Alzheimer’s disease (AD) is one of the most representative age-related neurodegenerative diseases accompanied by memory and cognitive deficits (Hippius and Neundörfer, 2003). Although Alois AD has been known for more than a century, there is still no effective treatment for it. Several factors contribute to AD. The most notable hypotheses for AD development are the amyloid hypothesis and the hyperphosphorylated tau hypothesis (Bloom, 2014; DeTure and Dickson, 2019). The deposition of both amyloid-β peptide (Aβ) and tau neurofibrillary tangles causes several pathological symptoms, including synaptic degradation, neuroinflammation, and neuronal cell loss, which induce memory and cognition impairment and finally develop into severe dementia (LeBlanc, 2005).

In addition to amyloid and hyperphosphorylated tau, calcium (Ca²⁺) dyshomeostasis is deeply implicated in the pathogenesis of AD (Bojarski et al., 2008; Cascella and Cecchi, 2021; Green and LaFerla, 2008). Ca²⁺ acts as an intracellular messengers and involves diverse signal transmission activities, including enzyme activation (through phosphorylation or dephosphorylation), exocytosis, gene transcription and programmed cell death (Baker et al., 2013). These actions of intracellular Ca²⁺ signaling in neurons affect neural growth, memory formation, and synaptic plasticity. In other words, Ca²⁺ dyshomeostasis causes damage and eventually leads to cell death in the hippocampus.

It has been reported that soluble Aβ aggregates and increases neuronal Ca²⁺ influx in patients with AD, resulting in Ca²⁺ overload in neurons (Berridge, 2011). As a result, resting Ca²⁺ increases lead to Ca²⁺ dysregulation, which includes several pathological features such as neuronal death and degeneration (Calvo-Rodriguez et al., 2020; Jadiya et al., 2019). Previously, we focused on these aspects and tried mediating AD pathogenesis by regulating Ca²⁺ levels through transient receptor potential vanilloid 1 (TRPV1), a Ca²⁺-permeable nonselective cation channel well-known for regulating pain and inflammation. A triple-transgenic AD (3xTg-AD^+/+) mouse model was used to characterize the anti-AD effect of TRPV1 deficiency (Kim et al., 2020). In that study, TRPV1 deficiency alleviated Aβ and Tau deposition and consequently improved memory and learning deficits in the 3xTg-AD^+/+ mouse model. Additionally, it was shown that Ca²⁺ was overloaded in 3xTg-AD^+/+ mice, resulting in memory and learning deficits. However, in TRPV1 receptor knockout 3xTg-AD mice (3xTg-AD^+/+/TRPV1^−/−), after normalized Ca²⁺ levels, memory and learning abilities were restored.

The hippocampus is a curved-shaped region in the middle of the brain, essential for learning and memory. Therefore, damage to the hippocampus can lead to severe memory deficits, including memory formation and consolidation. Decreased hippocampal synaptic density in Patients with AD has been reported, which is highly correlated with cognitive deficits (Scheff and Price, 2006). Additionally, several causes of neuronal apoptosis, such as Aβ accumulation, increased oxidative stress, and DNA fragmentation, have also been reported in the brain of Patients with AD (Calissano et al., 2009). These previous studies showed that apoptosis in the brain is closely related to cognitive ability in patients with AD.

Brain-derived neurotrophic factor (BDNF) is a major neurotrophic factor that regulates neuronal survival, differentiation, and plasticity (Miranda et al., 2019). The expression of BDNF and its receptor, tyrosine receptor kinase B (TrkB), is reduced in Patients with AD (Ferrer et al., 1999; Zheng et al., 2010). There is also evidence that Aβ directly or indirectly reduces BDNF levels, leading to memory and cognition deficits (Corona et al., 2010). Therefore, the expression levels of BDNF and TrkB are important for evaluating the neuroprotective effect of TRPV1 deficiency in AD models.

TrkB activation triggers the activation of several downstream pathways, including phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt), which in turn phosphorylates and activates cAMP response element binding protein (CREB) (Minichiello et al., 2002; Santos et al., 2010). ERK/MAPK (mitogen-activated protein kinase) is related to memory formation (Impey et al., 1999) and antiapoptotic effects (Park and Cho, 2006). Akt also plays an important role in neuroprotection (Zarneshan et al., 2022). CREB is a transcription factor that regulates the proliferation or survival of neurons by binding to cAMP-responsive elements, thereby modulating the transcription of the genes (Finkbeiner et al., 1997).

B-cell lymphoma 2 (Bcl-2) family proteins play a critical role in the apoptotic activity. The Bcl-2 family includes both the antiapoptotic protein Bcl-2 and the proapoptotic protein Bcl-2-associated X (Bax) or Bcl-2 homologous antagonist killer (Bak) (Shacka and Roth, 2005). When CREB is phosphorylated, it directly binds to the Bcl-2 family promoter, activating the signaling cascade (Meller et al., 2005). Bcl-2 forms a heterodimer with the apoptotic protein Bax and inhibits the activation of Bax, which regulates the activation of caspase-3 (Ku et al., 2011; Yin et al., 1994). Cleaved caspase-3 is known to be involved in the death of mammalian neural cells (D’Amelio et al., 2012). Activated caspase-3 cleaves cellular proteins, including poly (ADP-ribose) polymerase (PARP). Because PARP is a crucial enzyme in the repair of DNA breakage, its cleavage causes neurons to undergo apoptosis (Sairanen et al., 2009).

In this study, we aimed to understand how neurological parameters are related to the neuroprotective effect by preventing apoptosis in the hippocampus by using TRPV1 receptor knockout 3xTg-AD mice to explore the effect of TRPV1 deficiency in the hippocampus. It was found that TRPV1 deficiency inhibits hippocampal cell death by promoting the BDNF/TrkB signaling pathway. Furthermore, activating TrkB leads to the activation of CREB, which inhibits the activated caspase-3 dependent apoptosis.

Animals

3xTg-AD mice (kindly given by Professor Frank M. Laferla, UC-Irvine, Irvine, CA, USA) were homozygous for APPswe, TauP301L, and PS1M146V. These 3xTg-AD mice were mated with TRPV1^−/− mice (stock #003770; provided from Jackson Laboratories, Bar Harbor, ME, USA) and offspring with nine genotypes (3xTg-AD^−/−/TRPV1^+/+, 3xTg-AD^−/−/TRPV1^+/−, 3xTg-AD^−/−/TRPV1^−/−, 3xTg-AD^+/−/TRPV1^+/+, 3xTg-AD^+/−/TRPV1^+/−, 3xTg-AD^+/−/TRPV1^−/−, 3xTg-AD^+/+/TRPV1^+/+, 3xTg-AD^+/+/TRPV1^+/−, 3xTg-AD^+/+/TRPV1^−/−) were generated. Four groups were selected with or without AD pathology and TRPV1 depletion. The selected groups were 3xTg-AD^−/−/TRPV1^+/+ mice (wild type, WT), 3xTg-AD^+/+/TRPV1^+/+ mice, 3xTg-AD^+/+/TRPV1^+/− mice and 3xTg-AD^+/+/TRPV1^−/− mice. Mice in each group were raised until 12 months. Mice were maintained at 21°C ± 2°C, 12-h light:12-h dark photoperiod with specific pathogen-free conditions in ventilated cages and provided with free choice to consume food and water. All animal testing was performed following protocols approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-140410-12, SNU-140410-14, SNU-140625-2).

Animal dissection and tissue collection

Zoletil (50 mg/kg) was administered intraperitoneally to anesthetize the mice (n = 5) in each group before normal saline, and a 4% paraformaldehyde solution was transcardially administered. The brains were removed and postfixed in 4% paraformaldehyde overnight at 4°C, and routine-frozen sections (10 μm) were prepared for TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining and immunohistochemistry analysis. The other half of the mice in each group were sacrificed by decapitation, and their brains were rapidly collected and cut on an ice-cooled board. The hippocampus was dissected and stored at −80°C for TUNEL staining and western blotting analysis.

TUNEL staining

TUNEL staining was performed on the frozen-embedded sections using the in-situ cell death detection kit (Promega, USA) according to the standard protocol provided by the manufacturer. Apoptotic nuclei were visualized using the peroxidase-DAB reaction. The sections were counterstained with hematoxylin. TUNEL-positive cells in the hippocampus were analyzed in each of the 3 regions of Cornu Ammonis 3 (CA3), Cornu Ammonis 1 (CA1) (especially the soma of pyramidal neurons), and dentate gyrus (DG) sections per high-power field (×40). TUNEL staining fluorescence signal intensity was measured using the ImageJ analysis program (National Institute of Health, USA). Regions of interest were selected from the hippocampus’s CA3, CA1, and DG regions.

Western blotting

Five mice’s hippocampus tissues were removed, and they were all individually examined. There was no replication. Samples were prepared by homogenizing the hippocampus tissues. The protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, USA). Western blotting analyses were performed on 50 μg of protein. Samples were briefly separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (0.4 μm; Merck Millipore, USA). The membranes were blocked in a 5% bovine serum albumin (Bovogen Biologicals, Australia) in tris-buffered saline and Tween-20 (Junsei Chemical, Japan). Membranes were incubated overnight at 4°C with primary antibodies against BDNF (1:1,000; Santa Cruz Biotechnology, USA), p-TrkB (1:1,000; Cell Signaling Technology, USA), TrkB (80E3, 1:1,000; Cell Signaling Technology), p-Akt (Ser473, 1:1,000; Cell Signaling Technology), Akt (1:1,000; Cell Signaling Technology), p-ERK (E-4, 1:1,000; Santa Cruz Biotechnology), ERK (1:1,000; Santa Cruz Biotechnology), p-CREB (Ser133, D1G6, 1:1,000; Cell Signaling Technology), CREB (48H2, 1:1,000; Cell Signaling Technology), Bax (1:1,000; Santa Cruz Biotechnology), Bcl-2 (1:1,000; Santa Cruz Biotechnology), cleaved caspase-3 (1:500; Cell Signaling Technology), caspase-3 (1:500; Abcam, USA), cleaved PARP (1:1,000; Cell Signaling Technology), GAPDH (1:1,000; Cell Signaling Technology), and β-actin (1:5,000; Sigma, USA). After incubation with horseradish peroxidase-conjugated secondary antibodies (1:5,000; Sigma) for 1 h at room temperature, immunodetection was performed using an enhanced chemiluminescence detection kit (GE Healthcare, USA). The relative density of each band was measured using the ImageJ analysis program (National Institute of Health).

Statistical analysis

IBM SPSS Statistics for Windows (ver. 19.0; IBM, USA) was used for the statistical analyzes. The results were presented as mean ± SEM values. All the data were analyzed using the Fisher’s LSD (least significant difference).

TRPV1 deficiency reduces apoptosis in the hippocampus of 3xTg-AD mice

To investigate the neuroprotective effect of TRPV1 deficiency, we performed a TUNEL assay to detect hippocampal apoptosis in WT, 3xTg-AD^+/+/TRPV1^+/+, 3xTg-AD^+/+/TRPV1^+/−, and 3xTg-AD^+/+/TRPV1^−/− mice. CA3, CA1, and DG regions of the hippocampus were subjected. Nuclei of apoptotic cells in each region are shown in green fluorescence. The immunofluorescence results showed that TUNEL-positive apoptotic cells were remarkably increased in all hippocampus regions of 3xTg-AD^+/+/TRPV1^+/+ mice compared with WT mice. However, apoptotic cells tremendously decreased in all 3 regions of 3xTg-AD^+/+/TRPV1^−/− mice compared with 3xTg-AD^+/+/TRPV1^+/+ mice (Fig. 1A). Quantitative analysis also showed that TRPV1 deficiency significantly reduced apoptotic cells in CA3 (Fig. 1B), CA1 (Fig. 1C), and DG (Fig. 1D) regions by 67%, 67%, and 62%, respectively, compared with the same region in 3xTg-AD^+/+/TRPV1^+/+ mice. These results indicate that TRPV1 deficiency protects hippocampal neurons and neuroglial cells from apoptosis due to AD pathogenesis.

TRPV1 deficiency increases BDNF expression levels and promotes TrkB receptor phosphorylation in the hippocampus of 3xTg-AD mice

Western blotting analyses were conducted to examine whether TRPV1 deficiency increases the levels of BDNF and activation of its receptor protein TrkB. BDNF expression levels significantly decreased in 3xTg-AD^+/+/TRPV1^+/+ mice by 26% compared with 3xTg-AD^−/−/TRPV1^+/+ mice. However, BDNF expression levels significantly increased in 3xTg-AD^+/+/TRPV1^−/− mice by 625% compared with 3xTg-AD^+/+/TRPV1^+/+ mice (Figs. 2A and 2B). Similarly, p-TrkB expression levels proportional to TrkB and β-actin significantly decreased in 3xTg-AD^+/+/TRPV1^+/+ mice by 40% and 29%, respectively compared with 3xTg-AD^−/−/TRPV1^+/+ mice. p-TrkB expression levels also significantly increased in 3xTg-AD^+/+/TRPV1^−/− mice by 268% and 310% respectively compared with 3xTg-AD^+/+/TRPV1^+/+ mice (Figs. 2C and 2D). These results indicate that TRPV1 deficiency increases the levels of BDNF and promotes the phosphorylation of TrkB in the hippocampus of 3xTg-AD mice. As previously mentioned, BDNF is responsible for synaptic plasticity and neuronal survival and is reported to be impaired in patients with AD. In this respect, BDNF-signaling recovery is considered a promising therapeutic approach to treating AD (Fumagalli et al., 2006). Memantine, a well-known AD drug, has also been reported to reduce preclinical AD pathology by increasing BDNF protein levels, supporting the usefulness of our results (Folch et al., 2018).

TRPV1 deficiency increases the phosphorylation of ERK, Akt, and CREB

To investigate whether the activation of TrkB leads to activation of the downstream signaling of BDNF/TrkB, we measured the phosphorylation level of Akt and ERK. Akt and ERK independently mediate the BDNF/CREB pathway, where the downstream protein CREB plays an important role in memory formation by regulating BDNF (Saura and Valero, 2011). Western blotting revealed that the levels of p-Akt and p-ERK were decreased in the hippocampus of 3xTg-AD^+/+/TRPV1^+/+ mice compared to 3xTg-AD^−/−/TRPV1^+/+ mice, and the difference disappeared in 3xTg-AD^+/+/TRPV1^+/− and 3xTg-AD^+/+/TRPV1^−/− mice (Fig. 3A). The densitometry results show that TRPV1 deficiency induces the phosphorylation of Akt (Fig. 3B) and ERK (Fig. 3C) whereas the basal levels of Akt and ERK did not change. We then investigated the expression and phosphorylation level of CREB, which plays an important role in mediating the apoptosic pathway. In 3xTg-AD^+/+/TRPV1^+/+ mice, CREB phosphorylation levels decreased compare with WT. However, CREB phosphorylation levels in 3xTg-AD^+/+/TRPV1^−/− mice were significantly increased, similar to those in WT mice. Previous studies have reported that Akt and ERK mediate signal transduction in various neurodegenerative diseases, including AD (Rai et al., 2019). Similarly, our results indicate that TRPV1 deficiency in the hippocampus of 3xTg-AD mice has a neuroprotective effect by phosphorylating and activating Akt and ERK, thereby activating the CREB pathway (Figs. 3D-3F).

TRPV1 deficiency reduces the cleavage of caspase-3 and PARP in 3xTg-AD mice

As shown in Fig. 1, TRPV1 deficiency reduces apoptosis in the hippocampus of 3xTg-AD mice. As an executioner of apoptosis, caspase-3 is an essential regulator in AD progression (Asadi et al., 2022). In this regard, we investigated how TRPV1 deficiency reduces apoptosis via the regulation of caspase-3 in the hippocampus of 3xTg-AD mice. Western blotting revealed that the level of Bcl-2 was decreased, whereas Bax, cleaved-caspase-3, and cleaved PARP increased in the hippocampus of 3xTg-AD^+/+/TRPV1^+/+ mice compared with 3xTg-AD^−/−/TRPV1^+/+ mice. These differences disappeared in 3xTg-AD^+/+/TRPV1^+/− and 3xTg-AD^+/+/TRPV1^−/− mice (Fig. 4A).

The densitometry results showed that Bcl-2 expression levels significantly increased in 3xTg-AD^+/+/TRPV1^−/− mice compared with 3xTg-AD^+/+/TRPV1^+/+ mice. Consequently, expression levels of apoptotic factors Bcl-2 (Fig. 4B), Bax (Fig. 4C), cleaved caspase-3 (Fig. 4D), and cleaved PARP (Fig. 4E) decreased in 3xTg-AD^+/+/TRPV1^−/− mice, which indicates that TRPV1 deficiency blocks apoptosis by activating Bcl-2 and downregulating Bax, cleaved caspase-3 and cleaved PARP expression in the hippocampus.

Memory and cognition deficits are well-known symptoms of AD, and apoptosis in the brain contributes to most of these deficits. Aβ and tau neurofibrillary tangles play a major role in memory and learning deficits. However, Ca²⁺ dyshomeostasis is reported to be another major cause of AD pathogenesis (Wang et al., 2017b). Ca²⁺ plays a critical role as a signal carrier regulating synaptic signaling and neurotransmission. It has been reported that Ca²⁺ is overloaded in Patients with AD (Ichimiya et al., 1988) and the 3xTg-AD mouse model (Jadiya et al., 2019), leading to memory and behavior disorders.

In a previous study, TRPV1 deficiency effectively restored Ca²⁺ levels in the hippocampus and consequently decreased Aβ and Tau deposition, resulting in anti-AD effects (Kim et al., 2020). Interestingly, there are reports regarding the effect of TRPV1 activation on AD pathology in which TRPV1 activation was shown to be beneficial in other AD mouse models by alleviating cognitive impairments in APP23/PS45 double transgenic AD model mice at the age of 4 months (Du et al., 2020) and alleviating microglia dysfunction in APP/PS1 mice at the age of 7-8 months (Lu et al., 2021). As calcium is an important secondary messanger for cellular growth and death through signal transduction, adequate influx through TRPV1 activation might have positive effects up to a moderate level. However, we used more severe AD-state animal model with 3xTg-AD mice at the age of 12 months. In this model, Aβ aggregation occurs more severely and moreover, tau pathology also occurs. Accordingly, calcium dyshomeostasis was already highly formed and as a result we assumed that TRPV1 dificiency, not activation, has positive effects in the hippocampus.

TRPV1 induces proliferation through Ca²⁺ entry, ATP release, membrane purinoceptor 2 (P2Y2) receptor activation and EGFR (epidermal growth factor receptor) activity. Activation of TRPV1 results in phosphorylation and activation of ATM serine-threonine kinase, which induces Fas signal. Fas is a subgroup of the TNF-R (tumor necrosis factor receptor) family which trigger apoptotic cell death. As a result, this causes mitochondrial damage and induces endoplasmic reticulum stress, resulting in apoptosis in the hippocampus (Zhai et al., 2020).

Memantine, an N-methyl-D-aspartate receptor (NMDAR) antagonist and treatment for AD, has also been reported to have neuroprotective effects by blocking excessive calcium influx into cells (Robinson and Keating, 2006). NMDAR is a ion channel with a high calcium permeability, in which overexpression of NMDAR accelerates cognitive dysfunction. Interestingly, NMDAR and TRPV1 both mediate signal pathway through Ca²⁺ influx. The activation of TRPV1 increases Ca²⁺ influx and activates CaMKII/protein kinase C (PKC) pathway. NMDAR consequently activated by PKC, which induces additional Ca²⁺ influx, which result in apoptosis in the brain. Similary, TRPV1 is also activated by the increased Ca²⁺ influx due to NMDAR activation, which inferred that NMDAR and TRPV1 affect each other, not in one direction. These relationship is also shown when the treatment of TRPV1 agonist capsaicin promotes NMDAR activation, whereas treatment of TRPV1 antagonist capsazepine blocks NMDAR activation by inhibiting phosphorylation of NMDAR subunit 2B, which suppresses neuroinflammation (Lee et al., 2012).

Subsequent studies also supported this neuroprotective effect of memantine in reducing hippocampal apoptosis (Wang et al., 2018). Additionally, these studies showed that memantine reduced caspase-3 activity (Wang et al., 2017a) by preventing the loss of BDNF function (Tanqueiro et al., 2018) and activating the BDNF/TrkB/Akt/ERK/CREB pathway (Jantas et al., 2009; Takahashi et al., 2018; Zhu et al., 2015).

Apoptosis is a form of cell death that occurs through a series of molecular steps within the cell (Gorman et al., 2000). TUNEL assay results show that TUNEL-positive cells were significantly increased in the hippocampus’s CA3, CA1, and DG regions in 3xTg-AD^+/+/TRPV1^+/+ mice. This increase was almost completely prevented by TRPV1 deficiency in all 3 regions of the hippocampus (Fig. 1). These results indicate that TRPV1 deficiency exerts a neuroprotective effect by preventing apoptosis in the hippocampus.

It has been reported that Aβ induces NMDAR activation, leading to intracellular Ca²⁺ overload, which activates calpain and reduces BDNF levels (Tanqueiro et al., 2018; Vanhoutte and Bading, 2003). This study hypothesized that TRPV1 deficiency protects neurons by inhibiting apoptosis in the hippocampus through Ca²⁺ normalization. Western blotting revealed that TRPV1 deficiency restores the protein expression of BDNF and the phosphorylation level of its membrane receptor TrkB (Fig. 2), suggesting that BDNF is involved in TRPV1 deficiency-mediated neuroprotective effects. Subsequent western blotting results show that TRPV1 deficiency increases the BDNF downstream of the intracellular proteins Akt and ERK phosphorylation, which activates the CREB (Fig. 3).

When CREB is phosphorylated, p-CREB binds to the Bcl-2 promoter and increases the Bcl-2 expression (Meller et al., 2005). Bcl-2 then binds directly to Bax and inhibits activation and oligomerization of Bax (Fletcher et al., 2008). Therefore, the apoptotic effect of Bax is blocked by the forming pores in the mitochondria (Antonsson et al., 1997). Additionally, Bax plays an essential role in activating caspase-3 by releasing cytochrome c from mitochondria, which interacts with the APAF-1 (apoptotic protease activating factor-1) and activates caspase-3 (Cregan et al., 1999; Elena-Real et al., 2018; Wang, 2001; Yuan et al., 2003). When caspase-3 is cleaved and activated, it cleaves cellular proteins, including PARP, that can repair damaged DNA (Sairanen et al., 2009). Thus, CREB phosphorylation upregulates Bcl-2 and downregulates Bax expression, preventing apoptosis (Eimer and Vassar, 2013; Porter and Jänicke, 1999). In this study, expression levels of Bcl-2 increased, whereas expression levels of the apoptotic factors Bax, cleaved caspase-3, and cleaved PARP decreased in 3xTg-AD^+/+/TRPV1^−/− mice compared to 3xTg-AD^+/+/TRPV1^+/+ mice (Fig. 4).

Generally, these findings suggest that TRPV1 deficiency has neuroprotective effects through BDNF/CREB signaling that mediates the Bcl-2 family-related antiapoptotic pathway in the hippocampus (Fig. 5).

In conclusion, a proposed molecular mechanism of neuroprotection by TRPV1 deficiency in 3xTg-AD mice is shown in Fig. 5. TRPV1 deficiency normalizes Ca²⁺ levels in the 3xTg-AD mouse model (Kim et al., 2020). Consequently, TRPV1 deficiency upregulates the BDNF/TrkB and downstream Akt/ERK/CREB signaling pathways. This signaling upregulates the antiapoptotic factor Bcl-2, which causes the levels of the apoptotic factors Bax, cleaved caspase-3, and cleaved PARP to decrease. In conclusion, TRPV1 deficiency protects hippocampal neurons from hippocampal apoptosis due to AD pathogenesis in the 3xTg-AD mouse model via the BDNF/CREB signaling pathway. Additionally, the normalization of Ca²⁺ levels by TRPV1 deficiency may be a therapeutic target to suppress memory loss and learning deficits in AD pathogenesis.

This research was supported by intramural grants (2E31881) from the Korea Institute of Science and Technology (KIST) and the Basic Science Research Program (2018R1D1A1B07050182) from the Ministry of Education in Korea.

J.K. (Juyong Kim) and S.S. contributed equally to this work and were responsible for the study design and performing the experiments. J.K. (Juyong Kim) interpreted the results, and S.S. wrote the initial draft of the manuscript. J.K. (Jiyoung Kim) designed this study to investigate the role of cell membrane-bound Ca²⁺ transporting-TRPV1 deficiency on 3xTg-AD and helped generate multiple genotypes of TRPV1 and 3xTg-AD. J.K. (Jiyoung Kim) also helped identify the involvement of BDNF and apoptosis markers as key factors for TRPV1-deficient 3xTg-AD, which showed improved memory in a previous study. J.H.Y.P. revised the manuscript. J.-C.K. contributed to the interpretation of the results. K.W.L. was responsible for the concept and design of the study, and supervised the work.

The authors have no potential conflicts of interest to disclose.