Alzheimer’s disease (AD) is a neurodegenerative disease responsible for >80% of dementia cases worldwide. AD leads to a gradual loss of mental abilities and causes behavioral problems, including language issues, disorientation, mood swings, and an inability to manage self-care (Grande et al., 2022; Therriault et al., 2022). Characteristic pathological features of AD include amyloid plaque deposition, the development of neurofibrillary tangles, inflammation, and neuronal loss in specific brain regions (Park et al., 2020; Lin et al., 2023; Do et al., 2024). The cholinergic hypothesis suggests that an acetylcholine deficiency induces death of cholinergic neurons thus contributing to the cognitive decline observed in old patients with AD (Huh et al., 2014; Chen et al., 2022a). For the past twenty years, the only FDA-approved treatments for AD have been drugs developed based on the cholinergic hypothesis. These include drugs such as tacrine, donepezil, rivastigmine, and galantamine, which focus on enhancing the activity of the cholinergic neurotransmitter system by increasing levels of acetylcholine through the inhibition of acetylcholinesterase (Chen et al., 2022a). Although such drugs have therapeutic effects in patients with moderate AD, their effects are limited and accompanied by side effects such as diarrhea, nausea, insomnia, and vomiting (Shin et al., 2018; Chen et al., 2022a). There is also controversy over the effectiveness of the amyloid beta-targeted antibody treatment that was recently approved by the FDA due to its weak efficacy and side effects such as ARIA (Vaz et al., 2022). Therefore, developing more effective therapeutic agents with fewer side effects is needed for AD.

Recent studies demonstrated the involvement of epigenetic regulation, such as histone acetylation, in the pathogenesis of AD and has been used to develop subsequent treatments (Santana et al., 2023). Histone acetylation, regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), plays an essential role in regulating gene transcription (Wu et al., 2022). Since abnormal histone acetylation is involved in AD pathology, regulating HATs or HDACs could be a strategy for treating AD. In fact, HDAC inhibitors improved memory and cognition in a mouse model of AD (Kumar et al., 2022). In particular, suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor, rescues hippocampal-dependent spatial memory impairment in rodents (Athaide Rocha et al., 2023). The efficacy of Pan-HDAC inhibitors is not only based entirely on the regulation of histone acetylation; changes in various physiological activities within the cell through the regulation of the PTM of various non-histone proteins present in the cytoplasm also play significant roles (Wu et al., 2022). Therefore, applying selective HDAC inhibition to AD research improve the understanding of its therapeutic effects via histone and non-histone proteins.

HDAC6, a class IIb HDAC family member, acts primarily on cytoplasmic non-histone substrates, including α-tubulin, HSP90, and cortactin (Kim et al., 2020; Jo et al., 2022; Kumar et al., 2022). Previous studies demonstrated that HDAC6 plays a vital role in brain regions related to learning and memory, including the hippocampus and cortex (Chen et al., 2022b; Kumar et al., 2022; Lin et al., 2024). Furthermore, mounting evidence found increased HDAC6 levels in the brains of AD patients and that HDAC6 overexpression could contribute to AD pathogenesis (Zhang et al., 2024a). In addition, a previous study suggested that loss of endogenous HDAC6 could improve learning and memory deficits in a transgenic mouse model of AD (Bai et al., 2022; Zhang et al., 2024a). However, the role of HDAC6 in the cholinergic dysfunction underlying the pathogenesis of AD is not clear.

This study aimed to investigate the effects of (E)-3-(2-(4-fluorostyryl)thiazol-4-yl)-N-hydroxypropanamide (4-FHA), a novel HDAC6 inhibitor (Nam et al., 2019), on scopolamine-induced cognitive and memory impairments in mice. Since previous research results have proven that 4-FHA has specific selectivity and strong potency for HDAC6 (Nam et al., 2019), this study was designed to determine whether it has an in vivo pharmacological effect on cholinergic dysfunction. To this end, we used the Y-maze test, passive avoidance test (PAT), and Morris water maze test (MWMT). The novel object recognition test (NORT) and novel place recognition test (NPRT) served to assess the effects of 4-FHA on scopolamine-induced cognitive impairments. To confirm whether 4-FHA acts as an HDAC6 inhibitor in the hippocampus of scopolamine-administered mice, we evaluated the expression of acetylated (Ac) α-tubulin, a primary substrate of HDAC6, using Western blot. To determine the pharmacological mechanisms underlying the effects of 4-FHA, we evaluated the presence of alterations in signaling molecules associated with learning, memory, and synaptic transmission, including brain-derived neurotrophic factor (BDNF), extracellular signal-regulated kinase 1/2 (ERK), cAMP response element-binding (CREB), and choline acetyltransferase (ChAT) protein in the hippocampus.

Animals

Male Institute of Cancer Research mice (4-week-old, 21-23 g) were purchased from Koatech Co., Ltd. (Pyongtaek, Korea). Ten mice per cage (26×42×18 cm) were housed in a temperature- and humidity-controlled room (23°C ± 1°C and 55% ± 5%, respectively) under a 12 h light/dark cycle (light on 07:00-19:00) and with ad libitum access to food and water. After arrival, the mice were acclimatized for one week before experimental procedures. The mice were placed in an experiment room of ≥30 min before the behavioral tests. All animal care procedures followed the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University.

Drugs and chemicals

The novel compound 4-FHA was synthesized and provided by Professor Young Hoon Jung at the School of Pharmacy, Sungkyunkwan University (Suwon, Korea). 4-FHA exhibited potent inhibitory effects on HDAC6 (IC₅₀ value=42.98 nM) but not on HDAC1 (IC₅₀ value=5,432 nM); 4-FHA was approximately 126-fold more selective for HDAC6 than for HDAC1 (Nam et al., 2019). The HDAC inhibitory activity of 4-FHA was quantified using an HDAC enzymatic assay and performed by Professor Hyun-Ju Park at the School of Pharmacy, Sungkyunkwan University (Suwon, Korea). SAHA (Cayman Chemical, Ann Arbor, MI., USA) was used as a pan-HDAC inhibitor for class I and II HDACs, to compare the effects of selectively inhibiting HDAC6 through non-selective inhibition. Both drugs were dissolved in 5% DMSO and 5% Tween 80 in physiological saline. Scopolamine hydrochloride (Sigma-Aldrich, St Louis, MO, USA), a muscarinic receptor antagonist, was dissolved in physiological saline and used to induce cognitive and memory impairments in mice. 4-FHA, SAHA, and scopolamine were freshly prepared and administered intraperitoneally (i.p.) to mice.

Drug administration schedule

Scopolamine is known to be immediately exposed to the brain after administration and induces memory impairment by blocking the central cholinergic neurotransmission (Chen et al., 2022c; Wang et al., 2023; Choi et al., 2024). Therefore, we designed a preventive model to confirm the efficacy of 4-FHA. 4-FHA was administered in advance, followed by scopolamine administration, and each behavioral assessment was performed. To evaluate the effects of 4-FHA on scopolamine-induced cognitive and memory impairments in the Y-maze test, NORT, NPRT, and PAT (Fig. 1A), mice were treated with 4-FHA (0.03, 0.1, or 0.3 mg/kg, i.p.) or SAHA (5 mg/kg, i.p.) 30 min before scopolamine administration. Control and scopolamine-only mice received vehicle injections (5% DMSO and 5% Tween 80 in physiological saline). To induce cognitive and memory impairments, mice received scopolamine (0.5 mg/kg, i.p.) 30 min before all behavioral tests except for the PAT (day 6 in Fig.1A). Control mice received injections of physiological saline instead of scopolamine.

Figure 1. Drug administration schedule and experimental design. (A) Mice received 4-FHA (0.03, 0.1, or 0.3 mg/kg, i.p.), SAHA (5 mg/kg, i.p.), or vehicle 30 min before scopolamine (0.5 mg/kg, i.p.) administration. To induce cognitive and memory impairments, mice were administered scopolamine (0.5 mg/kg, i.p.) or saline (control) 30 min before all behavioral tests except on day 6 (PAT). (B) For the MWMT, the mice received 4-FHA (0.01, or 0.03 mg/kg, i.p.), SAHA (5 mg/kg, i.p.), or vehicle 30 min before scopolamine (0.5 mg/kg, i.p.) administration. To induce memory impairment, mice were administered scopolamine (0.5 mg/kg, i.p.) or saline (control) 30 min before the first trial of each of the five training sessions. The probe test was conducted without drug injection at 24 h after the final trial of the last training day. inj, injection.

To determine the effects of 4-FHA on scopolamine-induced memory impairment in the MWMT (Fig. 1B), mice were treated with 4-FHA (0.01, or 0.03 mg/kg, i.p.), SAHA (5 mg/kg, i.p.), or vehicle 30 min before scopolamine administration. To induce memory impairment, mice received scopolamine (0.5 mg/kg, i.p.) or saline (control) 30 min before the first trial of each of the five training days. However, no drug administration was performed for the probe test of the MWMT. From the Y-maze test to the PAT, each mouse completed the behavioral experiments from the least to the most stressful test.

Y-maze test

According to a previously described method (Ko et al., 2018), the Y-maze consisted of a horizontal maze (30 cm×5 cm×12 cm) with three arms (labeled A, B, and C). The floor and walls were constructed of dark gray polyvinyl plastic. Initially, all mice were placed in one arm. The number of alternations (i.e., consecutive entry sequences of ABC, CAB, or BCA but not BAB) and arm entries were manually counted for each mouse over 8 min. The percentage of alternation was assessed according to the following equation:

Percentage alternation=[(Number of alternations)/(Total arm entries-2)]×100

The Y-maze apparatus was wiped clean with 10% ethanol between trials.

Novel object recognition test and novel place recognition test

Using the method detailed in our previous work (Hur et al., 2023), the NORT and NPRT were conducted in an open field in an opaque plastic chamber (30×30×30 cm). On day 2 (Fig. 1A), the animals were habituated to an open field chamber for 10 min. On day 3, a training session of the NORT was performed 30 min after scopolamine administration. During this session, the mice were placed on the opposite side of two identical objects (7×3×3 cm) in an experimental chamber and allowed to explore for 10 min. During the test session (1 h thereafter), the mice were allowed to explore for another 10 min in the chamber where one of the objects had been replaced by a novel object. On day 4, the NPRT was performed without a separate habituation procedure. Similar to the training session for the NORT, that for the NPRT was conducted 30 min after scopolamine administration. During the test session, 1 h after the training session, the mice were allowed to explore for 10 min in the chamber where one of the objects was shifted diagonally. In both the NORT and NPRT, the animals explored the objects when facing them and when its nose was within 2 cm. The time spent exploring each object (T_familiar, T_novel) was used to calculate a discrimination index according to the following equation:

Discrimination index (%)=[(Tnovel-Tfamiliar)/(Tnovel+Tfamiliar)×100]

The experimental chamber and the objects were cleaned with 10% ethanol between tests.

Passive avoidance test

Following our established procedure (Ko et al., 2018), the step-through passive avoidance apparatus (O’Hara Co., Ltd., Tokyo, Japan) comprised a clear and a dark chamber separated by a guillotine door. The floors of both chambers (12×10×12 cm) were made of 2 mm stainless-steel rods spaced 0.5 cm. The transparent chamber was illuminated by a 50-W lamp fixed 1 m above both chambers. On day 5, a training session was completed 30 min after scopolamine administration. During the training session, mice were initially placed in the illuminated compartment. When a mouse entered the dark chamber, the door closed, and an electrical foot shock (0.5 mA for 3 s) was delivered through the stainless-steel rods. The test session was performed on day 6, 24 h after the training session. Again, mice were placed in the illuminated compartment during the test session. During training and test sessions, the time required for a mouse to enter the dark compartment was considered as the latency and recorded for up to 300 s (cut-off time). The apparatus was cleaned with 10% ethanol between trials.

Morris water maze test

The Morris water maze is a white circular pool (100 cm in diameter and 30 cm in height) with a featureless inner surface. The pool was filled with water and non-toxic, water-soluble black dye (25°C ± 1°C) and divided into four quadrants of equal area. A black platform (10 cm×10 cm) was centered in one of the four quadrants and submerged 0.5 cm below the water surface and invisible at water level. The pool was placed in a test room containing various prominent visual cues. The location of each swimming mouse, from the start position to the platform, was monitored by a computer-based video-tracking system (NeuroVision, Pusan, Korea). The MWMT procedure followed a previously described method with minor modifications (Kwon et al., 2013). Training sessions were performed 30 min after scopolamine administration. During training, each mouse underwent three daily trials for five consecutive days to find the platform in a single fixed location, with a 5 min intertrial interval. In each training trial, the mouse had 120 s to escape to the hidden platform. At that time, the escape latencies were recorded. This parameter was averaged for each trial session and mouse. The mouse’s entry point into the pool was the same in trials 1-3 but changed each day thereafter. Once the mouse located the platform, it could remain on it for 10 s. If the mouse did not locate the platform within 120 s, it was placed on the platform for 10 s and removed from the pool by the experimenter. In the probe test 24 h after the last training trial, mice were allowed to swim for 120 s without the platform and the swimming time in the pool quadrant where the platform had previously been placed was recorded.

Brain dissection and tissue preparation

Mice were sacrificed within 1 h after the PAT. Whole brain dissections were performed and harvested hippocampal tissues stored at −75°C until further use. The brain tissues were homogenized with an ice-cold lysis T-per tissue protein extraction buffer (Thermo Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitor cocktails (Roche Diagnostics, GmbH, Germany). After centrifugation at 1,000×g for 15 min, the supernatant was separated and stored at −75°C for Western blot analysis.

Western blot analysis

The protein concentration was determined using a protein assay kit (Thermo Scientific). Samples containing 15 μg of protein were separated on a 10-12.5% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride transfer membranes in a transfer buffer [25 mM Tris-HCl buffer (pH 7.4) containing 192 mM glycine and 20% v/v methanol] for 1 h, at 4°C. Then, the membranes were blocked with 5% non-fat milk containing 0.5 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween-20 for 1 h at room temperature. Next, the membrane was incubated with primary antibodies for β-actin (1:1,000, Santa Cruz Biotechnology, Dallas, TX, USA), Ac-α-tubulin (1:1,200, Millipore, Bedford, MA, USA), α-tubulin (1:10,000, Millipore), BDNF (1:500, Abcam, Cambridge, MA, USA), p-ERK 1/2 (1:1,000, Santa Cruz Biotechnology), ERK 1/2 (1:1,000, Santa Cruz Biotechnology), p-CREB (1:1,000, Abcam), CREB (1:1,000, Abcam), choline acetyltransferase (ChAT; 1:1,000, Invitrogen) and vesicular acetylcholine transporter (VAChT; 1:2000, Abcam) overnight at 4°C. After washing four times with Tris-buffered saline with 0.1% Tween-20 (TBST), the blots were exposed to goat anti-rabbit (1:5,000, Cell Signaling Technology, Danvers, MA, USA) or anti-mouse (1:10,000, GenDEPOT, Katy, TX, USA) horseradish peroxidase-conjugated secondary antibodies in TBST with 5% non-fat milk for 1 h at room temperature. Next, blots were washed four times in TBST buffer and the bands visualized using an enhanced chemiluminescence (ECL) detection method by incubation in a mixed solution of ECL reagents A and B (Dongin LS, Seoul, Korea) for 5 min at a 1:1 ratio and exposure to a photographic film (Kodak, Rochester, NY, USA) for a few minutes. The protein bands were quantified by densitometric analysis using ImageJ software (NIH, Bethesda, MD, USA).

Statistical analysis

Data are expressed as the mean ± standard error of the mean (SEM) and were analyzed using Prism 6.0 software (GraphPad Software, Inc., San Diego, CA, USA). Behavioral data were analyzed with a one-way analysis of variance (ANOVA) followed by Fisher’s LSD post hoc test, except for the escape latency of the MWMT which was analyzed using a two-way ANOVA followed by Fisher’s LSD post hoc test with drug treatment, day, and their interaction as independent factors. For Western blots, statistical analyses were performed using one-way ANOVA followed by Newman-Keuls post hoc comparison. The statistical significance was set at p<0.05.

4-FHA ameliorated scopolamine-induced memory impairment as shown by the Y-maze test

The Y-maze test was used to assess the effects of 4-FHA on scopolamine-induced memory impairment. Compared with the control group, scopolamine impaired the spontaneous alternative behavior (Fig. 2A, F _{(5, 54)}=7.92, p<0.001). Further, the scopolamine-induced decrease in spontaneous alternative behavior was ameliorated by 4-FHA (0.3 mg/kg; p<0.01) and SAHA (5 mg/kg) administration (p<0.05). On the other hand, scopolamine enhanced the number of arm entries compared with the control group (Fig. 2B, F _{(5, 54)}=7.59, p<0.001). In contrast, the 4-FHA (0.03, 0.1, and 0.3 mg/kg) and SAHA (5 mg/kg) groups showed no significant differences in the number of arm entries compared with the scopolamine group. These results suggest that, similar to SAHA, 4-FHA rescued the scopolamine-induced memory impairment without affecting locomotor activity in the Y-maze test.

Figure 2. Effects of 4-FHA on scopolamine-induced memory impairments in the Y-maze test. Mice received 4-FHA (0.03, 0.1, or 0.3 mg/kg, i.p.), SAHA (5 mg/kg, i.p.), or vehicle 30 min before scopolamine (0.5 mg/kg, i.p.) administration. To induce memory impairment, mice were administered scopolamine (0.5 mg/kg, i.p.) 30 min before the Y-maze test. The spontaneous alternative behavior (A) and number of arm entries (B) were quantified over 8 min. Data are shown as the mean ± SEMs (n=10 mice per group). *p<0.05 and ***p<0.001 compared to the control group. ^#p<0.05 and ^##p<0.01 compared to the scopolamine group. Con, control; n.s., no significant effect.

4-FHA improved scopolamine-induced cognitive impairments as shown in the NORT and NPRT

We evaluated the effects of 4-FHA on scopolamine-induced cognitive impairment using the NORT and NPRT. Scopolamine administration before training resulted in a significant decrease in the exploration time around the novel object in the NORT (Fig. 3A, F _{(5, 54)}=5.10, p<0.001). This scopolamine-induced cognitive impairment was recovered by 4-FHA (0.03, 0.1, and 0.3 mg/kg; p<0.05, p<0.05, and p<0.001, respectively) and SAHA (5 mg/kg) administration (p<0.05). On the other hand, scopolamine increased the total exploration time (increased locomotor activity) in the NORT compared with the control group (Fig. 3B, F _{(5, 54)}=3.09, p<0.05). Neither 4-FHA (0.03, 0.1, and 0.3 mg/kg) nor SAHA (5 mg/kg) caused a significant difference in the total exploration time compared with the scopolamine group. In addition, scopolamine administration decreased the exploration time around the object located in a new place in the NPRT (Fig. 3C, F _{(5, 54)}=3.77, p<0.05). Conversely, 4-FHA (0.1 and 0.3 mg/kg) administration improved scopolamine-induced cognitive impairment (p<0.05, both), as did SAHA (5 mg/kg) administration (p<0.001). Moreover, compared with the control group, none of the groups showed significant differences in the total exploration time in the NPRT (Fig. 3D, F _{(5, 54)}=0.63, p>0.05). These results demonstrate that, similar to SAHA, 4-FHA improved scopolamine-induced cognitive impairments without affecting locomotor activity in the NORT and NPRT.

Figure 3. Effects of 4-FHA on scopolamine-induced cognitive impairments in the NORT (A, B) and NPRT (C, D). Mice were treated with 4-FHA (0.03, 0.1, or 0.3 mg/kg, i.p.), SAHA (5 mg/kg, i.p.), or vehicle 30 min before scopolamine (0.5 mg/kg, i.p.) administration. To induce cognitive deficits, mice were administered scopolamine (0.5 mg/kg, i.p.) 30 min before the training trial. The testing trial was performed after 24 h. The discrimination index and total exploration time in the NORT (A, B) and NPRT (C, D) were calculated over 5 min. Data are presented as the mean ± SEMs (n=10 mice per group). *p<0.05 and ***p<0.001 compared to the control group. ^#p<0.05 and ^###p<0.001 compared to the scopolamine group. Con, control; n.s., no significant effect.

4-FHA rescued scopolamine-induced memory impairment as shown in the PAT

PAT was performed to investigate the effects of 4-FHA on the memory impairment induced by scopolamine. Despite the lack of significant differences between groups during the training session (Fig. 4A, F _{(5, 54)}=1.16), scopolamine administration decreased the step-through latency during the test session compared to the control group (Fig. 4B, F _{(5, 54)}=2.50, p<0.01). Furthermore, 4-FHA (0.1 mg/kg) administration (p<0.05) but not SAHA (5 mg/kg) could recover from the scopolamine-induced decrease in step-through latency time. These results indicate that 4-FHA improved scopolamine-induced memory impairment in the PAT.

Figure 4. Effects of 4-FHA on scopolamine-induced memory impairments in the PAT. Mice were treated with 4-FHA (0.03, 0.1, or 0.3 mg/kg, i.p.), SAHA (5 mg/kg, i.p.), or vehicle 30 min before scopolamine (0.5 mg/kg, i.p.) administration. To induce memory impairment, mice were administered scopolamine (0.5 mg/kg, i.p.) 30 min before the training session. The test session was conducted after 24 h. The step-through latency in training (A) and test (B) sessions was measured for 300 s. Data are shown as the mean ± SEM (n=10 mice per group). **p<0.01 compared to the control group. ^#p<0.05 compared with the scopolamine group. Con, control; n.s., no significant effect.

4-FHA restored scopolamine-induced memory impairment as indicated by the MWMT

Next, mice performed the MWMT. Compared with the control group, scopolamine administration delayed the escape latency time from day 1 to 5 (Fig. 5A, p<0.05). However, 4-FHA (0.03 mg/kg) administration, but not SAHA (5 mg/kg), significantly decreased the escape latency time from day 3 to day 5 compared with the scopolamine group (p<0.05). A two-way ANOVA revealed significant effects for drug treatment (F _{(4, 150)}=15.26, p<0.001) and training day (F _{(4, 150)}=5.37, p<0.001). However, the interaction of the two factors (F _{(16, 150)}=0.46, p>0.05) was not confirmed. Moreover, 4-FHA (0.01 mg/kg) administration decreased the escape latency time over the training sessions, although not significantly (p=0.053 on the last training day). Fig. 5B shows the distance traveled until the mice reached the hidden platform on the last training trial of day 5, and Fig. 5C shows the distance traveled in the target quadrant over 2 min. In the probe test, compared with the scopolamine group, 4-FHA (0.03 mg/kg) administration resulted in increased time in the target quadrant (Fig. 5D, F _{(4, 30)}=2.42, p<0.05). However, there were no significant differences in the average swimming speed (Fig. 5E, F _{(4, 30)}=0.73, p>0.05). These results show that 4-FHA ameliorated scopolamine-induced spatial memory impairment observed in the MWMT.

Figure 5. Effects of 4-FHA on scopolamine-induced memory impairments in the MWMT. Mice were treated with 4-FHA (0.01, or 0.03 mg/kg, i.p.), SAHA (5 mg/kg, i.p.), or vehicle 30 min before scopolamine (0.5 mg/kg, i.p.) administration. To induce memory impairment, mice were administered scopolamine (0.5 mg/kg, i.p.) 30 min before the first trial of each of the five training sessions. The probe test was performed 24 h after the final trial of the last training day. The escape latency (A) during the training session, time in the target quadrant (D), and speed (E) in the probe test were assessed for 120 s. The tracking figures in the final trial of the last training day (B) and the probe test (C) are shown as well. Data are presented as mean ± SEM (n=7 mice per group). *p<0.05, **p<0.01, and ***p<0.001 compared with the control group. ^#p<0.05 and ^##p<0.01 compared with the scopolamine group. Con, control; Scp, scopolamine.

4-FHA increased the expression of memory and synaptic transmission-related proteins in the hippocampus of scopolamine-treated mice

Among the numerous non-histone HDAC6 substrates, α-tubulin has been the most studied (Zhu et al., 2023), and its acetylation level is a standard pharmacodynamic marker of HDAC6 inhibitors. Thus, we quantified the expression of Ac-α-tubulin to determine whether 4-FHA inhibited HDAC6 in the brain of scopolamine-treated mice. Although scopolamine did not affect Ac-α-tubulin expression compared with the control group (Fig. 6A, F _{(5, 12)}=7.25, p>0.05), 4-FHA (0.03, 0.1, or 0.3 mg/kg) increased it when compared with the scopolamine group (p<0.05). Similarly, administration of SAHA (5 mg/kg), a pan-HDAC inhibitor, enhanced Ac-α-tubulin levels compared with the scopolamine group (p<0.05). This suggests that 4-FHA could act as an HDAC6 inhibitor in the hippocampus of scopolamine-treated mice.

Figure 6. Effects of 4-FHA on the expression of Ac-α-tubulin (A), brain-derived neurotrophic factor/extracellular signal-regulated kinase/cAMP response element-binding protein signaling pathway (B), and synaptic transmission proteins (C) in the hippocampus of scopolamine-treated mice. Mice were decapitated within 1 h after the test session of the PAT, and the hippocampus dissected for Western blot analysis. Data are presented as mean ± SEM (n=3–4 per group). *p<0.05 and **p<0.01 compared with the control group. ^#p<0.05, ^##p<0.01, and ^###p<0.001 compared with the scopolamine group. Con, control; Scp, scopolamine.

To clarify memory-related molecular mechanisms in the hippocampus of 4-FHA, we performed Western blot analysis targeting BDNF, p-ERK, and p-CREB. Injection of 4-FHA (0.03, 0.1, and 0.3 mg/kg) increased BDNF expression, which decreased with scopolamine (Fig. 6B, F _{(5, 18)}=12.61, p<0.01). In addition, 4-FHA (0.03, 0.1, and 0.3 mg/kg) rescued the phosphorylation levels of ERK and CREB reduced by scopolamine (F _{(5, 18)}=6.94, p<0.05 and F _{(5, 18)}=9.24, p<0.001, respectively). Moreover, SAHA (5 mg/kg) increased BDNF expression, and CREB phosphorylation levels of were reduced by scopolamine (p<0.001).

To observe the effects on synaptic transmission, we further quantified the expression of ChAT and VAChT by Western blot. Scopolamine decreased ChAT expression in the hippocampus of treated mice (Fig. 6C, F _{(5, 18)}=18.00, p<0.05), whereas 4-FHA and SAHA (5 mg/kg) injection (p<0.001) restored the decline (0.03, 0.1, and 0.3 mg/kg). Interestingly, VAChT expression was not affected by scopolamine or the other drugs.

These results show that, similar to SAHA, 4-FHA enhanced the expression of memory- and synaptic transmission-related proteins, including BDNF, phosphorylation of ERK and CREB, and ChAT in the hippocampus.

Recent studies demonstrated that HDACs could be an effective target for AD therapy, and various HDAC inhibitors have been applied for AD treatment (Li et al., 2021; Santana et al., 2023). Among them, SAHA (Vorinostat), a well-known pan-HDAC inhibitor was approved by the U.S. Food and Drug Administration for treating cutaneous T-cell lymphoma. However, severe side effects, including thrombocytopenia, anemia, and common side effects, such as fatigue, nausea, and diarrhea, have been reported (Li et al., 2024). In several studies, SAHA rescued scopolamine-induced spatial memory impairment in rats and recovered contextual memory deficits in CBP^+/− and AβPP^swe/PS1^△E9 mice (Alarcon et al., 2004; Kilgore et al., 2010; Yang et al., 2013). Therefore, we used SAHA as a positive control to investigate the applicability of 4-FHA as a therapeutic agent.

This study evaluated the effects of 4-FHA, a novel HDAC6 inhibitor, on cognitive and memory impairments induced by cholinergic dysfunction. Both 4-FHA at 0.3 mg/kg and SAHA at 5 mg/kg significantly increased the spontaneous alternation behavior in the Y-maze test compared with the scopolamine group, suggesting that 4-FHA or SAHA recovered scopolamine-induced memory loss. Consistent with previous studies, scopolamine enhanced locomotor activity in mice (Zhang et al., 2024a), as we found that scopolamine administration increased arm entries in the Y-maze test. However, neither 4-FHA nor SAHA significantly affected the scopolamine-induced locomotor activity in the Y-maze test. Therefore, 4-FHA, similar to SAHA, appears to rescue scopolamine-induced memory impairment without affecting locomotor activity.

In this study, the NORT and NPRT showed that, similar to SAHA, 4-FHA administration could improve scopolamine-induced cognitive impairment. However, the total exploration time in NORT increased in scopolamine-treated mice compared to control mice, suggesting that scopolamine increased locomotor activity. Furthermore, as shown in Fig. 5B, the total exploration time in NPRT did not differ between groups. This suggests environmental adaptation because NPRT was performed in the same chamber as the NORT. When mice are repeatedly exposed to new environments, the locomotor activity in the open field test decreases gradually because the mice get habituated to new environments (Ferreira et al., 2022).

4-FHA (0.1 mg/kg) improved scopolamine-induced memory impairment in PAT. In addition, in the MWMT, 4-FHA at 0.03 mg/kg recovered scopolamine-induced impairments in spatial learning from the third training day and spatial memory retrieval in the probe test. However, at 0.01 mg/kg, 4-FHA did not have significant effects, but caused decreasing escape latency time over training. In contrast, SAHA at 5 mg/kg did not improve scopolamine-induced memory impairments in PAT or MWMT. These results suggest that selective HDAC6 inhibition could be more effective than non-selective HDAC inhibition in recovering scopolamine-induced memory impairment.

Scopolamine, a non-selective muscarinic receptor antagonist, inhibits cholinergic neurotransmission by blocking acetylcholine receptors at synapses (Zhang et al., 2024a), resulting in cognitive and memory dysfunction in rodents. This antagonist has been shown to disrupt the BDNF-ERK-CREB signaling pathway, which is crucial for learning, memory, and synaptic plasticity (Balakrishnan et al., 2023; Choi et al., 2024). Additionally, scopolamine reduces the expression of ChAT in the rodent brain (Cheon et al., 2021). BDNF is a crucial regulator of synaptic functions, plasticity, neuronal cell survival, and neurogenesis (Jeon et al., 2012; De Vincenti et al., 2019; Wang et al., 2022). Indeed, increased hippocampal BDNF enhances spatial learning, memory, and cognitive functions (Jeon et al., 2012; Wang et al., 2022). In addition, downstream molecules of BDNF, such as ERK and CREB, are involved in hippocampus-dependent memory and synaptic function (Wang et al., 2022). Davis et al. reported that ERK activation was required to induce or maintain long-term potentiation and memory formation in the dentate gyrus (Davis et al., 2000). Furthermore, ERK can activate CREB, a transcription factor for BDNF, which enhances neuronal survival and cognitive function (Jeon et al., 2012; Wang et al., 2022). Lastly, ChAT is the enzyme responsible for synthesizing acetylcholine, a neurotransmitter essential for cognitive processes, including memory formation and recall (Shim et al., 2012; Yoon et al., 2022). Therefore, to investigate how 4-FHA rescues scopolamine-induced memory impairment, we assessed the expression of the memory-related pathway proteins BDNF, ERK, CREB, and ChAT in the hippocampus. Consistent with the effects of 4-FHA observed in our behavioral tests, 4-FHA at 0.03, 0.1, and 0.3 mg/kg significantly recovered the scopolamine-induced imbalance in the expression of BDNF, p-ERK (an activated form of ERK), p-CREB (an activated form of CREB) and ChAT in the hippocampus. Therefore, 4-FHA could improve scopolamine-induced memory impairment by activating BDNF/ERK/CREB and ChAT signaling.

Among HDAC6 substrates, α-tubulin, a key component of microtubules, has emerged as a potential target for treating neurodegenerative diseases (Kumar et al., 2022). Acetylation of α-tubulin enhances axonal transport by increasing microtubule stability, a critical factor in AD pathophysiology (Berth and Lloyd, 2023). Several studies have reported that the expression of α-tubulin acetylation is reduced in the brain tissue of AD patients and disease model animals (Hempen and Brion, 1996; Govindarajan et al., 2013; Zhang et al., 2015). HDAC6 inhibition increases the anterograde axonal transport of vesicles containing cargo proteins, including BDNF, by promoting α-tubulin acetylation (Dompierre et al., 2007; Xu et al., 2014). This inhibition has been reported to rescue learning and memory impairments in a mouse model of AD (Zhang et al., 2024a). In addition to α-tubulin acetylation, biological benefits of HDAC6 inhibition have been reported in several studies. HDAC inhibitors, such as sodium butyrate, have been shown to increase p-CREB levels in ischemic brain tissue (Kim et al., 2009). In an AD model using APOE4 and Aβ1-40 co-injection, HDAC6 inhibition significantly increased ChAT mRNA expression in the hippocampus (Ding et al., 2019). In this study, scopolamine did not affect the expression of α-tubulin acetylation. Considering these previous findings, the cognitive function and memory-enhancing effects of 4-FHA observed here may be attributed to BDNF and ChAT pathways regulation by HDAC6 inhibition rather than physiological responses related to α-tubulin acetylation.

Since SAHA has low blood-brain barrier permeability (Zhang et al., 2024b), the increased α-tubulin acetylation in the brain caused by a lower dose of 4-FHA suggests that 4-FHA has higher brain penetration ability. This can explain the better efficacy of 4-FHA in NORT, PAT, and MWMT. These potential advantages, coupled with the higher HDAC6 selectivity of 4-FHA, exhibit superior pharmacological effects in several behavioral assessments than SAHA.

The present results indicate that 4-FHA improves scopolamine-induced memory impairments and cognitive deficits in the Y-maze test, NORT, NPRT, PAT, and MWMT. Said improvements might result from 4-FHA’s enhancement of BDNF/ERK/CREB signaling and ChAT expression, which is reduced by scopolamine (Fig. 7). Therefore, 4-FHA could be a novel therapeutic candidate for improving the memory impairment and cognitive decline typical of AD.

Figure 7. Schematic diagram for potential pharmacological mechanisms of 4-FHA (created with BioRender.com).

This research was supported by the National Research Foundation of Korea (2022R1A6A1A03054419 and 2019R1A5A2027340).

The authors have no conflicts of interest to declare.