Alpha-Ketoglutarate improves brain cell connection problems in a mouse model of Alzheimer's disease

We used a mouse model of AD called APP/PS1, which expresses a mutated chimeric mouse/human amyloid precursor protein (APP) and the exon‐9 deleted variant of human presenilin 1 (PS1) under the control of a prion promoter element (APPSwe/PS1dE9) (Borchelt et al. 1997). Both mutations of APP and presenilin 1 are associated with early‐onset familial AD (Lanoiselée et al. 2017). APP/PS1 transgenic mice aged 4–5 months were used for all experiments. We have previously shown that mice at this age exhibit early pathological features of AD, including impaired LTP, memory deficits, increased amyloid beta (Aβ) plaque burden, microglial activation, and altered expression of genes related to synaptic plasticity and memory (Navakkode et al. 2021). All animals were housed under a 12/12 h light/dark cycle and supplied with food and water ad libitum. A total of 200 hippocampal slices prepared from 51 APP/PS1 and 52 WT mice were used for electrophysiological recordings. For western blotting, slices were collected at the end of the recording and snap frozen. All experimental procedures involving animals were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore.

For acute hippocampal slice preparation, mice were anesthetized using CO₂ and decapitated. The brain was immediately removed and transferred to cold (2°C–4°C) artificial cerebrospinal fluid (ACSF). Both the left and right hippocampi were isolated in cold ACSF and subsequently sliced into 400 μm transverse sections with a manual tissue chopper. For field electrophysiology, the slices were quickly transferred to an interface chamber (Scientific System Design, Ontario, Canada) which is maintained at 32°C, continuously perfused with oxygenated ACSF equilibrated with 95% O₂‐5% CO₂ (carbogen) (total consumption: 16 L/h) at a flow rate of 0.89 mL/min. Recordings were performed at 32°C, a temperature commonly used to preserve slice viability, maintain metabolic stability, and support stable physiological functions especially during long‐term recordings (Sajikumar et al. 2005). Carbogen (96% O₂ and 4% CO₂) was used to bubble the recording chamber and ACSF to ensure sufficient tissue oxygenation and maintain a stable pH (7.3–7.4). The ACSF contained the following (in mM): 124 NaCl, 4.9 KCl, 1.2 KH₂PO₄, 2.0 MgSO₄, 2.0 CaCl₂, 24.6 NaHCO₃, and 10 D‐glucose, equilibrated with 95% O₂/5% CO₂ (32 L/h; Li et al. 2017).

In all electrophysiological recordings, two‐pathway experiments were performed. Two monopolar lacquer‐coated, stainless‐steel electrodes (5 MΩ; AM Systems) were positioned at an adequate distance within the stratum radiatum of the CA1 region for stimulating two independent synaptic inputs S1 and S2 of one neuronal population (Figure 1A), thus evoking field excitatory post synaptic potential (fEPSP) from Schaffer collateral/commissural‐CA1 synapses. Pathway specificity was tested by using a previously described method (Li et al. 2017). For recording the fEPSP (measured as its initial slope function), one electrode (5 MΩ; AM Systems) was placed in the CA1 apical dendritic layer, and signals were amplified by a differential amplifier (model 1700; AM Systems). The signals were digitized by using a CED 1401 analog‐to‐digital converter (Cambridge Electronic Design) and monitored online.

After 3 h incubation, a synaptic I‐O curve (input–output curve) (afferent stimulation vs. fEPSP slope) was generated. Test stimulation intensity was adjusted to elicit a fEPSP slope of 40% of the maximal slope response for both synaptic inputs S1 and S2. Test stimulation comprising four 0.2‐Hz biphasic constant‐current pulses (0.1 ms per polarity) was used at each time point. In all experiments, a stable baseline was recorded for at least 30 min before plasticity induction. To induce late‐LTP, a strong tetanization (STET) protocol, consisting of three high‐frequency stimulations of 100 pulses at 100 Hz (single burst, stimulus duration of 0.2 ms per polarity) with an inter‐train interval of 10 min, was used. To induce early‐LTP, a weak tetanization (WTET) protocol consisting of a single stimulus train of 21 pulses at 100 Hz (stimulus duration of 0.2 ms per polarity) was used.

AKG was dissolved in water and then in ACSF to a final concentration of 1 mM. CaAKG was first dissolved in 0.1 M HCl and then in ACSF to a 1 mM concentration. AP‐5 or nifedipine (Tocris) was used at a concentration of 50 and 10 μM, respectively (dissolved in ACSF and 0.1% DMSO). We performed preliminary tests to determine an appropriate concentration of Ca‐AKG. We found that 1 mM Ca‐AKG had no significant effect on basal synaptic transmission in hippocampal slices from WT or APP/PS1 mice (data not shown), indicating that this concentration does not interfere with baseline neuronal function. Based on this, we selected 1 mM for all subsequent electrophysiological experiments.

IEM‐1460 (IEM; Tocris), an inhibitor of CP‐AMPARs, was dissolved in water and used at a final concentration of 30 μM (Park et al. 2016). mTOR inhibitor rapamycin (Tocris Bioscience, Bristol, England) was dissolved in DMSO to create a 1 mM stock and was diluted in ACSF to a final concentration of 1 μM. For stocks prepared in DMSO, the final DMSO concentration was kept below 0.1%, a concentration that was shown to not affect basal synaptic responses (Navakkode et al. 2004).

In field electrophysiological recordings, synaptic efficacy was assessed by measuring the slope of the fEPSP (in millivolts per millisecond). All data are presented as mean ± SEM. To evaluate statistical significance within a group, the Wilcoxon signed‐rank test (noted as Wilcox) was employed to compare the mean normalized fEPSPs at specific time points with the baseline fEPSP measured at −15 min. For comparisons between different groups, the Mann–Whitney U‐test (noted as U‐test) was used. Statistical significance was determined at P < 0.05. Nonparametric tests were chosen because a Gaussian normal distribution could not always be assumed, particularly due to the small sample sizes in each series and the analysis of extended recordings (Li et al. 2017; Navakkode et al. 2007). The variable "n" indicates the number of slices used in in vitro electrophysiology or the number of animals in Western blot experiments. For all in vitro and biochemical studies, slices from a minimum of 3–4 biological replicates were included. All graphs and statistical analyses were created using Graph Pad Prism 11.

After recording, hippocampal slices were carefully taken from the recording chamber, snap frozen in liquid nitrogen, and then stored at −80°C. Proteins were extracted from tissues using Tissue Protein Extraction reagent (T‐PER; Thermo Scientific, Waltham, MA, USA) followed by centrifugation for 5 min at 10,000 rpm at 4°C. The protein concentration was determined using the Bradford assay (Bio‐Rad, Hercules, CA, USA). Suitable amounts of protein were added to the sample buffer and heated at 95°C for 10 min before being separated by SDS‐polyacrylamide gels. Gels were transferred to Nitrocellulose membranes (Bio‐Rad, Hercules, CA, USA) for 2 h at 100 V or 30 V overnight at 4°C. The membranes were blocked with 5% w/v nonfat dry milk in 1X Tris‐buffered saline with Tween 20 (TBST) and then immunoblotted with primary antibodies. The primary antibodies with their concentrations used were as follows: rabbit anti‐LC3 (1:1000, Cell Signaling, Danvers, MA, USA, #3868S) and mouse anti‐tubulin (1:20,000; Sigma‐Aldrich, Darmstadt, Germany, #T6557). The membranes were incubated with respective secondary peroxidase‐conjugated antibodies. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) was used to generate signals for imaging. The amounts of all the proteins were quantified by densitometric measurement of Western blots using ImageJ (NIH software). The densitometric values of each blot were normalized to the amounts of tubulin, which served as the loading control.

We first evaluated the impact of CaAKG on hippocampal synaptic plasticity in male wild‐type (WT) mice (Figure 1B). Application of CaAKG in hippocampal slices from male WT mice led to stable potentiation lasting up to 240 min. Strong tetanisation (STET) in S1 (blue filled circles) resulted in a significant increase in fEPSP values from min 1 (p = 0.0039, Wilcoxon test; p = < 0.0001, U test) post‐ STET and remained significant until 240 min (p = 0.0039, Wilcoxon test; p = < 0.0001, U test) compared to its own baseline (at −15 min) or the corresponding control S2 (blue open circles) (n = 9). Next, we tested whether CaAKG shows a sex‐dependent effect in WT mice, so we tested its effects in female WT mice. In female WT mice (Figure 1C) STET in S1 (pink filled circles) in the presence of CaAKG led to an increase in fEPSP values at 1 min (p = 0.0312, Wilcoxon test; p = 0.0022, U test) and it remained stable and significant until 240 min (p = 0.0312, Wilcoxon test; p = 0.0022, U test) compared to its own baseline or the S2 (open pink circles) (n = 5).

To investigate whether CaAKG has a protective effect in AD mice, we first tested its effect in male APP/PS1 mice. In male APP/PS1 mice (Figure 1D) CaAKG rescued the impaired LTP. STET in S1 (blue filled circles) led to a significant potentiation that was stable from 1 min (p = 0.0156, Wilcoxon test; p = 0.0037, U test) until 240 min (p = 0.0078, Wilcoxon test; p = 0.0037, U test, (n = 8)). In female APP/PS1 mice (Figure 1E), STET in S1 (pink filled circles) in the presence of CaAKG led to a significant potentiation that was stable from 1 min (p = 0.0078, Wilcoxon test; p = 0.0002, U test) until 240 min (p = 0.0156, Wilcoxon test; p = 0.0030, U test (n = 8)).

Comparison of fEPSP values between WT male and WT female mice did not show any difference at any time points (comparison of Figure 1B,C). Interestingly, we observed that CaAKG exerted a more profound effect in APP/PS1 females compared to APP/PS1 males (Figure 1F; Figure 1D vs. 1E). Comparison of fEPSP values from input S1 of male and female APP/PS1 treated with CaAKG showed that females showed a higher potentiation compared to males from 5 to 50 min (5 min, p = 0.0019, U test; 30 min, p = 0.0499, U test; 50 min, p = 0.0379, U test).

Comparison between control male APP/PS1 mice with male APP/PS1 mice treated with CaAKG (Figure S1C vs. Figure 1D) shows that the values are significant from 120 min onwards (120 min, p = 0.0426, U test). In contrast, comparison of control female APP/PS1 mice with female APP/PS1 mice treated with CaAKG (Figure S1D vs. Figure 1E) shows that fEPSP values were statistically significant from 1 min onwards until 240 min (1 min, p = 0.0093, U test; 60 min, p = 0.0006, U test; 180 min, p = 0.0037, U test; 240 min, p = 0.0037, U test). These comparisons of control male and female APP/PS1 mice with the corresponding CaAKG treated ones clearly strengthen our findings that female APP/PS1 mice have a profound effect with CaAKG compared to male APP/PS1 mice. Control input S2 in all experiments (blue and pink open circles) showed stable fEPSP values throughout the recordings. Overall, our results indicate that CaAKG ameliorated synaptic deficits in APP/PS1 mice, with female AD mice having a more profound effect than male AD mice.

Next, we investigated whether pure AKG has any effect on LTP in APP/PS1 mice. Since CaAKG is a source of calcium (Ca²⁺) in addition to AKG and moreover Ca²⁺ ions play a significant role in cellular signaling, we used pure AKG as a control to see if AKG alone has a differential effect. We first examined the role of AKG in male WT hippocampal slices (Figure 2A). Bath application of AKG during the STET in hippocampal slices did not induce a significant effect. STET in S1 (blue filled circles) led to a potentiation that was statistically significant from 1 min (1 min, p = 0.0010, Wilcoxon test; p = 0.0007, U test) until 240 min (240 min, p = 0.0010, Wilcoxon test; p = 0.0007, U test (n = 7)). p values were not significant at any time points between control WT and those treated with AKG p < 0.05. When WT female (Figure 2B) hippocampal slices were bath applied with AKG during LTP, a long‐lasting, input‐specific potentiation was observed in S1 (pink filled circles), which was statistically significant from 1 min (1 min, p = 0.0312, Wilcoxon test; p = 0.0022, U test) until 240 min (240 min, p = 0.0312, Wilcoxon test; p = 0.0022, U test) (n = 5). No significant difference was found between female WT controls and those treated with AKG p < 0.05. To test whether AKG has any effect on AD mice, we used male APP/PS1 mice first. In male APP/PS1 (Figure 2C) mice, the application of AKG rescued the impaired plasticity. Induction of LTP with STET in S1 (blue filled circles) led to an increase in fEPSP values that remained stable from 1 min (1 min, p = 0.0156, Wilcoxon test; p = < 0.0001, U test) until 240 min (240 min, p = 0.0312, Wilcoxon test; p = 0.0238, U test) (n = 8). To study if AKG induces any sex‐dependent effects, we studied its effects in female AD mice. In female APP/PS1 mice (Figure 2D) also STET in S1 (pink filled circles), in the presence of AKG led to an increase in fEPSP values from 1 min (1 min, p = 0.0020, Wilcoxon test; p = < 0.0001, U test) until 240 min (240 min, p = 0.0020, Wilcoxon test; p = < 0.0001, U test (n = 10)).

No significant difference was found between WT males and females when compared to those treated with AKG (Figure 2A vs. 2B). fEPSP values were compared between male AD and female AD mice treated with AKG (Figure 2C,D). No significant changes were observed at any time points from 1 min until 240 min when comparing S1 between male and female APP/PS1 mice treated with AKG (1 min, p = > 0.9999, U test; 60 min, p = 0.5148, U test; 180 min, p = 0.1728, U test; 240 min, p = 0.0934, U test). But comparison of control male APP/PS1 mice with the male APP/PS1 treated with AKG (Figure 2E; Figure S1C vs. Figure 2C) showed statistically significant differences in fEPSP values from 45 min onward (45 min, p = 0.0007, U test). In contrast, comparison of control female APP/PS1 mice with female APP/PS1 mice treated with AKG (Figure 2F; Figure S1D vs. Figure 2D) showed fEPSP values that were statistically significant from 1 min onwards until 240 min (1 min, p = 0.0046, U test; 60 min, p = 0.000, U test; 180 min, p = 0.0001, U test; 240 min, p = 0.0001, U test). In all recordings, the control input S2, fEPSP values were stable throughout the recording (blue and pink open circles).

These results indicate that AKG restores input‐specific synaptic potentiation in hippocampal slices from APP/PS1 mice, with a potentially stronger restorative effect observed in female AD mice, but these differences were observed only when compared with its control untreated animals. Furthermore, a comparison between AKG and CaAKG‐treated slices revealed no differences p < 0.05, confirming that the Ca²⁺ in CaAKG did not have any additional effects in our studies.

Multiple studies have shown that inhibition of NMDARs completely blocks STET induced LTP, suggesting that NMDARs are critical for LTP (Dozmorov et al. 2006; Navakkode et al. 2007; Pananceau and Gustafsson 1997; Vickers et al. 2005). To test whether CaAKG‐facilitated LTP is NMDAR‐dependent, we blocked NMDARs with AP‐5 in both male WT and male APP/PS1 mice treated with CaAKG. When STET was applied in the presence of CaAKG and AP‐5 in male WT mice (Figure 3A), significant potentiation of fEPSP values was observed in S1 (blue filled circles) from 1 min (1 min, p = 0.0312, Wilcoxon test; p = 0.0002, U test) to 240 min (240 min, p = 0.0312, Wilcoxon test; p = 0.0048, U test) (n = 7). In APP/PS1 mice, the co‐application of CaAKG and AP‐5 also showed statistically significant LTP for 4 h (Figure 3B). STET in S1 (blue filled circles) induced potentiation that was statistically significant from 1 min (1 min, p = 0.0312, Wilcoxon test; p = 0.0043, U test) to 240 min (240 min, p = 0.0312, Wilcoxon test; p = 0.0152, U test (n = 8)). These results clearly suggest that CaAKG ameliorated LTP through an NMDAR‐independent mechanism. Ca²⁺ entry through LTCCs could serve as an alternative for NMDAR‐independent LTP; therefore, we tested whether CaAKG‐mediated LTP is dependent on LTCCs. An LTCC blocker, nifedipine, applied along with CaAKG during STET in S1 (blue filled circles) in male WT hippocampal slices did not affect late‐LTP (n = 7) (Figure 3C). However, in APP/PS1 mice (Figure 3D) application of CaAKG and nifedipine impaired late‐LTP, showing that in APP/PS1 mice, CaAKG mediated its effects via LTCCs. STET in S1 (blue filled circles) led to an increase in fEPSP values that were statistically significant from 1 min (1 min, p = 0.0039, Wilcoxon test; p = < 0.0001, U test) to 65 min (65 min p = 0.0273, Wilcoxon test) compared to its baseline, and from 70 min onwards it decayed to baseline (70 min p = 0.0547, Wilcoxon test) (n = 8). It remained statistically significant until 40 min (40 min, p = 0.0188, U test) when compared to S2 and decayed to baseline from 45 min (45 min, p = 0.0508, U test). Control potentials in S2 remained stable throughout the recording period in experiments (blue open circles). The bar graph (Figure 3E) compares male WT mice treated with CaAKG/AP‐5 and male APP/PS1 mice treated with CaAKG and AP‐5 at 1 min and 240 min (Figure 3A vs. 3B). The values show no significant difference between the groups at either time point (1 min, p = 0.1520, U test, 240 min, p = 0.0721, U test). The bar graph (Figure 3F) compares male WT treated with CaAKG and nifedipine and male APP/PS1 mice following CaAKG and nifedipine treatment (Figure 3C vs. 3D). There is no significant difference at 1 min; however, a significant difference is observed at 240 min (1 min, p = 0.7577, U test, 240 min, p = 0.0021, U test). Our findings indicate that CaAKG reinstates LTP in APP/PS1 mice through an NMDAR‐independent and LTCC‐dependent mechanism.

The Ca²⁺ conductance of CP‐AMPARs may have both physiological and pathological roles. Thus, we were interested in evaluating whether the effects of CaAKG involve the action of CP‐AMPARs and whether this is pathological or neuroprotective. First, we examined if inhibition of CP‐AMPAR affects late‐LTP in male WT mice (Figure 4A). IEM, an inhibitor of CP‐AMPARs, was bath applied for 60 min, 30 min before and 30 min after STET in S1 (blue filled circles). IEM had no effect on the induction and maintenance of LTP in WT slices. In the presence of IEM, STET led to an increase in fEPSP values that was statistically significant from 1 min (1 min, p = 0.0312, Wilcoxon test; p = 0.0022, U test (n = 7)) until 240 min (240 min, p = 0.0312, Wilcoxon test; p = 0.0087, U test). Next, we investigated the role of CP‐AMPARs in APP/PS1 mice (Figure 4B) by repeating the same experiment in male APP/PS1 mice. STET in S1 (blue filled circles) in the presence of IEM led to a significant potentiation at 1 min (1 min, p = 0.0234, Wilcoxon test; p = 0.0104, U test), which remained stable until 50 min when compared to its own baseline (p = 0.0391, Wilcoxon test), and 45 min when compared to control S2 (45 min p = 0.0281, U test (n = 7)). Potentiation decayed to baseline from 55 min (p = 0.0547, Wilcoxon test) and 50 min (50 min, p = 0.0104, U test). Next, CaAKG was co‐applied with IEM in WT hippocampal slices (Figure 4C). STET in S1 in the presence of CaAKG and IEM had no additional effect on WT slices. STET led to a potentiation in S1 (blue filled circles) that was statistically significant from 1 min (1 min, p = 0.0156, Wilcoxon test; p = 0.0082, U test) until 240 min (240 min, p = 0.0156, Wilcoxon test; p = 0.0012, U test (n = 7)). In contrast, in APP/PS1 mice, co‐application of IEM and CaAKG prevented the expression of persistent LTP by CaAKG (Figure 4D). This indicates that inhibition of CP‐AMPAR by IEM prevented the rescue of LTP by CaAKG in male APP/PS1 mice. STET in S1 (blue filled circles) led to a potentiation that was statistically significant from 1 min (1 min, p = 0.0039, Wilcoxon test; p = < 0.0001, U test) until 135 min (135 min p = 0.0039, Wilcoxon test) compared to its own baseline, but from 140 min onwards it decayed to baseline (140 min p = 0.0547, Wilcoxon test). It remained statistically significant until 125 min (125 min, p = 0.0244, U test (n = 9)) when compared to S2 and decayed to baseline from 130 min (130 min, p = 0.0503, U test). Control potentials remained stable throughout the recording period in all experiments (open blue circles). The bar graph (Figure 4E) compares male WT mice treated with IEM and male APP/PS1 mice treated with IEM (Figure 4A vs. 4B) at 1 min and 240 min. The values show no significant difference between the groups at 1 min (1 min, p = 0.1520, U test) while it shows a significant difference at 240 min (240 min, p = 0.0021, U test). The bar graph (Figure 4F) compares male WT treated with CaAKG and IEM and male APP/PS1 mice following CaAKG and IEM treatment (Figure 4C vs. 4D). There is no significant difference at 1 min; however, a significant difference is observed at 240 min (1 min, p = 0.6807, U test, 240 min, p = 0.0007, U test). Our results suggest that IEM blocks late‐LTP in APP/PS1 mice and that CaAKG does not rescue this impairment.

A recent study by Tumurbaatar showed that preserved autophagy is a key protective mechanism that maintains cognitive integrity in non‐demented AD pathology (Tumurbaatar et al. 2023). Activation of autophagy is known to rescue synaptic and cognitive deficits in both old and fragile X mice (Glatigny et al. 2019; Yan et al. 2018). This prompted us to check whether increasing autophagy could play a role in improving synaptic deficits in APP/PS1 mice.

Rapamycin is known to upregulate autophagy by inhibiting mammalian target of rapamycin (mTOR), a key regulator of translation initiation and long‐term synaptic plasticity. We first examined the effects of mTOR inhibition by rapamycin on late‐LTP in the CA1 area of the hippocampus from male WT mice (Figure 5A). Rapamycin was applied for 1 h during the tetanisation, from 30 min before to 30 min after the induction of late‐LTP by STET in S1 (blue filled circles). fEPSP values were statistically significant from min 1 (Wilcox test, p = 0.0078, U‐test, p = < 0.0001) until 75 min (Wilcox test, p = 0.0078 (n = 8)), before declining to the baseline potentials. Next, we studied the effect of mTOR inhibition on APP/PS1 mice (Figure 5B). Application of rapamycin, 30 min before and 30 min after STET in S1 (blue filled circles) rescued LTP in APP/PS1 mice. fEPSP values were statistically significant from 1 min (Wilcox test, p = 0.0312, U‐test, p = 0.0013) after STET until 240 min (Wilcox test, p = 0.0312, U‐test, p = 0.0007 (n = 7)). This indicates that enhancing autophagy has the potential to ameliorate synaptic deficits in AD mice, whereas in WT a balance of protein synthesis and degradation has to be maintained to facilitate long‐term synaptic plasticity (Fonseca et al. 2006).

Next to study if application of CaAKG has any impact on rapamycin‐treated slices in WT, rapamycin was coapplied with CaAKG for 1 h during late‐LTP. fEPSP values showed significant potentiation from 1 min (Wilcox test, p = 0.1250, U‐test, p = 0.0286) until 240 min (Wilcox test, p = 0.1250, U‐test, p = 0.0286) (n = 6) (Figure 5C). In APP/PS1 mice, induction of late‐LTP by STET in S1 (blue filled circles) in the presence of both rapamycin and CaAKG led to a statistically significant potentiation from 1 min (Wilcox test, p = 0.0156, U‐test, p = 0.0002) until 240 min (Wilcox test, p = 0.0312, U‐test, p = 0.0033 (n = 7) (Figure 5D)). Comparison of fEPSP values of S1 (blue filled circles) between APP/PS1 mice treated with rapamycin alone and those co‐treated with CaAKG (Figure 5B,D) showed no significant differences at any time points from 1 min until 240 min (p > 0.05). The control synaptic inputs S2 in both WT mice and APP/PS1 in all experiments remained stable throughout the entire recording period (blue open circles). The bar graph (Figure 5E) compares male WT mice treated with rapamycin and male APP/PS1 mice treated with rapamycin (Figure 5A vs. 5B) at 1 and 240 min. The values show no significant difference between the groups at 1 min (1 min, p = 0.6126, U test) while they show a significant difference at 240 min (240 min, p = 0.0003, U test). The bar graph (Figure 5F) compares male WT treated with CaAKG and rapamycin and male APP/PS1 mice following CaAKG and rapamycin treatment (Figure 5C vs. 5D). There is no significant difference at either 1 or 240 min (1 min, p = 0.4219, U test, 240 min, p = 0.6020, U test). These findings indicate that in WT mice, rapamycin blocked the maintenance of LTP while in APP/PS1 mice, rapamycin rescued the impaired LTP. With CaAKG, LTP remained intact in both WT and APP/PS1 mice. In APP/PS1 mice, CaAKG may act in synergy with rapamycin to enhance the impaired LTP .

To evaluate the impact of CaAKG on autophagy, we assessed levels of microtubule‐associated protein 1 light chain 3 isoform II (LC3‐II), a widely accepted marker of autophagosome formation. Western blot analysis was performed on hippocampal tissue from both APP/PS1 and wild‐type (WT) mice, with and without CaAKG treatment. Our results revealed a significant reduction in LC3‐II levels in untreated APP/PS1 mice (AD, light red bar) compared to CaAKG treated APP/PS1 slices (AD/CaAKG, dark red bar) (p = 0.0152, U‐test, n = 5), indicating impaired autophagic activity in the APP/PS1 mice and that CaAKG increases autophagy (Figure 6A,B). Although treatment with CaAKG did not elevate LC3‐II expression in WT slices treated with CaAKG slices (WT/CaAKG, dark blue bar) compared to their untreated WT (WT, light blue bar) (p = 0.6991, U‐test, n = 5). These findings suggest that CaAKG selectively enhances autophagic activity in APP/PS1 mice, which may account for the enhanced synaptic activity in AD mice.

Synaptic tagging and capture (STC) is impaired in both AD mice models and slices treated with Aβ (1–42) oligomers (Krishna et al. 2016; Li et al. 2017; Pavon et al. 2024; Sharma et al. 2017). Since CaAKG enhanced impaired LTP in AD mice, we set out to study if CaAKG can restore associative plasticity in APP/PS1 mice. To study the effect of CaAKG on STC, we employed the "strong before weak" paradigm, in which STET was delivered to synaptic input S1 prior to WTET in S2 with an interval of 60 min in WT mice (Figure 7A). After a stable baseline was obtained in the inputs S1 and S2, a STET was delivered to S1 (blue filled circles). One hour later, an early LTP which typically decays to baseline within 1–3 h‐ was induced in synaptic input S2 (blue open circles) by a weak tetanization (WTET). STET in S1 resulted in a statistically significant potentiation from 1 min (Wilcox test, p = 0.0005) until 240 min (Wilcox test, p = 0.0188 (n = 5)). After delivery of WTET, S2 expressed an immediate increase in fEPSP that was statistically significant from 1 min until the end of the recording (1 min, Wilcox test, p = 0.0307, 240 min, Wilcox test, p = 0.0171).

We repeated these experiments in APP/PS1 mice (Figure 7B). In both S1 and S2, the fEPSP values declined to baseline levels. STET in S1 (blue filled circles) induced a potentiation that was statistically significant from 1 min until 90 min (1 min, Wilcox test, p = 0.0156, 90 min, Wilcox test, p = 0.0469), but from 95 min onwards it was insignificant when compared to its own baseline until 240 min (95 min, Wilcox test, p = 0.0781, 240 min, Wilcox test, p = 0.9375). WTET in S2 (blue open circles) led to potentiation that remained significant from 1 min until 125 min (1 min, Wilcox test, p = 0.0156, 125 min, Wilcox test, p = 0.0469), but from 130 min onward, it was insignificant when compared to its own baseline until 240 min (95 min, Wilcox test, p = 0.0625, 240 min, Wilcox test, p = 0.15625) (n = 7).

To study the effects of CaAKG in mediating associative plasticity, we studied its effect in hippocampal slices from WT mice (Figure 7C). A stable baseline was recorded for 30 min after which STET was delivered to S1 (blue filled circles). Thirty minutes later, CaAKG was bath applied for 60 min. Sixty minutes after the STET in S1, a WTET was delivered in S2 (blue open circles), in the presence of CaAKG. WTET in S2 resulted in a long‐lasting LTP by STC. STET in S1 led to statistically significant results from 1 min until 240 min (1 min, Wilcox test, p = 0.0312, 240 min, Wilcox test, p = 0.0312). In S2, fEPSP values showed significant potentiation from 1 min until 240 min compared to its own baseline (1 min, Wilcox test, p = 0.0312; 240 min, Wilcox test, p = 0.0312) (n = 5).

Next, we examined whether CaAKG can restore long‐term associative plasticity in hippocampal CA1 of APP/PS1 mice. We conducted similar experiments as in Figure 7C in APP/PS1 mice (Figure 7D). A long lasting LTP was observed in both S1 and S2. STET was induced in S1, followed by WTET in S2 in presence of CaAKG. WTET in presence of CaAKG expressed late‐LTP, whereas STET in S1, which normally leads to a short lasting LTP (impaired LTP in APP/PS1 mice), was converted to late‐LTP by STC. fEPSP values were statistically significant at all‐time points compared to its own baseline (1 min, Wilcox test, p = 0.0039, 240 min, Wilcox test, p = 0.0078). WTET in S2 in the presence of CaAKG converted early‐LTP into late‐LTP. Similar to S1, fEPSP values were significant at all‐time points in S2 from 61 min (1 min after WTET to 240 min) (1 min, Wilcox test, p = 0.0156, 240 min, Wilcox test, p = 0.0078 (n = 9)). Our experiments show that CaAKG can not only facilitate, long‐ term plasticity but also promote associative plasticity in APP/PS1 mice.