Editor—Evidence suggests that inflammation plays important roles in Alzheimer's disease (AD)1 and postoperative cognitive dysfunction associated with anaesthesia and surgery.2 Dantrolene, the only drug used to treat malignant hyperthermia in anaesthesia practice, is neuroprotective in AD animal models, especially when administered intranasally as a nanoparticle formulation.3, 4, 5, 6 Although dantrolene has been shown to inhibit inflammation in a sepsis model,7 its effects and potential mechanisms on pathological inflammation and associated programmed cell death by pyroptosis in the CNS have not been investigated.
Dantrolene is an antagonist of ryanodine receptor (RyRs) Ca2+ channels, and potently inhibits excessive Ca2+ release from intracellular stores. The ability of dantrolene to upstream Ca2+ dysregulation owing to glutamate excitotoxicity in different neurodegenerative diseases and to stabilise intracellular Ca2+ homeostasis renders dantrolene a drug capable of inhibiting downstream mitochondrial dysfunction, oxidative stress, activation of the inflammatory pyroptosis pathway and associated pyroptosis, and synapse destruction.8 This is especially important when the number of RyRs is pathologically increased in AD patient brain.9 Dantrolene in a crystalline nanoparticle formulation (Ryanodex®, Eagle Pharmaceutical Company, Woodcliff Lake, NJ, USA) significantly increased its brain–blood concentration ratio, promoting its neuroprotective effects in the CNS while minimising its peripheral side-effects or organ toxicity.3,10 We investigated the effects of intranasal dantrolene nanoparticles on Ca2+ channels in the endoplasmic reticulum (ER) membrane (RyRs and InsP3 receptors [InsP3Rs]), inflammation and associated pyroptosis, and synaptic proteins in brains of 5XFAD AD model mice.
ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were followed. 5XFAD and wild-type (WT) B6SJLF1/J mice were treated with intranasal dantrolene nanoparticles (5 mg kg−1) daily, Monday to Friday, for 12 weeks continuously starting at 9 months of age. Blood and brain samples were harvested at 13 months of age, 1 month after completion of 12 weeks of intranasal dantrolene nanoparticle treatment. Blood biomarkers related to the function of the liver (alanine transaminase [ALT]), kidney (creatinine), and thyroid (thyroid-stimulating hormone [TSH]) were measured using ELISA. Changes in whole brain tissue proteins related to pyroptosis activation regulatory proteins (NLR family pyrin domain containing 3 [NLRP3], cleaved caspase-1, full-length and N-terminal of gasdermin D [GSDMD]), pyroptosis-related cytotoxic cytokines (IL-1β, IL-18), and synapse proteins (PSD-95, synapsin-1) were determined using immunoblotting using specific antibodies targeting various proteins in Supplementary Table 1. In addition, Ca2+ release channels on the ER membrane (RyRs-2 and InsP3R-1), oxidative stress represented by the lipid peroxidation byproduct malondialdehyde (MDA)- and 4-HNE modified proteins, other cytotoxic (IL-6, TNF-α) and cytoprotective (IL-10) cytokines, biomarkers for astrogliosis (GFAP) and microgliosis (IBA-1) were measured using immunoblotting. Body weights were monitored regularly.
As shown in Fig 1, compared with WT mice, pyroptosis regulatory proteins (NLRP3, cleaved caspase-1, N-terminal GSDMD) and pyroptosis-related cytotoxic cytokines (IL-1β, IL-18) were pathologically and significantly increased in 5XFAD mice brains. Intranasal dantrolene nanoparticle treatment inhibited the pathological increases of NLRP3 by 41% (0.79 vs 0.46, P=0.0021), cleaved caspase-1 by 58% (0.74 vs 0.31, P=0.0008), N-GSDMD by 43.8% (0.85 vs 0.48, P=0.0041), IL-1β by 29.9% (0.94 vs 0.66, P=0.0042), and IL-18 by 65.5% (0.82 vs 0.28, P=0.0003). Intranasal dantrolene nanoparticles also increased PSD-95 by 32.1% (1.03 vs 1.36, P=0.0035), and synapsin-1 by 32.5% (0.98 vs 1.30, P=0.0080) (Fig 1) in 5XFAD mice brains, and inhibited pathological increases in RyR-2 and InsP3R-1 (Supplementary Fig. S1), lipid peroxidation byproduct MDA-modified proteins and 4-HNE (Supplementary Fig. S2), cytotoxic (IL-6, TNF-α) vs cytoprotective (IL-10) cytokines (Supplementary Fig. S3), and biomarkers for astrogliosis (GFAP) and microgliosis (IBA-1) (Supplementary Fig. S4). Intranasal dantrolene nanoparticles for 12 weeks did not significantly change blood biomarkers for function of liver, kidney, or thyroid, nor did it affect body weight (Supplementary Fig. S5).
Fig 1.
Dantrolene inhibits pathological activation of the pyroptosis pathway and synapse loss in 5XFAD mice brains. Wild-type (WT) or 5XFAD mice at 9 months old were treated with intranasal dantrolene nanoparticles (IN-DAN, 5 mg kg−1), daily, Monday to Friday for 12 consecutive weeks. Mice brains were harvested at 13 months old. Representative immunoblots and associated statistical analysis were used to determine changes of critical regulatory proteins of pyroptosis activation pathway. (a and b): NLR family pyrin domain containing 3 (NLRP3); (c and d): cleaved caspase-1 (p-20); (e and f): gasdermin D full-length (GSDMD-FL); (g and h): gasdermin D N-terminal (GSDMD-NT); (i and j): IL-1β; (k and l): IL-18; (m and n): PSD-95; (o and p): synapsin-1 (SYN-1). GAPDH was used as the loading control. Data are mean (sd) from four separate mice brains (N=4) in each experimental group and were analysed using two-way anova followed by Tukey's multiple comparison test. P<0.05 is considered statistically significant.
These findings suggest that intranasal dantrolene nanoparticles significantly inhibit pathological increases of pyroptosis activation proteins and pyroptosis-related neurotoxic cytokines and synapse destruction in 5XFAD mice brains, providing a novel mechanism of dantrolene neuroprotection in AD. Intranasal dantrolene nanoparticle treatment for up to 3 months did not affect thyroid or kidney function, suggesting its chronic use might be safe, as supported by other studies.3,10,11 NMDA-type glutamate receptor overactivation by excessive glutamate and associated pathological elevation of cytosol and mitochondria Ca2+ concentration and ER Ca2+ depletion have been considered a primary upstream signal leading to different downstream pathologies in various neurodegenerative diseases.12 Dantrolene inhibited NMDA receptor-mediated elevation of cytosolic Ca2+ concentrations by up to 78% in primary cultured cerebral cortical neurones,13 suggesting dantrolene inhibition of NMDA receptor overactivation and subsequent excitotoxicity and associated pathology.
Dantrolene has been shown to be neuroprotective in multiple neurodegenerative diseases and some psychiatric disorders such as depression and anxiety.8,14 Recent studies have proposed that intranasal administration of dantrolene nanoparticles enhances dantrolene crossing the blood–brain barrier and increases dantrolene brain–blood concentration ratio, thus promoting its neuroprotective effects in the CNS while minimising peripheral side-effect or organ toxicity.3,10 Thus, intranasal dantrolene is expected to be neuroprotective in various neurodegenerative diseases, which is also convenient to be used in clinical studies considering dantrolene is already an approved drug. Intranasal esketamine, a recently approved drug for the treatment of major depression, is an NMDA receptor antagonist,15 which suggests that intranasal dantrolene nanoparticles might also treat major depression disorder. The limitation of the current study includes a missing WT mice group treated with dantrolene, low sample size, lack of behavioural studies of cognitive function, missing examination of amyloid pathology, neurogenesis, and measurement of Ca2+ concentrations.
In conclusion, intranasal dantrolene nanoparticles inhibited pathological increases of proteins in the pathway leading to programmed cell death by pyroptosis and synaptic protein loss in 5XFAD mice brains, without side-effects or organ toxicity in the liver, kidney, and thyroid with chronic use. These beneficial effects of intranasal dantrolene nanoparticles were associated with inhibition of pathological increases in RyR-2 and InsP3R-1 Ca2+ channels, oxidative stress, and biomarkers on gliosis and astrogliosis. This study provides a new mechanism of dantrolene neuroprotection in AD or other neurodegenerative diseases.
Authors’ contributions
Study design and conception: HW
Conducted experiments: PB, WZ, RS, KK, YW, GL
Data acquisition: PB, WZ, RS, KK, YW, GL, Data analysis: PB, WZ, RS, KK, YW, GL, HW
Manuscript preparation: PB, WZ, RS, KK, YW, GL, HW
Manuscript reviewed and approval of the final manuscript: all authors
Funding
The US National Institute on Aging (NIA: R01AG061447 and a NIA R01 Supplemental grant 3R01AG061447-03S1 to HW).
Declaration of interests
HW and GL are listed as inventors on US patent applications entitled ‘Intranasal Administration of Dantrolene for Treatment of Alzheimer's Disease’ in multiple countries by The Trustees of the University of Pennsylvania.
Acknowledgements
Studies performed in the laboratory of HW and should be attributed to the Department of Anesthesiology, University of Pennsylvania (Philadelphia, PA, USA). We appreciate technical support from Yutong Yi, an undergraduate student at UPENN.
Handling Editor: Hugh C Hemmings Jr
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bja.2025.02.018.
Appendix A. Supplementary data
The following is the Supplementary data to this article.
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