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Mol Brain
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Molecular Brain
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BioMed Central London

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10.1186/s13041-024-01137-y
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Felodipine attenuates neuroinflammatory responses and tau hyperphosphorylation through JNK/P38 signaling in tau-overexpressing AD mice
Hwang Jeong-Woo 12
Kim Jeongha 12
Park Jin-Hee 1
Nam Jinhan 1
Jang Ji-Yeong 123
Jo Aran 12
Lee Hyun-ju hjlee@kbri.re.kr

12
http://orcid.org/0000-0001-8877-8594
Hoe Hyang-Sook sookhoe72@kbri.re.kr

123
1 https://ror.org/055zd7d59 grid.452628.f 0000 0004 5905 0571 Department of Neural Development and Disease, Korea Brain Research Institute (KBRI), 61, Cheomdan-ro, Dong-gu, Daegu, 41068 Republic of Korea
2 https://ror.org/055zd7d59 grid.452628.f 0000 0004 5905 0571 AI-Based Neurodevelopmental Diseases Digital Therapeutics Group, Korea Brain Research Institute (KBRI), 61, Cheomdan-ro, Dong-gu, Daegu, 41062 Korea
3 https://ror.org/03frjya69 grid.417736.0 0000 0004 0438 6721 Department of Brain Sciences, Daegu Gyeongbuk Institute of Science & Technology, Daegu, 42988 Korea
2 9 2024
2 9 2024
2024
17 6211 7 2024
19 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
We previously demonstrated that felodipine, an L-type calcium channel blocker, inhibits LPS-mediated neuroinflammatory responses in BV2 microglial cells and wild-type mice. However, the effects of felodipine on tau pathology, a hallmark of Alzheimer's disease (AD), have not been explored yet. Therefore, in the present study, we determined whether felodipine affects neuroinflammation and tau hyperphosphorylation in 3-month-old P301S transgenic mice (PS19), an early phase AD mice model for tauopathy. Felodipine administration decreased tauopathy-mediated microglial activation and NLRP3 expression in PS19 mice but had no effect on tauopathy-associated astrogliosis. In addition, felodipine treatment significantly reduced tau hyperphosphorylation at S202/Thr205 and Thr212/Ser214 residues via inhibiting JNK/P38 signaling in PS19 mice. Collectively, our results suggest that felodipine significantly ameliorates tau hyper-phosphorylation and tauopathy-associated neuroinflammatory responses in AD mice model for tauopathy and could be a novel therapeutic agent for AD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13041-024-01137-y.

Keywords

Felodipine
Neuroinflammation
Tau
Microgliosis
Alzheimer's disease
KBRI24-BR-02-03 24-BR-02-12 23-BR-05-02 24-BR-03-01 24-BR-03-07 Hoe Hyang-Sook NSTCCL22061-100 Hoe Hyang-Sook NIPAH0301-24-1001 Hoe Hyang-Sook issue-copyright-statement© Min Zhuo, Bong-Kiun Kaang and BioMed central Ltd. 2024
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pmcMain text

The growing size of the older population is increasing the societal burden of Alzheimer's disease (AD), a degenerative brain disease [1]. Accumulating evidence suggests that abnormal regulation of calcium ion (Ca2+) channels is involved in development of neurovegetative disease [2, 3]. In particular, the failure of L-type calcium channels (LTCCs) is linked to aging and AD [4] and calcium imbalance can promote neurofibrillary tangle (NFT) formation and Aβ deposition [5]. The potent L-type calcium channel (LTCC) blocker felodipine is an FDA-approved drug for treatment of hypertension [6]. Interestingly, we recently found that felodipine significantly alleviates lipopolysaccharide (LPS)-evoked microglial activation, proinflammatory cytokine production, and spatial memory deficits in vitro and/or wild-type mice [7]. However, the effects of felodipine on tau pathology and tau-mediated neuroinflammatory responses have not been explored in a mouse model of AD.

In the present study, we investigated the effects of felodipine on neuroinflammation and tau hyperphosphorylation and its mechanisms of action in P301S transgenic mice (PS19), a model of AD overexpressing human mutant tau. To test this, Tau Tg PS19 mice were injected with vehicle (5% DMSO + 5% PEG + 5% Tween20 + 85% D.W., i.p.) or felodipine (5 mg/kg, i.p.) daily for 14 days, and immunofluorescence (IF) staining was conducted with an anti-Iba-1 and anti-GFAP antibody. Felodipine treatment significantly reduced Iba-1 fluorescence intensity, Iba-1-labeled area and the number of Iba-1-positive cells (Fig. 1A-B). However, felodipine injection did not alter GFAP fluorescence intensity in Tau Tg PS19 mice (Supplementary Fig. 1). These data suggest that felodipine administration suppresses tauopathy-mediated microgliosis in Tau Tg PS19 mice but not astrogliosis.Fig. 1 Felodipine treatment signficantly suppresses microgliosis, NLRP3 expression and tau hyperphosphorylation by regulating JNK/P38 signaling in Tau Tg PS19 mice. Three-month-old PS19 mice were injected with vehicle (5% DMSO + 5% PEG + 5% Tween20 + 85% D.W., i.p.) or felodipine (5 mg/kg, i.p.) daily for 14 days. A, C Immunofluorescence staining was performed with anti-Iba-1 and anti-NLRP3 antibodies. B Quantification of data in A (n = 23–24 brain slices from 6 mice/group). D Quantification of data in C (n = 24 brain slices from 6 mice/group). E The relative mRNA levels of the indicated genes were analyzed by real-time PCR (n = 9–10/group). F–G Western blotting of RIPA-soluble/insoluble brain lysates was conducted with anti-AT8 and anti-AT100 antibodies (n = 8 mice/group). H, J Immunofluorescence staining was performed with anti-AT8 and anti-AT100 antibodies. (I) Quantification of data in H (n = 24 brain slices from 6 mice/group). K Quantification of data in J (n = 23–24 brain slices from 6 mice/group). L–O Western blotting of brain lysates was conducted with anti-p-JNK, anti-JNK, anti-p-P38, anti-P38, anti-p-CDK5, anti-CDK5, anti-p-CaMKIIα, and anti-CaMKIIα antibodies (n = 8 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar = 100 µm

NLRP3 is an important molecular target for inhibiting neuroinflammatory responses [8]. Activation of NLRP3 expression results in upregulation of IL-1β, which induces NLRP3 inflammasome complex formation and accelerates AD progression [9]. The NLRP3 inflammasome activates Aβ-induced tau pathology and neurodegeneration in vivo [10, 11]. Activation of NLRP3 inflammasome requires Ca2+ signaling, which leads to IL-1β secretion. Interestingly, we recently found that injection of the L- and T-type calcium channel blocker lomerizine significantly inhibits LPS-induced NLRP3 expression in wild-type mice [12]. In this study, we thus examined whether the L-type calcium channel blocker felodipine modulates NLRP3 expression in a mouse model of AD. For this experiments, Tau Tg PS19 mice were injected with felodipine (5 mg/kg, i.p.) or vehicle daily for 14 days, and IF staining was performed with an anti-NLRP3 antibody. Felodipine administration significantly decreased NLRP3 fluorescence intensity in Tau Tg PS19 mice (Fig. 1C–D). In addition, felodipine treatment decreased NLRP3 mRNA levels in the cortex and hippocampus region of Tau Tg PS19 mice (Fig. 1E), suggesting that felodipine treatment may downregulate tauopathy-associated neuroinflammatory responses by inhibiting NLRP3 expression. However, we did not determine whether felodipine treatment regulates the NLRP3 inflammasome complex formation. Thus, it is possible that felodipine-treated Tau Tg PS19 mice may suppresses neuroinflammatory responses by regulating NLRP3 inflammasome complex formation. Other possibility is that felodipine may regulates other neuroinflammation-associated molecular targets to regulate neuroinflammatory responses in Tau Tg PS19 mice, thus we will address in a future study.

Tau hyperphosphorylation is a hallmark of AD and a major target of efforts to develop AD drugs. Abnormal phosphorylation of tau leads to the formation of NFTs, aggregates of hyperphosphorylated tau [13]. The association between calcium channels and tau was first suggested by reports that okadaic acid, a phosphatase inhibitor, activates LTCCs and increases tau phosphorylation [14, 15]. Here, we therefore investigated the effects of felodipine treatment on tau hyperphosphorylation in RIPA-soluble and RIPA-insoluble fractionation of cortex and hippocampus from Tau Tg PS19 mice. We found that felodipine treatment significantly reduced RIPA-soluble tau hyperphosphorylation at Ser202/Thr205 (AT8) and Thr212/Ser214 (AT100) residues in the cortex and hippocampus regions, but not RIPA-insoluble tau levels (Fig. 1F–G). To further confirm our findings as above, we conducted IF staining and found that felodipine-treated Tau Tg PS19 mice significantly decreased tau hyperphosphorylation at Ser202/Thr205 (AT8) and Thr212/Ser214 (AT100) in the cortex and hippocampus (Fig. 1H–K), suggesting that felodipine regulates tauopathy in early phase AD mice model. In this study, we did not examine whether felodipine administration inhibits NFT formation or whether felodipine reduces tau hyperphosphorylation in an aged Tau Tg PS19 mice. Thus, we will investigate the effects of felodipine on tau hyperphosphorylation and/or NFT formation in aged Tau Tg PS19 mice.

Tau kinase activity and JNK/P38 signaling are associated with hyperphosphorylation of tau [16]. Therefore, inhibiting tau kinase activity or JNK/P38 signaling is involved in suppressing tau inclusion therefore being an therapeutic strategy for AD treatment [17]. To address this, we investigated the effects of felodipine on JNK/P38 signaling and found that felodipine-treated Tau Tg PS19 mice significantly downregulated JNK phosphorylation in cortex and hippocampus (Fig. 1L). In addition, felodipine-treated PS19 mice showed decreased P38 phosphorylation in cortex but not in hippocampus (Fig. 1M). However, felodipine did not alter phosphorylation of tau kinases including CDK5 and CaMKIIα in hippocampus of PS19 mice (Fig. 1N–O). These data suggest that felodipine alleviates tauopathy by inhibiting JNK/P38 signaling in Tau Tg PS19 mice. Of course, it is possible that felodipine-treated Tau Tg PS19 mice modulate other tau kinases (i.e., DYRK1A and GSK3β) to alter tau pathology in a mouse model of AD. In future work, we will explore whether felodipine regulates tauopathy in L-type calcium channer (on target)-dependent manner by using AAV shRNA knockdown vector system in AD mice model. In addition, we will investigate the effect of felodipine on various AD pathologies including synaptic loss, mitochondrial dysfunction, autophagy malfunction, metal dyshomeostasis, hormonal imbalance, and oxidative stress in AD mice model. Furthermore, we will assess how the regulatory effect of felodipine on these AD pathologies affect cognitive function via multiple behavioral tests such as Y maze, novel object recognition test, passive avoidance test, and fear conditioning test in AD mice model.

In conclusion, we demonstrated that administration of felodipine, an LTCC blocker, inhibits tauopathy-mediated microglial activation and neuroinflammation-associated molecular target NLRP3 expression in Tau Tg PS19 mice. Importantly, felodipine treatment significantly reduced tau inclusion by suppressing JNK/P38 phosphorylation in Tau Tg PS19 mice. Collectively, our data suggest that felodipine treatment alleviates neuroinflammatory responses and tau pathology in a mouse model of AD.

Supplementary Information

Supplementary material 1

Abbreviations

AD Alzheimer’s disease

LTCCs L-type calcium channels

CDK Cyclin-dependent kinase

GSK3β Glycogen synthesis kinase 3 beta

JNK C-Jun N-terminal kinase

CaMKIIα Calcium/calmodulin-dependent protein kinase II α

Acknowledgements

The cryostat (Leica CM1850, Wetzlar, Germany) was supported by the Brain Research Core Facilities at the Korea Brain Research Institute (KBRI). We thank previous and current members of the neurodegenerative disease’s lab for editing and valuable comments on our manuscript and for technical assistance with in vitro and in vivo studies.

Author contributions

H.J.L and H.S.H. conceived and participated in the design of the study. J.W.H., J.H.K., J.H.P., J.H.N., J.Y.J., A.R.J., H.J.L and H.S.H. wrote the manuscript. J.W.H., J.H.K., J.H.P., H.J.L., and J.H.N. conducted in vivo experiments, immunofluorescence staining, western blot and real time PCR. J.W.H., J.H.K., J.H.P., J.H.N., J.Y.J., and A.R.J. performed statistical analysis. J.W.H., J.H.K., J.H.P., J.H.N., J.Y.J., and A.R.J. generated figures. All authors read and approved the final manuscript.

Funding

This work was supported by the KBRI basic research program through KBRI funded by the Ministry of Science, ICT & Future Planning (grant numbers 24-BR-02-03, 24-BR-02-12, 23-BR-05-02, 24-BR-03-01, 24-BR-03-07, H.S.H.), and a National Research Council of Science & Technology (NST) grant funded by the Korean government (CCL22061-100, H.S.H.). This work is also supported by the Korea Ministry of Science and ICT's Special Account for Regional Balanced Development for Commercialization supervised by the NIPA (National IT Industry Promotion Agency) to support digital medical devices for AI-based Neurodevelopmental disorders (H0301-24-1001).

Availability of data and materials

All data generated and/or analyzed during this study are included in this published article and its supplementary materials. Materials and methods are presented in the supplementary materials.

Declarations

Ethics approval and consent to participate

All experimental procedures were approved by the institutional biosafety committee (IBC) and performed in accordance with approved animal protocols of the Korea Brain Research Institute (KBRI, Approval No. IACUC-22-00046).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jeong-Woo Hwang, Jeongha Kim and Jin-Hee Park have contributed equally to this work.
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