was always an adjacent arm on either side (randomly chosen) of the study arm. The purpose of this design was to make two arms (study and choice arms) equally available at the time of the choice phase by preventing the situation in which the rat might easily avoid choosing a study arm by remembering the direction information of the study arm rather than the spatial cues. Eight trials per day were given with an inter-trial interval of 20 s.

Statistical analysis

The survival rate was determined by Kaplan-Meier survival analysis with a Mantel-Cox (log rank) test. Data were analyzed using one-way ANOVA, two-sample two-tailed Student’s t-test, or pair-sample Student’s t-test, where appropriate, with p < 0.05 taken as statistically significant. Data are presented as mean±SEM.

RESULTS

Enhanced gamma oscillations in the mPFC and amelioration of gamma activity by Bac in APP^-/- mice

The depolarizing state (up-state) is a self-organized ensemble of neurons maintaining balanced excitatory and inhibitory inputs [31, 32]. The continuous discharges of pyramidal neurons that receive inhibitory inputs from a large number of GABAergic interneurons result in cortical oscillatory activity, e.g., gamma oscillations [33], which is nested within the up-state [34]. Given that the up-state duration is significantly increased in the mPFC of APP^-/- mice [16] and that a tight relationship pertains between cortical up-down states and oscillatory activities, we reasoned that APP deficiency may alter oscillatory activity in the mPFC. Utilizing in vivo LFP recordings, we observed significantly increased power of gamma rhythm oscillations in APP^-/- mPFC, while delta (0.2–4 Hz) and theta (8–14 Hz) oscillations remained unchanged (Fig. 1A–C).

Fig. 1

Focal microinjection of Bac into mPFC abolishes the difference in extracellularly recorded in vivo gamma oscillations between WT and APP^-/- mice. A) Example of filtered LFP from a 1-2-month-old WT and APP^-/- mouse showing activity in the delta (0.2–4 Hz), theta (8–14 Hz), and gamma (30–100 Hz) bands. Above each trace is an expanded view of the section indicated by the dashed lines. The bottom shows power spectra of slow and delta (0.2–4 Hz), theta (8–14 Hz), and gamma (30–100 Hz) frequency ranges. Scale bars are shown on the right; delta, theta, and gamma time scales are the same. B, C) Quantification of mean delta and theta oscillation power shows no significant differences between APP^-/- and WT mice. D) Quantification shows increased gamma oscillation power in APP^-/- compared with WT mice and rescue of mean gamma oscillation power by Bac in APP^-/- mice (WT: n = 9 mice; APP^-/-: n = 9 mice). Insets show the reduced mean oscillation power caused by Bac administration in WT and APP^-/- mice. Each value represents the mean±SEM. ^*p < 0.05; two-way analysis of variance with the Bonferroni post-hoc test.

GABA_BR family members are G protein-coupled receptors (GPCRs), mainly distributed in the membranes of GABAergic and glutamatergic axonal terminals, which govern the release of neurotransmitters and regulate up-down states [20]. We evaluated the effect of a GABA_BR agonist, Bac, on gamma oscillations, and found that the Bac-sensitive component of gamma power increased significantly in the mPFC of APP^-/- mice (Fig. 1D), implicating an enhanced GABA_BR-mediated gamma oscillations in the absence of AβPP.

Identical number of PV-positive interneurons in the mPFC

Considering that gamma oscillations can be ind-uced by activating interconnected PV interneurons [33, 35], we next evaluated whether an alteration in numbers of PV-positive (PV⁺) cells plays a role in mediating abnormal gamma oscillations in APP^-/- PFC. However, immunofluorescent staining in the mPFC showed a similar number of PV⁺ cells in the two genotypes (Fig. 2A, B), excluding the possibility that altered inhibitory activity mediated by PV⁺ GABAergic interneurons contributes to increased gamma oscillations in the APP-deficient mPFC.

Fig. 2

Number of PV⁺ interneurons is identical in the mPFC of 1-2-month-old WT and APP^-/- mice. A) Representative images of PV⁺ cells in the mPFC of WT (left) and APP^-/- mice (right). B) Quantification shows an identical number of PV⁺ cells in the mPFC of APP^-/- mice (n = 29 sections from 6 mice) compared to WT (n = 13 sections from 5 mice). Each value represents the mean±SEM; two-sample two-tailed Student’s t-test.

Bac differentially inhibits mPFC neuronal firing in APP^-/- and WT mice

Pyramidal neuronal activities are necessary during the up-state [36, 37] where the gamma oscillations are related. Next, by evaluating the number of spikes, we compared the neuronal firing rate in the mPFC of WT and APP^-/- mice and examined the effect of Bac treatment (Fig. 3A, B). Although neuronal firing rate was identical in both mouse strains under baseline conditions (Fig. 3D), the effect of Bac on firing differed significantly (Fig. 3C-E). Briefly, a dose-response curve showed that APP^-/- mice required a higher dose of Bac to reduce neuronal firing (Fig. 3C). Moreover, neuronal firing decreased significantly 4 min after the addition of Bac, at a dose of 0.1 pMol, in WT but not in APP^-/- mice (Fig. 3D). The time-response curve showed that it took longer for 0.1 pMol Bac to suppress neuronal firing in APP^-/- mice (Fig. 3E). Together with the results of a previous study showing upregulated levels of GABA_BR expression [16], our data suggest there are increased numbers of GABA_BRs in the APP^-/- mPFC, and thus more Bac is required to achieve sufficient GABA_BR-mediated inhibition.

Fig. 3

Bac differentially inhibits neuronal firing in mPFC of 1-2-month-old WT and APP^-/- mice. A,B) Representative spike firing patterns of mPFC neurons measured before, during, and after applying the selective GABA_BR agonist, Bac, in WT (black) and APP^-/- (red) mice. Blue dots show the spikes. C) Bac inhibits spike firing in a dose-dependent manner in WT and APP^-/- mice. D) Quantification of spike firing before and after focal microinjection of Bac into mPFC does not rescue the spike firing rate (WT: n = 10 mice; APP^-/-: n = 10 mice). E) Summarized data of WT (black) and APP^-/- (red) animals showing the firing rate (4-min time window) before and after Bac injection. Each value represents the mean±SEM. ^*p < 0.05; two-way analysis of variance with the Bonferroni post-hoc test.

GIRK-activation mediates the effect of Bac on mPFC neuronal firing

A prevalent form of postsynaptic inhibition by GABA_BR is achieved through activation of a G protein, which activates inward-rectifier potassium channels (GIRKs), also known as internal rectified K+ Kir3 channels [20, 38]. We next evaluated whether or not the alteration in Bac inhibition of APP^-/- mPFC neuronal firing involves the GIRK pathway. A GIRK inhibitor, Teq, along with Bac, was injected into the mPFC of both mouse strains. The mPFC neuronal firing rate was then evaluated at different post-injection time points and compared with the baseline before injection (Fig. 4A, B). Intriguingly, Bac no longer suppressed neuronal firing in the mPFC of either WT or APP^-/- mice in the presence of Teq (Fig. 4C), suggesting that the inhibitory role of Bac on mPFC neuronal firing requires activation of the GIRK pathway in APP^-/- as well as WT mice.

Fig. 4

Bac-induced inhibition of mPFC neuronal firing requires activation of GIRK pathway. A, B) Representative spike firing patterns of mPFC neurons measured before, during, and after application of Bac and Teq (a GIRK inhibitor) in WT (black) and APP^-/- (red) mice. Blue dots show the spikes. C) Summarized data in WT (black) and APP^-/- (red) animals showing the firing rate (4 min time window) before and after Bac + Teq injection (WT, n = 10 mice; APP^-/-, n = 11 mice). Each value represents the mean±SEM; two-way analysis of variance with the Bonferroni post-hoc test.

Blockade of GS increases levels of GABA_BR in the mPFC of APP^-/- mice

Having observed an ameliorated effect of Bac on mPFC gamma oscillations (Fig. 1) and neuronal firing (Fig. 3) in APP^-/- mice, we speculated that upregulation of GABA_BR might compensate for reduced levels of GS and consequent reduction of GABA neurotransmitter in APP^-/- mice [16]. To test this hypothesis, we searched for chemicals that could either positively or negatively regulate GS activity, but found only a GS inhibitor, MSO, which blocks GS activity and increases glutamate release [39], and thus functions as a convulsant [40]. Strikingly, IP injection of MSO decreased levels of GABA_BR in WT mPFC (Fig. 5A), but further increased GABA_BR levels in the mPFC of APP^-/- mice (Fig. 5B). These results suggest that upregulation of GABA_BR expression is specific to the APP^-/- mice.

Fig. 5

Effect of MSO on survival rate and levels of GABA_BR. A,B) Representative immunoblots of PFC GABA_B1R expression in saline- or MSO-treated WT and APP^-/- mice. Quantification showing significantly reduced PFC GABA_B1R levels in MSO-treated WT mice and increased GABA_B1R levels in MSO-treated APP^-/- mice. C) Kaplan–Meier survival curves of WT and APP^-/- mice treated with MSO (log-rank test, p = 0.02) (IP, once daily for 7 consecutive days, 25 mg/kg), (WT: n = 10 mice; APP^-/-: n = 6 mice). D) Representative immunoblots of HEK293 cells transfected with GABA_B1b, GABA_B2, and hAPP695 constructs. GABA_B1R levels were elevated in the absence of hAPP695 constructs. Each value represents the mean±SEM of at least three samples per genotype. ^*p < 0.05; two-sample two-tailed Student’s t-test.

In support of the above western blotting results, APP^-/- mice injected with MSO demonstrated a significantly higher survival rate than WT controls (Fig. 5C), suggesting, again, stronger GABA_BR–mediated inhibition in the absence of AβPP, which compensates for MSO-induced hyperexcitation. To determine whether or not upregulation of GABA_BR is a direct consequence of APP deletion, a GABA_BR construct was transfected into the HEK-293 cell line, either alone or co-transfected with APP. We found that GABA_BR expression levels were significantly higher in cells transfected with GABA_BR alone than when GABA_BR was co-transfected with APP (Fig. 5D). Together, the results suggest that AβPP is critically involved in limiting GABA_BR expression.

Abnormal levels of mitochondrial proteins in the mPFC of APP^-/- mice

A possible connection between the accumulation of AβPP and its cleaved product, Aβ, and oxidative stress and mitochondrial dysfunction has been noted in cellular, transgenic, and human AD models [41 –45]. Collectively, these and other studies indirectly suggest the involvement of AβPP in mitochondrial dysfunction [46 –48]. We next tested whether the GABAergic abnormalities resulting from APP deficiency involve alterations in mitochondrial mitophagy. Western blotting analysis revealed significantly reduced levels of Pink-1, a mitochondrial serine/threonine-protein kinase encoded by the Pink1 gene [49, 50], in the mPFC of APP^-/- mice (Fig. 6A, B). In addition, levels of Drp-1, a Pink-1 interacting fission protein, decreased significantly, while levels of the fusion proteins Mfn-1 and Mfn-2 remained unchanged (Fig. 6A, B). To further explore the consequence of APP deficiency on mitochondrial function, we analyzed the levels of ATP-5A, a mitochondrial α-F1 subunit of ATP synthase [51], and nitrotyrosine, a marker of oxidative stress [52]. The mPFC of APP^-/- mice demonstrated significantly decreased and increased levels of ATP-5A and nitrotyrosine, respectively (Fig. 6B).

Fig. 6

Mitochondrial dysfunction in 1-2-month-old APP^-/- mice. A) Representative immunoblotting images of mPFC extracts from WT and APP^-/- mice. B) Quantification showing significantly reduced levels of Pink-1, Drp-1 and ATP-5A, and increased nitrotyrosine levels, in APP^-/- compared with WT mice, while Mfn-1 and Mfn-2 remain unchanged among groups. Each value represents the mean±SEM of at least six samples per genotype, with two repeats. ^*p < 0.05, ^**p < 0.01; two-sample two-tailed Student’s t-test.

Unchanged working memory in APP^-/- mice

One of the main clinical manifestations of AD patients is cognitive impairment involving working memory deficits [53], a mPFC-governed behavior [54, 55]. Accordingly, we asked whether abnormal GABA_BR-mediated activity and mitochondrial dysfunction in the mPFC would impair working memory in APP^-/- mice. Utilizing the delayed mat-ching space task [56], we observed no significant difference in spatial working memory between APP^-/- animals and WT controls (Fig. 7A, B), implying that abnormal molecular and cellular prefrontal activities due to APP deficiency occur earlier than behavioral deficits.

Fig. 7

Working memory remains unchanged in 1-2-month-old WT and APP^-/- mice. A) Representative tracking paths of 1-2-month-old WT and APP^-/- mice in baited arms. B) Quantification of correct choice percent revealed no significant differences between APP^-/- mice (n = 11) and WT mice (n = 10) (p = 0.140). Each value represent mean±SEM; two-sample two-tailed Student’s t-test.

DISCUSSION

AβPP is distributed in vesicular fractions of dendrites and axons [57], suggesting a role for AβPP in neuronal and synaptic activity. We report here that APP deficiency results in significantly enhanced gamma oscillations in the mPFC, which can be ameliorated by focal microinjection of a GABA_BR agonist, Bac. Moreover, it requires a higher dose and a longer time for Bac to suppress neuronal firing in the mPFC of APP^-/- mice, which is in line with a higher survival rate under hyperexcitatory conditions caused by blockade of GS and thus GABA synthesis by MSO. Furthermore, altered GABA_BR function is associated with abnormal levels of several mitochondrial proteins. This study uncovers a critical role for AβPP in the regulation of GABA_BRs and mitochondrial proteins and highlights the influence of these abnormalities on related neuronal and network activity.

Gamma oscillations are generated by the network of synaptic connections between neuronal populations [58, 59] and the balance between excitatory and inhibitory neuronal activities in these neural networks [60, 61]. Altered gamma band activity has been observed in multiple brain regions in several neurological and psychiatric disorders, including reduced spontaneous gamma synchronization in patients with AD and decreased gamma power in multiple AD mouse models [62 –64]. Optogenetically driving fast-spiking parvalbumin-positive (FS-PV)-interneurons at gamma frequencies (40 Hz) reduces levels of Aβ_1–40 and Aβ_1–42 isoforms in 5xFAD mice [62], implicating a critical role for PV neurons in abnormal gamma activity in AD pathogenesis. However, in this study, APP deficiency did not change the number of PV positive neurons; this, together with the fact that a previous report showed the number of GAD₆₇-GFP neurons to be unchanged, which indicates a similar total number of GABAergic neurons in the mPFC of APP^-/- mice [16], suggests that altered gamma oscillations in APP^-/- mice mice is attributed, at least in part, to enhanced GABA_BR-mediated inhibition.

It should be noted that a previous study revealed similar gamma oscillations in the dorsal PFC between 36-month-old APP-deficient and WT mice [65]. The dissimilarity may be due to differences in animal age and recording position since the present study conducted recordings in the mPFC of 1-2 month-old APP^-/- and WT controls. Single neurons in the mPFC are modulated by theta oscillations in the hippocampus [66, 67] and the hippocampal-prefrontal theta-gamma coupling is critically associated with memory performance and emotion [68 –70]. Aberrant inhibitory activity occurred in the hippocampus of 1-2 month-old APP^-/- mice [15 –17] and we observed significantly reduced hippocampal theta- and gamma-oscillations in this study (Supplementary Figure 1). Together, the results implicate possible abnormal coupling between hippocampal-theta and mPFC-gamma activity that may contribute, at least in part, to altered mPFC-gamma activity. Therefore, the dissimilarity of gamma oscillations between 36-month-old and 1-2-month-old APP^-/- mice suggest that other aging-related changes may diminish differences of gamma frequency oscillations.

GABA_BRs, metabotropic receptors for the inhi-bitory neurotransmitter GABA that belong to the GPCR family [20, 71], regulate presynaptic neurotransmitter release and postsynaptic membrane excitability [20], and play a critical role in gamma oscillations [72, 73]. Reduced overall GABAergic inhibition in the mPFC of APP^-/- mice is implicated by longer up-state duration, which indicates higher excitability [16]. We show in this study that APP-deficient mice develop stronger GABA_BR-mediated activity, which compensates for enhanced excitability and thus improves survival of the MSO-induced lethal hyperexcitatory condition. Furthermore, a GIRK inhibitor, Teq, similarly blocks the effect of Bac on mPFC neuronal firing in APP^-/- and WT mPFC, suggesting that Bac suppresses neuronal activity via activation of postsynaptic GABA_BR, which achieves inhibition through activation of GIRKs [20]. Deficits in GABA_BR-mediated inhibition have long been associated with various neuronal disorders [74, 75]. Given that many druggable targets for the treatment of diseases involve GPCRs, which mediate the therapeutic effects of ∼34%of marketed drugs [76], AβPP regulation of GABA_BR expression and function highlights the potential for targeting GPCRs, specifically GABA_BR, in the treatment of certain symptoms of AD.

Interestingly, treatment with the GABA_BR agonist baclofen mitigates neurotoxicity by targeting mitochondrial permeability transition pore (mPTP) opening [77], which is in line with our findings that baclofen ameliorated abnormal gamma oscillations in APP^-/- mice. Together, the results suggest a critical role of GABA_BR as an upstream factor in mediating normal mitochondrial activity that is crucial in maintaining proper neuronal and network function. Although exactly how APP deficiency results in GABA_BR and mitochondrial dysfunction is still unclear, the increase in GABA_BR and decrease in mitophagy protein Pink-1 levels, along with the subsequent reduction in Drp-1 levels, suggest a pivotal role for AβPP in maintaining the normal physiology of both mitochondria and GABA_BR.

Overexpression of mutant APP results in dysfunctional mitochondria, including increased mitochondrial fission protein, Drp-1, production [78, 79], leading to excessive ROS production and decreased energy output [80, 81]. In comparison, we found decreased levels of Drp-1 in APP deficient mice, suggesting a crucial role of AβPP in maintaining the proper expression and function of Drp-1. Various studies indicated that overexpressing of pathologic APP and APP751 changed mitochondrial structure and function [82, 83]. Given that AβPP is involved in mitochondrial translocation machinery [84], and Drp-1 plays a vital role in mitochondrial movement and quality control, AβPP seems necessary for the normal function of mitochondria. Moreover, neurons are highly energy-intensive, and their demand for ATP during neuronal transmission and network activity is primarily met by oxidative phosphorylation by mitochondria [85]. Fission uniquely facilitates the movement of mitochondria within axons and dendrites [85], whereas disruptions of this movement due to alterations in the mitochondrial fission process can impair neuronal activity. Therefore, abnormal levels of mitochondrial fission protein, Drp-1, and ATP-5A suggest that APP deficiency-induced mitochondrial dysfunction may contribute, at least in part, to neuronal network abnormalities seen in APP^-/- mice.

Taken together, this study offers an integrated biophysical, pharmacological, and neurochemical explanation for AβPP modulation of mPFC neural and network activity that involves abnormal GABA_BR-mediated inhibition associated with mitochondrial dysfunction. In addition, our data suggest GABA_BR as a potential therapeutic target under pathological conditions where cortical neural network activity is disturbed. Therefore, this study may provide a basis for understanding the molecular and cellular mechanisms of network function in the cerebral cortex, as well as an understanding of the pathogenesis and treatment of cortical network dysfunction in AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants from the Nat-ional Natural Science Foundation of China (NSFC) (31970915, 31871170, 81804197, 31771219), the Guangdong Natural Science Foundation for Major Cultivation Project (2018B030336001), and the Guangdong Grant ‘Key Technologies for Treatment of Brain Disorders’ (2018B030332001).

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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Amyloid-β Protein Precursor Deficiency Changes Neuronal Electrical Activity and Levels of Mitochondrial Proteins in the Medial Prefrontal Cortex

Abstract

Background:

Objective:

Methods:

Result:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Animals and surgery

In vivo extracellular recordings in the mPFC of anesthetized mice

Micro- and IP-injection

Plasmids, cell culture, and western blots

Immunofluorescent staining

Eight-arm radial maze test

Statistical analysis

RESULTS

Enhanced gamma oscillations in the mPFC and amelioration of gamma activity by Bac in APP-/- mice

Identical number of PV-positive interneurons in the mPFC

Bac differentially inhibits mPFC neuronal firing in APP-/- and WT mice

GIRK-activation mediates the effect of Bac on mPFC neuronal firing

Blockade of GS increases levels of GABA B R in the mPFC of APP-/- mice

Abnormal levels of mitochondrial proteins in the mPFC of APP-/- mice

Unchanged working memory in APP-/- mice

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Enhanced gamma oscillations in the mPFC and amelioration of gamma activity by Bac in APP^-/- mice

Bac differentially inhibits mPFC neuronal firing in APP^-/- and WT mice

Blockade of GS increases levels of GABA_BR in the mPFC of APP^-/- mice

Abnormal levels of mitochondrial proteins in the mPFC of APP^-/- mice

Unchanged working memory in APP^-/- mice